advance technology in autoimmunity tests · radiographic presence of pulmonary infiltrates or...
TRANSCRIPT
The world leader in serving science
Advance technology in autoimmunity tests Dr Chia-Ching Lin
Global marketing autoimmunity Immunodiagnostics division April 24th 2018
2
Sweden
bull Uppsala ndash Allergy
bull Global headquarters
bull PHC (Pharmaceutical and Healthcare Collaborations)
bull Helsingborg ndash Allergy
bull Allergon ndash Allergen raw material
Germany
bull Freiburg ndash Autoimmunity
Immunodiagnostics division - centers of excellence
3
The product range in autoimmunity
Connective tissue diseases
Rheumatoid arthritis
Anti-phospholipid syndrome
ANCA-associated vasculitis
Celiac disease
Inflammatory bowel diseases
Thyroid diseases
Autoimmune liver diseases
4
50 markers for gt 20 different
autoimmune diseases
5
Phadia Laboratory Systems
6
Pathogenesis of autoimmune diseases
bull Mostly T cells are the trigger
bull Autoantibodies are usually not triggers but useful markers
bull Genetic predisposition (specific HLA class II alleles)
bull More frequent in women ndash female hormones increase the risk disease often starts in times of hormonal changes
bull Possible triggers for AI diseases
bull Viral or bacterial infections (cross reactivity with common epitopes)
bull Wrong expression of MHC class II antigens of normal tissue cells
bull Vaccination
bull Antibiotics
bull hellip
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - ANCA-associated vasculitis
8
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
2
Sweden
bull Uppsala ndash Allergy
bull Global headquarters
bull PHC (Pharmaceutical and Healthcare Collaborations)
bull Helsingborg ndash Allergy
bull Allergon ndash Allergen raw material
Germany
bull Freiburg ndash Autoimmunity
Immunodiagnostics division - centers of excellence
3
The product range in autoimmunity
Connective tissue diseases
Rheumatoid arthritis
Anti-phospholipid syndrome
ANCA-associated vasculitis
Celiac disease
Inflammatory bowel diseases
Thyroid diseases
Autoimmune liver diseases
4
50 markers for gt 20 different
autoimmune diseases
5
Phadia Laboratory Systems
6
Pathogenesis of autoimmune diseases
bull Mostly T cells are the trigger
bull Autoantibodies are usually not triggers but useful markers
bull Genetic predisposition (specific HLA class II alleles)
bull More frequent in women ndash female hormones increase the risk disease often starts in times of hormonal changes
bull Possible triggers for AI diseases
bull Viral or bacterial infections (cross reactivity with common epitopes)
bull Wrong expression of MHC class II antigens of normal tissue cells
bull Vaccination
bull Antibiotics
bull hellip
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - ANCA-associated vasculitis
8
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
3
The product range in autoimmunity
Connective tissue diseases
Rheumatoid arthritis
Anti-phospholipid syndrome
ANCA-associated vasculitis
Celiac disease
Inflammatory bowel diseases
Thyroid diseases
Autoimmune liver diseases
4
50 markers for gt 20 different
autoimmune diseases
5
Phadia Laboratory Systems
6
Pathogenesis of autoimmune diseases
bull Mostly T cells are the trigger
bull Autoantibodies are usually not triggers but useful markers
bull Genetic predisposition (specific HLA class II alleles)
bull More frequent in women ndash female hormones increase the risk disease often starts in times of hormonal changes
bull Possible triggers for AI diseases
bull Viral or bacterial infections (cross reactivity with common epitopes)
bull Wrong expression of MHC class II antigens of normal tissue cells
bull Vaccination
bull Antibiotics
bull hellip
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - ANCA-associated vasculitis
8
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
4
50 markers for gt 20 different
autoimmune diseases
5
Phadia Laboratory Systems
6
Pathogenesis of autoimmune diseases
bull Mostly T cells are the trigger
bull Autoantibodies are usually not triggers but useful markers
bull Genetic predisposition (specific HLA class II alleles)
bull More frequent in women ndash female hormones increase the risk disease often starts in times of hormonal changes
bull Possible triggers for AI diseases
bull Viral or bacterial infections (cross reactivity with common epitopes)
bull Wrong expression of MHC class II antigens of normal tissue cells
bull Vaccination
bull Antibiotics
bull hellip
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - ANCA-associated vasculitis
8
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
5
Phadia Laboratory Systems
6
Pathogenesis of autoimmune diseases
bull Mostly T cells are the trigger
bull Autoantibodies are usually not triggers but useful markers
bull Genetic predisposition (specific HLA class II alleles)
bull More frequent in women ndash female hormones increase the risk disease often starts in times of hormonal changes
bull Possible triggers for AI diseases
bull Viral or bacterial infections (cross reactivity with common epitopes)
bull Wrong expression of MHC class II antigens of normal tissue cells
bull Vaccination
bull Antibiotics
bull hellip
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - ANCA-associated vasculitis
8
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
6
Pathogenesis of autoimmune diseases
bull Mostly T cells are the trigger
bull Autoantibodies are usually not triggers but useful markers
bull Genetic predisposition (specific HLA class II alleles)
bull More frequent in women ndash female hormones increase the risk disease often starts in times of hormonal changes
bull Possible triggers for AI diseases
bull Viral or bacterial infections (cross reactivity with common epitopes)
bull Wrong expression of MHC class II antigens of normal tissue cells
bull Vaccination
bull Antibiotics
bull hellip
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - ANCA-associated vasculitis
8
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - ANCA-associated vasculitis
8
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
8
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
9
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull cytoplasmic ANCA = c-ANCA
bull Antigen in most cases anti-proteinase 3
(PR3)
bull perinuclear ANCA = p-ANCA
bull Antigen in most cases anti-myeloperoxidase
(MPO)
bull sometimes other enzymes from
granulocytes but in these cases usually not
related to vasculitis
bull atypical ANCA
bull Not identifiable as p- or c-ANCA
bull Different antigens usually not specific for
ANCA-associated vasculitis
c-ANCA
staining the
whole
cytoplasm of
the
granulocytes
p-ANCA
staining only
the
surrounding
of the cell
nucleus
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
10
Recommendations from
1990
IIF ANCA as first-line test
all positives measured
on antigen-specific tests
Multicenter study 2016
IIF ANCA have a much
lower likelihood ratio than
antigen-specific tests
IIF as first-line test
Source see next page
Damoiseaux et al 2016 Ann Rheum Dis 201601
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
11
Damoiseaux J et al 2016
ldquoConsequently dual IIFantigen-specific immunoassay
testing of each sample is not necessary for maximal
diagnostic accuracy These results indicate that the
current international consensus on ANCA testing for AAV
needs revisionrdquo
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
12
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
13
New Consensus
Bossuyt et al 2017Nat Rev Rheumatol 13683
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
14
Will ANCA IIF be obsolete
bull For autoimmune vasculitis ANCA IIF is no longer deemed suitable as the
first screening test1
bull However for hepatitis and inflammatory bowel syndrome ANCA IIF still
might be of interest1
bull For these diseases antigen-specific tests such as EliA PR3S and EliA
MPOS are not of diagnostic use as in most cases other antigens are
responsible for the ANCA pattern1
1Bossuyt et al 2017Nat Rev Rheumatol 13683ndash692
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
15
bull What is the meaning behind the numbers
bull Example 10 IUml EliA MPOS (Cutoff = 5 IUml)
bull How high is the risk for the patient to have an autoimmune vasculitis
(Post-test probability)
How to interprete the test result
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
16
bull Likelihood Ratio in intervals of
antibody titer
bull A patient with relatively low
probability for vasculitis (eg
Radiographic presence of
pulmonary infiltrates or nodules)
bull How much information does a test
result give How much more
probable is vasculitis
How to interprete the test result
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533
EliA MPOs and EliA
PR3s
Positive
Likelihood
Ratio
Percentage of
vasculitis
patients in a
multicenter study
(n=1175)
0 - 21 IUml 01 10
21 ndash 49 IUml 335 8
50 ndash 160 IUml 12 18
160 ndash 1420 IUml 59 57
1420 ndash 1800 IUml infin 7
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
17
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 10 pre-test probability
10 pre-test probability
bull radiographic evidence of mucosal thickening
involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or
nodules or both
IUml
30
1
60
90
100
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
18
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How to interpret test results Example 85 pre-test probability
85 pre-test probability
bull radiographic evidence of mucosal thickening involving one or more sinuses
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
IUml
Bossuyt et al 2017 Rheumatology (Oxford) 56(9) 1533-41
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
The world leader in serving science
Modern technology used nowadays to help autoimmune disease diagnosis - Rheumatoid arthritis
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
20
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
EliA test panel for autoimmune diseases
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
21
Current ACR classification criteria
A score of at least 610 is needed for classification of a patient as having definite RA
Score
1 Joint involvement
1 large joint 0
2 ndash 10 large joints 1
1 ndash 3 small joints (with or without involvement of large joints) 2
4 ndash 10 small joints (with or without involvement of large joints) 3
gt10 joints (at least 1 small joint) 5
2 Serology (at least 1 test result is needed for classification)
Negative RF and negative ACPA 0
Low-positive RF or low-positive ACPA 2
High-positive RF or high-positive ACPA 3
3 Acute-phase reactants (at least 1 test result is needed for classification)
Normal CRP and normal ESR 0
Abnormal CRP or abnormal ESR 1
4 Duration of symptoms
lt6 weeks 0
6 weeks 1
ldquoantindashcitrullinated
protein antibody
(ACPA) (tested as
antindashcyclic
citrullinated peptide
[anti-CCP])rdquo
Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
22
CCP antibodies appear in early stage of disease
bull Anti-CCP may appear years before first symptoms occur
0
10
20
30
40
50
60
70
80
0246810
years before first symptoms
CC
P-2
po
sit
ive
Rantapaumla-Dahlqvist et al 2003 Arthritis Rheum 48 2741-2749
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
23
The target gain time
without treatment
treatment with biologicals
conventional treatment
treatment with biologicals
time window of
opportunity for
early efficient
treatment opened
by CCP
Joint
damage and
functional
disability
conventional treatment Diagnosis
Diagnosis
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
24
Antibody prevalence in associated disease(s) Rheumatoid Arthritis Associated Disease(s) Antibody
Prevalence []
EliA CCP Well Rheumatoid Arthritis 70-80
Juvenile Idiopathic Arthritis (but associated with
polyarticular manifestation)
0-15
Psoriatic Arthritis 7-16
EliA RF IgM Well
Rheumatoid Arthritis 70-80
Sjoumlgrenrsquos Syndrome 55-70
Systemic Lupus Erythematosus 15-35
Scleroderma 20-30
Mixed Connective Tissue Disease 50-60
Granulomatosis With Polyangiitis 5-20
Endocarditis Lenta 25-60
Chronic hepatitis Primary Biliary Cirrhosis 15-70
Tuberculosis 15
Bacterial Infections 5-60
Parasite infections 20-90
Viral Infections 15-65
K Conrad WS F Hiepe M J Fritzler Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3ed Pabst Science
Publishers 2015
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
25
Why is testing of RF still indicated
bull Combination of anti-CCP and RF IgM for a reliable diagnosis of RA
according to the diagnostic criteria1
bull Individual RF isotype measurement for a better prognosis of RA to help
the clinician in the treatment decision2
bull
bull RF isotypes with high titer have a good specificity for RA to differentiate
from other diseases13
1 Kay and Upchurch 2012 Rheumatology (Oxford)51 Suppl 65-9
2 Ingegnoli et al 2013 Dis Markers 35(6)727
3 Shiboski et al 2012 Arthritis Care Res (Hoboken) 64(4)475
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
26
More reliable diagnosis of RA through the combination of EliA CCP and EliA RF IgM IgA and IgG
Triple positivity of RF isotypes makes RA almost certain even in
CCP-negative patients
bdquoMeasurement of all 3 isotypes of RF may increase by 7- to 21-fold
the chance of making the serologic diagnosis of RAldquo2010)
Test results Interpretation
RF IgM RF IgA RF IgG CCP2 Probability for RA
+ + + - Almost certain
+ + + + Almost certain
+ + - + Almost certain
- - - + Very likely
+ - - + Very likely
+ + - - Likely
+ - - - Possible
Jaskowski et al 2010 J Rheumatol 137(8)1582
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
27
bull JIA comprises a heterogeneous group of rheumatic joint disease with an
onset in childhood (before 16th of age)
bull Autoantibodies are not considered to be of diagnostic help but have
relevance in differential diagnosis1
bull International League of Associations for Rheumatology classification of
juvenile idiopathic arthritis second revision Edmonton 20012
bull Systemic Arthritis
bull Oligoarthritis
bull Polyarthritis (RF-)
bull Polyarthritis (RF+)
bull Psoriatic arthritis
bull Enthestitis related arthritis
bull Undifferentiated arthritis
Juvenile idiopathic arthritis (JIA)
1 Schoenfeld and Meroni 2012 The general practice Guide to Autoimmune diseasesPabst Science Publishers
2 Petty et al 2004 J Rheumatol 31(2)390
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
28
bull Anti-CCP antibodies are associated with RF positive polyarticular course of
JIA
Brunner and Sitzmann Clin Exp Rheumatol 2006 24(4)449
Conrad et al Autoantibodies in Systemic Autoimmune Diseases A Diagnostic Reference 3rd Ed 2015
Tebo et al Pediatric Rheumatology 2012 1029
CCP in JIA
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
29
Thank you
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
The world leader in serving science
Say goodbye to the last-generation technology ndash How tests nowadays help autoimmune disease diagnosis
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
31
EliA test panel for autoimmune diseases
Rheumatoid Arthritis
EliA CCP IgG
EliA RF IgM
EliA RF IgA
EliA RF IgG
Vasculitis
EliA MPOS
EliA PR3S
EliA GBM
Anti-Phospholipid
Syndrome
EliA b2 Glycoprotein-I IgG
EliA b2 Glycoprotein-I IgM
EliA b2 Glycoprotein-I IgA
EliA Cardiolipin IgG
EliA Cardiolipin IgM
EliA Cardiolipin IgA
Connective Tissue Diseases
EliA CTD Screen
EliA SymphonyS
EliA dsDNA
EliA U1RNP
EliA RNP70
EliA SmDP
EliA Ro
EliA Ro52
EliA Ro60
EliA La
EliA Scl 70
EliA Jo-1
EliA CENP
EliA Rib-P
EliA PCNA
EliA PM-Scl
EliA Fibrillarin
EliA Mi-2
EliA ssDNA
EliA RNA Pol III
Celiac Disease
EliA Gliadin IgA
EliA Gliadin IgG
EliA GliadinDP IgA
EliA GliadinDP IgG
EliA Celikey IgA
EliA Celikey IgG
IBD
EliA Calprotectin2
EliA ASCA IgG
EliA ASCA IgA
Miscellaneous
EliA Anti-IgA
EliA Intrinsic Factor
EliA Parietal Cells
Thyroid
EliA anti-TG
EliA anti-TPO
EliA anti-TSH-R
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
32
autoimmune
connective tissue diseases
Prevalence Incidence
Sjoumlgrenlsquo s syndrome (SS) 05 -1 100 60 100000
systemic lupus erythematosus
(SLE)
3 - 400 100000 51 100000
Scleroderma 4 ndash 253 1000000 210 1000000
Dermatomyositis Polymyositis
(DMPM)
15 1000000 60 1000000
Mixed connective tissue disease
(MCTD)
50 100000 20 1000000
Prevalence and Incidence of connective tissue diseases (CTDs)
Schoenfeld et al 2006 Autoantibodies 2nd EdElsevier
Schoenfeld et al Diagnostic Criteria in Autoimmune Diseases 1st Ed Humana Press
Hochberg et al 2014 Rheumatology 6th Ed Elsevier
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
33
bull No single characteristic feature
bull Common symptom - nonspecific fatigue
bull A wide variety of symptoms may occur
bull fever
bull muscle and joint pain and stiffness
bull weakness
bull many other symptoms
bull specific andor non-specific autoantibodies could present
bull Multi-organs are affected especially skin joints lungs
bull Diagnosis mixture of the examination
laboratory results and image diagnostic aid the final diagnosis of connective tissue
diseases which will be made by the physicians
Difficulty of autoimmune connective tissue disorders diagnosis
Gordon and gross 2011 Connective tissue diseases Clinical Publishing
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
34
Fatigue
Hair fall
Oral Ulcer
Arthralgia
Raynaudlsquos phenomenon
Fevers
And many many more
SLE SLE- first symptoms
Hochberg MC1997 Arthritis Rheum 40 1725
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
35
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
36
bull Antibody against RoLa can cross the placenta and create a syndrome
called Neonatal lupus1
bull Could occur up to
bull 1-2 of infants from mothers with SLE12
bull 15-20 of infants from mothers with SLE and anti-Ro Ab12
bull Auto-antibodies directed against Ro52 kDa are associated with a higher
risk of congenital heart block (CHB)3
bull CHB is believed to affect approximately 2 of offspring exposed to anti-
Ro Ab4
Neonatal lupus
1 Hochberg et al 2014 Rheumatology 6th Ed Elsevier
2 Buyon and Clancy 2005 Dis Clin North Am 31(2)299
3 Sawalha and Harley 2004 Curr Opin Rheumatol 16(5)534
4 Brucato et al 2002 Lupus 11(11)716
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
37
SLE
Hochberg MC1997 Arthritis Rheum 40 1725
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
38
How are ANA detected
bull The most popular screening test for ANA is the indirect
immunofluorescence assay (IIF) using HEp-2 cells as substrate
bull IIF detects all ANA with high sensitivity
(except for Ro521 Ro601 Jo-123 and Rib-P4 antibodies)
bull What you get as result is a certain pattern
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
39
How can you differentiate ANA in IIF
A homogeneous
B quasihomogeneous
C fine speckled
D coarse speckled
E dense fine speckled
F centromeric Mariz et al 2011 Arthritis Rheum 63(1)191
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
40
Which Antibodies are responsible for these patterns
Chan et al 2015 Front Immunol 206412
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
41
The relevance of ANA-IIF
bull Antinuclear antibodies occur
bull in various autoimmune diseases
bull Connective tissue diseases (CTD)
bull Autoimmune hepatitis
bull Primary biliary cirrhosis
bull Rheumatoid arthritis
bull Addisonrsquos disease
bull Hashimoto thyroiditis
bull Type 1 diabetes mellitus
bull as well as in non-autoimmune diseases1
bull Cancer
bull Gastrointestinal diseases
bull Lung diseases
bull Skin diseases
bull Infections
bull ANA are positive in a considerable proportion of the healthy population2
ANA-IIF are not very specific for certain diseases
BUT ANA are mainly used to support diagnosis of CTDs
1 Malleson et al 2010 Pediatric Rheumatology 827
2 Satoh et al 2012 Arthritis Rheum64(7) 2319
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
42
What is the effect
Satoh et al 2012 Arthritis Rheum64(7) 2319
A sign of low test specificity
bull The overall prevalence of ANA in the
US population was 138 323 million
people while the prevalence of CTD is
lt05 or 15 million
bull There is a high degree of false positive
in the general population
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
43
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
ANA-IIF (120)
Sensitivity 89
Specificity 77
EliA CTD Screen
Sensitivity 74
Specificity 95
Test sensitivity 100 = 100 CTD patients identified
Test specificity 100 = 900 healthy individual excluded
Jeong et al 2017 PLoSONE 2(3
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
44
Which test provides higher diagnosis accuracy
Lab performs 1000 screen incidence for CTD is 10 so 900 patients have no CTD
and 100 of them have CTD
EliA CTD Sen 74 Spe 95
test POS test NEG
CTD 74 26
none-CTD 45 855
119 881
ANA-IIF Sen 89 Spe 77
test POS test NEG
CTD 89 11
none-CTD 207 693
296 704
PPV 30 NPV 98 PPV 62 NPV 97
Jeong et al 2017 PLoSONE 12(3
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
45
Does IIF detect all antibodies
bull HEp-2 and even HEp-2000 ( only spiked with Ro60 antigen) has a
problem to detect Ro52 and even Ro60 antibodies1
bull Jo-1 is difficult to detect by IIF23
bull Rib-P is difficult to detect by IIF4
1 Mahler et al 2014 J Immunol Res 315179
2 Hoffman et al 2002 Arthritis Res 4(Suppl 1) 84
3Loacutepez-Hoyos et al 2007 Ann N Y Acad Sci1109322
4 Mahleret al 2008 Arthritis Res Ther 10(6)R131
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
46
bull Pediatric rheumatologists have pointed out in the literatures that the ANA is a poor screening test and is being used inappropriately1234
bull the ANA test has such a high false-positivity rate that a positive test is of little if any clinical utility as a screening test and should not be ordered routinely to screen children with musculoskeletal complaints5
bull Its use should be limited to the diagnosis of SLE MCTD and similar systemic illnesses5
How about ANA-IIF in pediatric rheumatology
bull ANA-IIF has a problem in detecting some autoantibodies
bull ANA-IIF is not very specific
EliA CTD screen has higher clinical utility
1Deane et al 1995 Pediatrics 95892-5
2 McGhee et al 2002 Pediatrics 110354-9
3 Siegel 2003 Pediatr Rev 24320-1
4 Jarvis 2008 Pediatr Rheumatol Online J 619-23
5 Malleson et al Pediatric Rheumatology 2010 827
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
47
EliA CTD Screen can help detect specific CTDs1
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome and
systemic sclerosis
dsDNA Ro La Sm CENP-B U1RNP SCL-70 Jo-1
Antibodies 45 76 26 7 19 9 6 2
EliA CTD
Screenpositive
43 75 26 7 18 9 6 2
EliA CTD
Screenborderline
2 0 1 0 0 0 0 0
ANA-IIFpositive
1160
33 65 25 5 19 7 6 0
Confirmed antibodies (n=223) and their detection
Robier C et al 2016 Clin Chem Lab Med 54(8)1365
Test result positive single test should be ordered according to clinical
symptoms
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
48
EliA CTD Screen identifies the most common connective tissue diseases
Sjӧgrenrsquos
syndrome
Systemic lupus
erythematosus
Scleroderma Polymyositisdermatomyosi
tis
Mixed connective
tissue disease
Ro52 kDa 70-
10012
dsDNA 90
(active)15
CENP 70-90 limited9 Jo-1 25910 U1RNP 10078
Ro60 kDa 70-
10012
Ro52 kDa 40-5034 Scl-70 70 systemic7 Ro52 kDa 2386 RNP70 10078
La 35-7012 Ro60 kDa 40-5034 RNA Pol III 4-25
systemic19
Ro60 kDa 156
U1RNP 30-4078 Ro52 kDa 206 Mi-2 10-1523
SmD 20-3078 U1RNP (AC70) 8-1411-14 Pm-Scl 82122
Rib-P 15-2016 Ro60 kDa 66
La 6-155 Fibrillarin 6-820
PCNA lt518 Pm-Scl 32122
Polymyositisscleroderma (overlap syndrome)
Pm-Scl 242122
Mo
re c
om
mo
n gt
lt More common
1 Wenzel J et al British Journal of Dermatology 2001 2 Yoshimi R et al Clinical and Developmental Immunology 2012 3 van den Hoogen FHJ and van de Putte LBA Manual of Biological Markers of Disease 1996 pp C31 1-8 4 Reichlin M and Scofield RH Autoantibodies 1996 pp 783-788 5 Keech CL et al Autoantibodies 1996 pp 789-797 6 Dugar M et al Postgrad Med J 2010 7 Tan EM Immunologist 1999 8 Peng SL and Craft JE Autoantibodies 1996 pp 774-782 9 Craft J and Hardin J Dubois Lupus Erythematosus 1992 pp 216-224 10 Maddison PJ Autoantibodies 1996 pp 31-35 11 Kuwana M et al ArthritisRheum 1994 12 Reveille JD et al Semin Arthritis Rheum2001 13 Ihn H et al Clin Exp Immunol1996 14 Sharp GC et al N Eng J Med1976 15 Hochberg MC Arthritis Rheum 1997 16 Gerli L and Caponi L Autoimmunity 2005 17 Linnik MD et al Arthritis Rheum 2005 18 Mahler M et al Autoimmun Rev 2012 19 Nikpour M et al Arthritis Research amp Therapy 2011 20 Conrad K et al Autoantibodies in systemic autoimmune diseases - A diagnostic reference 21 Ho KT and Reveille JD Arthritis Res Ther 2003 22 Walker JG and Fritzler MJ Curr Opin Rheumatol 2007 23 Ghirardello A et al Clin Rev Allergy Immunol 2010
lt L
ess c
om
mo
n
Less common gt
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
49
Summary
bull CTDs are rare and diagnosis is complicated12
bull At low titres the chance of false positives with ANA-IIF increases3
bull Incorrect diagnosis can cause patients emotional and physical harm45
bull EliA CTD Screen offers equivalent sensitivity and superior specificity to
ANA-IIF and can help detect specific CTDs67
bull EliA CTD Screen has been successful as a first-line test in the real
world8
1 Hochberg MC et al 2014 Rheumatology sixth edition
2 Rasmussen A et al 2016 Rheumatology 55(7)1195-20
3 Op De Beeck K et al 2011 Autoimmun Rev10(12)801
4 Celińska-Loumlwenhoff M and Musiał 2012 J Psychiatria Polska 46(6)1029
5 Narain S et al 2004 Arch Intern Med164(22)2435
6 Otten HG et al 2017 Clin Exp Rheumatol 35(3)462
7 Robier C et al 2016 Clin Chem Lab Med 54(8)1365
8 Wood G et al 2016 Pathology in Practice 1747
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
50
Thank you
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
51
Spectrum of Autoimmune Diseases
Organ Specific Autoimmune Diseases Diabetes mellitus Typ I (juvenile diabetes)
Hashimoto Thyroiditis
Basedow
Celiac Disease
Goodpasture-Syndrome
Ulcerative Colitis Crohnacutes Disease
Primary Biliary Cirrhosis
Myasthenia Gravis
Sjoumlgrenacutes Syndrome
Dermato-Polymyositis
Vasculitis
Rheumatoid Arthritis
MCTD
Scleroderma
Systemic Lupus Erythematosus SLE
Systemic Autoimme Diseases
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
52
Conclusion
bull In more than 95 of all ANA requests the physician wants to know if
CTD plays a role in these patients
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
bull IIF results have only a limited clinical usefulness for the doctors
bull Other test methods can be used according the ACR
52
Is IIF still the first test to use
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
53
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
54
Is there a clinical usefulness of IIF results
bull Even high titres (1640) have only a positive predictive value of 35
for connective tissue diseases (CTDs)
bull ANA in IFA have a predictive value of 11 for SLE and 11 for other
CTDs
bull 4 of 5 ANA positive results cannot be traced back to antigens with
known clinical relevance
54
IIF results have only a limited clinical usefulness for
the doctors
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
55
EliA CTD Screen offers equivalent sensitivity and superior specificity to ANA-IIF12
EliA CTD Screen has a high sensitivity for Sjӧgrenrsquos syndrome
systemic sclerosis and mixed connective tissue disease2
n ANA-IIF 1100 EliA CTD Screen
Positive n
()
Sensitivity
()
Positive n
()
Sensitivity
()
SLE 28 28 (100) 100 21 (75) 80
SS 17 16 (94) 94 17 (100) 100
SSc limited 9 9 (100) 100 8 (889) 90
SSc 2 2 (100) 100 2 (100) 100
MCTD 4 4 (100) 100 4 (100) 100
Confirmed antibodies (n=223) and their detection
1 Otten HG et al Clin Exp Rheumatol 2017 2 Robier C et al Clin Chem Lab Med 2016
Further support from Jeong S et al PLoS ONE 2017 Op de Beeck K et al 2011 and van der Pol P et al Poster presented at Erasmus MC 2017
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
56
Clinical features of SLE
Definition Inflammatory rheumatic systemic disease with a
potential involvement of all organs
Sex Ratio male female = 1 9
Age Every age peaks at 15-25 and 40-50 years
Critical manifestations kidneys CNS
Most frequent cause of death Infections
Diagnosis 4 of 11 ACR-criteria have to be fulfilled
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
57
Criteria of SLE
Clinical features of SLE
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
58
SLE early skin lesions and butterfly rash
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
59
SLE Skin manifestations
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
60
SLE manifestations neuropsychiatric
bullanything possible
bullmild concentration disorder personality
change
bullepilepsy depression psychosis
bullbehaviour disturbances
bullstroke movement disorders
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
61
Scleroderma
Definition Fibrosing systemic disease with lesions of the
vessels leading to atrophy and fibrosis of almost all
organs (Fibrosis = proliferation of connective tissue)
Sex Ratio malefemale = 12
Age mostly adults peak at 40-50 years
Diagnosis ARA-criteria 1 main criterium (= symmetric
sclerodermal lesions of joints) and at least 2 of 3
minor criteria
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
62
Clinical features of systemic sclerosis
bull Raynaudlsquos phenomenon
bull Honeycomb lung
bull Diffuse skin systemic sclerosis with
pigmentation
bull Systemic sclerosis
telangiectasia (rat bites) small mouth
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
63
Clinical features of systemic sclerosis
bull Digital tip ulcers
bull Picture reference httpswwwstudybluecomnotesnotensclerodermadeck4903313
bull Fingertip pitting scares
bull Picture reference httpwwwhuidziektennlzakboekdermatosenstxtSclerodermiaGeneralisatahtm
bull Puffy fingers
bull Picture reference httpspicturesdoccheckcomcomphoto18450-scleroderma-hands-1
bull Systemic sclerosis trying to make fists
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
64
First symptoms
Fatique
Raynaudlsquos phenomenon
Swollen face and hands in the morning
Further course
Calcium deposits in the skin
Ulcerations of the fingers
Telangiectasis (small dilated blood vessels near the
surface)
Involvement of the lung in gt60
Scleroderma
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
65
Scleroderma
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
66
Dermatomyositis Polymyositis
Definitionacute or chronic inflammatory disease of
muscle and skin
Sex Ratio malefemale = 13
Age every age
Diagnosis 5 Criteria (5 manifestation of the skin
dermatomyositis) according to Bohan and Peter
The more criteria are fulfilled the clearer the diagnosis
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
67
Dermatomyositis Polymyositis
First symptoms
Fatique
Muscle weakness in shoulders pelvis or thighs
Further course
Symmetric pain
When skin is involved redness and swelling
Pain in joints
Difficulties with speech and swallowing
Prognosis
Depending on severity but often curable with steroids and
immunosuppression
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
68
Dermatomyositis Polymyositis
Criteria Dermatomyositis
bull Myopathic muscle weakness (Yes)
bull Serum skeletal muscle enzymes (High or
Normal)
bull Electromyographic findings (Myopathic)
bull Muscle enzymes High (up to 50 fold normal)
bull Muscle-biopsy findings (Perifascilular
perimysial or perivascular infiltrates
perifascilular artophy)
bull Rash of Calcinosis (present)
Criteria Polymyositis
bull Myopathic muscle weakness (Yes)
bull Muscle enzymes High (up to 50 fold normal)
bull Electromyographic findings (Myopathic)
bull Muscle-biopsy findings (primary inflammation with
CD8MCH-1 complexes and no vacuoles)
bull Rash of Calcinosis (absent)
No auto-antibody results required
Lancet 2003 Sep 20362(9388)971-82
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
69
Sjoumlgrenlsquos syndrome
Definition a chronic inflammatory disease of unknown cause
characterized by diminished lacrimal and salivary gland
secretion resulting in keratoconjunctivitis sicca and
xerostomia
Sex Ratio malefemale = 19
Age 30-40 Years
Diagnosis ACR-EULAR Classification Criteria for primary
Sjoumlgrenrsquos syndrome (pSS)
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
70
Sjoumlgrenlsquos syndrome
First symptoms
Fatique
Dry eyes
Due to Lessno lacrimal fluid saliva fluid the consequences
are
Frequent eye infection even up to blindness
Diffeculty with speech to swallow (Aphasia and dysphagia)
Intense caries
Involvement of other organs esp polyarthritis is possible
Secondary Sjoumlgrenlsquos syndrome
Complication of rheumatoid arthritis (10-15) less frequent
of SLE (1-3)
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
71
Sjogrenacutes Syndrome - criteria
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
72
The classification of SS applies to any individual who meets the inclusion
criteria does not have any condition listed as exclusion criteria and who
has a score ge 4 when summing the weights from the following items
Sjogrenacutes Syndrome - criteria
Arthritis Rheumatol 2017 Jan 69(1) 35ndash45
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
73
Mixed connective tissue disease MCTD
Definition Overlap syndrome with unclear characterisation a
syndrome with features of scleroderma rheumatoid arthritis
SLE and polymyositis-dermatomyositis and characteristic
high titre of U1RNP antibodies
Sex Ratio malefemale = 13
Age every age
Diagnosis 1 of 2 general symptoms antibodies to U1RNP 2
of 3 mixed symptoms (according to Kasukawa)
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
74
MCTD
First symptoms
Fatique
Raynaudlsquos phenomenon (often many years in advance)
Muscle weakness
Swollen hands and general swelling of the skin
Further course
At least 50 of patients develop a classical connective tissue
disease in the course of 10 years
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
75
MCTD - criteria
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
76
Summary
1 SLE systemic All organs can be involved
Most frequent joints general symptoms skin
Critical manifestationens kidney CNS
2 Systemic sclerosis calcium deposits in the skin and other organs
3 DermatomyositisPolymyositis muscle weakness often curable
4 Sjoumlgrenlsquos syndrome Exocrine glands especially lacrimal and
salivary glands Most often occurs as secondary disease
ANA-IIF is mentioned but not mandatory often specific antiobodies are
mentioned Sm dsDNA RNP70 SS-A SS-B Scl-70 CENP RNA Pol III
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
77
CTD tests
Screening Single Tests
Symphony U1RNP (RNP70 A C)
Sm
SS-ARo (60 kDa 52 kDa)
SS-BLa
Centromere B
Scl-70
Jo-1
dsDNA
Fibrillarin
RNA Pol III
Rib-P
PM-Scl
PCNA
CTD Screen Mi-2
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
78
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
79
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
80
Anti-ssDNA
Anti-ssDNA
Anti-dsDNA low avidity
Anti-dsDNA high avidity
not related to
any syndrome
related to SLE and
similar syndroms
related to SLE
Single stimulus Recurrentpersistent stimulation
somatic
mutations
time
Stimulation of
incompletely deleted
B-cells with inherent
specificity for dsDNA
conv
Elisa
Farr RIA EliA
Why this
Usage of graph kindly allowed by Prof OP Rekvig Tromso Norway
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
81
AdvantagesDisadvantages of Different Methods
dsDNA Abs Method Sensitivity Specificity
CLIFT
FARR RIA
ELISA
ELIA
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
82
Data from acuteinternalacute evaluation
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
83
Results out of this comprehensive analysis
activity Index (SLEDAI) Activity Group total neg pos pos in activity group
0 I 2 2 0
2 I 28 19 9
3 I 1 1 0
4 II 3 0 3
6 II 11 2 9
8 II 1 0 1
10 II 5 1 4
12 III 4 0 4
14 III 5 0 5
23 III 1 0 1
32 III 3 0 3
Total 64 25 39 609
290
850
1000
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
84
Detailed analysis - graph
SLE SLE Act Gr I SLE Act Gr II SLE Act Gr III01
1
10
100
1000
=
Eli
A d
sD
NA
in
IU
ml
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
85
The Importance of Specificity
Test positive Test
negative
Total
RA 148 52 200
Non-RA 147 9653 9800
Total 295 9705 10000
Prevalence 2 Sens 74 spec 985 (EliA CCP) Bizzaro N et al 2007
147 false positives potentially referred on to specialists andor treatment PPV = 50
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
86
The Importance of Specificity
Test positive Test negative Total
RA 146 54 200
Non-RA 392 9408 9800
Total 538 9558
10000
Prevalence 2 Sens 73 spec 96 (Inova CCP3) Bizzaro N et al 2007
245 patients more with a false positive result PPV = 27
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
87
The Importance of Specificity
Test positive Test negative Total
RA 108 92 200
Non-RA 1372 8428 9800
Total 1480 8520 10000
Prevalence 2 Sens 54 spec 86 (RF) Bizzaro N et al 2007
1225 patients more with a false positive result PPV = 7
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
88
CCP is much more specific than RF
Disease n CCP n in RF n in
SLE 77 8 10 19 25
Sjoumlgrenlsquos syndrome 156 22 14 80 51
scleroderma 148 6 4 22 15
myosits 11 3 27 1 9
ankylosing spondylitis 43 6 14 4 9
psoriatic arthritis 34 2 6 3 9
non-classified arthritis 103 11 11 5 5
osteoarthritis 15 1 7 3 20
fibromyalgia 22 3 14 4 18
total 609 62 102 150 246
Fabien et al Clin Rev Allerg Immunol 2008 3440-44
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
89
1997 ACR Classification Criteria for Lupus
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
90
1 J Wenzel R Gerdsen M Uerlich R Bauer T Bieber and I Boehm ldquoAntibodies targeting extractable nuclear antigens historical development and current knowledgerdquo British Journal of Dermatology vol 145 no 6 pp 859ndash867 2001
2 Ryusuke Yoshimi Atsuhisa Ueda Keiko Ozato and Yoshiaki Ishigatsubo Clinical and Pathological Roles of RoSSA Autoantibody System Hindawi Publishing Corporation Clinical and Developmental Immunology Volume 2012 Article ID
606195 12 pages doi1011552012606195
3 van den Hoogen FHJ van de Putte LBA (1996) Anti-U1snRNP antibodies and clinical associations In vanVenrooij WJ Maini RN (eds) Manual of Biological Markers of Disease pp C31 1-8 Kluwer Academic Publishers Dordrecht
4 Reichlin M Scofield RH (1996) SS-A (Ro) autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodiespp 783-788 Elsevier Amsterdam
5 Keech CL McCluskey J Gordon TP (1996) SS-B (La) autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 789-797 Elsevier Amsterdam
6 Dugar M Cox S Limaye V et al (2010) Diagnostic utility of anti-Ro52 detection in systemic autoimmunity Postgrad Med J 86 79ndash82
7 Tan EM (1999) Autoantibodies in Diagnosis and in Identifying Autoantigens Immunologist 7 85-92
8 Peng SL Craft JE (1996) Spliceosomal snRNPs autoantibodies In Peter JB Shoenfeld Y (eds)Autoantibodies pp 774-782 Elsevier Amsterdam
9 Craft J Hardin J (1992) Anti-snRNP Antibodies In Wallace DJ Hahn BH (eds) Dubois Lupus Erythematosus pp 216-224 Williams and Wilkens
10 Maddison PJ (1996) Aminoacyl-tRNA Histidyl (Jo-1) Synthetase Autoantibodies In Peter JB Shoenfeld Y (eds) Autoantibodies pp 31-35 Elsevier Amsterdam
11 Kuwana M Kaburaki J Okano Y Tojo T Homma M Clinical and prognostic associations based on serum antinuclear antibodies in Japanese patients with systemic sclerosis Arthritis Rheum 19943775ndash83
12 Reveille JD Fischbach M McNearney T Friedman AW Arnett FC GENISOS Study Group Systemic sclerosis in 3 US ethnic groups a comparison of clinical sociodemographic serologic and immunogenetic determinants Semin Arthritis
Rheum 200130332ndash346 doi 101053sarh200120268
13 Ihn H Sato S Fujimoto M Kikuchi K Igarashi A Soma Y Tamaki K Takehara K Measurement of anticardiolipin antibodies by ELISA using β2-glycoprotein I (β2-GPI) in systemic sclerosis Clin Exp Immunol 1996105475ndash479
14 Sharp GC Irvin WS May CM Association of antibodies to ribonucleoprotein and Sm antigens with mixed connective-tissue disease systemic lupus erythematosus and other rheumatic diseases N Eng J Med 19762951149ndash1154
15 Hochberg MC (1997) Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus Arthritis Rheum 40 1725
16 Gerli L Caponi L Anti-ribosomal P protein antibodies Autoimmunity 2005 3885-92
17 Linnik MD Hu JZ Heilbrunn KR et al (2005) Relationship between anti-double-stranded DNA antibodies and exacerbation of renal disease in patients with systemic lupus erythematosus Arthritis Rheum 52 1129-1137
18 Mahler M Miyachi K Peebles C Fritzler MJ The clinical significance of autoantibodies to the proliferating cell nuclear antigen (PCNA) Autoimmun Rev 2012 doi101016jautrev201202012
19 Nikpour M et al Prevalence correlates and clinical usefulness of antibodies to RNA Polymerase III in systemic sclerosis a cross-sectional analysis of data from an Australian cohort Arthritis Research amp Therapy 2011 13 R211
20 Conrad K Schoessler W Hiepe F Fibrillarin antibodies In Autoantibodies in systemic autoimmune diseases - A diagnostic reference Lengerich Pabst Science Publishers 78ndash79
21 Ho KT and Reveille JD (2003) The clinical relevance of autoantibodies in scleroderma Arthritis Res Ther 580-93
22 Walker JG and Fritzler MJ (2007) Update on autoantibodies in systemic sclerosis Curr Opin Rheumatol 19 580ndash591
23 Ghirardello A Zampieri S Tarricone E et al Cutting Edge Issues in Polymyositis Clin Rev Allergy Immunol 20101-11
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
91
New Consensus
A second PR3-MPO-ANCA or IIF
can be considered for negative
results in patients with a high clinical
suspicion
(to increase sensitivity) or in case of
low antibody levels (to increase
specificity) Take antibody level into
account
Source see previous page
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
92
bull Patients presenting with raised RF IgA developed more severe erosive disease
ndash They developed a greater number of erosions12
ndash These patients required much more pharmaceutical treatment1
bull The presence of RF IgA could justify more aggressive treatment at an early
stage1
but may predict a poor response to TNF inhibitors3
RF IgA has high prognostic value1
1 Teitsson I et al Ann Rheum Dis 1984 2 Eggelmeijer F et al Rheumatol Int 1900 3 Bobbio-Pallavicini F et al Ann Rheum Dis 2007
Further support from Tarkowski A and Nilsson L J Clin Lab Immunol 1983 Winska Willoch HW et al Scand J Rheumatol suppl 1988 Van Zeben D et al Ann Rheum
Dis 1987 Gioud-Paquet M et al Ann Rheum Dis 1987 Brik R et al Clin Exp Rheumatol 1990 Elkon KB et al Clin Exp Immunol 1981 Luacutepartviacuteksson BR et al Scand J
Rheumatol 1992 and Elson CJ et al Rheumatol Int 1985
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
93
Anti-Neutrophil Cytoplasmic Antibodies on indirect immunofluorescence assay (IIF)
bull Slides have very different qualities and
high lot-to-lot variation
bull classical c-ANCA pattern has a c-ANCA
pattern in both fixations formalin and
ethanol (mostly anti-PR3)
bull classical p-ANCA pattern is seen only on
ehtanol-fixed cells gives a c-ANCA
pattern on formalin (mostly anti-MPO)
bull most frequent atypical ANCA formalin
negative ethanol p-ANCA (seldom anti-
MPO)
c-ANCA
p-ANCA
in
ethanol-
fixed
granulo-
cytes
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
94
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 30 pre-test probability
30 pre-test probability
bull radiographic presence of pulmonary infiltrates or nodules or both
bull urinalysis demonstrating hematuria and red blood cell casts
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
95
0
02
04
06
08
1
0 02 04 06 08 1
Po
st-
test p
rob
ab
ility
Pre-test probability
EliA
0 - 21 21 - 5 5 - 16 16 - 142 142 - 180
How do interprete test result Example 50 pre-test probability
50 pre-test probability
bull Rapidly progressive glomerulonephritis
Source Bossuyt X et al 2017 Rheumatology 56(9) 1533-41
IUml
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489
96
Clinical syndromes associated with ANCA
Anti-MPO Anti-PR3
Disease Literature 1) Literature 1)
GPA (Wegener) 5-60 40-95
MPA 50- 70 25-30
EGPA
(Churg-Strauszlig)
30-40 9-30
Renal limited
vasculitis (eg NCGN)
50-70 25-30
bull PR3 antibodies are quite specific for GPA (Wegenerrsquos granulomatosis) but
may occur in other ANCA-associated vasculitides
bull MPO antibodies occur in all ANCA-associated vasculitides and in
vasculitis of the kidney but almost never in other diseases such as
infections non-ANCA-associated vasculitides or connective tissue
diseases
bull 1) Wiik AS Rheum Dis Clin N Am 201036479ndash489