© 2006 diagnostica stago - all rights reserved diagnostica stago laboratory diagnosis of the...

© 2006 Diagnostica Stago - All rights reservedDIAGNOSTICASTAGO

Laboratory Diagnosisof the

Anti-Phospholipid Syndrome (APS)

Olivier Morboeuf, Ph.D

Diagnostica StagoAsnières / Seine, France


+

APS

Clinical criteria

Sapporo criteria 1999(Wilson et al. Arthritis Rheum. 1999)

Laboratory criteria

auto-antibodies

ACALA

vascular thrombosis

pregnancy morbidity


Specificity of antiphospholipid antibodies(Galli et al. Lancet 1990; Mc Neil et al. 1990)

APS is a syndrome with a wrong name !

auto-antibody

anionic phospholipids

target protein

auto-antibody



Possible target proteins for antiphospholipid antibodies binding to


2-glycoprotein I prothrombin • protein S• protein C• EPCR• HMWK

• factor XII• Annexin V• TFPI• complement factor H• phospholipases• tPA …


2-glycoprotein I

2-glycoprotein I

• structure : 5 "sushi" domains

• synthesis : liver

• function : "in vitro" anticoagulant

• congenital deficiency : asymptomatic


Generally accepted as the clinical relevant antibodies

Antibodies against 2-glycoprotein I

2-glycoprotein I

binding site foranionic phospholipids


Antibodies against 2-glycoprotein I(de Laat et al. Blood 2006; 107 : 1916-1924)

Auto-antibodies increase the affinity of -glycoprotein I

for anionic phospholipids

I

II

III

IVV

auto-antibody2GPI

I

II

III

IVV

conformationalchange

affinity(surface-dependent) I

II

III

IVV

Kd 2GPI170 nM

I

II

III

IVV

I

II

III

IV

stabilisation Kd dimer 2GPI5 nM

V


LA, ACA and anti-2GPI antibodies are related antibodies with overlapping specificity but they are not

identical antibodies

ACALA

anti-2GPI

2GP1


0,1 1 10 100 1000

LA/ACA/a2GPI Any thrombosis

ACA/a2GPI Abortions

LA Any thrombosis

Antibody positivity Clinical scenario

ACA/a2GPI Any thrombosis

ACA Any thrombosis

a2GPI Any thrombosis

(Pengo et al. Thromb. Haemost. 2005; 93 : 1147-1152)

Antiphospholipid Profile & APS: Multivariate Analysis of 100 Patients

OR, 95% CI


Revised Sapporo criteria 2006(Miyakis et al. J. Thromb. Haemost. 2006)

+

APS

Clinical criteria Laboratory criteria

ACALAvascular thrombosis

pregnancy morbidity

anti-2GPI


Revised Sapporo criteria 2006Laboratory Criteria

(Miyakis et al. J. Thromb. Haemost. 2006)

LA : - present in plasma- 2 or more occasions, 12 weeks apart- detected according ISTH guidelines

ACA : - IgG and/or IgM- titer > 40 GPL (or MPL) or > 99th percentile- present in plasma or serum- 2 or more occasions, 12 weeks apart- measured by standardized ELISA

anti-2GP1 : - IgG and/or IgM- titer > 99th percentile- present in plasma or serum- 2 or more occasions, 12 weeks apart- measured by standardized ELISA

and/or

and/or


APS patient classificationbased on laboratory

findings(Miyakis et al. J. Thromb. Haemost. 2006)

2 main categories:

• Category I:

→ More than one laboratory criteria present (LA, ACA , anti-2GP1)

• Category II :

→ IIa : LA present alone→ IIb : ACA present alone→ IIc : anti-2GP1 present alone


Proposed pathogenic mechanisms of antiphospholipid antibodies

(Franchini Clin. Lab. 2006)

• Interference with the function of the coagulation cascade- inhibition of 2-glycoprotein 1- decreased activation of PC- inhibition of AT activity- inhibition of fibrinolysis- inhibition of annexin V binding- upregulation of tissue factor activity

• Activation of endothelial cells- increased expression of adhesion molecules- increased expression of tissue factor- secretion of proinflammatory cytokines


Proposed pathogenic mechanisms of antiphospholipid antibodies

(Franchini Clin. Lab. 2006)

• Activation of platelets and stimulation of platelet aggregation- increased synthesis of thromboxane A2

• "Heparin-induced thrombocytopenia"-like mechanism- vascular damage with exposure to negatively charged phospholipids- formation of immune complexes between APA & 2-glycoprotein 1 bound to membrane phospholipids with further vascular damage and platelet activation


Lupus

Anticoagulant

(LA)

Detected by phospholipid-

-dependent clotting tests

AntiphospholipidAntibodies

(ACA, anti-2GPI)

Detected by ELISA

Laboratory diagnosis of APS


Lupus

Anticoagulant

(LA)

Detected by phospholipid-

-dependent clotting tests


Laboratory diagnosis of LA

• No screening reagent is 100% sensitive and 100% specific

• International recommendations by the Scientific Subcommittee (SSC) on LA


LA testing flow chart following international recommendations

normal plasma +patient plasma

Mixing study

Incubated mixing study

Corrects

Abnormal

Factor assays

factor deficiency

Corrects

Specific inhibitor

Test 1

Test 2

Confirmatory

tests

LA confirmed

Failure to correct

Positive

Negative

Phospholipiddependance

Stop evaluation for LA

- Sensitive to LA- 2 different principles- Low PL concentration

Test 1

Screening

tests

Test 2

Normal

Normal


LA DiagnosisScreening tests


(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)

" PTT-LA is the most sensitive APTT reagent "

APTT reagents sensitivitytowards LA

0

20

40

60

80

100

PTT-LA V W X Y Z

APTT

PTT-LA Silimat PTT-A Platelin APTT ActinFSL


Screening test :DRVV Screen Reagent

- Insensitive to FVII, FXII, FXI, FIX and FVIII deficiencies

- Insensitive to FVIII inhibitors- Insensitive to anticoagulants within therapeutic

ranges

- High specificity :

- Screen reagent : - RVV = FX activator

- phospholipid- calcium- heparin inhibitor


" PTT-LA (87% positive) and DRVVT (59% positive) may be discordant, but the combination of these two tests provide a very sensitive procedure, and immediately available."

0

20

40

60

80

100

PTT-LA V W X Y Z

DRVVT

APTT

Combination of APTT and DRVV tests for LA screening


(Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)


0

20

40

60

80

100


KCT

DRVVT

APTT

P. Cauchie. High sensitivity of a new APTT reagent in Lupus anticoagulant detection. XIVth congres of the ISTH, Thromb Haemost, 1993, 69,N°6.

Combination of APTT, DRVVt and KCT for LA screening


LA DiagnosisConfirmatory tests


Confirmatory test : Staclot® LASensitivity and specificity close to 100%

• Hexagonal phase phospholipids• Sensitive and specific to LA

– Diagnoses LA at low titer– Eliminates false positive

• Insensitive to heparin– Allows direct diagnosis of LA in heparinized

patients

• Insensitive to factor deficiencies– Allows LA testing in warfarin patients, patients with

specific factor deficiency or inhibitor

(Triplett et al. Thromb. Haemost. 1993; 70, 787-793)


Confirmatory test :DRVV Confirm reagent

Confirm reagent :

- RVV = FX activator- phospholipid +++- calcium- heparin inhibitor


AntiphospholipidAntibodies

(ACA, anti-2GPI)

Detected by ELISA


Minimal requirements for APA ELISAs(Tincani et al. Thromb. Res. 2004; 114 : 553-558)

• to run the samples in duplicate

• to determine the cut-off level in each laboratory with at least 50 samples from normal subjects

• to calculate the cut-off level in percentiles

• to use humanized monoclonal antibodies as calibrators and controls (proposal : HCAL for IgG and EY2C9 for IgM)

• to use arbitrary units (eg. MAU/GAU; MPL/GPL)

standardization = decrease the inter-laboratory variability


Screening for Antiphospholipid antibodies :Asserachrom® APA Screen

Qualitative determination of APA (IgG, IgM & IgA)

Screening for ACA/APA : Asserachrom® APA IgG,M

Quantitative determination of APA (IgG and/or IgM)

Asserachrom® APA line

cardiolipinphosphatidylserinephosphatidic acid

2GP1

1/ TMB+ wait for5 min.

2/ H2SO4sampledil. 1:101

30 min.room temp.+ wash (5X)

anti-human IgG,M,(A)coupled withperoxidase

30 min.room temp.+ wash (5X)

read at450 nm

APA


Asserachrom® APA line

Screening for anti-2GP1 antibodiesAsserachrom® Anti-2GP1 IgG

Quantitative determination of anti-2GP1 antibodies (IgG)

Asserachrom® Anti-2GP1 IgM Quantitative determination of anti-2GP1 antibodies (IgM)

human 2GP1

1/ TMB+ wait for5 min.

2/ H2SO4sampledil. 1:101

1 hourroom temp.+ wash (5X)

anti-human IgG or IgMcoupled withperoxidase

1 hourroom temp.+ wash (5X)

read at450 nm

anti-2GP1


LA correlates best with thrombotic complications

2Glycoprotein 1 is the relevant antigen in the diagnostic assays (LA,

ACA, anti-2GP1)

Anti-2GP1 assay is more specific than ACA assay for APS diagnosis

Revised Sapporo laboratory criteria should demonstrate an improvement of

APS diagnosis

Conclusion


Thank youfor

your attention !

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