congreso internacional de anticuerpos antifosfolipidos. rio de janeiro 2013 grupo de trabajo de saf:...

Congreso Internacional de Anticuerpos Antifosfolipidos.

Rio de Janeiro 2013

Grupo de trabajo de SAF: diagnostico de laboratorio y nuevos marcadores

1955 - 2013

Subgroup 1: Harmonization of aCL and anti-b2GPIReference material: Dr Rohan Willis• To establish international consensus units (IU) for the

measurement for aβ2GPI antibodies• RM serum was assigned an IU value• Sent with 30 samples to six commercial companies (INOVA,

Bio-Rad, Corgenix, Phadia, Human and Instrumentation Laboratory)

• Tested on 8 kits eight, according to an approved protocol • linearity, unit equivalency and commutability (CLSI guideline

C53-A)

APS Task force 3:Laboratory diagnostics and trends

• The new polyclonal and monoclonal RMs for IgG and IgM aβ2GPI are an excellent and promising tools for harmonizing aβ2GPI IgG and IgM results across different methods

• These studies contribute significantly to the much-needed standardization of aβ2GPI immunoassays

Monoclonal reference material: HCAL

• MoRM demonstrates excellent

linearity and produces results

highly correlated with other

calibrant materials

• Commutability studies suggest

that this material is suitable for

use in a wide array of aβ2GPI

assays

Subgroup 1: Harmonization of aCL and anti-b2GPI

APS Task force 3:Laboratory diagnostics and trends

Low titers/cut off: Dr Gabriella Lakos

• Clinical view has changed: diagnostic antibodies risk factors. Risk associated to the “presence” of antibodies-Quantitative risk is to be studied

• New analytical technologies have introduced a new level of variability

• Low, medium and high antibody level cannot be defined as a universally applicable value as they are “assay specific”, even when using the same units

Subgroup 2: Lupus anticoagulant

APS Task force 3:Laboratory diagnostics and trends

Weak Lupus anticoagulant: Prof Thomas Ortel

“weak” positive LA results should be considered positive when making clinical decisions

• We have no data to state that “weak” positive results are not clinically significant

• we have no data to state at what level of detection a lupus anticoagulant becomes “weak”

• we have assays that vary in their sensitivity in detecting these “weak” antibodies

Subgroup 2: Lupus anticoagulant

APS Task force 3:Laboratory diagnostics and trends

Lupus anticoagulant testing: Prof Philip de Groot• Mixing is only necessary when there is a suspicion of APS

but the screen is negative

• Low levels of clotting factors and β2-Glycoprotein I (prothrombin) can mask the presence of the auto-antibodies

Prop

osal

APS Task force 3:Laboratory diagnostics and trends

APS Task force 3:Laboratory diagnostics and trends

GRADE: QUALITY OF EVIDENCEThe extent to which our confidence in an estimate of the

treatment effect is adequate to support a particular recommendation

4 categories of quality:• High• Moderate• Low• Very low

Balshem H et al. J Clin Epidemiol 2011 64 (4): 401–6. http://www.cc-ims.net/gradepro . GRADE handbook for grading quality of evidence and strength of recommendation

• High Quality: low probability of further research completely changing the presented conclusions

• Moderate Quality: Estimate lies close to the true value, but further research may completely change the conclusions

• Low Quality: Estimate and the true value may be substantially different. Further research is likely to change the presented conclusions completely

• Very low quality: The authors do not have any confidence in the estimate

Grade: Quality of evidence

Determinants of qualityWhat lowers quality of evidence?

Methodological limitations

Inconsistency of results

Indirectness of evidence

Imprecision of results

Publication bias

APS Task force 3:Laboratory diagnostics and trends

Publ

ishe

d Ig

A aC

L

12 descriptive studies showing +ve associations with APS

SG3: IgA aPL testsProf Angela Tincani

Publ

ishe

d Ig

A aC

L

16 descriptive studies showing no associations with APS

Publ

ishe

d Ig

A an

ti-b

2GPI

14 descriptive -1 case control studies showing +ve associations with APS

Published IgA anti-b2GPI

4 descriptive studies showing no associations with APS

Unpublished IgA aPL

4 descriptive studies showing no associations with APS1 animal model

SG3: IgA aPL testsDr Anne Tebo

IgA anti-domain IV-V b2GPI

4 descriptive studies showing correlation with IgA aCL and associations with APS

Subgroup 3: IgATesting for IgA aCL:• Can contribute to identifying patients with thrombosis and

pregnancy morbidity

Testing for IgA anti-b2GPI:• Can contribute to the assessment of risk for thrombosis and/or

pregnancy morbidity in APS• Can contribute to a better identification of patients with APS

Testing for domain IV-V anti-b2GPI:• Correlation with IgA aCL and some associations but only 4

descriptive studies

Level of evidence III-Low quality evidence

APS Task force 3:Laboratory diagnostics and trends

Published: Dr Anne Tebo

4 descriptive studies showing correlation with aCL and associations with APS

SG4: Tests for antibodies to negatively charged phospholipids

Unpublished: Dr Mittermayer Santiago

Studies report aPhL specificity and sensitivity against aCL

SG4: Tests for antibodies to negatively charged phospholipids

aPE: Dr Marc Lambert

2 studies showing association

6 studies showing no association

SG4: Tests for antibodies to negatively charged phospholipids

Subgroup 4: Other aPL and aPETesting for other negatively charged aPL:• aPI and aPS may identify additional women with recurrent

pregnancy loss

Testing for aPhL:• More specific than standard aCL discriminating better APS

from non-APS• Confirmatory tests? Alternative to aCL?

Level of evidence III-Low quality evidence

APS Task force 3:Laboratory diagnostics and trends

Subgroup 4: Other aPL and aPETesting for aPE:• Most of the studies do not support an association between

aPE and thrombosis or PM, making the assumption of “NO NEED TO TEST” a valid one.

• However, the level of evidence is even low for this recommendation and further well designed studies may probably change the presented conclusions completely

Level of evidence III-Very Low quality evidence

APS Task force 3:Laboratory diagnostics and trends

SG5: Tests for antibodies to prothrombin and aPS/PT

Antiprothrombin detection

irradiated plate

PT

aPT

PS

non-irradiated plate

PT

aPS/PT

non-irradiated plate

ab

protein

IgG aPT

200100504030201054321

IgG

aPS

-PT

200

100

504030

20

10

543

2

1

aPT vs. aPS-PT

Intrinsic Pathway

Extrinsic Pathway

VIICommon Pathway

VIIIa

TF

Prothrombin

Fibrinogen Fibrin

X Xa

Thrombin

Va

IXa

VIIa

Protein C

APC

Protein S

XIa

XIIa

Fragment 1+2

Ca++

PL

Prothrombin antigen level

Prothrombin antigen activity%

Bertolaccini et al. Thromb Hemost 2013

APS Thrombosis Pregnancy loss

Antibodies AUC OR [CI 95%] p AUC OR [CI 95%] p AUC OR [CI 95%] p

LA+aCL .612 3.22 [1.41-7.36] 0.0041 .620 3.04 [1.67-5.52] 0.0002 .613 1.70 [0.99-2.91] 0.0543

LA+aCL+anti-β2GPI .612 3.22 [1.41-7.36] 0.0041 .620 3.04 [1.67-5.52] 0.0002 .613 1.70 [0.99-2.91] 0.0543

LA+aCL+anti-β2GPI+aPS/PT .610 1.69 [0.89-2.96] NS .599 3.05 [1.61-5.75] 0.0004 .620 4.03 [1.50-10.79] 0.0033

LA+aCL+aPS/PT .610 1.70 [0.88-2.97] NS .599 3.04 [1.67-5.52] 0.0002 .620 4.03 [1.50-10.79] 0.0033

LA +anti-β2GPI+aPS/PT .712 3.73 [1.82-5.38] 0.0001 .709 3.75 [2.13-6.62] 0.0001 .677 4.82 [2.17-10.72] 0.0007

aCL+anti-β2GPI+aPS/PT .614 1.76 [1.04-2.99] 0.0357 .606 2.79 [1.54-5.08] 0.0006 .612 3.13 [1.31-7.46] 0.0076

Anti-β2GPI+aPS/PT+aPT .658 3.63 [2.07-6.36] 0.0001 .643 3.35 [1.91-5.88] 0.0001 .643 3.38 [1.59-7.19] 0.001

aPT+aPS/PT+aCL .590 1.70 [1.00-2.89] 0.0482 .589 2.58 [1.38-4384] 0.0026 .578 2.34 [0.98-5.60] 0.0516

aPT+aPS/PT+LAC .618 2.31 [1.36-3.93] 0.0018 .609 2.58 [1.47-4.54] 0.0009 .619 2.87 [1.32-6.22] 0.006

Anti-β2GPI+aPE+aPS/PT .627 2.49 [1.46-4.24] 0.0007 .609 2.58 [1.47-4.54] 0.0009 .632 3.29 [1.48-7.32] 0.0024

aPE+aPS/PT+LAC .610 2.08 [1.23-3.52] 0.0062 .612 2.65 [1.50-4.68] 0.0007 .611 2.68 [1.24-5.81] 0.0104

aPE+aPS/PT+aCL .581 1.59 [0.94-2.81] NS .582 2.44 [1.30-4.57] 0.0049 .591 2.81 [1.12-7.07] 0.0234

Diagnostic accuracy of combinations

Thrombotic risk in SLE

Unpublished: Prof Tatsuya Atsumi

6 studies on aPT

9 studies on aPS/PT

2 in-vitro studies

Published aPT: Dr Ricardo Forastiero

30 retrospective studies 4 cross-sectional 2 case-control 1 prospective

11 studies: 10 retrospective

1 case control

Published aPS/PT: Dr Ricardo Forastiero

Subgroup 5: aPT and aPS/PTTesting for aPT:• Results widely differ between groups suggesting a true

difference• Most data retrospective studies• Not possible to identify the role of aPT alone• Lack of multivariate adjustment

Level of evidence IIILow quality evidence

APS Task force 3:Laboratory diagnostics and trends

Subgroup 5: aPT and aPS/PTTesting for aPS/PT:• Can contribute to assess the risk of thrombosis• Can contribute to a better identification of patients with APS• Multivariate analysis confirm aPS/PT as independent risk

factors• Results do not substantially differ between groups• Association with LA deserves further study

Level of evidence IIILow/Moderate quality evidence

APS Task force 3:Laboratory diagnostics and trends

SG6: Tests for antibodies to Domain I

Anti-b2GPI

de Laat et al. Nat Clin Pract Rheumatol 2008

De Laat et al. Blood 2005

2 types of anti-b2GPI:

A - recognize domain I

- cause lupus anticoagulant activity

- are associated with thrombosis

B - heterogeneous reactivity for all domains

De Laat et al. Blood 2006

• Pathogenic anti-b2GPI bind to a cryptic epitope

in domain I of b2GPI

• This epitope (G40-R43) is only expressed after

the molecule suffers a conformational change

Anti-domain I antibodies• Present in 243/442 patients (55%)

• 83% had thrombosis [OR3.5 (2.3-5.4)]

• Associated with pregnancy morbidity

Published: Prof Anisur Rahman

11 studies

SG6: Tests for antibodies to Domain I

Unpublished: Dr Gabriela Lakos

13 studies – 3 on animal models

SG6: Tests for antibodies to Domain I

Subgroup 6: Antibodies to domain I• Cross-sectional retrospective data show that IgG anti-DI

positivity is associated with thrombosis and pregnancy morbidity

• Animal models show pathogenicity• No prospective data• Frequency of patients positive for DI and negative for anti-b2GPI is not established

• Value of anti-DI as the sole antibody still to be determined

Level of evidence III-Low/Moderate quality evidence

APS Task force 3:Laboratory diagnostics and trends

How do we assess the risk

• Full thrombophilia screen• History of other autoimmune diseases• Other cardiovascular risk factors• Presence of aPL

• LA is the strongest risk factor Galli et a. Blood 2003

• Double or triple aPL positivity the riskPengo et al. JTH

2010

• Testing for LA+anti-b2GPI+aPS/PT has the best diagnostic accuracy Sciascia et al. JTH 2012

SG7: aPL as risk factors

Subgroup 7: aPL as risk factors

APS Task force 3:Laboratory diagnostics and trends

Assessing risk in APS: the global APS score - Savino Sciascia

• GAPSS is score model based on six clinical factors that has been proven to represent the “probability” or likelihood of having thrombosis or pregnancy loss in SLE

• Derived from the combination of independent risk factors for thrombosis and pregnancy loss:

- aPL profile (including criteria and non-criteria aPL)- conventional cardiovascular risk factors- autoimmune antibodies profile.

• Risk score derived from the combination of independent risk factors • Each variable was assigned points proportional to its regression coefficient

Development and Validation of GAPSS

• Patients were filtered by the criterion of the diagnosis in order to equally distribute the disease prevalence (SLE and APS, SLE and aPL positivity or SLE alone)

• Efficacy of randomization was confirmed by computing the prevalence of the variables in the 2 sets where no statistical differences were found

Development and Validation of GAPSS(n=211)

Thrombosis+ve Thrombosis-ve(n=106)

Thrombosis+ve Thrombosis-ve(n=105)

Higher values of GAPSS were seen in patients who experienced thrombosis compared to those with pregnancy loss alone

GAPSS in PAPS (N=62)

Patients with thrombotic recurrences showed higher values of GAPSS

GAPSS in PAPS (N=62)

GAPSS in PAPS (N=62)

GA

PS

S

GAPSS: prospective validation

Increased GAPSS (entry vs. last visit) was seen in patients who developed thrombosis

P3-28

Subgroup 7: aPL as risk factors

APS Task force 3:Laboratory diagnostics and trends

Assessing risk in APS: the global APS score - Savino Sciascia

• GAPSS is a valid tool for risk stratification for thrombosis and its recurrences

• GAPSS has been prospectively validated as a valid tool for accurate prediction of thrombosis in SLE patients with aPL

• The application of GAPSS leads to a substantial improvement in risk prediction of thrombosis or pregnancy loss

Subgroup 7: aPL as risk factors

APS Task force 3:Laboratory diagnostics and trends

Designing the perfect study: how best to assess risks-Robert Roubey

Proposed Action Points:

• Identify and develop collaborations with existing large, population-based, prospective cohorts with data on thrombosis, pregnancy outcomes

• Evaluate traditional and newer aPL tests

• Proper analysis of aPL tests as continuous variables with attention to analytical sensitivity

• Consider, analyze, and test combinations of aPL tests, e.g., “triple positivity,” global aPL score

• Cluster analysis, techniques from microarray analysis, etc.

• Confirm risk models on out-of-sample data

Task force membersSubgroup 1 Harmonisation of aCL and anti-b2GPI Subgroup 5 Antibodies to prothrombin and aPS/PT

Pierluigi Meroni Angela Tincani Tatsuya Atsumi Vittorio Pengo

Maria Orietta Borghi Gabriella Lakos Olga Amengual Luis Lopez

Katrien Devreese Melissa Snyder Ricardo Forastiero Ken Oku

Rohan Willis Nigel Harris Gary Norman Maria Orietta Borghi

Richard Wong Piet Meijer PierLuigi Meroni Savino Sciascia

Subgroup 2 Lupus anticoagulant Subgroup 6 Antibodies to domain 1 of B2GPI

Thomas Ortel Pierre Meijer Anisur Rahman Angela Tincani

Philip deGroot Elaine Gray Ian Giles Hilde Kelchterman

Dorothy Adcock Vittorio Pengo Charis Pericleous Michael Mahler

Jeffrey Dlott Helen Wilmot Gabriella Lakos Savino Sciascia

Subgroup 3 IgA aPL Jacob Rand John Ioannou

Gary Norman Angela Tincani Bas de Laat Angela Tincani

Anne Tebo Laura Andreoli PierLuigi Meroni Jacob Rand

Rohan Willis David Murray Subgroup 7 aPL as risk factors for thrombosis

Michelle Petri Yu Zuo Robert Roubey

Subgroup 4 Test for antibodies to negatively charged PL K Otomo

Dawn Wagenknecht William Kutteh Savino Sciascia

Nigel Harris Benjamin Leader Vittorio Pengo

Anne Tebo Marc Lambert Jakub Swadźba

Savino Sciascia Mittermayer Santiago