Congreso Internacional de Anticuerpos Antifosfolipidos. Rio de Janeiro 2013 Grupo de trabajo de SAF: diagnostico de laboratorio y nuevos marcadores

Subgroup 1: Harmonization of aCL and anti-  2GPI Reference material: Dr Rohan Willis To establish international consensus units (IU) for the measurement for aβ 2 GPI 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 aβ 2 GPI assays

Subgroup 1: Harmonization of aCL and anti-  2GPI 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 Proposal

APS Task force 3: Laboratory diagnostics and trends

APS Task force 3: Laboratory diagnostics and trends GRADE: QUALITY OF EVIDENCE The 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 (4): 401–6. 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 quality What 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

Published IgA aCL 12 descriptive studies showing +ve associations with APS SG3: IgA aPL tests Prof Angela Tincani

Published IgA aCL 16 descriptive studies showing no associations with APS

Published IgA anti-  2GPI 14 descriptive -1 case control studies showing +ve associations with APS

Published IgA anti-  2GPI 4 descriptive studies showing no associations with APS

Unpublished IgA aPL 4 descriptive studies showing no associations with APS 1 animal model SG3: IgA aPL tests Dr Anne Tebo

IgA anti-domain IV-V  2GPI 4 descriptive studies showing correlation with IgA aCL and associations with APS

Subgroup 3: IgA Testing for IgA aCL: Can contribute to identifying patients with thrombosis and pregnancy morbidity Testing for IgA anti-  2GPI: 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-  2GPI: 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 aPE Testing 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 aPE Testing 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 abababab protein

aPT vs. aPS-PT

Intrinsic Pathway Extrinsic Pathway VII Common Pathway VIIIa TF Prothrombin FibrinogenFibrin 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

APSThrombosisPregnancy loss AntibodiesAUCOR [CI 95%]pAUCOR [CI 95%]pAUCOR [CI 95%]p LA+aCL [ ] [ ] [ ] LA+aCL+anti-β2GPI [ ] [ ] [ ] LA+aCL+anti-β2GPI+aPS/PT [ ]NS [ ] [ ] LA+aCL+aPS/PT [ ]NS [ ] [ ] LA +anti-β2GPI+aPS/PT [ ] [ ] [ ] aCL+anti-β2GPI+aPS/PT [ ] [ ] [ ] Anti-β2GPI+aPS/PT+aPT [ ] [ ] [ ]0.001 aPT+aPS/PT+aCL [ ] [ ] [ ] aPT+aPS/PT+LAC [ ] [ ] [ ]0.006 Anti-β2GPI+aPE+aPS/PT [ ] [ ] [ ] aPE+aPS/PT+LAC [ ] [ ] [ ] aPE+aPS/PT+aCL [ ]NS [ ] [ ] 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/PT Testing 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 III Low quality evidence APS Task force 3: Laboratory diagnostics and trends

Subgroup 5: aPT and aPS/PT Testing 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 III Low/Moderate quality evidence APS Task force 3: Laboratory diagnostics and trends

SG6: Tests for antibodies to Domain I

Anti-  2GPI de Laat et al. Nat Clin Pract Rheumatol 2008

De Laat et al. Blood types of anti-  2GPI: 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-  2GPI bind to a cryptic epitope in domain I of  2GPI 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 ( )] 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-  2GPI 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 risk Pengo et al. JTH 2010 Testing for LA+anti-  2GPI+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+veThrombosis-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

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 members Subgroup 1 Harmonisation of aCL and anti-  2GPI Subgroup 5Antibodies to prothrombin and aPS/PT Pierluigi MeroniAngela TincaniTatsuya AtsumiVittorio Pengo Maria Orietta BorghiGabriella LakosOlga AmengualLuis Lopez Katrien DevreeseMelissa SnyderRicardo ForastieroKen Oku Rohan WillisNigel HarrisGary NormanMaria Orietta Borghi Richard WongPiet MeijerPierLuigi MeroniSavino Sciascia Subgroup 2Lupus anticoagulantSubgroup 6Antibodies to domain 1 of B2GPI Thomas OrtelPierre MeijerAnisur RahmanAngela Tincani Philip deGrootElaine GrayIan GilesHilde Kelchterman Dorothy AdcockVittorio PengoCharis PericleousMichael Mahler Jeffrey DlottHelen WilmotGabriella LakosSavino Sciascia Subgroup 3IgA aPLJacob RandJohn Ioannou Gary NormanAngela TincaniBas de LaatAngela Tincani Anne TeboLaura AndreoliPierLuigi MeroniJacob Rand Rohan WillisDavid MurraySubgroup 7aPL as risk factors for thrombosis Michelle PetriYu ZuoRobert Roubey Subgroup 4Test for antibodies to negatively charged PLK Otomo Dawn WagenknechtWilliam KuttehSavino Sciascia Nigel HarrisBenjamin LeaderVittorio Pengo Anne TeboMarc LambertJakub Swadźba Savino SciasciaMittermayer Santiago