Anti-Phospholipid Syndrome (APS) Laboratory Diagnosis of the Anti-Phospholipid Syndrome (APS) Olivier Morboeuf, Ph.D Diagnostica Stago Asnières / Seine, France

(Wilson et al. Arthritis Rheum. 1999) Sapporo criteria 1999 (Wilson et al. Arthritis Rheum. 1999) Laboratory criteria Clinical criteria auto-antibodies vascular thrombosis + LA ACA pregnancy morbidity APS

APS is a syndrome with a wrong name ! Specificity of antiphospholipid antibodies (Galli et al. Lancet 1990; Mc Neil et al. 1990) auto-antibody auto-antibody target protein anionic phospholipids anionic phospholipids APS is a syndrome with a wrong name !

Possible target proteins for antiphospholipid antibodies binding to anionic phospholipids b2-glycoprotein I prothrombin protein S protein C EPCR HMWK factor XII Annexin V TFPI complement factor H phospholipases tPA …

b2-glycoprotein I structure : 5 "sushi" domains synthesis : liver function : "in vitro" anticoagulant congenital deficiency : asymptomatic b2-glycoprotein I

Generally accepted as the clinical relevant antibodies Antibodies against b2-glycoprotein I binding site for anionic phospholipids b2-glycoprotein I Generally accepted as the clinical relevant antibodies

Antibodies against b2-glycoprotein I (de Laat et al Antibodies against b2-glycoprotein I (de Laat et al. Blood 2006; 107 : 1916-1924) I II III IV V auto-antibody b2GPI I II III IV V stabilisation Kd dimer b2GPI 5 nM affinity (surface-dependent) I II III IV V Kd b2GPI 170 nM I II III IV V conformational change Auto-antibodies increase the affinity of b2-glycoprotein I for anionic phospholipids

LA, ACA and anti-b2GPI antibodies are related antibodies with overlapping specificity but they are not identical antibodies b2GP1 LA ACA anti-b2GPI

Antiphospholipid Profile & APS: Multivariate Analysis of 100 Patients 0,1 1 10 100 1000 LA/ACA/ab2GPI Any thrombosis ACA/ab2GPI Abortions LA Any thrombosis Antibody positivity Clinical scenario ACA/ab2GPI Any thrombosis ACA Any thrombosis ab2GPI Any thrombosis OR, 95% CI (Pengo et al. Thromb. Haemost. 2005; 93 : 1147-1152)

+ Revised Sapporo criteria 2006 APS Laboratory criteria (Miyakis et al. J. Thromb. Haemost. 2006) Laboratory criteria Clinical criteria vascular thrombosis + LA ACA pregnancy morbidity anti-b2GPI APS

Revised Sapporo criteria 2006 Laboratory Criteria (Miyakis et al. J. Thromb. Haemost. 2006) LA : - present in plasma - 2 or more occasions, 12 weeks apart - detected according ISTH guidelines and/or 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 and/or anti-b2GP1 : - IgG and/or IgM - titer > 99th percentile - present in plasma or serum - 2 or more occasions, 12 weeks apart - measured by standardized ELISA

APS patient classification based on laboratory findings (Miyakis et al. J. Thromb. Haemost. 2006) 2 main categories: Category I: → More than one laboratory criteria present (LA, ACA , anti-b2GP1) Category II : → IIa : LA present alone → IIb : ACA present alone → IIc : anti-b2GP1 present alone

Proposed pathogenic mechanisms of antiphospholipid antibodies (Franchini Clin. Lab. 2006) Interference with the function of the coagulation cascade - inhibition of b2-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 & b2-glycoprotein 1 bound to membrane phospholipids with further vascular damage and platelet activation

Laboratory diagnosis of APS Lupus Anticoagulant (LA) Detected by phospholipid- -dependent clotting tests Antiphospholipid Antibodies (ACA, anti-b2GPI) Detected by ELISA

Detected by phospholipid- -dependent clotting tests 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

Incubated mixing study Stop evaluation for LA - Sensitive to LA - 2 different principles - Low PL concentration Test 1  Screening tests Test 2 Normal LA testing flow chart following international recommendations normal plasma + patient plasma  Mixing study Incubated mixing study Corrects Abnormal Factor assays factor deficiency Specific inhibitor Test 1 Test 2  Confirmatory tests LA confirmed Failure to correct Positive Negative Phospholipid dependance

LA-sensitive APTT reagent Screening test : PTT-LA LA-sensitive APTT reagent Ph. Cauchie : " PTT-LA is the most sensitive APTT reagent " (Cauchie et al., Thromb. Haemost. 1993; 69,N°6) J. Arnout : “ PTT-LA clearly showed the highest responsiveness among the APTT reagents. It is clear that the LA sensitivity of the APTT largely depends on the reagents used” (Arnout at al., Thromb. Haemost. 1999; 81: 929)

LA Diagnosis Screening tests

APTT reagents sensitivity towards LA PTT-LA Silimat PTT-A Platelin APTT ActinFSL " PTT-LA is the most sensitive APTT reagent " (Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)

Screening test : DRVV Screen Reagent - RVV = FX activator - phospholipid - calcium - heparin inhibitor - High specificity : - Insensitive to FVII, FXII, FXI, FIX and FVIII deficiencies - Insensitive to FVIII inhibitors - Insensitive to anticoagulants within therapeutic ranges

Combination of APTT and DRVV tests for LA screening PTT-LA Silimat PTT-A Platelin APTT ActinFSL " 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." (Cauchie et al. Thromb. Haemost.; 1993, 69 : 6)

Combination of APTT, DRVVt and KCT for LA screening P. Cauchie. High sensitivity of a new APTT reagent in Lupus anticoagulant detection. XIVth congres of the ISTH, Thromb Haemost, 1993, 69,N°6.

LA Diagnosis Confirmatory tests

(Triplett et al. Thromb. Haemost. 1993; 70, 787-793) Confirmatory test : Staclot® LA Sensitivity 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 - RVV = FX activator - phospholipid +++ - calcium - heparin inhibitor

Incubated mixing study Test 1: Screening tests Test 2: Stop evaluation for LA Normal LA testing flow chart following international recommendations PTT-LA Abnormal normal plasma + patient plasma Mixing study 100% specificity SSC on LA, 1995 STA Staclot DRVV Screen Failure to correct Test 1: Test 2: Confirmatory tests Incubated mixing study Corrects Staclot LA Factor assays factor deficiency Corrects Positive LA confirmed 100% sensitivity Cauchie P. Thromb. Haemost, 1993. STA Staclot DRVV Confirm Negative Specific inhibitor

Antiphospholipid Antibodies (ACA, anti-b2GPI) 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

Asserachrom® APA line 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) cardiolipin phosphatidylserine phosphatidic acid b2GP1 1/ TMB + wait for 5 min. 2/ H2SO4 sample dil. 1:101 30 min. room temp. + wash (5X) anti-human IgG,M,(A) coupled with peroxidase read at 450 nm APA

Asserachrom® APA line Screening for anti-b2GP1 antibodies Asserachrom® Anti-b2GP1 IgG Quantitative determination of anti-b2GP1 antibodies (IgG) Asserachrom® Anti-b2GP1 IgM Quantitative determination of anti-b2GP1 antibodies (IgM) human b2GP1 1/ TMB + wait for 5 min. 2/ H2SO4 sample dil. 1:101 1 hour room temp. + wash (5X) anti-human IgG or IgM coupled with peroxidase read at 450 nm anti-b2GP1

Correlation between Stago calibrators and HCAL / EY2C9 (Woodhams et al. Thromb. Res. 2004; 114 : 669)

Conclusion LA correlates best with thrombotic complications b2Glycoprotein 1 is the relevant antigen in the diagnostic assays (LA, ACA, anti-b2GP1) Anti-b2GP1 assay is more specific than ACA assay for APS diagnosis Revised Sapporo laboratory criteria should demonstrate an improvement of APS diagnosis

Thank you for your attention !