The antiphospholipid syndrome:The clinical importance of detecting antiphospholipid antibodies by immunoassays

Antiphospholipid syndrome (APS) is an autoimmune disease characterised by thrombosis and pregnancy complications with the presence of so-called antiphospholipid (aPL) antibodies. The detection of these antibodies is extremely important to establish the diagnosis of APS. This dissertation investigated the importance of testing for aPL antibodies in the APS laboratory diagnostics. Furthermore, existing aPL antibodies were reconsidered. APL antibodies not included in the classification criteria of APS have also been investigated. An important finding was that for a patient who has had thrombosis, fewer tests are needed when thrombotic APS is suspected. In addition, this study was able to conclude that the diagnosis for APS depends partly on the laboratory test used. Finally, a specific group of aPL antibodies targeting prothrombin (an important protein in blood clotting) has been shown to activate platelets and potentially contribute to the increased risk of thrombosis in patients with APS. The results provide insight into the mechanism of APS that can contribute to choosing the right anticoagulants

Antiprothrombin antibodies induce platelet activation : a possible explanation for anti‐FXa therapy failure in patients with antiphospholipid syndrome?

Background Arterial and venous thrombosis are both common in antiphospholipid syndrome (APS). Recent studies have shown that anti-factor Xa (FXa) therapy in APS patients leads to a greater number of patients with arterial thrombosis than with warfarin. We hypothesize that this may be due to the lowering of prothrombin levels by warfarin. Objectives To investigate whether antiprothrombin antibodies induce platelet aggregation and to identify the platelet receptors involved. A second aim was to investigate the effect of reduced prothrombin levels on antiprothrombin antibody-induced platelet aggregation. Methods Enzyme-linked immunosorbent assays were performed to measure binding of antiprothrombin antibodies to prothrombin fragment 1+2 and prothrombin. Platelet aggregation assays in washed platelets were performed. Fc gamma RIIA was immunoprecipitated and tyrosine-phosphorylated Fc gamma RIIA was measured by western blot. Results The antiprothrombin antibodies 28F4 and 3B1 had lupus anticoagulant (LAC) activity and caused platelet aggregation in the presence of Ca2+ and prothrombin. Antiprothrombin antibodies without LAC activity did not activate platelets. Inhibition of Syk and Src kinases and Fc gamma RIIA blocked platelet aggregation. Fab and F(ab')(2) fragments of 28F4 were unable to induce platelet aggregation. Immunoprecipitations showed that whole 28F4 immunoglobulin G induced tyrosine phosphorylation of Fc gamma RIIA. Platelet aggregation was significantly reduced when prothrombin levels were reduced from 1 mu M to 0.2 mu M. Conclusions Antiprothrombin antibodies with LAC activity are able to activate platelets via Fc gamma RIIA. Decreased prothrombin levels resulted in less antiprothrombin antibody-mediated platelet aggregation. This may explain the lower incidence of arterial thrombosis in patients treated with warfarin than with anti-FXa therapy

Detection of anti-cardiolipin and anti-β2glycoprotein I antibodies differs between platforms without influence on association with clinical symptoms

Background: The anti-phospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with persistent presence of anti-phospholipid antibodies (aPL). Laboratory criteria include aPL detection by coagulation tests for lupus anticoagulant (LAC) or solid phase assays measuring anti-beta 2 glycoprotein I (a beta 2GPI) or anticardiolipin (aCL) immunoglobulin (Ig) G/IgM antibodies. External quality control programs illustrate that commercially available aPL assays produce variable results. Objective: We aimed to investigate the agreement and diagnostic accuracy of solid phase assays. Materials and Methods: In thismulti-centre study, 1,168 patient samples were tested on one site for aCL and a beta 2GPI IgG/IgM antibodies by four solid phase test systems. Samples included APS patients, controls and monoclonal antibodies (MoAB) against different epitopes of beta 2GPI. LAC was determined by the local centre. Results: aCL IgM assays resulted in the most discrepancies (60%), while aCL IgG and a beta 2GPI IgM assays resulted in lower discrepancies (36%), suggesting better agreement. Discrepant samples displayed lower median aPL titers. Dependent on the solid phase test system, odds ratios (ORs) for thrombosis and pregnancy morbidity ranged from 1.98 to 2.56 and 3.42 to 4.78, respectively. Three platforms showed lower sensitivity for MoAB directed against the glycine (Gly) 40-arginine (Arg) 43 epitope of domain I of beta 2GPI. Conclusion: Poor agreement was observed between different commercially available aCL and a beta 2GPI IgG/IgM assays, hampering uniformity in the identification of aPL-positive patients. Clinical association was globally concordant between solid phase test systems considering results of the four aPL together. An assay sensitive in detecting the MoAB against Gly40-Arg43 of domain I of beta 2GPI reached the highest OR for thrombosis

The Significance of Antibodies against Domain I of Beta-2 Glycoprotein I in Antiphospholipid Syndrome

The antiphospholipid syndrome (APS) is characterized by vascular thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPLs). Progress is being made in understanding the pathogenesis of the syndrome, but difficulties persist in the identification of patients at risk for thrombosis and/or pregnancy morbidity. Beta-2 glycoprotein I ( (2) GPI), a plasma protein consisting of five sushi domains, is thought to be the main antigenic target of aPLs. Antibodies recognizing domain I of (2) GPI are predominantly present in patients with an elevated risk of thrombosis, whereas antidomain IV/V antibodies are found in nonthrombotic autoimmune diseases. Indeed, domain I antibodies proved to be pathogenic in multiple studies. Retrospective studies have provided evidence for an added clinical value of antidomain I antibodies in the risk stratification of patients with APS. Still, wide ranges of odds ratio exist between studies, probably due to differences in the study and control population, and detection methods used. Despite the proven pathogenicity of antidomain I antibodies and their correlations with clinical manifestations of APS, heterogeneity of the current studies has prohibited their acceptance in the official diagnostic criteria. Well-designed large longitudinal prospective studies with available and new, preferentially functional, assays for the risk stratification of patients with APS are required

Added value of antiphosphatidylserine/prothrombin antibodies in the workup of thrombotic antiphospholipid syndrome: Communication from the ISTH SSC Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibodies

Background Diagnosis of antiphospholipid syndrome (APS) requires persistent presence of lupus anticoagulant (LAC), anticardiolipin (aCL) IgG/IgM, or anti-β2 glycoprotein I (aβ2GPI) IgG/IgM antibodies. Other antiphospholipid antibodies (aPL) such as antiphosphatidylserine/prothrombin antibodies (aPS/PT) are promising in assessment of thrombotic APS (TAPS). Aim To evaluate the added value of aPS/PT IgG and IgM in TAPS. Material and methods aPS/PT IgG/IgM, aCL IgG/IgM, aβ2GPI IgG/IgM, and LAC were determined in 757 patients (TAPS and controls). aPS/PT cut-off values were calculated, aPS/PT titers and positivity were compared between TAPS and controls, type of thrombosis, and antibody profiles. Likelihood ratios (LR), odds ratios (OR) and aPL-score were determined. Results aPS/PT IgG and IgM were associated with TAPS and triple positivity. In-house calculated cut-offs were higher for IgM (43 units), compared to manufacturer’s cut-off (30 units). Thresholds of 90 (IgG)/200 (IgM) units were determined as high-titer cut-off. Higher aPS/PT titers were observed in triple positive patients and showed higher LR and OR for TAPS. aPS/PT was independently associated with TAPS when adjusted for aCL/aβ2GPI, but not when adjusted for LAC. In isolated LAC positive patients, aPS/PT was positive in 27.1% TAPS patients and in 77.3% patients with autoimmune disease. Diagnostic value of aPL-score did not differ with and without including aPS/PT. Conclusion aPS/PT positivity, especially with high antibody titer, is associated with TAPS diagnosis. Analysis on top of current laboratory criteria is not essential in TAPS diagnosis, but aPS/PT could be useful in patients with thrombosis and a double positive aPL profile (aCL+/aβ2GPI+)

Semiquantitative interpretation of anticardiolipin and antiβ2glycoprotein I antibodies measured with various analytical platforms: communication from the ISTH SSC subcommittee on Lupus Anticoagulant/Antiphospholipid antibodies

Background Antiβ2glycoprotein I (aβ2GPI) and anticardiolipin (aCL) IgG/IgM show differences in positive/negative agreement and titers between solid phase platforms. Method specific semiquantitative categorization of titers could improve and harmonize the interpretation across platforms. Aim To evaluate the traditionally 40/80 units thresholds used for aCL and aβ2GPI for categorization into moderate/high positivity with different analytical systems, and to compare with alternative thresholds. Material and methods aCL and aβ2GPI thresholds were calculated for two automated systems (chemiluminescent immunoassay (CLIA) and multiplex flow immunoassay (MFI)) by ROC-curve analysis on 1108 patient samples, including patients with and without APS, and confirmed on a second population (n=279). Alternatively, regression analysis on diluted standard material was applied to identify thresholds. Thresholds were compared to 40/80 threshold measured by an enzyme linked immunosorbent assay (ELISA). Additionally, likelihood ratios (LR) were calculated. Results Threshold levels of 40/80 units show poor agreement between ELISA and automated platforms for classification into low/moderate/high positivity, especially for aCL/aβ2GPI IgG. Agreement for semiquantitative interpretation of aPL IgG between ELISA and CLIA/MFI improves with alternative thresholds. LR for aPL IgG increase for thrombotic and obstetric APS based on 40/80 thresholds for ELISA and adapted thresholds for the other systems, but not for IgM. Conclusion Use of 40/80 units as medium/high thresholds is acceptable for aCL/aβ2GPI IgG ELISA, but not for CLIA and MFI. Alternative semiquantitative thresholds for non-ELISA platforms can be determined by a clinical approach or by using monoclonal antibodies. Semiquantitative reporting of aPL IgM has less impact on increasing probability for APS

The (non‐)sense of detecting anti‐cardiolipin and anti‐β2glycoprotein I IgM antibodies in the antiphospholipid syndrome

International audienceThe antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of lupus anticoagulant (LAC), anti-cardiolipin (aCL) and/or anti-β2glycoprotein I (aβ2GPI) antibodies of the immunoglobulin G/immunoglobulin M (IgG/IgM) isotype. However, the role of aCL and aβ2GPI IgM as a serologic marker in APS is debated.Objectives: We aimed to assess the diagnostic and clinical value of IgM antiphospholipid antibodies (aPL) in APS within the classification criteria.Patients/methods: Our multicenter study comprised 1008 patients, including APS patients and controls. Anti-CL and aβ2GPI IgG and IgM antibodies were detected with four commercially available solid phase assays.Results: Positivity for aCL and/or aβ2GPI antibodies was significantly correlated with thrombosis and pregnancy morbidity, independent of the isotype and solid phase assay. Higher odds ratios were obtained for IgG compared to IgM positivity. Isolated IgM was rare in thrombotic APS, but more frequent in obstetric APS, ranging from 3.5% to 5.4% and 5.7% to 12.3%, respectively, dependent on the solid phase assay. In a multivariate logistic regression analysis of aPL, IgM positivity was found to be associated with pregnancy morbidity. However, detection of IgM was not independently associated with thrombosis. Combined positivity for LAC, IgG, and IgM was highly associated with thrombosis and pregnancy morbidity.Conclusions: Our data support testing for aCL and aβ2GPI IgM in women suspected of obstetric APS. However, no added value was found for testing IgM in patients suspected of thrombotic APS. Still, IgM aPL might be useful as a second-line test to improve thrombotic risk stratification