Evaluation of phosphatidylserine-dependent antiprothrombin antibody testing for the diagnosis of antiphospholipid syndrome: results of an international multicentre study.

Objective A task force of scientists at the International Congress on Antiphospholipid Antibodies recognized that phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT) might contribute to a better identification of antiphospholipid syndrome (APS). Accordingly, initial and replication retrospective, cross-sectional multicentre studies were conducted to ascertain the value of aPS/PT for APS diagnosis. Methods In the initial study (eight centres, seven countries), clinical/laboratory data were retrospectively collected. Serum/plasma samples were tested for IgG aPS/PT at Inova Diagnostics (Inova) using two ELISA kits. A replication study (five centres, five countries) was carried out afterwards. Results In the initial study ( n = 247), a moderate agreement between the IgG aPS/PT Inova and MBL ELISA kits was observed ( k = 0.598). IgG aPS/PT were more prevalent in APS patients (51%) than in those without (9%), OR 10.8, 95% CI (4.0-29.3), p < 0.0001. Sensitivity, specificity, positive (LR+) and negative (LR-) likelihood ratio of IgG aPS/PT for APS diagnosis were 51%, 91%, 5.9 and 0.5, respectively. In the replication study ( n = 214), a moderate/substantial agreement between the IgG aPS/PT results obtained with both ELISA kits was observed ( k = 0.630). IgG aPS/PT were more prevalent in APS patients (47%) than in those without (12%), OR 6.4, 95% CI (2.6-16), p < 0.0001. Sensitivity, specificity, LR + and LR- for APS diagnosis were 47%, 88%, 3.9 and 0.6, respectively. Conclusions IgG aPS/PT detection is an easily performed laboratory parameter that might contribute to a better and more complete identification of patients with APS.

Multiple antiphospholipid antibodies positivity and antiphospholipid syndrome criteria re-evaluation.

The antiphospholipid syndrome (APS) is characterized by the presence of aPL and thrombosis and/or pregnancy morbidity. The last APS laboratory classification criteria include the presence of at least one of the antiphospholipid antibodies (aPL) [lupus anticoagulant (LA), anticardiolipin (aCL) and/or anti-ß2 glycoprotein I antibodies (aß2GPI)] and introduced the concept of subclassification of APS patients into two different categories of aPL assay positivity (combination or single aPL). Several studies have recently shown that the risk for thrombosis increases with each additional aPL detected. We found that the presence of IgG antibodies to ß2GPI and/or prothrombin increased thrombotic risk in patients with LA and/or aCLin a prospective study. Various studies have recently demonstrated that patients with triple positivity (LA, aCL and aß2GPI) are at the highest risk for venous and arterial thrombosis and for obstetric complications. In retrospective but also in prospective studies the rate of thrombotic recurrence was high in subjects with triple positivity even while on anticoagulant therapy. In addition, the occurrence of a first thrombotic event in asymptomatic carriers of triple positivity was higher than in those with single aPL positivity. The inclusion of the detection of antibodies against domain I of ß2GPI and/or antibodies to prothrombin would probably help to further identify more clinically relevant aPL. Based on the last findings, there are some proposals to consider only patients with triple positivity as definite APS (thrombotic and obstetric).

Evaluation of different immunoassays for the detection of antiphospholipid antibodies: report of a wet workshop during the 13th International Congress on Antiphospholipid Antibodies.

BACKGROUND: The performance and standardization of anticardiolipin (aCL) and anti-ß2 glycoprotein I antibodies (aß2GPI) tests for the confirmation of diagnosis of antiphospholipid syndrome (APS) remain a matter of debate and concern. We evaluated the performance of different ELISAs and other new immunoassays for the detection of aCL and aß2GPI in a wet workshop at the 13th International Congress on Antiphospholipid Antibodies in Galveston, TX (April 13th, 2010, APLA 2010). METHODS: Aliquots of 26 un-identified APS or persistently aPL positive serum samples and 21 controls (9 from healthy individuals and 5 from patients with infectious diseases and 7 with various autoimmune diseases) were distributed to all participants/groups. All serum samples were evaluated in various aCL and aß2GPI ELISAs, a chemiluminescent immunoassay, a fluoro-enzyme immunoassay, and in a multiplexed immunoassay system. Monoclonal and polyclonal calibrators were also evaluated. RESULTS: Although not all the assays reported the titers of aCL and aß2GPI in the same units, the correlation of positive titers among the assays was good. All aCL and aß2GPI tests showed excellent clinical sensitivities, specificities and positive predictive values and good agreement with respect to the levels of the IgG and IgM antibodies, regardless of assay type, or whether tests were done using automated or "manual" systems. CONCLUSIONS: New methodologies for the detection of aPL look promising and comparable to currently approved ELISA tests. This study provides evidence of progress of efforts of harmonization of tests used to detect aPL.

New guidelines for lupus anticoagulant: sensitivity and specificity of cut-off values calculated with plasmas from healthy controls in mixing and confirmatory tests.

INTRODUCTION: The updated guidelines for lupus anticoagulant (LA) diagnosis indicate locally calculate the cut-off values of the index of circulating anticoagulant (ICA) and the clotting time in seconds (s) for mixing studies and % of correction (%C) for confirmatory tests. We assess sensitivity (SEN) and specificity (SPC) of the cut-off values obtained as the 99th percentile from 60 plasmas of healthy individuals. METHODS: We analysed 647 plasmas from patients studied in the last 3 years, and results were revaluated according to the new criteria and cut-off values. Four hundred and three had LA, and 75 of them were under oral anticoagulants (OA). We performed three screening tests: activated partial thromboplastin time (APTT), diluted Russell viper venom time (dRVVT) and dilute prothrombin time (dPT), and previous diagnosis was carried out using our home-made cut-off calculated by receiver operating characteristics curves. We reanalysed the mixing and confirmatory data of APTT/dRVVT, the tests selected in the new guidelines. To evaluate SPC, 244 plasmas (160 OA and 84 congenital deficient patients) were studied. RESULTS: Considering mixing studies, the cut-off values demonstrate that SEN of ICA-APTT was 94% and of clotting time in second (s) 83%, with an SPC of 77% and 84%, respectively. For ICA-dRVVT, SEN was 72% and for clotting time in second (s) 77%, with SPC of 98% and 84%, respectively. The cut-off values for %C for confirmatory APTT show good SEN 82% and high SPC 96%; for confirmatory dRVVT lower SEN 77%, but a SPC of 100%. CONCLUSION: The combination of mixing and confirmatory tests interpreted according to the new guidelines can clearly differentiate LA from other coagulopathies.

'Non-criteria' aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, TX, USA, April 2010.

Abstract: Current classification criteria for definite APS recommend the use of one or more of three positive standardized laboratory assays, including anticardiolipin antibodies (aCL), lupus anticoagulant (LA), and antibodies directed to ß(2)glycoprotein I (anti-ß(2)GPI) to detect antiphospholipid antibodies (aPL) in the presence of at least one of the two major clinical manifestations (i.e., thrombosis or pregnancy morbidity) of the syndrome. Several other autoantibodies shown to be directed to phospholipids and/or their complexes with phospholipids and/or to proteins of the coagulation cascade, as well as a mechanistic test for resistance to annexin A5 anticoagulant activity, have been proposed to be relevant to APS. A task force of worldwide scientists in the field discussed and analyzed critical questions related to 'non-criteria' aPL tests in an evidence-based manner during the 13th International Congress on Antiphospholipid Antibodies (APLA 2010, 13-16 April 2010, Galveston, Texas, USA). This report summarizes the findings, conclusions, and recommendations of this task force.

An international multicentre-laboratory evaluation of a new assay to detect specifically lupus anticoagulants dependent on the presence of anti-beta2-glycoprotein autoantibodies.

BACKGROUND: Antiphospholipid syndrome (APS) is diagnosed by the simultaneous presence of vascular thrombosis and/or pregnancy morbidity and detection of antiphospholipid antibodies in plasma. OBJECTIVES: We have shown that prolongation of clotting time by anti-beta2-glycoprotein I (beta2GPI) antibodies correlates better with thrombosis than a positive classic lupus anticoagulant (LAC) assay in a single center study. To confirm or falsify this finding we have conducted a multicenter study. METHODS AND RESULTS: In 325 LAC-positive samples, we found that the beta2GPI-dependent LAC correlated 2.0 times better with thrombosis than the classic LAC assay. Although significant, this was a minimal improvement compared with the 'classic' LAC. It was published that calcium influences the behavior of anti-beta2GPI antibodies in coagulation assays. To investigate whether calcium plays a role in the present study, we divided the patient population into two groups: (i) blood was collected in 0.109 m sodium citrate and (ii) blood was drawn in 0.129 m sodium citrate as anticoagulant. We found that a positive result with the beta2GPI-dependent LAC assay correlated better with thrombosis [odds ratio (OR): 3.3, 95% confidence interval (CI) 1.9-5.8] when 0.109 m sodium citrate was used compared with 0.129 m sodium citrate (OR: 0.4, 95% CI 0.1-1.1). CONCLUSION: We were able to confirm in an international multicenter study that a positive result in a beta2GPI-dependent LAC assay correlates better with thrombosis than the classic LAC assay, but that the assay needs further study as it is sensitive to external factors such as the sodium citrate concentration used as anticoagulant in the test sample.

Prothrombotic mechanisms based on the impairment of fibrinolysis in the antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a clinical autoimmune disorder characterised by thrombosis, venous or arterial, and recurrent pregnancy morbidity in association with the persistence of antiphospholipid antibodies (aPL). The clinical variety of aPL ranges from asymptomatic individuals to those with multiple organ thromboses and failure developing over a short period, also known as catastrophic APS. An increasing number of phospholipid-binding proteins with crucial functions in the regulation of blood coagulation and fibrinolysis are targeted by APS-related autoantibodies. Disruption of fibrinolysis is one of the proposed pathophysiological mechanisms for the APS. There are some experimental data for an association between impaired overall fibrinolytic activity and autoimmune aPL; however, evidence is still inconclusive and more studies are needed in this area. In this article, we review the evidence by which aPL may disturb fibrinolysis.

A prospective study of antibodies to beta2-glycoprotein I and prothrombin, and risk of thrombosis.

Antiphospholipid syndrome (APS) is a clinical autoimmune disorder characterized by thrombosis/pregnancy morbidity associated with the persistence of lupus anticoagulant (LA) and/or anticardiolipin (aCL) antibodies. We assessed the contribution of antibodies to beta2-glycoprotein I (anti-beta2GPI) and prothrombin (anti-PT) to the thrombotic risk in a cohort of 194 consecutive patients with persistent LA and/or aCL. Median follow-up was 45 months. A total of 39 patients (20.1%) had one documented episode of thrombosis during follow-up. Eleven of these patients had no previous thrombosis before enrollment in the study and 28 had recurrences of thrombosis. There were 21 venous and 18 arterial thrombotic events and the overall incidence of thrombosis was 5.6% per patient-year. After multivariate analysis, the male sex (P = 0.025), a previous thrombosis (P < 0.01), the presence of anti-beta2GPI (P = 0.001), and the presence of anti-PT (P = 0.03) remained as independent risk factors for recurrent thrombosis. Only IgG anti-beta2GPI and anti-PT were associated with an increased risk of thrombosis (P < 0.01 and P = 0.005). Patients testing positive for anti-beta2GPI had a higher rate of thrombosis than did antiphospholipid patients without anti-beta2GPI (8.0% vs. 3.1% per patient-year). Similarly, a higher rate of thrombosis was found in patients with positive anti-PT compared with patients without anti-PT (8.6% vs. 3.5% per patient-year). Considering only the group of 142 LA positive patients, the highest incidence of thrombosis was found in LA patients positive for both anti-beta2GPI and anti-PT (8.4% per patient-year). In conclusion, the presence of IgG anti-beta2GPI and anti-PT in patients with LA and/or aCL and mainly in those with LA predicts a higher risk of thromboembolic events.

Circulating levels of tissue factor and proinflammatory cytokines in patients with primary antiphospholipid syndrome or leprosy related antiphospholipid antibodies.

The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies (aPL) in patients with thromboembolic complications. In APS, most aPL are autoantibodies to beta2-glycoprotein I and prothrombin, which play a major role in the APS pathogenesis. Nevertheless, antibodies with the same antigen specificity are also found in aPL patients with leprosy, in whom thromboembolic complications are uncommon. The in vivo upregulation of the tissue factor (TF) pathway and the imbalance of cytokines have been proposed as potential mechanisms of thrombosis in the APS. We measured the circulating levels of TF, interleukin 6 (IL-6), IL-6 receptor (sIL-6R), tumor necrosis factor (TNF-alpha) and interferon gamma (IFN-gamma) in 83 patients with autoimmune aPL (42 with and 41 without clinical features of definite primary APS), 48 leprosy patients (33 with aPL) and 48 normal controls. There was a trend (P = 0.06) to higher median sTF in patients with autoimmune aPL (139 pg/mL) compared with leprosy patients (103.5 pg/mL) and controls (123 pg/mL). In addition, the frequency of raised sTF levels (> 187 pg/mL) was significantly higher in the group with autoimmune aPL [22.9% (APS 21.4%, non-APS 24.4%)] but not in leprosy (10.4%) compared with controls (4.2%). Elevated levels of IL-6 and TNF-alpha and a trend to lower IFN-gamma were found in patients with definite APS. Leprosy patients with aPL, however, had increased TNF-alpha and IFN-gamma but normal IL-6 levels. Levels of sIL-6R did not differ between controls and either patients with autoimmune aPL or leprosy. The different cytokine profiles as well as differences in circulating levels of TF might contribute to the high thrombotic risk found in patients with autoimmune aPL but not in leprosy related aPL patients.

Catastrophic antiphospholipid syndrome and Kikuchi-Fujimoto disease: the first case reported.

The case of a man with diagnosis of Kikuchi-Fujimoto disease (KFD) and catastrophic antiphospholipid syndrome (CAPS) is reported. He presented prolonged fever, lymphadenopathies, arthralgia, weight loss, hepatosplenomegaly and positive IgM for cytomegalovirus. While he was empirically treated with tuberculostatic drugs, he suddenly developed systemic inflammatory response syndrome, multiple organ failure and distal necrosis. On suspicion of severe sepsis, antibiotics, corticoids and recombinant human activated protein C (XIGRIS) were administrated. Exhaustive laboratory searching was negative. Histopathologic examinations of lymph node first disclosed malignant lymphoma but later KFD was confirmed. One month later, laboratory tests showed the presence of antiphospholipid antibodies (aPL). The patient was discharged after two months of hospitalization. This case exhibits a KFD complicated by definite CAPS. Cytomegalovirus could be involved in the development of both CAPS and KFD. Because of the severity of the case, we believe that XIGRIS noticeable improved the altered coagulation profile associated with CAPS.

Cardiac disease in the antiphospholipid syndrome: recommendations for treatment. Committee consensus report.

The Committee reviewed cardiac involvement in the antiphospholipid antibody syndrome. The Committee's recommendations are: Valve abnormalities: anticoagulation is recommended for symptomatic patients with valvulopathy. Prophylactic antiplatelet therapy may be appropriate for asymptomatic patients (recommended by 13/17 experts in an independent review). Committee members disagreed whether corticosteroid therapy is helpful, but agree that distinguishing among presumptive valvulitis (valve thickening on echocardiogram), valve deformity and vegetations is important, as treatment implications may differ. Occlusive arterial disease (angina, myocardial infarction): the Committee recommends aggressive treatment of all risk factors for atherosclerosis (hypertension, hypercholesterolaemia, smoking) and liberal use of folic acid, B vitamins and cholesterol-lowering drugs (preferably statins). Hydroxychloroquine for cardiac protection in APS patients may be considered. The Committee also recommends warfarin anticoagulation for those who have suffered thrombosis in the absence of atherosclerosis, but recognizes that developing data may support the use of antiplatelet agents instead. Intracardiac thrombi: the Committee recommends intensive warfarin anticoagulation, and consultation with cardiac surgeons when appropriate. Ventricular dysfunction: the Committee has no recommendations on this aspect of cardiac disease. Pulmonary hypertension: the Committee recommends intensive anticoagulation with warfarin and clinical trials of bosentan, epoprostenol and other new agents.

Autoimmune antiphospholipid antibodies impair the inhibition of activated factor X by protein Z/protein Z-dependent protease inhibitor.

The hemostatic process is tightly regulated by several antithrombotic mechanisms. Among them, protein Z (PZ)-dependent protease inhibitor (ZPI) potently inhibits factor (F)Xa in a manner dependent on calcium ions, phospholipids and PZ. Autoimmune antiphospholipid antibodies (aPL) are mainly directed against phospholipid-binding plasma proteins such as beta2-glycoprotein I (beta2GPI) and prothrombin, and are known to interfere with phospholipid-dependent hemostatic pathways. In this study, we investigated whether purified aPL are able to interfere with inhibition of FXa by PZ/ZPI. beta2GPI modestly delayed the FXa inactivation by PZ/ZPI and most isolated aPL-IgGs were found to further increase the inhibitory potential of beta2GPI on PZ/ZPI activity. Without beta2GPI, the PZ/ZPI activity was unaffected by the addition of aPL-IgG. As PZ deficiency is hypothesized to lead to a prothrombotic state, we performed a case-control study to measure plasma levels of PZ and ZPI in 66 patients with autoimmune aPL and 152 normal controls. The prevalence of low PZ levels (below the 5th percentile of controls) was significantly greater in the 37 patients with definite antiphospholipid syndrome (APS) (24.3%) but not in the 29 aPL patients not fulfilling the criteria for APS (10.3%) compared with the normal group (4.6%, P < 0.001 vs. APS). ZPI antigen levels were similar in patients with aPL and normal controls. Concomitant PZ deficiency increased by approximately sevenfold the risk of arterial thrombosis in aPL patients. Taken together, these data suggest that the PZ/ZPI system is commonly impaired in aPL patients thus probably increasing the thrombotic risk.

High titers of autoantibodies to tissue factor pathway inhibitor are associated with the antiphospholipid syndrome.

As the activity of the tissue factor pathway inhibitor (TFPI) may be impaired in patients with antiphospholipid antibodies (aPL), 162 aPL patients were evaluated for autoantibodies to recombinant TFPI (anti-TFPI) using an optimized ELISA. Anti-TFPI (>18 U mL(-1) for IgG and/or > 15 U mL-1 for IgM) were detected in 54 patients with aPL (33.3%) and in three out of 79 normal controls (3.8%, P < 0.0001). Among aPL patients, the prevalence of positive anti-TFPI was 38.3 and 28.4% in those with or without diagnosis of definite antiphospholipid syndrome (APS). Patients with definite APS had a significantly greater frequency of high titer (>50 U mL(-1)) anti-TFPI than aPL patients from the no definite APS group (18.5% vs. 6.2%, OR 3.7, P= 0.017). Most aPL recognized full-length TFPI, but not a truncated form of TFPI lacking the C-terminus of the molecule. Isolated IgGs from subjects with anti-TFPI impaired the dose-dependent inhibitory effect of TFPI on factor Xa activity in the presence, but not in the absence of phospholipid vesicles. Thus, aPL with high titer anti-TFPI limit TFPI action and are associated with the APS.

Link between anti-CD36 antibodies and thrombosis in the antiphospholipid syndrome.

OBJECTIVE: Some studies have previously suggested the involvement of antibodies directed against CD36 (anti-CD36) in the pathogenesis of thrombosis. The aim of this study was to evaluate the prevalence of anti-CD36 in patients with antiphospholipid antibodies (aPL) and its relationship with thrombosis. METHODS: Anti-CD36 were tested using an indirect MAIPA assay in 62 patients with autoimmune aPL but without SLE; there were 38 with and 24 without thrombosis. Nineteen patients with thrombosis served as an aPL(-) control group and 58 healthy subjects as the normal control group. RESULTS: 15 of 62 aPL patients (24.2%) but only 1 of 58 (1.7%) normal controls had anti-CD36 (p < 0.0005). As compared to normal controls, the prevalence of anti-CD36 was significantly higher in aPL patients with (26.3%, p < 0.0005) or without thrombosis (20.8%, p < 0.01). Anti-CD36 were significantly more frequent in aPL patients with thrombosis than in thrombosis aPL(-) subjects (26.3% vs 0%, p = 0.02). The presence of anti-CD36 seems to be more frequent in aPL patients with recurrent thrombosis than in those with a single episode (36.8% vs 15.8%). CONCLUSION: The presence of anti-CD36 is highly prevalent in patients with autoimmune aPL with a trend to being more frequent in patients with recurrent episodes of thrombosis.

Central retinal vein occlusion and thrombophilia risk factors.

The purpose of the present study was to investigate the role of risk factors predisposing to thrombosis in patients with central retinal vein occlusion (CRVO). We prospectively examined 37 consecutive patients with CRVO, and 144 healthy controls, for major and potential inherited and acquired thrombophilic risk factors. Among them, only the prevalence of hyperhomocysteinaemia (10/37, 27.0%) and antiphospholipid antibodies positivity (5/37, 13.5%) were significantly higher in patients with respect to controls (5.5%, P < 0.001 and 2.1%, P < 0.01, respectively). Both hyperhomocysteinaemia and antiphospholipid antibodies seem to be associated with CRVO. A search for acquired thrombophilia is advisable in patients with CRVO.