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.

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.

Major and potential prothrombotic genotypes in a cohort of patients with venous thromboembolism.

Factor V Leiden (FVL) and the prothrombin 20210A (PT-20210A) variant are well-known risk factors for venous thromboembolism (VT). The thermolabile variant (TT) of the methylenetetrahydrofolate reductase (MTHFR) gene, and homozygosity for the 4G allele of the promoter region of the plasminogen activator inhibitor-1 (PAI-1) are potential genetic polymorphisms that have not been consistently associated with increased risk of VT. A case-control study was performed on 192 consecutive unrelated patients referred for evaluation of thrombophilia because of VT and 200 healthy controls. FVL was found in 10.4% of patients compared to 3.0% of controls, while 6.3% of patients were carriers of the PT-20210A allele compared to 2.0% of controls. The adjusted odds ratios (OR) were 5.92 and 4.03 for FVL (P=.001) and the PT-20210A (P=.033), respectively. The prevalence of homozygotes for MTHFR (TT) and PAI-1 (4G/4G) among patients and controls were 13.7% versus 13.0% and 21.6% versus 23.5%, respectively (P=ns). A total of 121 patients underwent a complete screening for FVL, the PT-20210A, protein C (PC), protein S (PS), antithrombin III (ATIII), levels of factor VIII, and antiphospholipid antibodies (aPL). In 59 patients (48.8%) at least one defect was found, being a single defect in 55 and combined defects in 4 patients. Plasma levels of homocysteine (Hcy) were measured in 138 patients and 144 controls. Subjects from both groups carrying the MTHFR-TT variant had higher Hcy levels than those with the normal genotype. Hyperhomocysteinemia (HHcy) by itself is a risk factor for VT (OR 4.92, P<.0001). We conclude that FVL and the PT-20210A are risk factors for VT as well as Hcy levels, but the MTHFR and PAI-1 polymorphisms do not appear to be associated with VT in our country.

Lupus anticoagulant, high levels of anticardiolipin, and anti-beta2-glycoprotein I antibodies are associated with chronic thromboembolic pulmonary hypertension.

OBJECTIVE: To evaluate the prevalence of lupus anticoagulant (LAC) and anticardiolipin antibodies (aCL), and that of anti-beta2- glycoprotein I (anti-beta2-GPI) and prothrombin antibodies in patients with pulmonary hypertension (PH). METHODS: Fifty-four consecutive patients with PH were studied: 23 with primary, 20 secondary, and 11 chronic thromboembolic PH. LAC was diagnosed by screening and confirmatory coagulation tests, while aCL, anti-beta2-GPI, and prothrombin antibodies were measured by ELISA. RESULTS: Prevalence of aPL was higher in patients with chronic thromboembolic PH compared to the other 2 groups. The prevalence in chronic thromboembolic PH vs primary and secondary PH was: LAC 63.6 vs 13.0 and 10.0%, p < 0.001; aCL-IgG 54.5 vs 17.4 and 15.0%, p < 0.02; anti-beta2-GPI-IgG 36.4 vs 0 and 0%, p < 0.001; and prothrombin antibodies-IgG 36.4 vs 8.7 and 5.0%, p < 0.05. No differences between groups were found for any antibody of IgM isotype. Antibodies detected in patients with primary and secondary PH were of low titer, so considering only moderate or high titers these differences were greater for aCL-IgG (odds ratio, OR 24.6, confidence interval, CI 3.0-282, p = 0.0004) and IgM (OR 35.0, CI 2.9-1692, p = 0.0007) and remained significant for anti-beta2-GPI-IgG (OR = undefined, p = 0.006). Multivariate analysis showed that only LAC and aCL-IgG at moderate or high levels were independent variables associated with chronic thromboembolic PH. CONCLUSION: The presence of LAC, moderate or high levels of aCL-IgG, or anti-beta2-GPI-IgG was strongly associated with that of chronic thromboembolic PH. These data are in agreement with the close relationship observed among these 3 variables and thromboembolism in patients with aPL.

Relationship of anti beta2-glycoprotein I and anti prothrombin antibodies to thrombosis and pregnancy loss in patients with antiphospholipid antibodies.

The lupus anticoagulant (LA) and anticardiolipin antibodies (aCL) are clinically relevant because of their association with thrombosis and pregnancy loss. The group of antiphospholipid antibodies (aPL) includes antibodies primarily directed against various phospholipid-binding proteins, mainly beta2-glycoprotein I (beta2GPI) and prothrombin. Some studies suggest that there is an association between the presence of anti beta2GPI antibodies (alphabeta2GPI) of IgG isotype and thrombosis. Therefore, aPL defined according to the plasma protein to which they are directed appear to be more appropriate for the evaluation of their clinical importance. Using home-made ELISAs we evaluated the presence of alphabeta2GPI and antiprothrombin antibodies (anti-II) of both isotypes (IgG and IgM) in a group of 233 patients with LA and/or aCL. Forty-four women had a history of pregnancy loss, 45 patients had a history of venous thrombosis (VT) and 32 of arterial thrombosis (AT). Patients from the autoimmune group (systemic lupus erythematosus and antiphospholipid syndrome) had a higher prevalence of alphabeta2GPI and/or anti-II than those from the miscellaneous group. In the univariate analysis, a significant association was shown between the presence of alphabeta2GPI-IgG (OR 3.2; 95% CI 1.5-6.6) and previous VT, but not AT. Anti-II were related to VT but the multivariate analysis showed that alphabeta2GPI-IgG are the only independent risk factor for VT (OR 3.0; 95% CI 1.3-6.2). The presence of alphabeta2GPI-IgM correlates well with a history of pregnancy loss (OR 2.6; 95% CI 1.1-6.1). The coagulation tests profile showed that the clotting assays were more prolonged in patients having aCL, alphabeta2GPI or anti-II. But a higher prevalence of abnormal results was only found for the dilute Russell viper venom time in patients with VT, as compared to those without thrombosis (94.4% vs. 58.7%, p <0.02). The measurement of alphabeta2GPI of both isotypes could help to identify aPL-positive patients with a higher risk for thrombosis and pregnancy loss, although this association should be confirmed by prospective studies.

Pregnancy loss and autoantibodies against phospholipid-binding proteins.

OBJECTIVE: To evaluate the relationship between antibodies against beta 2-glycoprotein I or prothrombin and pregnancy losses in women with antiphospholipid antibodies. METHODS: Women with antiphospholipid antibodies, (lupus anticoagulant and/or anticardiolipin antibodies), with (n = 41) and without (n = 61) a history of pregnancy loss were evaluated. Thirty-one out of the forty-one patients with pregnancy loss had early miscarriages (at less than 13 weeks) and ten patients had late miscarriages. Immunoglobulin (Ig)-G and IgM anti-beta 2-glycoprotein I and anti-prothrombin antibodies were measured by an enzyme-linked immunosorbent assay method. RESULTS: A significant association between pregnancy loss and positive IgM anti-beta 2-glycoprotein I antibodies was found (odds ratio 2.6; 95% confidence interval 1.03, 6.6; P = .043). Women with late pregnancy loss had higher levels of both IgG and IgM anti-beta 2-glycoprotein I antibodies compared with controls (P < .05). There was a good correlation between anticardiolipin and anti-beta 2-glycoprotein I antibodies levels (IgG: r = 0.75; IgM: r = 0.73). In contrast, there was no correlation between the levels of anticardiolipin or anti-beta2-glycoprotein I antibodies and the levels of anti-prothrombin antibodies. Furthermore, the presence of anti-prothrombin antibodies was not associated with a history of pregnancy loss. CONCLUSION: The result of our study shows that there is a relationship between the presence of IgM anti-beta 2-glycoprotein I and previous miscarriages in women with anti-phospholipid antibodies.

Evaluation of recently described tests for detection of the lupus anticoagulant.

It is known that lupus anticoagulants (LA) are antibodies which interfere with phospholipid-dependent coagulation tests, but due to the heterogeneity of LA and the differences in sensitivity of reagents and tests, the diagnosis of LA remains difficult. Recently, Triplett et al. (26) have proposed a new test based on two venoms, Textarin (T) and Ecarin (E), that activate prothrombin but differ in their phospholipid requirements. By testing this new assay we have evaluated 36 patient plasmas containing LA according to standard tests (activated partial thromboplastin time, dilute Russell viper venom time and platelet neutralization procedure) and our results confirm a high sensitivity for LA of the T/E test. In addition, we observed a greater sensitivity of the tissue thromboplastin inhibition test using a recombinant thromboplastin instead of a human placenta thromboplastin. Our study also showed that the T/E test seems to be a useful assay in confirming the diagnosis of LA in patients with an unexplained prolonged APTT.