B. de Laat

The association between circulating antibodies against domain I of beta2-glycoprotein I and thrombosis: an international multicenter study

Summary.  Background: Diagnosis of the antiphospholipid syndrome (APS) is difficult as a result of limited specificity of existing assays for detecting clinically relevant antiphospholipid antibodies. Anti‐beta2‐glycoprotein I (beta2GPI) antibodies play a central role in the disease process of APS. Objectives: We have investigated the relation between antiphospholipid antibodies with specificity for domain I of beta2GPI and thrombosis/pregnancy morbidity in an international multicenter study. Patients/methods: Four hundred and seventy‐seven patients derived from nine different centres met the inclusion criterion of having anti‐beta2GPI antibodies in their plasma/serum. Clinical data and results of tests for lupus anticoagulant, anti‐cardiolipin antibodies and anti‐beta2GPI antibodies were established at the different centres of inclusion. After being re‐tested for the presence of IgG and/or IgM anti‐beta2GPI antibodies, the samples were tested for the presence of IgG‐directed against domain I of beta2GPI and results were correlated with the thrombotic and obstetric history. Results: Re‐testing for the presence of anti‐beta2GPI antibodies resulted in inclusion of 442/477 patients. IgG class anti‐domain I antibodies were present in plasma of 243/442 patients (55%). 201/243 (83%) had a history of thrombosis. This resulted in an odds ratio of 3.5 (2.3–5.4, 95% confidence interval) for thrombosis. Anti‐domain I IgG antibodies were also significantly correlated with obstetric complications [odds ratio: 2.4 (1.4–4.3, 95% confidence interval)]. Conclusion: In this multicenter study, the detection of IgG antibodies that are directed against domain I of beta2GPI proved to be more strongly associated with thrombosis and obstetric complications than those detected using the standard anti‐beta2GPI antibody assay.

Testing for antiphospholipid antibodies with solid phase assays: guidance from the SSC of the ISTH

K . M. J . DEVREESE ,* S . S . P I ERANGEL I ,† B . DE LAAT ,‡ A. TR IPODI ,§ T . ATSUMI ¶ and T . L . O r t e l , ** FOR THE SUBCOMMITTEE ON LUPUS ANT ICOAGULANT/PHOSPHOL IP ID/DEPENDENT ANT IBODIES *Coagulation Laboratory, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, Ghent, Belgium; †University of Texas Medical Branch, APLS Laboratory, Galveston, TX, USA; ‡Department of Biochemistry, Synapse BV, Maastricht University, Maastricht, the Netherlands; §Department of Clinical Sciences and Community Health, Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Universit a degli Studi di Milano and IRCCS Maggiore Hospital Foundation, Milan, Italy; ¶Department of Medicine II, Hokkaido University Graduate School of Medicine, Sapporo, Japan; and **Departments of Medicine and Pathology, Duke University Medical Center, Durham, NC, USA

Detection of lupus anticoagulant in the presence of rivaroxaban using Taipan snake venom time

G. M. A . VAN O S ,* B . DE LA AT , * § P . W. KAMPHUISEN ,– J . C . M. ME I J ERS – and P H. G . D E GRO O T* *Laboratory of Clinical Chemistry and Haematology, University Medical Center, Utrecht; Department of Experimental Vascular Medicine, Academic Medical Center, University of Amsterdam; Sanquin Research, Amsterdam; §Synapse, CARIM, Maastricht University Medical Center, Maastricht; and –Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

In vitro assessment, using thrombin generation, of the applicability of prothrombin complex concentrate as an antidote for Rivaroxaban

Rivaroxaban has been approved as an antithrombotic agent for prevention of venous thromboembolism with specific indications. At present no antidote is appointed and no guidelines have been formulated for the measurement of Rivaroxaban reversal.

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

Summary.  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.

Annexin A5 polymorphism (−1C→T) and the presence of anti-annexin A5 antibodies in the antiphospholipid syndrome

Background: Annexin A5 is thought to have a role in the pathophysiology of the antiphospholipid syndrome (APS)—a syndrome characterised by recurrent thrombosis and pregnancy morbidity. Objective: To investigate whether anti-annexin A5 immunoglobulin (Ig)M or IgG antibodies, or the −1C→T polymorphism of annexin A5, is a risk factor for thrombosis or miscarriage, and whether the −1C→T polymorphism is correlated with APS. Methods: A cohort study was carried out with a population of 198 patients with primary APS, systemic lupus erythematosus or lupus-like disease. For the detection of anti-annexin A5 antibodies and the measurement of annexin A5 plasma levels, ELISA-type methods were used. The annexin A5 −1C→T mutation was detected by restriction fragment length polymorphism. Results: 71 patients were positive for annexin A5 IgM or IgG antibodies, of whom 53 patients were positive for anti-annexin A5 IgG antibodies and 27 of 198 patients were positive for anti-annexin A5 IgM antibodies. The prevalence of IgM or IgG anti-annexin A5 antibodies was not significantly associated with thrombosis or miscarriage on multivariate analysis. The prevalence of the −1C→T mutation in the annexin A5 gene (46/198 patients) was significantly associated with miscarriage (odds ratio 2.7, 95% confidence interval 1.1 to 6.7, independent risk factor). Conclusion: The detection of anti-annexin A5 antibodies does not seem relevant for estimating the risk for thrombosis or miscarriage in APS. The −1C→T mutation was an independent risk factor for miscarriage, which is independent of APS.

High-avidity anti-β2 glycoprotein I antibodies highly correlate with thrombosis in contrast to low-avidity anti-β2 glycoprotein I antibodies

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Mixing studies in lupus anticoagulant testing are required at least in some type of samples

According to the ISTH guidelines for lupus anticoagulant (LAC) testing, the second step in the three‐step procedure (screening, mixing, and confirmation) is the mixing test, which improves the discrimination between the presence of an inhibitor and coagulation factor deficiencies such as those occurring in patients receiving vitamin K antagonists (VKAs).

Lupus anticoagulant: performance of the tests as recommended by the latest ISTH guidelines

Summary.  Objectives: Lupus anticoagulant (LA) is clinically the most relevant among all antiphospholipid antibody tests. Recently, new guidelines for LA detection were published. The objective of this retrospective cohort study was to compare tests recommended under these guidelines with other methods used for LA detection. Methods: The study group consisted of 336 subjects suffering from various autoimmune diseases. We used activated partial thromboplastin time (aPTT), diluted Russell viper venom time (dRVVT) and diluted prothrombin time (dPT) tests for LA detection together with a ratio between sensitive and insensitive aPTT reagent. We also tested if LA was dependent on β2glycoprotein I (β2GPI) using one of the recently described methods. Results: All LA tests performed were associated with a history of thrombosis. The highest odds ratio (OR) for thrombosis was found for β2GPI‐dependent LA but sensitivity was low (OR = 8.4; specificity/sensitivity = 98%/15%). All LA tests showed a much stronger association with thrombosis than with pregnancy failure. Conclusions: LA tested by aPTT and/or dRVVT (at least one out of two tests positive), as recommended by the guidelines, was associated less strongly with a history of thrombosis (OR = 4.1) than either of these tests separately (OR = 5.0 and 4.3, respectively). With both tests positive (‘double LA positivity’) the association with thrombosis was stronger (OR = 6.5) compared with only one positive test. In fact, ‘double LA positivity’, detected by combinations of any of the tests studied, was markedly associated with a history of thrombosis.

Thrombin generation assay using factor IXa as a trigger to quantify accurately factor VIII levels in haemophilia A

Summary.  Background: The available methods for measuring factor VIII (FVIII) activity suffer reportedly from lack of sensitivity and precision in the < 1 IU dL−1 range. This precludes correlation of clinical phenotype with FVIII levels. Objectives: To study a possible association between clinical phenotype in patients with FVIII levels < 1 IU dL−1. Methods/Results: The FIXa‐driven FVIII assay (FVIII‐CAT) has a detection limit of 0.05 IU dL−1. For the range of 0–2 IU dL−1 FVIII, the intra‐assay coefficient of variation (CV) is around 2% and the inter‐assay CV is about 8%. We tested 30 hemophiliacs with FVIII:C between < 1 and 6 IU dL−1 as measured in the one‐stage clotting assay using the FVIII‐CAT assay. For genetic defects related to moderate hemophilia, the FVIII‐CAT test finds FVIII levels that are in good agreement with those determined with the one‐stage assay. Of the 21 hemophilic patients with FVIII < 1 IU dL−1, four patients exhibited a mild bleeding phenotype. When we applied TF‐initiated thrombin generation, patients with a mild clinical phenotype showed significantly higher endogenous thrombin potentials. Conclusion: The novel developed FVIII assay measures accurately FVIII levels below 1 IU dL−1. Its application demonstrated that the clinical heterogeneity in individuals with < 1 IU dL−1 FVIII is not associated with their FVIII level.