Dilute Russell Viper Venom Research Articles

Thrombophilic factors in idiopathic intracranial hypertension: a report of 51 patients and a meta-analysis

Several studies have suggested that thrombophilic risk factors are more prevalent in individuals with idiopathic intracranial hypertension (IIH), and that a prothrombic state may be involved in the etiopathogenesis of this disease. We examine thrombophilic factors in a group of patients with IIH in relation to obesity. In addition, we reviewed the relevant literature and performed a meta-analysis. Thrombophilia work-up was performed on 51 patients with IIH at least 1 month following their first episode. Samples for the analysis of factor V Leiden (FVL), prothrombin gene variant (PGV) G20210A and methylenetetrahydrofolate reductase (MTHFR) were available in an additional 30 patients, that is 81 patients in all. Meta-analysis was performed. Of the 51 patients 40 were obese. Increased concentrations of fibrinogen, D-Dimer, factor VIII, factor IX and factor XI were found in 15, 7, 7, 6 and 2 patients, respectively, all obese. The circulating anticoagulant, measured by dilute Russell's viper venom time (dRVVT assay), found mainly in obese. All 51 patients were negative for the anticardiolipin antibody (IgG immunoglobulin G) and IgG anti-beta2 glycoprotein I. In the meta-analysis antiphospholipid antibodies were significantly associated with IIH [odds ratio (OR) of 4.25 (1.68-12.60)], similar to the association with high factor VIII [OR = 16.17 (2.87-91.01)], higher plasminogen activator inhibitor-1 (PAI-1) levels [OR = 6.91 (2.28-20.91)], and high lipoprotein (a) [LP(a)] [OR = 3.54 (1.54-8.70)]. Obesity often observed in IIH patients is frequently linked with thrombophilic factors. Thus, we believe that dysmetabolism could be the thrombophilic target for treatment in patients with IIH.

Usefulness of Silica Clotting Time for Detection of Lupus Anticoagulants

The presence of lupus anticoagulants (LA) is a strong risk factor for thrombosis in antiphospholipid syndrome. We investigated the usefulness of addition of silica clotting time (SCT) to the pre-existing dilute Russell's viper venom test (dRVVT) for detection of LA. Also, we analyzed differences in the thrombotic features and the characteristics of antiphospholipid antibodies between dRVVT and SCT. A total of 167 patients positive for LA or anti-cardiolipin (anti-CL) antibody and 76 healthy controls were enrolled. The dRVVT and SCT were used for detection of LA. Anti-CL, anti-beta2-glycoprotein I (anti-beta2 GPI) and anti-prothrombin (anti-PT) antibodies were measured using commercial ELISA kits. In detection of thrombosis, the sensitivity of the combined test of SCT and dRVVT was 56.4%, which was higher than that of dRVVT alone (46.2%) or SCT alone (23.1%). The specificity of the combined test (80.9%) was comparable to that of dRVVT (81.9%). Also, odds ratio for predicting thrombosis was higher in the combined test than in dRVVT or SCT alone. When normalized LA ratio of the two tests was compared, the group of patients with higher ratio of SCT showed significantly higher prevalence of recurrent abortion and higher positivity of IgG types of anti-CL, anti-beta2 GPI and anti-PT than the group with higher ratio of dRVVT. Addition of SCT to dRVVT can improve the detection sensitivity of thrombosis in LA test. And the high normalized LA ratio of SCT may be a useful parameter for detection of recurrent abortion.

Current clinical and laboratory practice for the investigation of the antiphospholipid syndrome: findings from the 2008 Australasian antiphospholipid antibody survey

The antiphospholipid syndrome (APS) is an autoimmune condition characterised by vascular thromboses and/or pregnancy morbidity, and its diagnosis currently requires laboratory evidence for the presence of antiphospholipid antibodies (aPL). aPL are identified using a large number of laboratory procedures based on one of two distinct test processes, namely 'solid' or 'liquid' phase assays. The former include anticardiolipin antibodies (aCL) and anti-beta-2-glycoprotein-I antibodies (aB2GPI), while the latter are centred on clot-based tests used to identify the lupus anticoagulant (LA). Depending on their clinical presentation, affected individuals might be seen by a variety of clinical specialties including general physicians and general practionners, with a potentially wide variation in the aPL assays requested. The current report summarises findings from the '2008 Australasian antiphospholipid antibody survey', a simple 5-step survey that assessed current clinical and laboratory practice in the investigation of APS. The survey was despatched via various clinical and scientific professional bodies. Responses were received from 130 scientific and clinical personnel, primarily haematology based (94/130; 72%) or immunology based (34/130; 26%). Most respondents (97/130; 75%) ordered or recommended tests for solid phase aPL testing, and most also attempted to grade these tests and their isotypes. Most were familiar with aCL and aB2GPI testing, and tended to request primarily IgG and IgM isotypes of these antibodies. Only a small number of respondents requested/recommended IgA isotype testing of these antibodies or the other solid phase aPL assays (e.g., anti-prothrombin). A similar number of respondents (104/130; 80%) also ordered or recommended tests for LA, and most also attempted to grade these tests and their isotypes. Some discipline-related biases were also evident, in that 32/34 (94%) of immunology-based respondents identified that they ordered or recommended specific solid phase tests for aPL, whereas only 62/94 (66%) of haematology-based respondents did so. In contrast, 83/94 (88%) of haematology-based respondents identified that they ordered or recommended specific LA test procedures, whereas only 18/34 (53%) of immunology-based respondents did so. To our knowledge, this report represents the first ever attempt to survey a wide range of clinical and scientific personnel regarding ordering and recommending practices for aPL testing, and provides a snapshot of current clinical and laboratory practice for the investigation of APS in Australia and New Zealand. Most respondents to our survey still consider the immunoglobulin G (IgG) aCL test to be a useful first-line solid phase aPL test, and the dilute Russell's viper venom time (dRVVT) assay to be the most useful LA test.

Clinical Significance of Indeterminate Lupus Anticoagulant (LAC) Results.

Abstract 3988 Poster Board III-924 Abstract: Introduction The International Society of Thrombosis and Hemostasis (ISTH) criteria for the diagnosis of the lupus anticoagulant (LAC) include: Screening test that demonstrates the prolongation of a phospholipid-dependent (PL-D) clotting time; mixing test that confirms the presence of an inhibitor; the confirmation that the inhibitor is PL-D and exclusion of other coagulopathies. Test results that do not fulfill all the criteria are considered indeterminate. These indeterminate results are common (Kottke-Marchant et al. J Thromb Haemost. 2007; 5 Supplement 2: P-M-455), still there is no published data regarding clinical significance. Patients/methods This study investigated the prevalence of thrombotic events in an initial cohort of unselected patients (n=256) from one tertiary hospital in the United States, who were tested for LAC and other antiphospholipid (aPL) antibodies from a 2 month period in 2006. The laboratory results (PT/INR, aPTT, dilute Russell's viper venom time (DRVVT), STACLOT and platelet neutralization (PNP)) were evaluated. The profile included 3 separate PL -D assays (DRVVT confirm, STACLOT, PNP). Samples containing heparin (>0.1U/ml) were pre-treated with Hepadsorb. The LAC profile was considered indeterminate if PL test results were positive, but without a positive aPTT or DRVVT mixing study. The initial cohort included 83 patients with indeterminate results. From this group, 18 patients were excluded: Four due to incomplete data, 2 due to high heparin level (anti Xa>1.0 U/ml), 5 due to other prothrombotic etiologies and 7 with other positive aPL antibodies. For an assessment of thrombotic history, we performed retrospective chart reviews and tabulated all Sapporo clinical features, malignancy and auto-immune disorders within 5 years before and 2 years after the index laboratory testing. Events that did not fulfill diagnostic criteria for thrombosis, ischemic events or obstetrical complications were excluded. The final analysis sample included 65 patients with indeterminate LAC, 106 with negative and 27 with positive LAC. Results The final indeterminate LAC cohort included 65 patients, with mean follow-up of 18 months. Malignancy was present in 29% and autoimmune disease in 25% of patients. The most common thrombotic events were deep vein thrombosis (DVT) (28%), cerebral ischemic stroke (14%) and pulmonary embolism (14%). When compared to those with negative tests, indeterminate group patients were more likely males, relatively older, and more likely to have DVT, superficial thrombosis (ST) or myocardial infarction (MI) (P= 0.049, 0.021, 0.044, 0.005 and 0.045 respectively). Concurrent coumadin (warfarin) therapy was more prevalent in the indeterminate group, but it did not reach statistical significance (p=0.15). There was no statistical significant difference in the prevalence of cancer or autoimmune disease (P=0.19 and 0.48 respectively). In the multivariate analysis model none of the previous variables reached any statistical significance between the two groups. When compared the above clinical variables between indeterminate results and positive LAC results groups from the same cohort, we failed to show any major statistically significant differences. We noticed very poor retesting rate in the indeterminate group during the follow up period of 2 years (15% only). Conclusions Indeterminate results are common among patients referred for LAC testing. When compared to those with negative results, patients with indeterminate results are more likely to have a history of DVT, superficial thrombosis or MI, but none of the clinical variables reached statistical significance in a multivariate model. On the other hand, patients in the indeterminate group shared demographic and clinical profiles with those in the positive results group. This further highlights the need to study the clinical significance of indeterminate LAC results in a prospective study. Disclosures: No relevant conflicts of interest to declare.

Anticoagulation Management in a Patient With Antiphospholipid Antibodies Requiring Repeat Sternotomy

THE MANAGEMENT OF anticoagulation for cardiopulmonary bypass (CPB) in patients with antiphospholipid antibodies is challenging because antiphospholipid antibodies increase the risk for arterial and venous thrombosis and affect commonly used in vitro coagulation assays such as the kaolin-activated coagulation time. 1 Petri M. Epidemiology of the antiphospholipid antibody syndrome. J Autoimmun. 2000; 15: 145-151 Crossref PubMed Scopus (391) Google Scholar , 2 Asherson R.A. Khamashta M.A. Ordi-Ros J. et al. The primary antiphospholipid syndrome: Major clinical and serological features. Medicine (Baltimore). 1989; 68: 366-374 Crossref PubMed Scopus (990) Google Scholar , 3 Finazzi G. Brancaccio V. Moia M. et al. Natural history and risk factors for thrombosis in 360 patients with antiphospholipid antibodies: A four-year prospective study from the Italian Registry. Am J Med. 1996; 100: 530-536 Abstract Full Text PDF PubMed Scopus (395) Google Scholar , 4 Galli M. Finazzi G. Bevers E.M. et al. Kaolin clotting time and dilute Russell's viper venom time distinguish between pro-thrombin dependent and beta 2 glycoprotein I dependent antiphospholipid antibodies. Blood. 1995; 86: 617-623 PubMed Google Scholar

In vitro Effects of Detergent Sclerosants on Antithrombotic Mechanisms

To investigate the in vitro effects of detergent sclerosants on antithrombotic pathways. Proteins C, S and antithrombin (AT) were assayed in normal plasma treated with increasing concentrations of sodium tetradecyl sulphate (STS) and polidocanol (POL). Activated protein C (APC) was investigated by mixing normal plasmas with sclerosants and testing with the activated partial thromboplastin time (APTT) and dilute Russell's viper venom time in the presence and absence of APC. The effect on factor Xa (FXa), heparin and enoxaparin was investigated using chromogenic anti-FXa and APTT methods. High concentration (>0.6%) STS significantly destroyed proteins C, S and AT whereas POL only caused a mild reduction in PC and AT and a moderate (60%) reduction in PS levels. STS potentiated the anticoagulant effect of APC while POL increased APC resistance. STS mimicked AT and demonstrated significant anti-Xa and anti-IIa activity. STS demonstrated a similar anticoagulant profile to heparin but was 1000x weaker. It also significantly potentiated the anticoagulant effect of heparin while POL had less effect. STS and POL demonstrated quite distinct and sometimes opposite effects on the antithrombotic mechanisms assayed. These effects were concentration-dependent and in general, STS had the greatest effect on antithrombotic proteins.

Plasma from Patients with a Lupus Anticoagulant Induce Tissue Factor Activity in Monocytes of Whole Blood-a Possible Risk Factor for Thrombosis

BACKGROUND: The presence of a lupus anticoagulant (LA) in a patient with a thromboembolic event often requires life-long oral anticoagulant. Yet, long-term oral anticoagulant therapy is not without risks. In addition, many individuals are discovered to have a LA but have not yet had a thromboembolic event. It is desirable to identify those at high risk of developing thromboembolic diseases and those unlikely to do so. Circulating monocytes are capable of generating functionally active tissue factor (TF) under many pathologic conditions. We hypothesize that patients with a lupus anticoagulant whose plasmas induce significant tissue factor activity on monocytes may be at higher risk of thrombosis than those whose plasmas do not.METHOD: To test the hypothesis, we collected consecutive leftover plasma samples that tested positive for the presence of a lupus anticoagulant by the Dilute Russell's Viper Venom Test from a clinical coagulation laboratory (DTL) over a four years period. These plasma samples were tested for their ability to induce TF activity in monocytes by recombining the test plasma samples with blood cells from freshly drawn blood, anticoagulated with Fragmin (heparin) and recalcified followed by incubation for 2 hrs at 37°C, a method previously described by Østerud et al (Blood Coagul Fibrinolysis 1990; 1:41–6). Seventeen plasma samples from healthy donors were also tested as control. The test laboratory (BØ) did not know the clinical diagnosis of these patients at the time of testing. The test results were then matched with the corresponding patients' clinical diagnosis.RESULTS: A total of 112 patients' plasma samples were tested, of which clinical information was available for 108 patients. The clinical data were used to divide the patients into two groups: those with thromboembolic diseases and those without at the time of testing. The results from TF activity induction data were also divided into two groups: those with significant TF induction activity and those without. The mean tissue factor activity detected with normal donors was 0.20 mU/106 monocytes, S.D= 0.20, range 0.02–0.5. A cut off value for positive tissue factor induction was chosen to be >1.0 (>4 S.D.). A contingency table was then constructed as follow:ThrombosisTF Activity InductionNoYesNegative2815Positive2243A Fisher's exact test analysis was carried out, which shows an Odd Ratio of 3.65 and a relative risk of 1.92, p=0.0017. In an additional on going study, IgGs were purified from 22 patients with significant thromboembolic diseases and of whom the plasmas were strongly positive for a lupus anticoagulant by the DRVVT test. Seventeen out of 22 of the IgG preparation induced significant TF activity on monocytes but failed to induce TF activity in neutrophils.CONCLUSIONS:Significant TF induction activity in a patient with a lupus anticoagulant is associated with an odd ratio of developing thrombosis of 3.6.IgGs from a majority, but not all, of patients with thromboembolic diseases and a lupus anticoagulant induce significant TF activity on monocytes.We propose that measurement of TF induced on monocytes of whole blood by plasma from an individual with a lupus anticoagulant may provide a method to evaluate potential risk of thrombosis in that patient.

The Activated Seven Lupus Anticoagulant Assay Detects Clinically Significant Antibodies

Lupus anticoagulants are a heterogeneous group of autoantibodies detected by their effects on phospholipid-dependent coagulation assays. Persistent lupus anticoagulants are associated with thrombotic disease, but not all are clinically significant. Antibody heterogeneity and reagent and test variability dictate that at least 2 tests, of different types, should be used to screen lupus anticoagulants. The objective of this study was to investigate whether the activated seven lupus anticoagulant assay detects clinically significant antibodies. Eighty-two patients with antiphospholipid syndrome (APS) and 32 with systemic lupus erythematosus + positive for activated seven lupus anticoagulant and who were without thrombosis, who were positive by activated seven lupus anticoagulant assay, were investigated for lupus anticoagulants by dilute Russell's viper venom time, dilute activated partial thromboplastin time, and Taipan snake venom time, and for anticardiolipin antibodies. Fifty-seven of the APS patients were positive for lupus anticoagulants in multiple assays, 25 in activated seven lupus anticoagulant alone. Fourteen of the latter group were previously positive in other antiphospholipid antibodies assays, and 11 had only been positive for lupus anticoagulants by activated seven lupus anticoagulant. Twenty-eight had elevated anticardiolipin antibodies, 6 of whom were from the group that was positive in activated seven lupus anticoagulant only. Eight of the systemic lupus erythematosus + lupus anticoagulants (without thrombosis) patients were positive for lupus anticoagulant by activated seven lupus anticoagulant alone and had only been positive in activated seven lupus anticoagulant previously, and none had elevated anticardiolipin antibodies. The remaining 24 patients were lupus-anticoagulant positive in multiple assays, and 9 had elevated anticardiolipin antibodies. Dilute Russell's viper venom time and Dilute activated partial thromboplastin time are widely used to detect lupus anticoagulants and are considered to detect clinically significant antibodies. Activated seven lupus anticoagulant detected antibodies in APS patients who were positive by these assays and also lupus anticoagulants undetectable by the dilute Russell's viper venom time/dilute activated partial thromboplastin time reagents used, demonstrating its utility as a first-line or second-line assay.

Performance of Platelin LS and dilute Russell's viper venom for the screening of lupus anticoagulant in patients with venous thromboembolism

Lupus anticoagulant is associated with thrombosis and pregnancy morbidity, and its detection is of major clinical importance. The nature and concentration of phospholipids strongly influence the sensitivity of activated partial thromboplastin time (aPTT) reagents to lupus anticoagulant. We investigated the ability of Platelin LS, an aPTT reagent, to screen lupus anticoagulant among 94 patients with venous thromboembolism by comparing its performance with the dilute Russell viper venom time (dRVVT). Twenty-four patients had an abnormal aPTT and dRVVT, whereas 37 only had a prolonged dRVVT. In users of oral anticoagulants (n = 56), the dRVVT prolonged more frequently than the aPTT (98.2 vs 39.3%, P < 0.0001). After the mixing study, seven patients maintained abnormal aPTT and dRVVT ratios, five of whom had prolonged mixture with both tests. The agreement in the mixing study between aPTT and dRVVT was substantial (kappa = 0.78, 95% confidence interval = 0.48-1.00). Except for one patient, the aPTT screened all cases that demonstrated phospholipid dependency of their inhibitor during the confirmatory procedure with the dRVVT. In conclusion, the aPTT using Platelin LS was highly associated with the presence of lupus anticoagulant detected by the dRVVT among patients with venous thromboembolism, and could be reliably employed as a screening assay for lupus anticoagulant.

Further evidence of false negative screening for lupus anticoagulants

Introduction Accurate and timely detection of lupus anticoagulants (LA) is of diagnostic and prognostic importance due to the association of persistent LA with thrombotic disease. Antibody heterogeneity and assay variability complicate LA detection and weak antibodies can go undetected. Methods Screen and confirm assays on equal volume mixing studies were performed on known LA using dilute Russell's viper venom time (DRVVT), dilute activated partial thromboplastin time (DAPTT) and activated seven lupus anticoagulant (ASLA) assay. Two established calculations for phospholipid dependence were applied to ascertain whether any antibodies diluted to within screening test reference ranges maintained a significant difference between screen and confirm results sufficient to imply LA activity. We then performed confirmatory tests on neat plasma samples from patients with thrombotic disease whose screening tests were within reference ranges. Results Forty nine of 155 DRVVT positive LA were conventionally positive in the mixing studies and 8 of the 106 negative mixing studies showed significant screen and confirm test discordance. This was the case for 21 of 56 negative DAPTT mixing studies and 2 of 39 negative ASLA mixing studies. Performance of confirm assays on the neat plasma samples with screen results within reference ranges revealed possible LA activity in 19 of 166 DRVVT results, 63 of 184 DAPTT results and 9 of 117 ASLA results. Conclusions LA activity can be demonstrated by assessment of screen and confirm data irrespective of screening test elevation above a reference range. Other workers have demonstrated this phenomenon in APTT using different study designs and it may be that standard interpretation criteria warrant re-assessment.

Anti-prothrombin antibodies predict thrombosis in patients with systemic lupus erythematosus: a 15-year longitudinal study.

To evaluate the role of anti-prothrombin (anti-PT) antibodies in predicting thrombosis in patients with systemic lupus erythematosus (SLE). An inception cohort of 101 SLE patients (12 males, 89 females; mean age 30 +/- 8 years), was considered. Clinical and laboratory evaluations were regularly performed during a 15-year follow-up (median 108 months) with a special focus on thromboembolic events. Serum samples were collected at time of diagnosis and at least once a year thereafter. IgG and IgM anti-PT, anti-cardiolipin (aCL) and anti-beta(2)glycoprotein I (beta(2)GPI) antibodies were measured by enzyme-linked immunosorbent assay (ELISA); lupus anticoagulant (LAC) was assayed by the dilute Russell's viper venom time and activated partial thromboplastin time tests. The analytical specificity of anti-PT ELISA was investigated. The timing of thrombosis occurrence was calculated using the Kaplan-Meier method. In the 15-year follow-up, thrombosis occurred in 14 out of the 101 patients: venous thrombosis in nine cases and arterial thrombosis in five. IgG and/or IgM anti-PT, anti-beta(2)GPI and aCL antibodies, and LAC activity were detected in ten, nine, seven, and nine cases, with sensitivity for thrombosis of 71.4%, 64.3%, 50% and 64.3%, respectively. Thrombosis-free survival was 90% at 5 years and 85.8% at 10 and 15 years, respectively. Thrombosis was predicted by anti-PT (P = 0.001), anti-beta(2)GPI antibodies (P = 0.002) and LAC activity (P = 0.001). Moreover, the risk of thrombosis progressively increased with the number of positive antiphospholipid antibody tests. The presence of four positive antibody tests was associated with a risk of thrombosis thirtyfold higher than in their absence. This longitudinal study shows that IgG anti-PT antibodies are predictors of thrombosis in SLE patients.

Combining Taipan snake venom time/Ecarin time screening with the mixing studies of conventional assays increases detection rates of lupus anticoagulants in orally anticoagulated patients

BackgroundOral anticoagulation compromises conventional lupus anticoagulant (LA) screening assays. Mixing studies can counteract the oral anticoagulant effect but the dilution reduces sensitivity and can generate false negative results. A firm diagnosis can be made from mixing studies when an elevated screen ratio is accompanied by a confirm ratio that generates significant correction to demonstrate phospholipid dependence, but also returns into the reference range, indicating complete normalisation of the oral anticoagulant effect. Taipan snake venom time (TSVT) with Ecarin time (ET) as a confirmatory test comprises an oral anticoagulant insensitive LA detection system and this study investigates the potential impact on detection rates when coupled with mixing studies on standard assays.MethodsEighty patients known to have LA who were receiving oral anticoagulation were tested with TSVT/ET and 1:1 mixing studies with normal plasma by dilute Russell's viper venom time (DRVVT) and dilute activated partial thromboplastin time (DAPTT) to assess detection rates by single and multiple assays.ResultsThirty three of the 80 samples from known LA positive patients were positive in all three assays and 15 were positive in combinations of DRVVT, DAPTT or TSVT/ET. The remainder were positive in only one assay; 12 by DRVVT, 4 by DAPTT and 16 by TSVT/ET. Although all DRVVT and DAPTT positive mixing studies generated significant correction of the screen ratio by the confirm ratio, not all confirm ratios corrected back into the reference range. This was the case for 87.5% of the DRVVT results, 44.7% of the DAPTT results and 13.3% of the TSVT/ET positive mixing tests.ConclusionAddition of TSVT/ET screening for LA in orally anticoagulated patients could increase diagnostic efficacy either by detecting antibodies diluted in the mixing tests of conventional assays or those that do not react in DRVVT or DAPTT. Additionally, TSVT/ET can affirm the presence of a LA where conventional assay mixing tests may not have fully counteracted the oral anticoagulant effect but confirmatory test correction suggests the presence of a LA.

Detection of acquired resistance to activated protein C associated with antiphospholipid antibodies using a novel clotting assay

Antiphospholipid antibodies (aPA) frequently interfere with the protein C pathway. This manifests as acquired activated protein C (APC) resistance in the absence of factor V Leiden and has been proposed as a putative mechanism for the pathogenesis of the antiphospholipid syndrome (APS). We have developed a Russell's viper venom test, performed with and without activation of endogenous protein C, which is sensitive to aPA-associated APC resistance. Results were reported as the endogenous APC ratio (EAPCr); the ratio of the two clotting times normalized against pooled normal plasma. Forty-four patients with aPA, anticardiolipin and/or lupus anticoagulant, including 34 with a history of thrombosis or pregnancy morbidity; a control group of aPA-negative patients; and 26 healthy normals were studied. EAPCr (mean, SD) was significantly higher in APS patients (1.94, 0.58) than normals (0.98, 0.12) or controls (1.14, 0.19; P < 0.00001). Elevated EAPCr (> 1.22) occurred in 91% of aPA-positive patients, predominantly due to resistance to APC (87%) rather than prolonged basal clotting times alone (15%). Significant correlation was observed between the EAPCr value and dilute Russell's viper venom time (rs = 0.44, P = 0.003), IgG anticardiolipin (rs = 0.54, P = 0.002), protein S (r = -0.46, P = 0.01) and activated partial thromboplastin time-based APC resistance (r = -0.61, P = 0.001). There was no significant relationship between EAPCr and protein C concentration, anti-beta2-glycoprotein-I (anti-beta2GPI) or IgM anticardiolipin. Purified aPA IgG caused a dose-dependent increase in APC resistance when added to normal plasma. We conclude that aPA-associated acquired APC resistance is a common feature of APS and may be independent of anti-beta2GPI.

Comparison of aPTT (Platelin LS®) and Dilute Russell's Viper Venom (Viperquik LA Test®) for the Screening of Lupus Anticoagulant in Patients with Venous Thromboembolism.

INTRODUCTION: To rule out the presence of LA antibodies, two or more assays that are sensitive to these antibodies must be negative. The dilute Russell's Viper Venom Time (dRVVT) and the activated partial thromboplastin time (aPTT) using sensitive reagents have been employed to detect LA. The aim of this study was to assess the concordance level of aPTT using Platelin LS and dRVVT as screening tests to identify LA in patients with venous thromboembolism (VTE).METHODS: 94 patients (58 women, 62%) with VTE were evaluated. The detection of circulating inhibitor with Platelin LS and dRVVT was based on a prolonged clotting time and a prolonged 1:1 mixture of sample plasma and normal pooled plasma. The confirmation of the phospholipid dependency was performed only with dRVVT.RESULTS: Among the 94 patients, an abnormal test was found in 24 patients (26%) with aPTT and in 61 patients (65%) using dRVVT. After mixing tests, aPTT ratio remained long in 25% of patients with abnormal aPTT, and in 9.8% with long dRVVT. Five patients had a prolonged mixing study identified by both tests, which resulted in a substantial agreement between the two tests (Kappa= 0.78). Confirmatory tests for LA were positive in 5 out of the 6 patients with long dRVVT mixture, resulting in a prevalence of LA detected by dRVVT of 5.3%. The 5 patients with LA detected by dRVVT also had prolonged mixture with aPTT.CONCLUSION: Our results indicate that aPTT with Platelin LS is highly associated with the presence of LA detected by dRVVT and may be suitable as a screening test for LA in patients with VTE.

The Prevalence and Clinical Significance of IgA Antiphospholipid Antibodies (aPL).

AIMS: We investigated the prevalence and clinical relevance and significance of the various immunoglobulin (Ig) isotypes of the different aPLs and lupus anticoagulant (LAC).METHOD: Our study registry for the year 2003 included 472 patients who had a complete antiphospholipid antibody laboratory work up and clinical evaluation. 137 had the presence of LAC, detected by the dilute Russell's viper venom time (Dade Behring, Marburg, Germany), PTT-LA (Diagnostica Stago, Asnieres, France), or hexagonal phospholipid neutralization test (Diagnostica Stago, Asnieres, France); 211 had elevated titers of antibodies of IgG, IgM or IgA isotypes against cardiolipin (aCL), phosphatidylserine (aPS), or β2-glycoprotein-I (aβ2GPI), detected by ELISA (Corgenix, Inc, Westminister, CO, USA). 204 had no evidence of either LAC or aPL.RESULTS: Of the patients with aPL, 67 (32%) had elevated titers of IgG, 126 (60%) had IgM, and 118 (56%) had IgA. Thrombotic events occurred in 61% (41 of 67), 52% (65 of 126), and 61% (72 of 118) of the patients with IgG, IgM, and IgA respectively. Patients with IgG and IgA isotypes of aCL, aPS and aβ2GPI had higher rates (58–67%) of thrombotic events (see table 1) than patients with IgM isotypes (46–50%). Stepwise logistic regression analysis identified elevated titer of IgA of any aPL as an independent risk factor for thrombosis (see table 2), even in the absence of LAC. Thrombotic events observed include deep venous thrombosis (25%), pulmonary embolism (12%), cerebrovascular accident (50%), myocardial infarction (8%), and peripheral arterial occlusion (3%); pregnancy complication, retinal vascular thrombosis, and dialysis access thrombosis were also seen.CONCLUSION: Our study demonstrates that aPL of IgA isotype is prevalent and significantly associated with thrombotic events.Proportions of Patients with Thrombosis amongst various aPLs and Ig IsotypesaCLaPSaβ2GPITotalIgG28/43 (65%)23/36 (64%)13/21 (62%)*41/67 (61%)IgM17/37 (46%)*28/58 (48%)45/90 (50%)*65/126 (52%)IgG15/26 (58%)*22/33 (67%)54/91 (59%)*72/118 (61%)Total44/79 (56%)58/105 (55%)85/159 (53%)118/211 (56%)* Two-sided P < 0.05 by McNemar's test for equality of two dependent proportionsRisk Factors for ThrombosisAntibodyOdds Ratio95% Wald Confidence LimitLAC1.6271.073–2.466*IgG regardless of LAC1.3640.781–2.384IgM regardless of LAC0.8740.569–1.344IgA regardless of LAC1.5871.012–2.490*IgG LAC negative1.7900.863–3.714IgM LAC negative0.8780.515–1.499IgA LAC negative1.8121.035–3.172** p < 0.05 by the Wald chi-square test. Hosmer and Lemeshow goodness-of-fit test was used to assess the model fit. N=472 for the 1st four analyses, N=335 for the last three analyses.

Distinguishing Antiphospholipid Antibodies from an Acquired Factor Inhibitor: A Novel Laboratory Approach.

Distinguishing between antiphospholipid antibodies (APA) and an acquired factor inhibitor can sometimes be difficult and complicated by the fact that high titer APA can interfere with in vitro phospholipid dependent coagulation tests, including factor assays and Bethesda titers. Incorrect diagnosis can lead to inappropriate management. The following case illustrates these problems and demonstrates a novel means to rapidly distinguish between APA and a factor inhibitor. A 78 year old man with a 3 year history of myelodysplastic syndrome was admitted for worsening pancytopenia. His PTT was prolonged (68 seconds) and did not correct with a mixing study. His PT was also elevated at 18 seconds. D-dimer and fibrinogen levels were normal. Some factor assays were ordered at the time showing factors IX (3%), II (103%), V (110%), VII (85%), and X (114%). Lupus anticoagulants (LA) were detected by the dilute Russell's Viper Venom test as well by a positive Staclot®LA test using standard methods. Anticardiolipin antibodies were detected by enzyme-linked immunosorbent assay (ELISA) and showed an elevated IgM level and a normal IgG level. Standard dilutions at 1:2 to 1:8 (patient plasma:buffer) were performed to evaluate for possible dilutional effects on the factor IX assays. On several attempts, the dilutional effect could not be adequately assessed because all clotting times obtained were off the standard curve. A Bethesda assay for factor IX inhibitor was then performed, using a standard procedure, indicating an anti-factor IX titer of 28 Bethesda units. Due to the unlikelihood for coexistence of APA with an acquired factor inhibitor, we used a novel method to determine if this patient plasma contained an extremely high titer of interfering APA that may have been responsible for a falsely low factor IX level determined by a phospholipid-dependent clotting assay. We postulated that diluting patient plasma with a significant amount of pooled normal plasma (PNP) would effectively decrease APA titer while not changing the clotting factor level. At a dilution of the patient plasma mixed with PNP where the factor level reaches 20-30% in a standard factor assay, further dilutional effects of this mix with buffer will be evident if the patient in fact has an interfering APA. We first serially diluted the patient plasma with PNP until we obtained a 25% factor IX activity. This occurred at a 1:64 dilution (patient:PNP). Standard dilutional assays with buffer were then performed on this mix (patient plasma mixed with PNP). These assays demonstrated 40% activity at a 1:2 dilution, 96% activity at a 1:8 dilution, and 128% activity at a 1:64 dilution. The results conclusively demonstrated the presence of an interfering APA that resulted in a falsely low detected level of factor IX. To determine if high titer APA were interfering with other phospholipid based assays, factor VIII and XI levels were drawn and showed levels of 2% and 3%, respectively. Chromogenic assays, ELISA-based techniques, and phospholipid neutralization have been developed to help differentiate between APA and factor inhibitors. However, this novel approach is potentially simpler, more rapid and less expensive, and is currently being validated on other patients with high titer APA to help validate these results.

The dilution effect of equal volume mixing studies compromises confirmation of inhibition by lupus anticoagulants even when mixture specific reference ranges are applied

Introduction One of the recommended criteria for the laboratory diagnosis of lupus anticoagulants (LA) is demonstration of inhibitory activity. This is confirmed by performing mixing tests with normal plasma, usually in a 1:1 ratio, and demonstrating persistence of an abnormal clotting time in the screening test with significant confirmatory test reduction. However, the mixing with normal plasma can dilute the antibodies to undetectable levels and generate apparent negative results. No guidelines or consensus exist in how to interpret mixing study results. Patients and methods The present study assessed the 1:1 mixing study results from 600 patients with a thrombotic history positive for LA demonstrated in neat plasma by individual assays, or combinations, of dilute Russell's viper venom time, dilute activated partial thromboplastin time, activated seven lupus anticoagulant assay and Taipan snake venom time, plus confirmatory tests. Mixing tests were assessed initially using locally derived neat plasma reference ranges and subsequently with mixture specific ranges. Results The mixture specific ranges had lower upper limits. Of the total LA positive results, 32.5% were positive in the mixing studies when neat plasma reference ranges were applied, and a further 11.2% demonstrated LA activity when using the mixture specific ranges. The remaining 56.3% had been diluted such that they did not elevate the screening test above the upper limit of normal or generated minimal prolongation with an insignificant difference between the screen and confirmatory test result sufficient to confirm LA activity. Conclusions The significant impact of the dilution effect in 1:1 mixing studies emphasises the limitations of mixing studies as a vehicle for confirmation of inhibition by LA antibodies.

Antiphospholipid antibodies in pediatric patients with prolonged activated partial thromboplastin time during infection

Objective To investigate the close association between different antiphospholipid antibodies (aPL) caused by infection and their appearance together with a prolonged activated partial thromboplastin time (aPTT). Methods Sera from 122 children were evaluated in this study. Thirty-seven children with mild to medium prolonged aPTT (>37.2 s) and elevated C-reactive protein (CRP) levels during various forms of infections (group 2), 18 children without infections (group 3) but with mild to medium prolonged aPTT and 13 children with infections (group 4) and with elevated CRP-level as well as a control group (group 1) of 54 patients without any infection and normal aPTT and negative CRP levels were investigated with commercially available ELISA tests (AESKU.Diagnostics, Wendelsheim, Germany) for the presence of antibodies directed against cardiolipin (CL), phosphatidylserine (PS) and β2-glycoprotein I ( β2GPI). The cutoff for positive results was defined with the healthy, aged matched control group (group 1) using the mean OD values plus 2 standard deviations. The lupus anticoagulant (dilute Russell's Viper Venom time, dRVVT) and coagulation Factor XII were determined with routine tests (Dade Behring). Results Detection of at least one antibody to phospholipids was possible in 89.2% of group 2. It could be shown that IgM anti- β2GPI antibodies were found in 27 (59.5%) of group 2, but only in 1 (5.6%) of group 3 ( p=0.024) and only in 4 (7.4%) of the controls ( p=0.014). The presence of IgG-anti- β2GPI antibodies showed no significant difference in the different groups. Furthermore, children of groups 2, 3 and 4 had statistically significant higher levels of antibodies against PS IgG and PS IgM than controls. Also, antibodies to CL of the IgG-type were more frequently detected in children of group 2 than in controls ( p=0.038). Detection of CL-IgM antibodies did not reach a significant level in the comparison of the different groups. Conclusion During commonly acquired infections elevation of aPL of nearly all types seems to be a common process. Mild prolongation of aPTT might reflect this presence of aPL in the course of the infectious disease. Our data suggest that there exists no differences in specificity in comparison to the “pathogenic” aPL but the presence over time might be the trigger for the autoimmune activity to begin.

Idiopathic Intracranial Hypertension: Associations with Thrombophilia and Hypofibrinolysis in Men

The existence of an association between idiopathic intracranial hypertension (IIH) and coagulation disorders in men was assessed prospectively. Microthrombi, associated with thrombophilia-hypofibrinolysis, occlude arachnoid sinus villi, thus reducing resorption of cerebrospinal fluid, leading to IIH. Ten consecutively referred men with IIH, nine whites, one African American, median age 36 years, were 2 to 1 matched by age and race by healthy male controls. Polymerase chain reaction assays were done for four thrombophilic and one hypofibrinolytic gene mutations: G1691A factor V Leiden, G20210A prothrombin, C677T MTHFR, platelet glycoprotein IIb/IIIa (PL A1/A2), and 4G/5G polymorphism of the plasminogen activator inhibitor (PAI-1) gene promoter. Coagulation measures in plasma included dilute Russel's viper venom time (dRVVT), activated partial thromboplastin time (aPTT), the lupus anticoagulant, factor VIII, factor XI, plasminogen activator inhibitor activity (PAI-Fx), protein C antigenic, protein S total (antigenic), protein S free (antigenic), antithrombin III (functional), and resistance to activated protein C (RAPC). Tests performed on serum included anticardiolipin antibodies, homocysteine, and Lp(a). The body mass index was 40 kg/m(2) or greater (extremely obese) in two men, 30 to 40 kg/m(2) (obese) in three, and was 25 to 30 kg/m(2) in five (overweight). Cases differed from controls for inherited 4G4G homozygosity of the PAI-1 gene, four of 10 (40%) vs. one of 20 (5%), Fisher's p [p(f)]= .031, and for high levels (>21.1 U/mL) of the hypofibrinolytic PAI-1 gene product, PAI-Fx, 5 of 10 (50%) vs. one of 18 (6%), p(f) = .013. Thrombophilic factor VIII was high (> or = 150%) in three of 10 (30%) cases vs. zero of 16 (0%) controls, p(f)=. 046. The thrombophilic lupus anticoagulant was present in two of 10 (20%) cases vs. zero of 32 (0%) controls, p(f) = .052. Heritable hypofibrinolysis and heritable and acquired thrombophilia appear, speculatively, to be treatable etiologies of IIH in men. Understanding contributions of hypofibrinolysis and thrombophilia to the development of IIH should facilitate development of novel new approaches to treat this often-disabling neurologic disorder.

Factor XII auto-antibodies present in a patient with a B-cell lymphoma

A 64-year-old woman was transferred for investigation of a mediastinal mass, biopsy of which showed a diffuse large B-cell lymphoma. She was also found to have an antiphospholipid antibody. The pre-operative coagulation screen showed a prolonged activated partial thromboplastin time, 71.3 s (normal range, 26-36 s), which was not corrected by the addition of normal plasma. The dilute Russell's viper venom time was positive. Anti-cardiolipin assay was strongly positive, immunoglobulin M was 153 AU; immunoglobulin G was normal, 3.1 AU. Assays of factors VIII, IX and XI showed higher concentrations with increasing dilutions in one-stage factor assays from 1: 10 to 1: 80 suggestive of an inhibitor. Factor XII was 9 U/dl and results were unaffected by increasing dilution, suggesting specific antibodies to factor XII. The factor XII antigen was 40 U/dl. The patient had immunoglobulin M auto-antibodies to factor XII.