Dilute Russell Viper Venom Time Research Articles

Laboratory testing of anticoagulants: the present and the future

This review provides an update on laboratory testing and monitoring for existing and emerging anticoagulants, starting with an overview of haemostasis and the routine coagulation tests currently employed within most haemostasis laboratories, including the prothrombin time (PT)/international normalised ratio (INR) and the activated partial thromboplastin time (APTT). Current anticoagulant therapy and laboratory monitoring is then discussed in terms of benefits and limitations, followed by a similar brief discussion of the new and emerging anticoagulants. The main focus, however, is laboratory testing related to vitamin K antagonists, heparin, lepirudin and the new agents dabigatran etexilate and rivaroxaban. Although the newer agents do not require laboratory monitoring, laboratory testing will occasionally be required, and pathology laboratories should become proactive in developing appropriate strategies. The tests most likely to fulfill this role are the ecarin clotting time (or chromogenic alternatives), and the chromogenic anti-Xa assay. Nevertheless, the dilute Russell viper venom time (dRVVT) assay may provide another alternative, and existing routine tests are also likely to be utilised for the foreseeable future, potentially also for laboratory testing of the new anticoagulants, albeit perhaps in modified form.

Effect of Telavancin (Vibativ) on Routine Coagulation Test Results

Telavancin (Vibativ, Astellas Pharma US, Deerfield, IL) is a lipoglycopeptide antibiotic that has activity against gram-positive microorganisms, but also has the ability to bind to artificial phospholipids found in coagulation reagents. Normal pooled plasma was spiked with telavancin to obtain concentrations of 0, 12.5, 25, 50, 75, 100, 125, and 150 μg/mL of drug. Samples were tested using 3 different prothrombin time/international normalized ratio (INR) and activated partial thromboplastin time (aPTT) reagent systems, as well as for fibrinogen level, thrombin time, D-dimer level, dilute Russell viper venom time (DRVVT), protein C activity, and protein S activity. There was no effect of telavancin seen with non-phospholipid-dependent assays: fibrinogen level, thrombin time, and D-dimer testing. All INR and aPTT systems demonstrated concentration-dependent increases in clotting times, with Innovin (Siemens Healthcare Diagnostics, Deerfield, IL) INRs the most dramatic. False-positive DRVVT ratios started at 12.5 μg/mL of telavancin, with no effect on protein C or protein S levels until the telavancin level reached more than 100 μg/mL.

No Thrombotic Risk in Patients with Asymptomatic Antiphospholipid Antibodies

Abstract 5254 Aim of the study.Antiphospholipid antibodies (APA) are well known risk factor for thromboembolic events and/or obstetric complications. They may be found incidentally in patients without thrombotic complications (asymptomatic APA), and they often lead to an incorrect suspicion of hemorrhagic diathesis, as well as to an unnecessary disqualification from surgical procedures or withdrawal from a proper antithrombotic prophylaxis during these procedures. The aim of the study was to register venous and/or thrombotic events in a group of patients with asymptomatic APA, diagnosed according to the international guidelines (Myakis et all. 2006). Material and methods.The study group consisted of 25 patients (18 women and 7 men) of the mean age of 46 years (20 – 75 years). Concomitantly 9 of them had other autoimmune disorders (no systemic lupus erythematosus) and 4 had neoplasms. Among risk factors for arterial thrombosis 5 patients had hypercholesterolemia, 5 – hypertension, 4 were smokers and 4 were obese (BMI >30 kg/m2). None of the patients had hereditary thrombophilia (antithrombin, protein C or protein S deficiency, factor V Leiden, prothrombin G20210A mutation, increased activity of factor VIII). Family history of venous thromboembolic disease has been noticed in 4 patients and of arterial thrombosis in 6 patients. The observation lasted for 3 to 127 months (mean 35 months). Results.The number and percent of patients (n = 25) with abnormal results 997337of different laboratory diagnostic assays for APA:AssessmentaPTT - n (%)dRVVT - n (%)ACA - n (%)anti-β2-GPI - n (%)initial24 (96)23 (92)11 (44)10 (40)after 12 weeks24 (96)22 (88)9 (36)11 (44)aPTT-activated partial thromboplastin time; dRVVT - diluted Russell viper venom time; ACA-anticardiolipin antibodies; β2-GPI – anti-β2–glycoprotein I antibodiesAbnormal results of more than one assay in any combination were found in 16 patients, lupus anticoagulant alone in 9 patients. Only 4 out of 25 patients have taken aspirin - 75mg daily, in the other 4 a prophylactic dose of low molecular weight heparin was administered temporarily because of surgery.During observation time no venous or arterial thrombotic events occurred in the study group. On a basis of a limited number of previous studies that predominantly included systemic lupus erythematosus patients, aPL-positive patients without previous thrombosis have a 0% to 3.8% annual incident thrombosis risk (Barbhaiya M, Erksan D. Curr Rheumatol Rep. 2011; 13: 59–69).In two patients asymptomatic APA disappeared. Conclusion:independently of the type and quantity of asymptomatic antiphospholipid antibodies, there were no venous or arterial thromboembolic events in the group of patients observed for meanly 35 months. Disclosures:No relevant conflicts of interest to declare.

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

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. 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. 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%. The combination of mixing and confirmatory tests interpreted according to the new guidelines can clearly differentiate LA from other coagulopathies.

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

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. 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 β(2) glycoprotein I (β(2) GPI) using one of the recently described methods. All LA tests performed were associated with a history of thrombosis. The highest odds ratio (OR) for thrombosis was found for β(2) GPI-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. 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.

Detection of Lupus Anticoagulant In Women with Obstetric complications

AbstractAbstract 3185 Introduction:Lupus anticoagulant (LA) is one of the laboratory criteria for the diagnosis of antiphospholipid syndrome. To detect the presence of LA, ISTH recommends performing two assays based on different principles. Our LA profile includes dilute Russell viper venom time (dRVVT) and two different activated partial thromboplastin time (aPTT) reagents.The aim of the study was to evaluate sensitivity and specificity of aPTT and dRVVT in women with a history of pre-eclampsia, intra-uterine growth restriction, early recurrent abortion, late fetal loss and placental abruption collectively termed “placenta mediated complications”. Materials and Methods:We studied a total of 247 patients and 287 samples in a 10 months period. Based on consensus criteria, samples were retested within a period of three months. 62/247 plasmas samples were collected during pregnancy.Laboratory studies: Screening tests: aPTT was performed using PTT-LA (Stago) and an aPTT home made reagent with diluted cephalin. dRVVT was performed using Russel viper venom RVV (Stago) and diluted cephalin. Negative control plasma was prepared according to ISTH LA recommendations for the mixing tests. Confirmatory tests: We performed a home made reagent for platelet neutralization procedure (PNP)-aPTT and dRVVT; Rosner Index and % of Correction as criteria for mixing and confirmatory tests interpretation.Statistical evaluation was performed by ROC method using EP Evaluator 9.9 software. Results:172/287 samples (59.9%) were negative, while 115/287 samples (40.1%) were positive for LA. 44/115 samples (38.26%) were positive for both tests (aPTT and dRVVT), 60/115 samples (52.17 %) were positive only for dRVVT and 11/115 samples (9.57%) were positive only for aPTT.LA was confirmed in 9/62 pregnant women samples (14.5 %). 7 (11.3%) had abnormal dRVVT, the remaining 2 samples were positive for both tests.Table for PP–Assay(UCA)/Cut offSensitivity (SE)Specificity (SP)aPTT (Cephalin)0.812/43 seg57.494.8aPTT (PTT–LA, Stago)0.813/42 seg58.391.9dRVVT (Cephalin)0.964/45 seg87.0100 Conclusions:Our results demonstrated: 1- High prevalence of dRVVT positive plasmas 104/115 (90.43%); 2- Despite showing a low performance regarding sensitivity when compared to dRVVT, both aPTT tests were able to detect LA in 47.83% of the positive studies. 3- According to our experience, considering this initial cohort of selected patients, the use of dRVVT as the first and aPTT as the second screening test may help improve the performance for the diagnosis of LA. Disclosures:No relevant conflicts of interest to declare.

Lupus Anticoagulant Testing

Lupus anticoagulant (LAC) testing is important for evaluating patients with antiphospholipid syndromes and hypercoagulable states. We reviewed results of proficiency testing challenges (n = 5) distributed by the North American Specialized Coagulation Laboratory Association to examine LAC testing performed by participating laboratories. The activated partial thromboplastin time (APTT) and dilute Russell viper venom time (dRVVT) constituted major testing methods. In screening studies, LAC-sensitive APTT methods were more sensitive to weak LAC than dRVVT-based methods but less specific. In confirmatory testing, dRVVT methods performed better, but performance was LAC-dependent. The highest false-negative confirmatory test results were obtained for the platelet neutralization procedure. Noncompliance with recommendations for LAC testing by the International Society on Thrombosis and Haemostasis was high (8%-38%), with the majority of noncompliant laboratories failing to report results of mixing studies. These data provide new insights into LAC testing in North America and identify opportunities for standardization.

Monitoring plasma heparin concentration in a patient with antiphospholipid syndrome

Antiphospholipid syndrome (APS) is an acquired thrombophilia characterized by the occurrence of arterial and venous events as well as recurrent miscarriages [1, 2]. In the revised classification criteria for APS, lupus anticoagulant (LAC), anti-cardiolipin antibody (aCL), and anti-b2 glycoprotein-I antibody (ab2GPI) have been utilized as laboratory criteria [1]. Concerning the LAC assay, prolongation of a phospholipid-dependent clotting time such as activated partial thromboplastin time (aPTT) has been commonly used as a screening test [3]. Once patients are diagnosed as having APS, they are recommended to receive prolonged antithrombotic therapy, especially with warfarin [4, 5]. We encountered a patient with APS who could not receive oral warfarin because of malabsorption, and it was also difficult to perform conventional therapeutic monitoring of the heparin dosage using aPTT because of the baseline prolongation of aPTT due to LAC. A 63-year-old female who had a history of recurrent miscarriages was admitted to our hospital because of severe abdominal pain on September 19, 2009. Radiologic examination revealed an arterial thrombus at the trunk of the superior mesenteric artery (SMA), and she was treated with intravenous urokinase and unfractionated heparin. However, 2 days later, she complained of severe abdominal pain again. She underwent emergent laparotomy and was diagnosed with extended intestinal necrosis ranging from the upper jejunum to transverse colon. Therefore, she received surgical resection of the necrotizing intestine followed by colostomy and common hepatic artery bypass grafting. Her abdominal pain resolved, but she became dependent on parenteral nutrition due to malabsorption as a result of short bowel syndrome after extended enterocolectomy. Intravenous heparin was continued until postoperative day (POD) 13 to prevent graft occlusion. aPTT (PTT-LA , Sysmex, Kobe, Japan, normal range 24.2–34.1 s.) was prolonged to more than 100 s. even after withdrawing anticoagulation with heparin. On POD 23, she was positive for LAC on the phospholipid-dependent coagulation test using the diluted Russell viper venom time (dRVVT; Gradipore , MBL, Nagoya, Japan) (Table 1). Both aCL and b2GPI dependent aCL (aCL/b2GPI) showed significantly high titers of [120 U/mL (normal range \ 10 U/mL) and 71.6 U/mL (normal range \ 3.5 U/mL), respectively, in her T. Inaba (&) N. Fujita Department of Infection Control and Laboratory Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan e-mail: inaba178@koto.kpu-m.ac.jp

Amphipathic peptides can act as an anticoagulant by competing with phospholipid membranes for blood coagulation factors

An ideal amphipathic peptide (IAP), composed of simply lysine and leucine residues in a 1:2 ratio (K 7L 15), specifically prolongs in vitro coagulation assays that use phospholipids, such as the activated partial thromboplastin time (APTT). The main hypothesis of the present work is that IAP’s anticoagulant effect occurs by competing with phospholipid membranes in in vitro coagulation reactions. We verified this hypothesis by employing different phospholipid-dependent coagulation assays, such as the APTT, the dilute prothrombin time (dPT) and the dilute Russell viper venom time (dRVVT) with both low and high amounts of phospholipids. We show that coagulation times are prolonged by IAP in a concentration-dependent manner, and that this prolongation is abrogated by adding excess phospholipid, demonstrating a phospholipid dependence for this inhibition. Using an ELISA-based binding assay, we show IAP inhibits the binding of one of the vitamin K-dependent coagulation factors, factor X, to phospholipid membranes. This is further confirmed with fluorescence spectroscopy, where the interaction of IAP and factor X is inhibited by phospholipid. In summary, this work demonstrates that IAP can act as an anticoagulant by impairing the interaction of coagulation factors with phospholipid membranes and provides a paradigm for the development of novel anticoagulants.

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.

The utility of the Taipan snake venom assay in assessing lupus anticoagulant status in individuals receiving or not receiving an oral vitamin K antagonist

The objective of the present study was to evaluate the Taipan snake venom time (TSVT) assay in the detection of lupus anticoagulant in patients receiving or not receiving oral vitamin K antagonists. Blood samples were collected from individuals requiring investigation for the presence of a lupus anticoagulant. The TSVT test was run in parallel with the dilute Russell viper venom time (dRVVT) assay and anticardiolipin antibody assays. Results were assessed by Bayesian statistics. Two thousand and five plasma samples were obtained from 1335 individuals. One thousand, one hundred and eleven individuals were not receiving oral vitamin K antagonists; of these, 451 had a lupus anticoagulant by conventional testing, and the TSVT showed a sensitivity of 22%, specificity of 98%, positive predictive value of 80%, negative predictive value of 78%, positive likelihood ratio of 12.3 and a negative likelihood ratio of 0.790. Two hundred and ten of the 224 patients receiving oral vitamin K antagonists were known to have a persistent lupus anticoagulant prior to oral anticoagulation. In this setting, TSVT had a sensitivity of 39% and specificity of 93%, but the dRVVT had a 50% false-positive rate, which meant that despite having a higher sensitivity at 61% than the TSVT, its specificity was only 53%. TSVT is a specific but insensitive assay for the lupus anticoagulant in individuals not receiving oral vitamin K antagonists, when assessed against the dRVVT as the gold standard. It can be argued that despite the TSVT's poor sensitivity in patients taking oral vitamin K antagonists, its high specificity and positive predictive value make it a preferable assay to the dRVVT to diagnose lupus anticoagulant in this group.

Diluted Russell viper-venom time improves identification of antiphospholipid syndrome in a lupus anticoagulant-positive patient population

The objective of this retrospective study was to evaluate the potential ability of diluted Russell viper-venom time (dRVVT) to identify antiphospholipid syndrome (APS) in a lupus anticoagulant (LA)-positive patient population, already selected by other LA clotting tests. Our cohort of positive LA patients was first identified in our outpatients population by the following sensitive LA-detecting tests: Rosner index, diluted prothrombin time (dPT) and Rosove index. Then the 227 consecutive LA-positive patients were tested for dRVVT with the same blood sample. Anticardiolipin (aCL) and anti-beta(2)-glycoprotein-I (beta(2)GPI) autoantibodies assays were also performed. APS using Sapporo clinical criteria revised at Sydney, was found in 116 of these 227 consecutive LA-positive patients. Results of the different tests were analysed statistically. Using univariate analysis, dRVVT, dPT, IgG aCL and IgG anti-beta(2)GPI autoantibodies were significantly associated with APS. The receiver operating-characteristics (ROC) curve defined the best cut-off value for dRVVT ratio at 1.61 with a good specificity (78%) and a lower sensitivity (53%). A multivariate analysis using a binary logistic procedure, retained the dRVVT ratio (> or = 1.61) and IgG anti-beta(2)GPI autoantibodies (> 15 USG) as being associated with APS (p = 0.018; odds ratio [OR] 2.39; 95% confidence interval [CI] 1.2-4.7, and p = 0.0001; OR 3.2; 95% CI 1.5-6.5, respectively). To conclude, these results agree with the need for LA criteria favouring specificity over sensitivity. The use of a threshold around 1.6 for dRVVT ratio should help discriminate APS from non-APS patients.

Elevated first trimester soluble fibrin polymer is associated with adverse pregnancy outcome in thrombophilic patients

Elevated first trimester soluble fibrin polymer is associated with adverse pregnancy outcome in thrombophilic patients

In a Prospective Inceptional Cohort Study of Children with Thrombosis, a Persistently Positive Dilute Russell Viper Venom Time Is Associated with Thrombus Progression or Recurrence and Multiple Antiphospholipid Antibodies

BACKGROUND: Antiphospholipid antibodies (APA) that persist ≥12 weeks in adults with acute thrombosis are predictive of thrombus recurrence. The significance of APA in children with thrombosis is unclear. This study was developed to examine the relationship of a persistently positive lupus anticoagulant (≥ 12 weeks), assessed by the dilute Russell Viper Venom Time (dRVVT), to adverse thrombus outcomes including progression or recurrence. In addition, the relationship of a persistently positive dRVVT to the prevalence and titer of several other APA in children with thrombosis were examined.METHODS: Data from a consented prospective inceptional cohort study of pediatric thrombosis and thrombophilia (COMIRB 05–0339) were extracted for this analytic project. Eligibility included age ≤ 21 years at presentation, objectively confirmed thrombus, dRVVT within 4 weeks of presentation and repeated ≥ 12 weeks later. Patients with a persistently positive dRVVT (ratio of Screen/Confirm ≥1.2) were classified as having antiphospholipid syndrome (APS positive), and those with a negative dRVVT were classified as APS negative. Twelve or more weeks from presentation, patient plasmas were tested in ELISA assays for the presence and titer of IgM and IgG antibodies directed against protein C (PC), protein S (PS), prothrombin (II), β-2-Glycoprotein I (B2GPI), and cardiolipin (ACA). A euglobulin clot lysis assay (ELT) and hexagonal (II) phase phospholipid assay (STACLOT LA, Diagnostica Stago, Inc.) were also performed.RESULTS: The cohort included 122 cases with thrombosis. Of these, 35 failed eligibility due to transient dRVVT positivity or lack of blood sampling within the specified time period. Of the remaining 87 patients, 43 were APS positive and 44 were APS negative. APS positivity was associated with longer duration of therapy (p<0.001) and multiple autoantibodies at ≥ 12 weeks from presentation (p=0.04). APS was associated with higher titers of several antibodies, including those directed against: B2GPI (IgG, p=0.01), II (IgG, p=0.02), PC (IgG, p=0.03; IgM, p<0.001). Despite the longer duration of therapy for these patients, APS was highly associated with thrombus progression or recurrence (47% in APS positive versus 8% in APS negative, p=0.001). Thrombus progression or recurrence was further associated with positivity for ACA IgM, Anti-II IgM and the STACLOT LA (p=0.02 for each), and additionally with higher titers of antibodies against B2GPI (IgG, p=0.03), II (IgM, p=0.05), PC (IgM, p=0.04), and PS (IgM, p=0.03).CONCLUSION: In this pediatric prospective inceptional cohort study, APS (defined as a persistence of positive dRVVT 12 weeks or greater in children affected with thrombosis), was found in more than one third of children and was associated with higher prevalence and titers of several APA. In spite of longer duration of therapy, children with APS had a significantly higher rate of thrombus progression or recurrence. APA testing in children with thrombosis is important to predict outcomes. Future clinical trials must consider APS status in evaluating risk-stratified therapy for thrombotic diseases in children.

Comprehensive Antiphopholipid Antibody Testing in Children with Acute Thromboembolism: Preliminary Findings from a Prospective Inceptional Cohort Study.

BACKGROUND: The significance of the lupus anticoagulant (LA) and other antiphospholipid antibodies (APA) in children with thromboembolism (TE) has not been as clearly defined in children as among adults.OBJECTIVE: To characterize TE presentation and prothrombotic risk factors among children with TE who are positive for the LA using the dilute Russell Viper Venom Time (dRVVT) as a screening test.METHODS: Children with acute TE were consecutively enrolled in a prospective inceptional cohort study and a compehensive APA testing battery was serially employed. APA testing included StaClot-LA, as well as ELISA for IgG and IgM binding to prothrombin, protein C, protein S, cardiolipin and β2GP1.RESULTS: 34 children who were dRVVT positive within 3 months of acute thrombosis were evaluated with the entire APA battery on 1 to 5 occasions over 3 months to 4 years. Multiple APA were detected in 20 (59% of) study participants (5 tests, n=1; 4 tests, n=2; 3 tests, n=6; 2 tests, n=11; 1 test, n=14). Findings were: dRVVT (100%), anti-prothrombin (50%), StaClot (18%), anti-protein S and ACA (12% each), anti-β2GP1 (6%) and anti-protein C (3%). TE presentation and prothrombotic risk factors were compared between these 20 children with multiple APA and 14 children who were dRVVT positive only (Tables 1 and 2). Children with LA-associated TE who had multiple APA were more likely to present with parenchymal sites of thrombosis (p=0.03) and appeared less likely to have otherwise-idiopathic TE (i.e., no additional prothrombotic risk factors identified), although the latter trend was not statistically significant.CONCLUSIONS: The majority of children with acute TE and a positive dRVVT in whom a comprehensive battery of APA is performed demonstrate multiple APA, among which anti-prothrombin antibodies are the most common. When compared to children positive for dRVVT only, children with LA-associated thrombosis who have multiple APA are particularly predisposed to parenchymal thrombotic events and may be more likely to experience otherwise-idiopathic TE. Future investigations will evaluate the risk of recurrent TE in children with thrombosis relative to findings on serial comprehensive APA testing.Table 1Thrombus Sites*, by APA Group.APA groupLimb DVTExtensive PECSVTParenchymal thrombosisArterial thrombosisTotalMultiple APA8 (40%)6 (30%)2 (10%)6 (30%)3 (15%)20 (100%)dRVVT only7 (50%)4 (29%)0 (0%)0 (0%)3 (21%)14 (100%)* Some patients had >1 site. Abbreviations: DVT=deep venous thrombosis; PE=pulmonary embolism; CSVT=cerebral sinovenous thrombosis. Arterial thrombosis includes ischemic arterial stroke.Table 2Prothrombotic Risk Factors, by APA Group.APA GroupInfectionSurgery/TraumaSLEOther*NoneTotalMultiple APA4 (20%)3 (15%)2 (10%)4 (20%)7 (35%)20 (100%)dRVVT only2 (14%)1 (7%)0 (0%)9 (64%)2 (14%)14 (100%)* Includes anatomic anomaly, obesity, sedentary condition, oral contraceptive use, genetic thrombophilia, central venous catheter, and cardiac disease. Abbreviation: SLE=systemic lupus erythematosus.

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.

Survey of lupus anticoagulant diagnosis by central evaluation of positive plasma samples.

To determine whether the diagnosis of lupus anticoagulant (LAC) in a large cohort of positive patients was confirmed at a reference laboratory. Over a 1-year period, each participating center collected samples from LAC-positive patients. Plasma was filtered and kept deep-frozen until it was sent on dry ice to the reference laboratory by express courier. Centers returned detailed laboratory information and clinical data from each patient. The reference laboratory screened plasma samples by diluted Russell viper venom time (dRVVT) and kaolin clotting time (KCT). When these were prolonged, 1:1 mixing studies were carried out, and confirmatory tests were performed as appropriate. Positive samples were further tested by thrombin time (TT). The presence of heparin was checked by measuring antifactor Xa activity when TT was prolonged. Negative samples were tested by activated partial thromboplastin time using hexagonal phospholipids. Plasma samples from 302 patients from 29 anticoagulation clinics were analyzed. LAC was excluded in 71 samples (24%), because dRVVT and KCT screening test results were normal (34) or reversed to normal by mixing studies (35). The remaining two samples were considered negative because they contained heparin. LAC-negative patients showed different characteristics from those in whom diagnosis was confirmed. They were significantly older (49.7 vs. 45.0 years, P < 0.03), were more often first diagnosed (66% vs. 41%, P < 0.001), and were more frequently judged as mild in LAC potency (60% vs. 25%, P < 0.0001). Moreover, anticardiolipin and anti-beta(2)-glycoprotein I antibody values were more often normal in LAC-negative (82%) than in LAC-positive (42%) samples (P < 0.0001). LAC-positive samples identified by both dRVVT and KCT (146/231, 63%) showed a LAC potency that was significantly stronger than that in samples in which LAC diagnosis was made by a single test. A false-positive LAC diagnosis is not uncommon across specialized centers. Patients' characteristics and a complete antiphospholipid antibody profile may help to identify these individuals.

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.

Aortic valve calcification in systemic lupus erythematosus

Aortic valve calcification is associated with atherosclerosis in the general population. We investigated the prevalence of and associates of aortic valve calcification in systemic lupus erythematosus (SLE). One-hundred and ninety-nine SLE patients enrolled in a clinical trial had aortic valve calcification assessed by helical CT. The patients had a mean age of 44.3 +/- 11.4 years and were 92% female, 61% Caucasian, 34% African-American, 2% Asian and 2% Hispanic. Aortic valve calcification was present in 1.5%, whereas coronary calcium was found in 43% and carotid plaque in 17%. Among cardiovascular risk factors, hs-CRP (P = 0.0592), fibrinogen (P = 0.0507), and lipoprotein(a) (P = 0.0250), were associated with aortic valve calcification. Prednisone use (P = 0.049) and use of methotrexate (P = 0.0174) were also associated with aortic valve calcification. Aortic valve calcification was associated with antiphospholipid antibody positivity (0.0287) (lupus anticoagulant, by dilute Russell viper venom time). It was not associated with coronary calcium or carotid plaque. Aortic valve calcification, although rare in SLE, was associated with some novel cardiovascular risk factors and with a marker of hypercoagulability (lupus anticoagulant). In contrast to the general population, aortic valve calcification in SLE is not associated with subclinical measures of atherosclerosis, such as coronary calcium or carotid plaque.