Dilute Russell Viper Venom Research Articles

Is lupus anticoagulant testing with dilute Russell’s viper venom clotting times reliable in the presence of inflammation?

BackgroundTesting for lupus anticoagulant (LA) is not recommended in case of inflammation as C-reactive protein (CRP) can interfere in vitro with the phospholipids present in the activated partial thromboplastin time test used to detect an LA. However, the potential interference of an acute phase protein (ie, CRP) in LA testing using the dilute Russell’s viper venom (DRVV) test is poorly studied. ObjectivesTo study the effect of inflammation, as evidenced by increased CRP levels, on DRVV tests. MethodsFirst, a retrospective analysis (2013-2023) was performed: data on all LA workups were retrieved, and the association between CRP levels and DRVV screen, mix, and confirm clotting times was studied. Second, data on DRVV panels and CRP levels were extracted from 2 prospective studies involving intensive care unit patients to study the association between both variables. Third, CRP was added to normal pooled plasma at 6 relevant concentrations (up to 416 mg/L) to study the association between CRP itself and DRVV coagulation times. ResultsIn the retrospective analysis, DRVV screen and confirm clotting times significantly increased as CRP increased (increase of 0.11 seconds and 0.03 seconds per 1 mg/L increase of CRP level, respectively). In the prospective analysis, only DRVV screen was prolonged with high CRP levels (increase of 0.06 seconds for a 1 mg/L increase in CRP level); DRVV screen/confirm ratio was also increased with high CRP levels. In vitro, the addition of CRP did not significantly increase any DRVV clotting times. ConclusionLA testing should be performed with much caution in the presence of inflammation as it may be associated with prolongation of both activated partial thromboplastin time and DRVV clotting times.

Taipan snake venom time has high sensitivity for lupus anticoagulants in non-anticoagulated, triple positive antiphospholipid syndrome patients.

Dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) are the mainstay assays in lupus anticoagulant (LA) detection yet they have limitations, particularly in relation to interferences and specificity. The recently validated Taipan snake venom time (TSVT) screening with ecarin time (ET) confirmatory assays overcome many of those limitations due to the innate specificity engendered from direct prothrombin activation, and insensitivity to the effects of vitamin K antagonists (VKA). The present study aimed to further evidence diagnostic utility of TSVT/ET by performing them in samples from 116 nonanticoagulated patients with established triple-positive antiphospholipid syndrome (APS). Samples were identified in three expert centres who performed dRVVT, APTT and solid phase antiphospholipid antibody assays with reagents from a variety of manufacturers. All samples additionally received TSVT/ET analysis using standardised reagents. Ninety seven of 116 (83.6%) were dRVVT- and APTT-positive, 85/97 (87.6%) of which were TSVT/ET-positive, 9/116 (7.8%) were dRVVT-positive only, 6 of which were TSVT/ET-positive, and 10/116 (8.6%) were APTT-positive only, 5 of which were TSVT/ET-positive. 96/116 TSVT/ET-positivity returned a high sensitivity for LA of 82.8%. Low coefficients of determination revealed weak relationships between LA potency and anticardiolipin and anti-β2-glycoprotein I antibody titres for all three LA assays. TSVT/ET has high sensitivity for the clinically significant LA found in triple positive APS patients. TSVT/ET can establish multiple LA assay positivity in nonanticoagulated patients negative for one of dRVVT or APTT, and is the only assay pairing insensitive to VKAs, the recommended anticoagulation for APS.

Domain Specificity of Anti-Prothrombin Antibodies That Express Lupus Anticoagulant Activity

Background: The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by the persistent presence of antiphospholipid antibodies (aPL). aPL are a heterogeneous group of autoantibodies directed against negatively charged phospholipids or phospholipid-binding proteins. Some of these autoantibodies exert lupus anticoagulant (LAC) activity, a laboratory phenomenon characterized by the prolongation of phospholipid-dependent coagulation tests. Recently, our group identified that LAC-positive anti-prothrombin antibodies activate platelets via the platelet receptor, FcγRIIA. However, a lot is still unclear regarding the mechanism by which these antibodies cause LAC and why some, but not all, antibodies in this heterogeneous group express LAC activity. Furthermore, experimental data on the binding site of these pathogenic anti-prothrombin antibodies are lacking. Aims: In this study we aimed to investigate the binding domain of LAC-positive monoclonal anti-prothrombin antibodies and APS patient derived anti-prothrombin antibodies. Method: Recombinant fragments of prothrombin were designed and produced in Chinese hamster ovary cells. Prothrombin fragment 1, fragment 2 and fragment 1.2 were coated on ELISA plates to study epitope specificity. Two LAC-positive monoclonal anti-prothrombin antibodies (3B1 and 28F4), two LAC-negative anti-prothrombin monoclonal antibodies (11H2 and 9C6), isolated anti-prothrombin antibodies derived from APS patients and plasma from APS patients were studied. A modified dilute Russell's viper venom time (DRVVT) test was used to evaluate whether the recombinant prothrombin constructs could neutralize the effect of LAC-positive monoclonal and patient derived anti-prothrombin antibodies. Furthermore, a fragment 1.2 nanobody was produced by immunizing Llama's followed by phage display. The ability of this nanobody to compete with LAC-positive monoclonal and patient derived anti-prothrombin antibodies for the prothrombin binding site was tested using a modified DRVVT test. Results: LAC-positive monoclonal anti-prothrombin antibodies recognized prothrombin fragment 2. Interestingly, LAC-negative anti-prothrombin antibodies bound exclusively to prothrombin fragment 1. In addition, LAC-ratios of patient samples correlated significantly with anti-fragment 2 antibody levels ( p<0.05, ρ=0.33), but not with anti-fragment 1 antibody levels. Furthermore, incubation with the prothrombin F1.2 nanobody almost completely reversed the prolonged DRVVT clotting time in plasma containing monoclonal and patient derived LAC-positive anti-prothrombin antibodies, but not in plasma containing LAC-positive anti-β2GP1 antibodies. This neutralising effect was also observed when the monoclonal anti-prothrombin antibody was incubated with prothrombin fragment 2. In contrast, the LAC-activity of anti-prothrombin antibodies was not inhibited when incubated with prothrombin fragment 1. Conclusion: Our data show that LAC-positive anti-prothrombin antibodies react with fragment 2 of prothrombin. Furthermore, a novel nanobody directed against prothrombin fragment 1.2 and prothrombin fragment 2 were able to neutralize a prothrombin-dependent LAC.

Differentiation of Acquired Hemophilia a from Lupus Anticoagulant Using Global Coagulation Assays and Magnesium-Dependent Activated Partial Thromboplastin Time Assay

CONCLUSION Rapid and reliable differentiation between bleeding and thrombotic tendency in cases with prolonged activated partial thromboplastin time (APTT) remains challenging in clinical practice, in particular, distinguishing cases positive for coagulation factor inhibitor or lupus anticoagulant (LA). To differentiate acquired hemophilia A (AHA) from LA using three global coagulation assays: rotational thromboelastometry (ROTEM), thrombin generation assay (TGA), and clot waveform analysis (CWA); also to validate the effectiveness of magnesium-dependent APTT (APTT-Mg) assay. This method uses APTT-Mg and normal APTT using Ca to screen for LA, and APTT-Mg/APTT ratio is smaller in LA. We included patients with prolonged APTT without congenital coagulation factor deficiency who were referred to our department between November 2019 and September 2021. APTT prolongation was defined as >35 s (normal range: 25-35 s) measured routinely with Coagpia APTT-N (Sekisui Medical). The AHA group had a confirmed diagnosis and underwent treatment of AHA. The LA group was divided into LA1 (possible LA) and LA2 (definite LA). LA1 was defined by any of the following conditions: (1) index of circulation anticoagulant ≥15 in the cross-mixing test performed with Coagpia APTT-N; (2) positive dilute Russell's viper venom time (DRVVT) phospholipid neutralization method using Gladipore LA (Medical & Biological Laboratories); (3) positive APTT phospholipid neutralization test using Hemosil SCT (Instrumentation Laboratory); (4) positive for anti-CL antibody; (5) positive for anti-CLβ2GPI antibody; or (6) positive for phosphatidylserine-dependent anti-PT antibody. LA2 was defined by index of circulation anticoagulant >12.4 in the cross-mixing test performed by Thrombocheck APTT-SLA (Sysmex Corporation), or item (2) or (3) above, in accordance with criteria of LA in the International Society of Thrombosis and Haemostasis. LA1 and LA2 did not overlap, because cases that did not meet the conditions of LA2 were treated as LA1, and global coagulation assays were performed in all LA cases. In the global coagulation assays, 12 healthy volunteer samples were tested and regarded as the normal goroup. For the APTT-Mg assay, equal volumes of 25 mmol MgCl 2 and 25 mmol CaCl 2 were used instead of the normally used CaCl 2, and Thrombocheck APTT-SLA was used to measure APTT-Mg, which was then divided by the normal APTT concentration measured with the same APTT reagent. Ten patients in the AHA, 12 in the LA1, and 32 in the LA2 groups were eligible. ROTEM showed that clotting time in the non-activated rotational thromboelastometry assay (NATEM) mode was >3600 s (minimum 1363 s) for AHA, 501 s (IQR: 430-632 s) for LA1, 533 s (IQR: 443-641 s) for LA2, and 500 s (IQR: 429-645 s) for the normal group. TGA showed that peak thrombin concentration was 16 nM (IQR: 13-29 nM) in the AHA group, 242 nM (IQR: 175-273 nM) in the LA1 group, 174 nM (IQR: 129-259 nM) in the LA2 group, and 190 nM (IQR: 125-288 nM) in the normal group. In the CWA, Ad|min1| was 2.1 (IQR: 1.7-3.2) in the AHA group, 8.7 (IQR: 8.0-9.5) in the LA1 group, 6.7 (IQR: 5.8-8.4) in the LA2 group, and 10.3 (IQR: 9.8-10.6) in the normal group. APTT-Mg/APTT was 1.23 (IQR: 1.18-1.29), 1.26 (IQR: 1.23-1.38), and 1.05 (IQR: 0.95-1.20) in the AHA, LA1, and LA2 groups, respectively. ROTEM, TGA, and CWA effectively differentiated between the LA1/2 and AHA groups. Clotting time in non-activated rotational thromboelastometry assay mode (ROTEM) and peak height (TGA) were better at distinguishing high-titer LA cases from LA-positive AHA cases. APTT-Mg/APTT could be suitable for distinguishing AHA from high-titer LA; however, it may not be useful for differentiating AHA from low-titer or suspected LA.

Effect of residual platelets in frozen-thawed plasma on results of dilute Russell's viper venom time assay for lupus anticoagulant testing.

To determine the impact of residual platelets on dilute Russell's viper venom time (DRVVT) assay in frozen-thawed plasma submitted for lupus anticoagulant (LAC) testing. We measured platelet counts in frozen-thawed samples submitted for LAC testing and evaluated the association between platelet count and the DRVVT screening time and ratios. We also spiked platelets into a LAC-positive sample to observe the effect on the DRVVT. Progressive increase in platelet count resulted in a statistically significant shortening of the DRVVT assay results on plasma after 1 freeze-thaw cycle. A similar effect was noted on the LAC-positive sample. Residual platelets in plasma samples result in shortening of DRVVT assay after 1 freeze-thaw cycle. This may result in a false-negative LAC test result.

A-140 Evaluating the Performance of Various Lupus Anticoagulants Tests Among Different Coagulation Platforms

Abstract Background Lupus Anticoagulants (LAs), one of the antiphospholipid antibodies, are risk factors for arterial or venous thrombosis with complications such as stroke, acquired thrombophilia, and pregnancy loss. Detection of LAs is challenging for laboratories as the heterogeneity of antibodies and epitopes on protein in the phospholipids. Thus, no single test is sensitive for all LAs, and the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee (ISTH) recommended laboratory should provide two test systems based on different principles, which dilute Russell's viper venom time (dRVVT) should be as the major test, and partnered with LA-sensitive activated partial thromboplastin time (aPTT). There are many different mechanisms of tests proposed by various manufacturers, however, the comparison of different platforms by different manufacturers on LAs remains unknown. In this study, we aimed to evaluate the performance of various LA tests among different platforms. Methods A total of 76 specimens from September to December 2022 were collected. dRVVT (dRVVTIL) and silica clotting time (SCTIL) were performed by ACL TOP 500 analyzer (Instrumentation Laboratory, US). In addition, ellagic acid-based aPTT (Siemens Dade Actin FSL reagent, aPTTAFSL), silica-based aPTT (Siemens Pathromtin SL reagent, aPTTPSL), and dRVVT (dRVVTsysmex) were performed by Sysmex CN-6000 analyzer (Sysmex Corporation, Japan). Analytical performances, agreements, and McNemar's test were calculated. Results All 76 specimens were originally tested by dRVVTIL and SCTIL, and showed that 30 specimens were positive for both assays, 6 were only positive for dRVVT, 5 were only positive for SCT, and 35 specimens were negative for both assays. In addition, all samples were further tested by dRVVTsysmex. Compared with the integrated use of dRVVTIL and SCTIL, dRVVTsysmex showed 100.0% (41/41) positive agreement and 91.4% (32/35) negative agreement. 3 specimens could only be tested positive on the dRVVTsysmex platform, of which one patient was diagnosed with systemic lupus erythematosus (SLE), another patient was diagnosed with infertility, and the other patient was diagnosed with antiphospholipid syndrome (APS). To further evaluate the performance of aPTT on LA screening, 44 specimens with either dRVVTIL, SCTIL and dRVVTsysmex positive were considered LA-positive and further tested by different aPTT assays. The positive agreement was 65.9% (29/44; 95% CI: 50.1–79.5%) and 77.3% (34/44; 95% CI: 62.2–88.5%) on aPTTAFSL and aPTTPSL, respectively, however, there was no significant difference between the positive agreement of two assays (P = 0.13). Conclusion Compared with the integrated use of dRVVTIL and SCTIL, dRVVTsysmex could achieve the same detection rate on LA. Furthermore, aPTTAFSL and aPTTPSL showed no significant difference between the positive agreements for LA-positive specimens.

Lupus anticoagulant laboratory diagnosis by applying the 2020 ISTH-SSC guidelines

BackgroundThe ISTH-SSC guidelines for lupus anticoagulant (LA) testing recommend using in-house determined cut-off values, pooled normal plasma (PNP) for ratio normalization, and a ratio for the mixing test interpretation. They strongly support the mixing step role in the diagnostic process. ObjectivesTo investigate and compare the LA testing results and interpretations obtained following the ISTH-SSC guidelines or the available alternatives. Patients/methodsBlood samples for LA testing from 462 consecutive patients were evaluated for screening, mixing and confirmatory tests. The analysis focused on the interpretation differences between using (1) the in-house cut-off values versus the manufacturer's cut-off values, (2) a normalized ratio calculated using PNP at each run versus the mean of the reference interval, (3) a normalized ratio versus the index of circulating anticoagulant to interpret the mixing step, and (4) a two-step versus three-step procedure. ResultsLA testing outcomes were comparable when using the in-house and manufacturer's cut-off values. More positive dilute Russell's viper venom (DRVV) time results were obtained with the normalized ratio based on PNP than with the mean of the reference interval. Overall, the mixing test results obtained with the normalized ratio and the index of circulating anticoagulant showed a good agreement. Among the 97 DRVV Screen test-positive samples, 33 and 89 were classified as LA-positive with the 3-step and the 2-step procedure, respectively. ConclusionsThe cut-off value used and the way to normalize ratios had a limited impact. Conversely, it is important to understand the mixing test characteristics to maximize its diagnostic potential.

Lupus Anticoagulant Testing: Dilute Prothrombin Time (dPT).

Lupus anticoagulants (LA) rarely affect routine prothrombin time assays because the high phospholipid (PL) content in thromboplastin reagents tends to overwhelm the antibodies. Dilution of thromboplastin to create a dilute prothrombin time (dPT) screening test renders the assay sensitive to the presence of LA. Technical and diagnostic performances are enhanced if recombinant thromboplastins are employed in place of tissue-derived reagents. Presence of an LA cannot be deduced from an elevated screening test alone since other coagulation disturbances can prolong clotting times. Confirmatory testing with less dilute or undiluted thromboplastin reveals the PL-dependent nature of LA by reducing the clotting time relative to that of the screening test. Where appropriate, such as a known or suspected coagulation factor deficiency, mixing tests are valuable in correcting factor deficiencies and evidencing inhibitory properties of LA, to increase diagnostic specificity. Although LA testing is commonly restricted to dilute Russell's viper venom time and activated partial thromboplastin time, dPT is sensitive to LA unreactive in those assays, and inclusion in routine testing increases detection rates of clinically significant antibodies.

New CMC Algorithm for Evaluation of Lupus Anticoagulant

Introduction: Lupus Anticoagulant (LA) is an autoantibody that in vitro inhibit phospholipid-dependent coagulation reactions in the absence of specific coagulation factor inhibition Primary laboratory criteria for LA detection include prolonged clotting time of a phospholipid dependent test, failure of correction on mixing study, shortening of the clotting time in the presence of high phospholipid concentrations & absence of other inhibitors. (1) According to guidelines,for diagnosing LA two tests based on different principles should be used- dRVVT(Dilute Russel's Viper Venom Time) should be the first test considered. Second test should be a sensitive aPTT (Activate Partial Thromboplastin Time).LA is a multistep procedure with screening, mixing and confirmation tests. Devreese et al recently concluded that integration of screening and confirmation in a single procedure and skipping the mixing tests result in a poor diagnostic efficacy for LA (2).LA is an in-vitro inhibitor and elevated Screening test without mixing studies does not prove inhibitor effect & it wastes time & resources to perform confirmatory tests skipping the mixing. Hence, we devised a new algorithm- Christian Medical College (CMC) Algorithm as shown in figure-1 which starts with DRVVT Screen Mix. To identify interferences due to anticoagulants like heparin and fibrin degradation products (FDP) thrombin time (TT) was also included. If Screen Mix is normal APTT Screen Mix was performed. Aim of our new algorithm was to reduce false positives and avoid interferences in LA Materials and methods: Four hundred and ninety-one (491) samples were analysed retrospectively for a period of 5 months from January 2022 to May 2022. The new algorithm was incorporated into STA R Max2 (Diagnostica Stago) analyser and automatic calculation of ratios and interpretation of results were done based on flow charts. LA testing was performed using DRVVT screen and confirm reagents (STA-Staclot DRVV Screen and STA-Staclot DRVV Confirm) and/or APTT reagent (STA-PTT Synthasil). LA test was considered positive when Screen/ Confirm(S/C) ratio was >1.2. We compared this result to the three hundred forty seven(347) samples, over a period of 4 months(September 2021 - December 2021) analyzed in old algorithm where DRVVT screen & APTT were done as the initial steps instead of DRVVT mixing studies as the 1st step. Results: In the new algorithm result were classified into 7 categories. Category 1 had normal DRVVT screen Mix and normal APTT; these represent true negative cases comprising 68.8 % of the cases. Category 2 had normal DRVVT screen Mix, prolonged APTT Screen Mix with normal TT. 1 case in this category had other Antiphosholipid Antibody (APLA) negative & repeat testing for LA was advised. Category 3 had normal DRVVT Screen mix, prolonged APTT Screen Mix with prolonged TT. There was 1 case in this category, who was on heparin, APLA antibody was negative. Repeat testing was advised at a later date. Category 4 had prolonged DRVVT Screen Mix, normal S/C ratio , with normal TT. There were 61 cases (12%) in this category among which 5 cases had normal DRVVT Screen with prolonged Mix Category 5 had Prolonged DRVVT Screen Mix, with normal S/C Ratio, prolonged TT and this category had 13 (2 %) cases. On further evaluation they were found to be on anticoagulants. Category 6 had prolonged S/C Ratio with normal TT, which are the true positive cases, n=67 (13.6%). There were 4 cases where Screen mix was more prolonged than Screen, and Screen fell below the cut off & could have been missed if mixing step was not done Category 7 had prolonged S/C ratio with prolonged TT. There were 10 cases (2%) in this category. On further evaluation 6 patients had history of anticoagulants and 2 had features of DIC. These 8 patients could represent false positive LA and would have been misclassified if TT was not done. Old algorithm had 20 % positive cases & 80 % negative cases. Since the algorithm were incorporated into STA R Max2 hands on time decreased from 240 minutes to 80 minutes. Cost of the reagents were similar by 2 algorithms. Conclusion: The new algorithm gives better clarity in classification of patients into true positive and true negative & detection of interference due to heparin as thrombin time is also done in parallel. Possible false positive cases could be identified at the onset even before repeat confirmatory lupus testing. This study concludes the importance of mixing studies in avoiding false negatives. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Lupus Anticoagulant Titer Is Associated with Elevated Anti-Phosphatidyl-Serine/Prothrombin Antibody Levels

Background Laboratory diagnosis of the antiphospholipid antibody syndrome (APS) requires evidence of persistently positive lupus anticoagulant (LAC) activity in clot-based assays and/or the presence of medium-high titer anticardiolipin (aCL) and/or anti-beta 2 glycoprotein I (aB2GPI) antibodies. Although aCL and aB2GPI methodologies provide quantitative information about the antibody titer, LAC testing is interpreted in a qualitative fashion (presence versus absence). Objectives In this study, we describe a method for measuring the LAC titer and evaluate the relationship between the LAC titer, other antiphospholipid antibodies and clinical outcomes. Methods Residual plasma from 51 LAC-positive samples (50 unique patients) were included in this study. To estimate LAC-titers, samples were tested undiluted and subjected to serial (dependent) twofold dilutions in pooled normal plasma (PrecisionBiologic) up to a final dilution of 1:128, as appropriate. The dilute Russell's viper venom screening time (DRVVT screen, LA Check, PrecisionBiologic) and activated partial thromboplastin screening time (APTT, SynthasIL, Werfen) were measured for each dilution. A four-parameter logistic model of the dilution factor versus the clotting time was used to interpolate the LAC-titer at the upper limit of normal for both assays (APTT of 37s and DRVVT screen ratio of 1.2). Measurements of aCL, aB2GPI and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) were performed using ELISA methodologies (QUANTA Lite, Werfen). Available clinical data were extracted from the electronic medical record. Continuity corrected chi-square test was used as a categorical test of association and quantile regression was used to compare medians for interval/ratio data across subgroups (Stata v17). Results There was a strong positive linear relationship between the APTT and DRVVT titers (Pearson's correlation coefficient, r=0.92, Figure 1A). There was only a moderate relationship between the APTT titer and the APTT screen in the undiluted sample (r=0.50) and almost no relationship between the DRVVT titer and the DRVVT screen ratio or DRVVT screen/confirm ratio in the undiluted sample (r=0.06 and r=0.13, respectively). Data-driven partitioning identified two DRVVT titer groups: low-titer (<10) and high-titer (≥10). Empirical cutpoint estimation (Youden) identified an APTT titer threshold of 5 for predicting the DRVVT titer group. Among patients with available clinical history, 15 of 25 (60%) low-DRVVT-titer and 16 of 20 (80%) high-DRVVT-titer patients had thromboembolic/obstetric events (p=0.26). Autoimmune diagnoses were more prevalent in the low-DRVVT-titer group (19 of 25 patients, 13 with SLE) than the high-DRVVT-titer group (8 of 20 patients, 3 with SLE, p=0.03). Comparing the low and high-DRVVT-titer groups, median aCL IgM (p=0.27), aCL IgG (p=0.99), aB2GPI IgM (p=0.72) and aB2GPI IgG (p=1.00) titers were not different. High-DRVVT-titer samples had a significantly higher median aPS/PT IgM compared to low-DRVVT-titer samples (p <0.01, Figure 1B) but there was no difference observed for IgG aPS/PT (p=0.57, Figure 1B). Conclusions APTT and DRVVT screening times are not strongly correlated with the corresponding APTT or DRVVT LAC-titer and may not indicate LAC potency. High DRVVT LAC-titers were strongly associated with IgM aPS/PT levels, but not with aCL, aB2GPI or IgG aPS/PT levels. Further research is required to evaluate the association between the LAC-titer and recurrent thrombosis in APS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application Value of Combined Detection of Anti-β2-GPI, ACL, and Lupus Anticoagulant in the Diagnosis of Patients with Antiphospholipid Syndrome.

To investigate the application value of combined detection of anti-beta 2-glycoprotein I antibody (anti-β2-GPI), anti-cardiolipin antibody (ACL), and lupus anticoagulant (LA) in the diagnosis of patients with antiphospholipid syndrome (APS). 30 APS patients in our hospital between Jan. 2020 and Jan. 2021 were chosen as the experimental group, and 30 healthy persons with normal physical examination during the same period were selected as the control group The anti-β2-GPI and ACL indexes of both groups were detected by enzyme-linked immunosorbent assay (ELISA), with the LA levels tested by modified dilute Russell's viper venom time (dRVVT) and LA ratio calculated. The diagnostic efficacy of single detection and combined detection was analyzed by plotting the receiver operating characteristic (ROC) curve. The serum indexes in the experimental group were remarkably higher than those in the control group (P < 0.001). ROC curve analysis suggested that in the diagnosis of APS, the area under the ROC curve by detecting anti-β2-GPI, ACL, LA ratio alone and simultaneously were 0.517, 0.583, 0.683, and 0.817 respectively, and the combined detection of the three had remarkably higher sensitivity and specificity than those of each single detection. The indexes of anti-β2-GPI, ACL, and LA ratio were highly expressed in APS patients, and the combined detection of the three has high diagnostic value and can effectively screen and assist the diagnosis of APS.

Association of coagulation markers and antiphospholipid antibodies with ischemic and bleeding risk in patients with atrial fibrillation undergoing percutaneous coronary intervention

Abstract Background Antiphospholipid antibodies and other coagulation markers are associated with ischemic risk in patients with coronary artery disease without oral anticoagulation (OAC). Aim To assess the association of antiphospholipid antibodies (aPL) and conventional markers of coagulation with ischemic and bleeding risk in patients with atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI). Patients and methods In this prospective single center observational cohort study, patients with AF and an indication for OAC were enrolled after PCI. Dilute Russell's viper venom time (DRVVT) was used to determine lupus anticoagulants (LA) in direct OAC-free plasma. Anti-cardiolipin IgG (aCL), IgM and anti-beta2GP-1-IgG (aβ2GP1) were analyzed by enzyme linked immunosorbent assay (ELISA). Fibrinogen C (FIBC), d-dimers and prothrombin fragments 1 and 2 (PF1+2) were measured in citrated plasma. Immature platelet fraction (IPF [%]) and absolute (IPF abs. [103/μl]) were measured in EDTA-blood. The primary ischemic outcome was defined as time to major adverse cardiovascular events (MACE: all-cause death, myocardial infarction, or stroke) assessed at 6 months. Bleeding was defined according to the International Society of Thrombosis and Haemostasis (ISTH) and the Bleeding Academic Research Consortium (BARC). Results 158 patients were enrolled between May 2020 and May 2021. The median age was 78 years (interquartile range, IQR 72–82), 111 (70%) were male, and 39 (25%) presented with acute coronary syndrome. All Patients were treated with clopidogrel and OAC, 145 (92%) in addition with acetylsalicylic acid (ASA). 32 patients (20%) had ≥1 antiphospholipid antibody (aPL; LA: 19 [12%], aCL: 14 [9%], aβ2GP1: 2 [1%]). D-dimers were elevated in 74 patients (47%), FIBC was increased in 40 (25%) and PF1+2 in 68 patients (43%). IPF [%] was elevated in 28 (18%) and IPF abs. [103/μl] in 11 (7%). The presence of aPL was neither significantly associated with MACE, nor with bleeding risk. Elevated d-dimers were significantly associated with higher risk for MACE (HR=5.1, 95% CI [1.1; 23.4], p=0.04), major ISTH bleeding events (HR= 6.9, 95% CI [1.5; 30.8], p=0.01) and BARC bleeding type 3 or 5 (HR=6.4, 95% CI [1.4; 28.7], p=0.02). Increased levels of FIBC were associated with risk of MACE (HR= 3.6, 95% CI [1.1; 12.0], p=0.03 (Table 1). Conclusion In patients with AF undergoing PCI, high levels of d-dimers and fibrinogen C indicate an increased ischemic risk. Elevated d-dimers are associated with higher risk for bleeding. aPL positivity was not significantly associated with outcome possibly due to low sample size. A combined panel of biomarkers might be suitable to assess ischemic and bleeding risk in patients with AF following PCI. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Faculty of Medicine, Freiburg

Lupus anticoagulant associates with thrombosis in patients with COVID‐19 admitted to intensive care units: A retrospective cohort study

BackgroundThrombosis is a frequent and severe complication in patients with coronavirus disease 2019 (COVID‐19) admitted to the intensive care unit (ICU). Lupus anticoagulant (LA) is a strong acquired risk factor for thrombosis in various diseases and is frequently observed in patients with COVID‐19. Whether LA is associated with thrombosis in patients with severe COVID‐19 is currently unclear. ObjectiveTo investigate if LA is associated with thrombosis in critically ill patients with COVID‐19. Patients/MethodsThe presence of LA and other antiphospholipid antibodies was assessed in patients with COVID‐19 admitted to the ICU. LA was determined with dilute Russell's viper venom time (dRVVT) and LA‐sensitive activated partial thromboplastin time (aPTT) reagents. ResultsOf 169 patients with COVID‐19, 116 (69%) tested positive for at least one antiphospholipid antibody upon admission to the ICU. Forty (24%) patients tested positive for LA; of whom 29 (17%) tested positive with a dRVVT, 19 (11%) tested positive with an LA‐sensitive aPTT, and 8 (5%) tested positive on both tests. Fifty‐eight (34%) patients developed thrombosis after ICU admission. The odds ratio (OR) for thrombosis in patients with LA based on a dRVVT was 2.5 (95% confidence interval [CI], 1.1–5.7), which increased to 4.5 (95% CI, 1.4–14.3) in patients at or below the median age in this study (64 years). LA positivity based on a dRVVT or LA‐sensitive aPTT was only associated with thrombosis in patients aged less than 65 years (OR, 3.8; 95% CI, 1.3–11.4) and disappeared after adjustment for C‐reactive protein. ConclusionLupus anticoagulant on admission is strongly associated with thrombosis in critically ill patients with COVID‐19, especially in patients aged less than 65 years.

Testing for Lupus Anticoagulants.

Lupus anticoagulant (LA) is one of the three criteria antiphospholipid antibodies (aPLs) employed in classification, and by default diagnosis, of antiphospholipid syndrome (APS). Detection of LA is not via calibrated assays but is based on functional behavior of the antibodies in a medley of coagulation assays. A prolonged clotting time in a screening test is followed by demonstration of phospholipid dependence and inhibitory properties in confirmatory and mixing tests, respectively, which are modifications of the parent screening test. Complications arise because no single screening test is sensitive to every LA, and no test is specific for LA, because they are prone to interference by other causes of elevated clotting times. Several screening tests are available but the pairing of dilute Russell's viper venom time (dRVVT) with LA-sensitive activated partial thromboplastin time (aPTT) is widely used and recommended because it is proven to have good detection rates. Nonetheless, judicious use of other assays can improve diagnostic performance, such as dilute prothrombin time to find LA unreactive with dRVVT and aPTT, and the recently validated Taipan snake venom time with ecarin time confirmatory test that are unaffected by vitamin K antagonist and direct factor Xa inhibitor anticoagulation. Expert body guidelines and their updates have improved harmonization of laboratory practices, although some issues continue to attract debate, such as the place of mixing tests in the medley hierarchy, and areas of data manipulation such as assay cut-offs and ratio generation. This article reviews current practices and challenges in the laboratory detection of LA.

Lupus anticoagulant: a clinical and laboratory diagnostic dilemma

Lupus anticoagulants are a group of diverse autoantibodies that interfere in vitro in phospholipid-dependent clotting tests, and inhibit both the common and intrinsic pathways of coagulation. Paradoxically, they are implicated to cause hypercoagulability, thrombotic events in vivo in varied clinical settings like obstetrics and oncology. A 56-year female was referred to the laboratory with complaint of repeated de novo clotting of drawn plasma samples. She was a post-operative case of surgery for superior mesenteric venous thrombosis, and a previously diagnosed case of squamous cell carcinoma of the buccal mucosa, not on treatment. The patient was evaluated clinically for new onset hypercoagulability and the history of exposure to heparin was negative, which ruled out heparin induced thrombocytopenia. The differential diagnosis were lupus anticoagulant, staphylococcal septicemia, and thrombotic thrombocytopenic purpura. On advanced work up, inhibitor screen was negative, dilute Russell's viper venom time was positive.

Stability of coagulation parameters in plasma samples at room temperature after one freeze/thaw cycle.

Sample freezing is a part of routine laboratory tasks because some coagulation parameters are analysed in batches to optimize reagent consumption. The coagulation parameter stability in fresh and frozen samples has been described, but data are scarcer after thawing. This study objective was to determine the stability of the main coagulation parameters (from blood withdrawn on siliconized CTAD tubes and double-centrifuged) after one freeze/thaw cycle to generate procedures for appropriate handling, storage and testing. Prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, D-dimers, clotting factors (F), protein C, protein S, antithrombin, lupus anticoagulant (LA)-sensitive aPTT and diluted-Russel's viper venom time (dRVVT) were assessed in 60 plasma samples (n=30, normal range and n=30, outside the normal range). Thirty samples from anticoagulated patients [unfractionated heparin (UFH), low-molecular weight heparin (LMWH), apixaban or rivaroxaban] were assessed using specific anticoagulant assays. Frozen samples were thawed, and assays were performed at 15min, 2, 4 and 6h after thawing. The coagulation parameter stability was assessed with the method of rejection limit. After thawing, aPTT, PT, fibrinogen, D-dimers, FII, FV, FX, FIX, FXI, FXII, PC and UFH anti-Xa activity remained stable for at least 6h, FVII for 5h, PS, AT, dRVVT screen assay and LMWH anti-Xa activity for 4h, and LA-sensitive aPTT and apixaban-specific anti-Xa activity for 3h. FVIII, dRVVT confirm assay and rivaroxaban specific anti-Xa activity were stable for 2h. These results suggest that sample stability for some haemostasis assays is limited after thawing.

Performance Characteristics of DOAC-Remove for Neutralization of the Effects of Apixaban and Rivaroxaban in Lupus Anticoagulant Assays.

This study established the performance characteristics of DOAC-Remove for neutralization of the effects of rivaroxaban and apixaban in lupus anticoagulant (LAC) testing. Normal donor, LAC control, and patient samples were spiked with rivaroxaban or apixaban to simulate their effects on the dilute Russell's viper venom time (dRVVT), activated partial thromboplastin time (APTT), and dilute prothrombin time (dPT). Anti-Xa activity was measured after spiking and after DOAC-Remove neutralization. Accuracy, complex precision, and reference interval verification were evaluated. DOAC-Remove neutralized rivaroxaban and apixaban concentrations as high as 415 ng/mL and 333 ng/mL, respectively. Percentage positive and negative agreement between the baseline and postneutralization interpretations were 75% or higher for the dRVVT and APTT methods but not for the dPT method. Coefficients of variation (CVs) were 10% or less for all assays except the Staclot-LA delta, which had a standard deviation of 2.5 seconds or CV of 25% or less depending on the level. The laboratory's reference intervals were verified for the dRVVT and APTT assays after DOAC-Remove treatment but not for the dPT assays. DOAC-Remove appears to have acceptable performance characteristics for neutralizing the effects of rivaroxaban and apixaban in the dRVVT and APTT methods but not in the dPT method.

Detection of direct oral anticoagulants with the dilute Russels viper venom time – observations in a real life setting

Abstract Background The detection of direct oral anticoagulants (DOACs) in emergency settings still remains challenging, especially when dedicated anti-Xa or anti-IIa tests are not readily available or the specific DOAC is unknown. The dilute Russel's viper venom time (dRVVT) is widely known in lupus anticoagulant (LA) testing and may also provide helpful information regarding the residual antithrombotic activity of DOACs. Yet real world data describing the effect of DOACs on the dRVVT is scarce. Objective The study aimed to evaluate the sensitivity and specificity of dRVVT for different DOAC plasma levels. Methods A total of 80 patients were recruited – 20 patients for each approved DOAC (apixaban, edoxaban, rivaroxaban and dabigatran). Blood plasma was sampled before (baseline), 6 and 12 hours after DOAC-intake and at plasma peak time. DRVVT was measured using the LA1 screening assay for lupus anticoagulant. Plasma levels were measured by calibrated anti-Xa or anti-IIa tests. Additionally, activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured. Results All DOACs significantly prolonged the dRVVT. The effects were more pronounced at higher DOAC plasma levels. The area under the receiver operating characteristic (ROC) curve regarding a plasma level cut-off of 30 ng/ml was 0.92 (95% confidence interval (CI) 0.823 to 1.00) for apixaban, 0.97 (95% CI 0.902 to 1.00) for edoxaban, 0.87 (95% CI 0.649 to 1.00) for rivaroxaban and 0.96 (95% CI 0.871 to 1.00) for dabigatran. Conclusion A dose dependent effect of all approved direct oral anticoagulants (DOACs) on the dilute Russel's viper venom time (dRVVT) was documented in our real world setting, while relevant DOAC plasma levels did not affect standard coagulation tests. The negative predictive value for the dRVVT was high even at low DOAC plasma levels. The dRVVT could therefore be helpful to rule out relevant antithrombotic plasma activity caused of DOACs in emergency situations. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Universität Leipzig Figure 1. ROC curves

Snake Venoms in Diagnostic Hemostasis and Thrombosis.

Snake venoms have evolved primarily to immobilize and kill prey, and consequently, they contain some of the most potent natural toxins. Part of that armory is a range of hemotoxic components that affect every area of hemostasis, which we have harnessed to great effect in the study and diagnosis of hemostatic disorders. The most widely used are those that affect coagulation, such as thrombin-like enzymes unaffected by heparin and direct thrombin inhibitors, which can help confirm or dispute their presence in plasma. The liquid gold of coagulation activators is Russell's viper venom, since it contains activators of factor X and factor V. It is used in a range of clotting-based assays, such as assessment of factor X and factor V deficiencies, protein C and protein S deficiencies, activated protein C resistance, and probably the most important test for lupus anticoagulants, the dilute Russell's viper venom time. Activators of prothrombin, such as oscutarin C from Coastal Taipan venom and ecarin from saw-scaled viper venom, are employed in prothrombin activity assays and lupus anticoagulant detection, and ecarin has a valuable role in quantitative assays of direct thrombin inhibitors. Snake venoms affecting primary hemostasis include botrocetin from the jararaca, which can be used to assay von Willebrand factor activity, and convulxin from the cascavel, which can be used to detect deficiency of the platelet collagen receptor, glycoprotein VI. This article takes the reader to every area of the diagnostic hemostasis laboratory to appreciate the myriad applications of snake venoms available in diagnostic practice.

Centralized Laboratory Testing for Inhibitors in the U.S. Hemophilia Treatment Centers Network: Testing Protocol and Results on 10,178 Specimens

Background: The incidence and prevalence of inhibitors among hemophilia patients in the U.S. has not been determined. The Centers for Disease Control and Prevention (CDC) has established a national inhibitor surveillance program called the Community Counts Registry for Bleeding Disorders Surveillance, through a collaboration with the American Thrombosis and Hemostasis Network and the U.S. Hemophilia Treatment Center (HTC) Network, to collect data and specimens to monitor inhibitors.Methods: A centralized laboratory was established at the CDC to perform inhibitor tests using a protocol based on accepted methods and published findings from a pilot project, the Hemophilia Inhibitor Research Study. Specimens are collected annually and when an inhibitor is detected or suspected locally from patients with hemophilia A (HA), hemophilia B (HB), and Type 3 von Willebrand disease (VWD3) who have received treatment and are enrolled at one of 134 HTCs. In the testing protocol, a modified Nijmegen-Bethesda assay with preanalytical heat inactivation (CDC-NBA) for factor VIII (FVIII) or factor IX (FIX) is first performed. On specimens with low positive or “gray zone” results of 0.5-1.9 Nijmegen-Bethesda units (NBU) for FVIII and 0.3-0.9 NBU for FIX, chromogenic Bethesda assay (CBA) for FVIII, fluorescence immunoassay (FLI) for anti-FVIII or anti-FIX antibodies, and dilute Russell's viper venom test (DRVVT) for lupus anticoagulants are performed. Repeat specimens are requested for positive or ambiguous results in patients with negative history.Results: Initial results on 10,178 specimens from 5,931 HA patients, 1,541 HB patients, and 134 VWD3 patients are shown in the Table. Additional testing on 141 low positive or “gray zone” FVIII specimens showed 74% to be CBA positive and 26% to be CBA negative. Of those CBA positive, 99% were also positive for IgG1 or IgG4 by FLI and were classified as true positives (104/141, 74%). Of CBA-negative specimens, 10 were also IgG1 and IgG4 negative and were classified as false positives (10/141, 7%). FLI results were positive on 26 CBA-negative specimens, which could not be classified (26/141, 18%). Final results on classified specimens are shown in the Table. Fourteen specimens were DRVVT positive, 13 of which were also CBA and FLI positive.Follow up was conducted on two groups of specimens: Arm 1 included specimens with positive or ambiguous results at CDC from patients who were reported to have no history of inhibitor, and Arm 2 included specimens from patients with new inhibitors locally detected at the HTC within the study period. In Arm 1, of 366 positive or ambiguous FVIII specimens, 20 were from patients with negative history and 17 of 18 repeated were confirmed; and of 44 positive FIX specimens, 3 were from patients with negative history and 2 of 3 repeated were confirmed. In Arm 2, 38 locally detected positive FVIII specimens and 6 locally detected positive FIX specimens were confirmed.Conclusions: From December 2013 through December 2016, newly positive test results on 55 FVIII and 8 FIX patients were identified and confirmed in two specimens, at least one of which was tested at the central laboratory. A cohort of 6,456 patients with negative history who were confirmed negative by central testing will be followed prospectively for inhibitor development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.