Dilute Russell Viper Venom Time Research Articles

The utility of the dilute prothrombin time in the interpretation of antiphospholipid syndrome testing.

To evaluate the utility of the dilute prothrombin time (DPT) in diagnosing antiphospholipid syndrome (APS), alone and when paired with the dilute Russell viper venom time (DRVVT). Dilute prothrombin time and DRVVT testing was performed on plasma samplesspiked with apixaban or rivaroxaban, or depleted of vitamin K-dependent clotting factors. A retrospective analysis of all functional APS testing results over a 44-month period at the University of Chicago Medical Center was performed. In spiking studies, the screening clotting time in the DPT (DPTS) is more sensitive to deficiency of vitamin K-dependent factors than is the screening clotting time in the DRVVT (DRVVTS). The converse is true for factor Xa direct oral anticoagulant (DOAC)-spiked plasma. In a 44-month retrospective analysis, only 2.6% of clinical APS panels showed isolated positivity in the DPT-based system. Comparing the DPT-based system with the DRVVT-based system showed utility in identifying false-positive DRVVT results due to anticoagulation. A DRVVTS/DPTS ratio of 0.785 or lower predicted an international normalized ratio of 1.5 or higher (sensitivity, 86.3%; specificity, 60.4%; likelihood ratio, 2.18). Conversely, a DRVVTS/DPTS ratio of 1.165 or higher was the optimal cutoff for predicting the identification of factor Xa DOAC (sensitivity, 61.8%; specificity, 77.8%; likelihood ratio, 2.78). Within the data set that had full DRVVT and DPT results, parameters were identified that could further improve identification of samples with anticoagulation interference. Dilute prothrombin time lupus anticoagulant assayis rarely the sole laboratory functional evidence for APS, but when combined with the DRVVT, the DPT can serve as an effective screen for common anticoagulant interference.

Dilute Russell viper venom time interpretation: Influence of lot changes and normalization.

Dilute Russell viper venom time interpretation: Influence of lot changes and normalization.

Reply to "Dilute Russell viper venom time interpretation: influence of lot changes and normalization".

Reply to "Dilute Russell viper venom time interpretation: influence of lot changes and normalization".

Antiphospholipid Syndrome and Lupus Anticoagulant: Where do we stand today ?

Antiphospholipid Syndrome and Lupus Anticoagulant: Where do we stand today ?

Pearls and Pitfalls in the Measurement of Direct Oral Anticoagulants.

Due to their widespread use, testing for direct oral anticoagulants (DOACs) has become urgent in certain clinical situations. Screening based on widely available, rapid, and simple hemostasis assays such as prothrombin time, activated partial thromboplastin time, or even diluted Russel Viper venom time may provide sufficient evidence of "over-coagulation" and could be used "in small/peripheral/spoke laboratories" as an emergency strategy, but is not thought to be reliable for driving clinical decision making. Given their good correlation with plasma concentration, urine dipsticks may be considered a valuable alternative for emergency screening, although their performance is dependent on renal function, may vary depending on the time since the last urination, and there may be problems of interfacing with the laboratory/hospital information system. Separation methods based on liquid chromatography and mass spectrometry may be clinically questionable, since they measure the concentration rather than the actual inhibitory effect of DOACs, are relatively expensive, cumbersome and time consuming, and therefore seem unsuitable for most conditions requiring urgent clinical decision making. A proposed approach therefore involves establishing a network of routine clinical laboratories, designating a reference center where DOAC tests could be available 24/7, establishing a clear diagnostic care pathway for ordering the tests from the laboratory and standard operating procedures for performing them, the use of the diluted thrombin time for dabigatran and anti-FXa assays (drug-calibrated) for rivaroxaban, apixaban, and edoxaban, as well as providing expert advice throughout the testing process, from ordering to interpretation of results.

To normalize or not?: Dilute Russell viper venom time testing.

We conducted a comparison between the nonnormalized dilute Russell viper venom time (dRVVT) screen/confirm ratio (SCR) in patient plasma and the normalized SCR obtained using reference pooled plasma. The aim was to assess the impact of normalization on the lupus anticoagulant (LA) status in our patient population. In our retrospective analysis, we included a total of 464 patients who underwent dRVVT testing. For those with positive screens, mixing studies were performed, followed by confirmatory testing. Additionally, the dRVVT of reference pooled plasma was measured. A positive conventional (nonnormalized) or normalized SCR was defined as an SCR greater than or equal to 1.2. In total, 5.6% (26) of the 464 samples tested were confirmed positive for LA by both methods, out of which 12 had a clinical history of thrombosis. Although a statistically significant difference between the 2 groups (P = .0096) was found, the magnitude of absolute mean SCR differences (bias) was 0.04 (2.51%). There was 100% concordance of testing results between the 2 groups. The lupus anticoagulant status by the dRVVT assay was not changed based on normalization. Normalization was of no clinical benefit in our patient population; therefore, there was no need for the extra calculation step. Normalization may be useful for intermethod and interlaboratory studies and not for within-method LA detection.

Quality Analysis of Lupus Anticoagulant Testing: Indeterminate and Interference

Abstract Lupus anticoagulant (LA) testing is a commonly performed test within hemostasis laboratories. Criteria for diagnosing a LA based on the International Society on Thrombosis and Hemostasis are: prolonged phospholipid-dependent (PD) clotting test, evidence of an inhibitor, evidence the inhibitor is PD, and exclusion of specific inhibitors. At our academic institution, we analyzed Lupus anticoagulant results to establish common sources of drug interference (DI), identify causes of indeterminate results, and evaluate if repeat testing was performed on patients with an initial indeterminate test. At this institution, Lupus anticoagulant evaluation includes activated partial thromboplastin time (aPTT), Anti-Xa activity, Thrombin Time (TT), diluted Russell Viper Venom Time, hexagonal phase phospholipid assay, and Platelet Neutralization Procedure. Samples with Anti-Xa activity >0.1 IU/mL and/or elevated TT are treated with hepzyme; if the Anti-Xa activity or TT does not correct, testing is not completed. Samples with elevated aPTT undergo an immediate and incubated mixing study (MS). Samples are considered positive (POS) for LA if there is a positive screening test (+ST), +MS, and evidence of PD in at least one assay. Tests are resulted as indeterminate (IND) with a +ST and either +MS or +PD. LA results are reported as unlikely (UNL) if only a single phospholipid panel is positive without +ST or +MS, and negative (NEG) if the ST, MS, and PD assays are all negative. Patients with indeterminate LA testing are recommended to have repeat LA testing after 12 weeks. A total of 3025 cases were identified over the 18-month period (July 2020 – December 2021). The majority of cases were interpreted as NEG (2310; 76%) followed by 8% POS (229), 6% IND (174), and 1% UNL (34). While the majority of IND met the ST (99%) and PD (71%) criteria, only 32% were +MS, indicating failure of an inhibitor detection in the mixing study is the major factor preventing an IND from becoming positive. Within the IND group, only 25% (43) underwent the recommended repeat LA testing. The results of repeated 43 IND tests are as follows: 16 NEG, 4 POS, 19 IND, 3UNL, 1 DI. While a significant proportion of IND repeated testing became negative, a majority remained IND, arguing for more comprehensive LA testing. This quality study demonstrates the majority of patients at this institution who receive an interpretation of IND LA do not undergo the recommended repeat testing, underscoring the importance of follow-up and education to standardize care. Prospective studies maybe needed to elucidate the clinical significance of IND LAs. Additionally, 278 (9%) cases were canceled due to drug interference, the majority (192; 69%) due to direct oral anticoagulants (DOAC). Testing for LA in patients actively receiving DOACs should be discouraged. Alternatively, methods to remove DOAC interference should be standardized.

Performance of a direct oral anticoagulant removal reagent, DOAC-StopTM, for lupus anticoagulant testing

Abstract Objective Routine use of direct factor Xa inhibitor (direct oral anticoagulant; DOAC) therapy necessitates a modified lupus anticoagulant (LAC) testing method for this population. To remove interfering DOACs in specimens, DOAC-StopTM treatment was used on LAC specimens to assess for antiphospholipid syndrome (APS) without altering patient’s anticoagulation treatment. Methods DOAC-StopTM was evaluated with our LAC testing panel with dilute Russell viper venom time (dRVVT), and Silica Clotting Time (SCT) assays, both of which activate coagulation. DOAC-StopTM beads were used for pretreatment by addition and rocking for 5 minutes, followed by 5000 rpm centrifugation for 5-minutes. Treated plasma supernatant was used for a screen, confirm (addition of phospholipid), 50:50 mix (addition of reagent plasma with coagulation factors), and confirm 50:50 mix phases of testing. The Total Ratio (TR; ratio between screen and confirm ratios relative to mean of normal ranges) was calculated and used per the package insert for interpretation. Implementation data was assessed for interpretation differences between groups with and without DOAC-StopTM treatment. Results Patients being tested for APS by LAC assay with and without DOAC therapy were included. DOACs included apixaban or rivaroxaban. Primary interpretations included no evidence of a LAC (NELAC), evidence of LAC, and results more consistent with coagulation factor deficiency. The effectiveness of DOAC removal was established by spiking NELAC samples with apixaban and treating with DOAC-StopTM, resulting in removal of anti-factor Xa activity. An apixaban spiking study with NELAC and LAC patient specimens with LAC testing before and after DOAC-StopTM treatment demonstrated unchanged interpretations. In a test modification study, Rivaroxaban resulted in positive dRVVT interpretations in nine of ten patients. All but one became negative after DOAC-StopTM treatment. Two SCT interpretations became positive after treatment. Apixaban resulted in two positive dRVVT interpretation in nine patients, one of which became negative after DOAC-StopTM treatment. SCT interpretations remained unchanged. Review of implementation data (n=78) with nine patients on DOACs having DOAC-StopTM treatment demonstrated similar patterns of test interpretation. Interpretations included: “no evidence of a LAC” in 53% (n=41) without and 55% (n=5) with DOAC-StopTM, “Does not meet criteria” in 17% (n=13) without and 11% (n=1) with DOAC-StopTM, “Evidence of a LAC” in 21% (n=16) without and 33% (n=3) with DOAC-StopTM. The remaining eight patients without DOAC-StopTM treatment had interpretations including factor deficiency, warfarin effect, equivocal, or concerning for interference. Conclusion Patients on DOACs benefit from DOAC-StopTM –enable LAC testing for APS assessment. Data analysis demonstrates that DOAC-StopTM effectively removes DOAC interference in the majority of tests with similar rates of major interpretation categories. LAC testing and interpretation for patients on DOAC therapy shows promise with novel approaches and reagents such as DOAC-StopTM, though interpretative challenges with false-positives and false-negatives continue.

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.

Variable Performance of Lupus Anticoagulant Testing: The Australasian/Asia-Pacific Experience.

Lupus anticoagulant (LA) is one of three tests identified as laboratory criteria for definite antiphospholipid syndrome (APS). The other two tests are anticardiolipin antibody (aCL) and anti-β2-glycoprotein I (aβ2GPI) antibody. The presence of LA is assessed using clot-based tests, while the presence of aCL and aβ2GPI is assessed by immunological assays. Since no test can be considered 100% sensitive or specific for LA, current guidelines recommend using two different clot-based assays reflecting different principles, with the dilute Russell viper venom time (dRVVT) and activated partial thromboplastin time (aPTT) recommended. Initially, LA-sensitive reagents are used to screen for LA, and then, in "screen-positive" samples, LA-"insensitive" reagents are used to confirm LA. Because LA assays are based on clot detection, anything that can interfere with fibrin clot development may affect test results. In particular, in addition to LA, the tests are also sensitive to the presence of a wide range of clinical anticoagulants, reflecting preanalytical issues for testing. We provide updated findings for LA testing in our geographic region, using recent data from the Royal College of Pathologists of Australasia Quality Assurance Programs, an international external quality assessment program with approximately 120 participants. Data show a wide variety of assays in use, especially for aPTT testing, and variable outcomes in reported numerical values with these assays when assessing proficiency samples. dRVVT testing mostly comprised reagents from three main manufacturing suppliers, which also showed differences in numerical values for the same homogeneous tested samples. Nevertheless, despite the use of different test reagents and processes, >98% of participants correctly identified LA-negative samples as LA-negative and LA-positive samples as LA positive. We hope our findings, reflecting on the heterogeneity of test processes and test data, help improve diagnostic testing for LA in the future.

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.

Antiphospholipid antibodies in convalescent plasma of donors recovered from mild COVID-19 infection.

Passive immunization by the infusion of convalescent plasma (CP) obtained from patients who have recently recovered from COVID-19, thus having antibodies to severe acute respiratory syndrome coronavirus 2, is a potential strategy to reduce the severity of illness. A high prevalence of antiphospholipid antibodies (APLA) in patients with COVID-19 has been reported during the pandemic, raising a concern whether the use of CP could increase the risk of thrombosis in transfused patients. We aimed to evaluate the prevalence of APLA in COVID-19 CP (CCP) in order to assess the potential prothrombotic influence of transfused CCP to COVID-19 patients. We studied the prevalence of APLA in 122 CCP samples collected from healthy donors who recovered from mild-COVID-19 at two time periods: September 2020-January 2021 (defined as 'early period' samples) and April-May 2021 (defined as 'late period' samples). Thirty-four healthy subjects unexposed to COVID-19 were used as controls. APLA were present in 7 of 122 (6%) CCP samples. One donor had anti-β2-glycoprotein 1(anti-β2GP1) IgG, one had anti-β2GP1 IgM and five had lupus anticoagulant (LAC) using silica clotting time (SCT), all in 'late period' donors. In the control group, one subject had anti-β2GP1 IgG, two had LAC using dilute Russell viper venom time (dRVVT) and four had LAC SCT (both LAC SCT and LAC dRVVT in one subject). The low prevalence of APLA in CCP donors reassures the safety of CCP administration to patients with severe COVID-19.

COMPARATIVE ANALYSIS OF HAEMATOLOGICAL, IMMUNOHAEMATOLOGICAL PARAMETERS AND CLINICAL CHEMISTRY PROFILE AMONG LUPUS ANTICOAGULANT POSITIVE AND LUPUS ANTICOAGULANT NEGATIVE COVID-19 CASES

Introduction: Coronavirus disease 2019 (COVID-19) is characterised by a pro-coagulant state that can lead to thromboembolic events. A high prevalence of Lupus Anti-coagulant has been shown in several studies, that may atleast partially explain the procoagulant prole of COVID-19. Few people infected by Coronavirus have become seriously ill while others showed little or no signs or symptoms related to COVID-19. This study was undertaken to analyze Haematological, Immunohaematological parameters and clinical Chemistry prole among LA positive and LA negative COVID -19 cases. Objectives: 1. To assess clinical, hematological and coagulation prole in COVID-19 lupus anticoagulant positive cases and lupus anticoagulant negative cases .. 2. To correlate ndings amongst lupus anticoagulant positive and negative cases. 3. To assess inammatory status amongst Covid-19 lupus anticoagulant positive and negative cases with markers like Crp-q, LDH, D-Dimer and Ferritin. Materials and Methods: The study was conducted as a prospective observational study with search data forms from the case records of patients hospitalized as ICU/ non- ICU COVID-19 cases in MGM Hospital and Medical College, Aurangabad during the pandemic for a total duration of 2 years from December 2020 to December 2022. A total of 90 patients with conrmed diagnosis of COVID-19 by RTPCR/RAT were studied. To assess Lupus Anti-Coagulant positivity- Dilute Russel Viper venom time (DRVVT) technique is used for established COVID-19 cases Conclusion: In conclusion, the current study conrmed no statistical correlation for parameters such as age, gender, comorbidities (hypertension, diabetes mellitus, COPD, IHD) in LA positive and LA negative cases. There was no statistical correlation found for ANA, RA factor, Inammatory markers and Coagulation prole among LA positive and LA negative cases. Neutrophil : Lymphocyte ratio and Lymphocyte : CRP ratio did not statistically differ in LA positive and LA negative cases. Complications (stroke, IC bleed, DVT, PTE) were observed in 16.27% LA negative patients while none of the above complications were seen in LA positive patients. Further large scale studies are required to comment on differences in Haematological, Immunohaematological and Clinical Chemistry prole between LA positive and LA negative cases.

Quantitative Risk for Single-Positive Lupus Anticoagulant Results With Different Anticoagulants.

Clinical experts recommend against testing for lupus anticoagulant (LAC) during anticoagulation. We quantitated the risk of a single-positive dilute Russell viper venom time (dRVVT) result or partial thromboplastin time-based phospholipid neutralization (PN) result on anticoagulation. Any anticoagulation led to a fourfold greater likelihood of single-positive results, primarily by rivaroxaban (odds ratio [OR] = 8.6) and warfarin (OR = 6.6), resulting in a positive dRVVT test with a normal PN test. Heparin and apixaban were twofold more likely to show single-positive results, but enoxaparin did not show significant single positivity. Our results quantitatively support experts' avoidance of LAC testing during anticoagulation.

Evaluating the Relationship Between Antiphospholipid Antibodies and COVID-19 Severity.

Coronavirus 2 (COVID-19) has emerged as a new global pandemic, causing severe acute respiratory syndrome. Furthermore, the existence of antiphospholipid (APL) antibodies (Abs) and ultimately patient death may be linked to the occurrence of thrombotic events in patients with COVID-19. We aimed to investigate if there was a link between the presence of APL Abs and the severity of COVID-19 disease in patients at the Vali-Asr Hospital in Zanjan from June to July 2021. Real-time PCR was used to diagnose COVID-19 in 76 hospitalized patients. A total of 38 patients were hospitalized in the internal medicine ward and another 38 people were admitted to the intensive care unit of the Vali-Asr Educational Hospital in Iran's Zanjan region. Lupus anticoagulant (LAC) detection was done using the dilute Russell viper venom time method, and tests for anticardiolipin (ACL) Abs, IgG and IgM, and anti-beta2 glycoprotein 1 Abs, IgG and IgM, were done on blood and plasma samples of linked patients using the enzyme-linked immunosorbent assay technique. SPSS 24 was used to analyze data. Our findings showed that the presence of LAC was associated with disease severity in COVID-19 patients (p = 0.001). However, there was no significant relationship between APL Abs and mortality in patients affected with COVID-19. The evaluation of APL Abs, particularly LAC, in COVID-19 patients appears to be helpful in predicting the severity of the disease.

Choosing Wisely Ten Years on: Inpatient Thrombophilia Testing

Introduction The American Society of Hematology (ASH) Choosing Wisely campaign have created thoughtful conversations between providers and patients in recommending care that is supported by evidence to reduce duplicative testing. However, some have raised concerns regarding the impact on patient care. We provide our institutional experience in exploring the clinical feasibility of the recommendation on reducing unnecessary inpatient thrombophilia testing. Methods This was a retrospective review of patients admitted with a diagnosis of venous thromboembolism (VTE) across the 3 hospitals in Central PA from September 2021 to December 2021. Pregnancy was an exclusion criterion. Thrombophilia workup included tests for factor V Leiden mutation, activated protein C resistance, protein S activity, antithrombin deficiency, prothrombin gene mutation, anti-cardiolipin antibodies, dilute Russell viper venom time, lupus anticoagulant, anti-beta2-glycoprotein antibodies. The cost of testing was extrapolated by institutional hospital cost reports. Descriptive statistics included reporting the categorical variables as number and percent. The categorical variables were compared between groups with the use of Fisher's exact test or the chi-square test. Results During our study period, 310 patients had new VTE. Of those, 47.9% had pulmonary embolism, 33.7% upper or lower extremities deep vein thrombosis (DVT), 1.6% splanchnic DVT, 14.6% multiple DVTs, and 2.2% DVTs in other locations. The median age was 64. Provoked factors were found in 269 patients (86.7%) and 41 patients (13.2%) had unprovoked VTE. Overall, 33 out of 310 patients (10%) underwent thrombophilia workup with a total of 175 tests performed. More thrombophilia testing was ordered in unprovoked group (29.3% vs 7.8%, p=0.0003) compared to provoked. There was no difference between the amount of inpatient hematology consults in both groups (33.83% vs 47.50%, p=0.0920). Of the hematology consult, 19 out of 118 patients (16%) had thrombophilia testing recommended by hematologist. The cost of all tests ordered outside of hematology recommendation was $34,083 (avg $344 per person). Of the group that got tested regardless of provoking factors, there was no difference between hospital length of stay (2-12 days vs 3-12 days, p= 0.0665). Anticoagulation choice was also not affected by thrombophilia testing between the two groups. Discussion Our experience found that many thrombophilia work up were costly and did not impact quality patient care. Despite the relatively low frequency of inpatient workup (10%), eliminating testing could save our health system approximately $136,332 per year ($34,083 per quarter). Therefore, we support ASH's campaign in reducing inpatient thrombophilia testing. We aim to work with our administration to provide education in reducing unnecessary testing as a part of a quality improvement initiative.

Lupus anticoagulant testing for patients on direct oral anticoagulants

Abstract As use of direct factor Xa inhibitor (direct oral anticoagulant; DOAC) therapy increases, a modified lupus anticoagulant (LAC) testing method was studied to remove interfering DOACs, allowing clinicians to evaluate patients for antiphospholipid syndrome (APS) without altering anticoagulation treatment. We evaluated the use of DOAC-StopTM on our LAC testing panel with two assays: dilute Russell viper venom time (dRVVT), a factor X activator, and Silica Clotting Time (SCT), an intrinsic coagulation pathway activator. The plasma specimens were pretreated with DOAC-STOPTM beads by mixing on a rocking platform for 5 minutes and centrifugation at 5000 rpm for 5 minutes. The treated plasma supernatant was used for each step of the assay including the screen, confirm (addition of phospholipid), 50:50 mix (addition of reagent plasma with coagulation factors) and confirm 50:50 mix. The total ratio (ratio between screen and confirm ratios relative to mean of normal ranges) was calculated and used per the package insert in the positive or negative interpretation of each LAC testing panel. The study included patients with one or more of the following characteristics identified during coagulation sign-out and chart review: no evidence of a LAC (NELAC), evidence of a LAC, and whether on apixaban or rivaroxaban therapy. The effectiveness of DOAC removal was characterized with an apixaban spiking experiment for NELAC and LAC patient specimens which showed no change in LAC testing panel interpretation before and after DOAC-STOPTM treatment. A second experiment with apixaban-spiked NELAC samples demonstrated removal of anti-factor Xa activity upon comparison of pre- and post-treatment with DOAC-STOPTM. Of 10 patients taking rivaroxaban, nine had positive dRVVT interpretations; eight of these specimens changed to negative interpretations after DOAC-STOPTM treatment. For SCT, two positive interpretations remained positive, while two of the remaining eight negatives became positive after treatment. Of nine patients taking apixaban, two had positive dRVVT interpretations, one of which became negative post-treatment. Of the seven negative interpretations, one became positive post-treatment. For SCT, three were positive pre-treatment and seven were negative with no change post-treatment. Providing a LAC testing option for patients on DOACs may be of benefit to patients and clinicians in APS assessment, eliminating the risk of suspending anticoagulation therapy. Our study shows DOAC-STOPTM effectively removes DOAC interference in the majority of patient specimens studied, though LAC testing and interpretation in the setting of a high-complexity patient population on DOAC therapy remains challenging with occasional false positives and false negatives.

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

Confirming Choosing Wisely: Inpatient thrombophilia testing.

22 Background: The American Society of Hematology (ASH) Choosing Wisely campaign have created thoughtful conversations between providers and patients in recommending care that is supported by evidence to reduce duplicative testing. However, some have raised concerns regarding the impact on patient care. We provide our institutional experience in exploring the clinical feasibility of the recommendation on reducing unnecessary inpatient thrombophilia testing. Methods: This was a retrospective review of patients admitted with a diagnosis of venous thromboembolism (VTE) across the 3 hospitals in Central PA from September 2021 to December 2021. Pregnancy was an exclusion criterion. Thrombophilia workup included tests for factor V Leiden mutation, activated protein C resistance, protein S activity, antithrombin deficiency, prothrombin gene mutation, anti-cardiolipin antibodies, dilute Russell viper venom time, lupus anticoagulant, anti-beta2-glycoprotein antibodies. The cost of testing was extrapolated by institutional hospital cost reports. Descriptive statistics included reporting the categorical variables as number and percent. The categorical variables were compared between groups with the use of Fisher’s exact test or the chi-square test. Results: During our study period, 310 patients had new VTE. Of those, 47.9% had pulmonary embolism, 33.7% upper or lower extremities deep vein thrombosis (DVT), 1.6% splanchnic DVT, 14.6% multiple DVTs, and 2.2% DVTs in other locations. The median age was 64. Provoked factors were found in 269 patients (86.7%) and 41 patients (13.2%) had unprovoked VTE. Overall, 33 out of 310 patients (10%) underwent thrombophilia workup with a total of 175 tests performed. More thrombophilia testing was ordered in unprovoked group (29.3% vs 7.8%, p = 0.0003) compared to provoked. There was no difference between the amount of inpatient hematology consults in both groups (33.83% vs 47.50%, p = 0.0920). Of the hematology consult, 19 out of 118 patients (16%) had thrombophilia testing recommended by hematologist. The cost of all tests ordered outside of hematology recommendation was $34,083 (avg $344 per person). Of the group that got tested regardless of provoking factors, there was no difference between hospital length of stay (2-12 days vs 3-12 days, p = 0.0665). Anticoagulation choice was also not affected by thrombophilia testing between the two groups. Conclusions: Our experience found that many thrombophilia work up were costly and did not impact quality patient care. Despite the relatively low frequency of inpatient workup (10%), eliminating testing could save our health system approximately $136,332 per year ($34,083 per quarter). Therefore, we support ASH’s campaign in reducing inpatient thrombophilia testing. We aim to work with our administration to provide education in reducing unnecessary testing.