Russell's Viper Venom Research Articles

Using Electronic Best Practice Alerts to Improve Thrombophilia Testing Based on ASH Choosing Wisely Guidelines

IntroductionThrombophilia testing is costly, and results can be misleading in the setting of acute venous thromboembolism (VTE) or anticoagulation use. It is frequently ordered despite limited impact on clinical decision making. American Society of Hematology (ASH) Choosing Wisely guidelines recommend against thrombophilia testing in patients with VTE in the setting of major transient risk factors (surgery, trauma, prolonged immobility).MethodsA best practice alert (BPA) was implemented in our electronic medical record system to promote awareness of the ASH Choosing Wisely guidelines. The BPA showed the text of the ASH Choosing Wisely guidelines onscreen whenever thrombophilia testing was ordered for a patient with a diagnosis of pulmonary embolism (PE) or deep venous thrombosis (DVT). It was up to the clinician to decide whether the Choosing Wisely guidelines applied in a particular clinical scenario and whether to follow the BPA recommendation and cancel the thrombophilia test or to override the BPA and order testing.Thrombophilia testing orders which would trigger the BPA were protein C and S activity levels, lupus anticoagulant, dilute Russell viper venom test, homocysteine levels, Factor V Leiden, prothrombin gene mutation and activated protein C resistance.The BPA began displaying to providers in the outpatient setting on 7/1/2016 and in the inpatient setting on 9/1/2016. The pre-BPA period was defined as the 12 months prior to activation of the outpatient BPA (7/1/2015 to 6/30/2016). The post-BPA period was defined as the 7 months from the activation of the inpatient BPA (9/1/2016 to 3/31/2017). We reviewed the number of BPAs triggered as well as the actions taken by providers (follow or override). We also compared the total number of thrombophilia tests in the outpatient and inpatient settings in the pre- and post-BPA periods.ResultsThe BPA appeared onscreen 140 times in the outpatient setting and 35 times in the inpatient setting during the study period. The BPA was followed 17.1% of the time in the outpatient setting and 100% of the time in the inpatient setting. Thrombophilia testing volumes stayed unchanged in the outpatient setting (471.5 tests per month pre-BPA vs. 471.6 tests per month post-BPA, p=1.00). In contrast, there was a significant decrease in the average number of thrombophilia tests per month (101.1 vs. 73.3, p=0.038) in the inpatient setting after the implementation of the BPAs. During the study period, inpatient censuses were not statistically different in the pre-BPA and post-BPA periods.DiscussionWe found that the thrombophilia BPA was followed at a very high rate in the inpatient setting compared to the outpatient setting. The BPA is meant to remind providers of the ASH Choosing Wisely guidelines, but providers must still use clinical judgment to determine whether the guideline applies in a given clinical scenario. The high rate of compliance in the inpatient setting suggests that in many cases, thrombophilia testing is being ordered on inpatients with transient risk factors, and that a brief reminder message can effectively change provider behavior. On the other hand, BPA compliance was relatively low in the outpatient setting, though this could be appropriate based on the clinical scenarios which outpatient providers encounter. Future analyses will include manual chart review to determine the appropriateness of BPA overrides in the outpatient setting, as well as a thematic analysis of BPA override comments.There was a significant reduction in the average number of inpatient thrombophilia tests ordered per month in the post-BPA period compared to the pre-BPA period. Although the inpatient thrombophilia BPA had a high rate of compliance, it was only shown 35 times during the study period. Thus the BPA messages alone were unlikely to account for the significant change in total volume of thrombophilia testing. Prior research on blood transfusion BPAs suggests that BPAs can have an educational impact, causing providers to become more aware of guidelines and thus actually reducing the frequency of triggered BPAs over time. Thus, each BPA message may have a multiplicative effect, preventing future unnecessary tests in addition to the original order. DisclosuresNo relevant conflicts of interest to declare.

Is Lupus Anticoagulant a Significant Feature of COVID-19? A Critical Appraisal of the Literature.

The term "lupus anticoagulant (LA)" identifies a form of antiphospholipid antibodies (aPLs) causing prolongation of clotting tests in a phospholipid concentration-dependent manner. LA is one of the laboratory criteria identified in patients with antiphospholipid (antibody) syndrome (APS). The presence of LA in patients with APS represents a significant risk factor for both thrombosis and pregnancy morbidity. There have been several reports of similarities between some of the pathophysiological features of COVID-19 and APS, in particular the most severe form, catastrophic APS. There have also been many reports identifying various aPLs, including LA, in COVID-19 patients. Accordingly, a very pertinent question arises: "Is LA a feature of COVID-19 pathology?" In this review, we critically appraise the literature to help answer this question. We conclude that LA positivity is a feature of COVID-19, at least in some patients, and potentially those who are the sickest or have the most severe infection. However, many publications have failed to appropriately consider the many confounders to LA identification, being assessed using clot-based assays such as the dilute Russell viper venom time, the activated partial thromboplastin time (aPTT), and the silica clotting time. First, most patients hospitalized with COVID-19 are placed on anticoagulant therapy, and those with prior histories of thrombosis would possibly present to hospital already on anticoagulant therapy. All anticoagulants, including vitamin K antagonists, heparin (both unfractionated heparin and low-molecular-weight heparin), and direct oral anticoagulants affect these clot-based assays. Second, C-reactive protein (CRP) is highly elevated in COVID-19 patients, and also associated with severity. CRP can also lead to false-positive LA, particularly with the aPTT assay. Third, persistence of aPL positivity (including LA) is required to identify APS. Fourth, those at greatest risk of thrombosis due to aPL are those with highest titers or multiple positivity. Most publications either did not identify anticoagulation and/or CRP in their COVID-19 cohorts or did not seem to account for these as possible confounders for LA detection. Most publications did not assess for aPL persistence, and where persistence was checked, LA appeared to represent transient aPL. Finally, high titer aPL or multiple aPL positivity were in the minority of COVID-19 presentations. Thus, at least some of the reported LAs associated with COVID-19 are likely to be false positives, and the relationship between the detected aPL/LA and COVID-19-associated coagulopathy remains to be resolved using larger and better studies.

Impact of a commercially available DOAC absorbent on two integrated procedures for lupus anticoagulant detection

BackgroundLupus anticoagulant (LA)-detection in anticoagulated patients is an unmet need, which becomes even more cogent with the introduction of direct oral anticoagulants (DOAC) that may lead to false-positive results. AimsWe aimed to investigate the effect of a commercially available DOAC absorbent on residual drug concentrations, and on integrated procedures for LA-detection. MethodsBlood from patients treated for atrial fibrillation with either dabigatran (n = 39), rivaroxaban (n = 55), apixaban (n = 47) or edoxaban (n = 47) were collected at peak and trough, and centralized for testing with two LA integrated procedures [i.e., the silica clotting time (SCT) and dilute Russel viper venom (dRVVT)] before and after DOAC absorbent exposure. ResultsThe commercially available DOAC absorbent investigated in this study proved effective in reducing the concentrations of all the investigated DOAC, although small residual drug was detected after absorption, especially in patients on edoxaban.Results mimicking LA were observed in patients on DOAC before absorbent exposure, especially for rivaroxaban when testing was performed with dRVVT (88% rate at peak and 20% at trough) and much less with SCT (12% at peak and 8% at trough). The correspondent rate of results mimicking LA in patients on rivaroxaban after exposure was reduced [dRVVT (peak 8%, trough 4%); SCT (peak and trough 8%)], but not abolished. ConclusionsOverall, in vitro DOAC absorbance by active charcoal compounds is a useful laboratory tool for LA-detection in patients on DOAC. Caution should however be exerted when used in daily practice.

Practical Considerations and Testing Nuances for the Detection of Lupus Anticoagulant: Do Low Phospholipid Screen Results, Assay Type, and Test Ratio Matter?

Lupus anticoagulant (LA) detection requires (1) prolongation of a phospholipid (PL)-dependent clot-based screening assay, (2) noncorrection upon adding normal pooled plasma, and (3) a confirmatory PL dependency test. Paired LA assays run screening and confirmatory tests simultaneously, with their test ratio (TR) or differences used to evaluate test results. We evaluated patients whose paired testing demonstrated PL dependence suggestive of LA, yet the low PL screen was not prolonged. Clinical and laboratory parameters are compared across (1) true positive (screen prolonged, TR positive) vs borderline (screen not prolonged, TR positive); (2) low-, moderate-, and high-TR subgroups; and (3) dilute Russell viper venom time (dRVVT) vs silica clotting time (SCT). Borderline samples are not statistically different from true positives in their rate of repeat LA positivity or association with other anti-PL antibodies. Compared with true positives, borderline dRVVT is more frequent in pregnancy, women, and younger age. Elevated activated partial thromboplastin time is more frequent in true-positive dRVVT and SCT vs borderline and with an increasing dRVVT TR. LA persistence is more frequent with an increasing SCT TR. In addition, dRVVT true positivity is more frequent with thromboembolic events, while SCT is more frequent with autoimmunity and pregnancy complications. Negative low-PL screens may not necessarily lack LA. A reevaluation of the laboratory criteria for LA detection may be needed.

A Diagnostic Solution for Lupus Anticoagulant Testing in Patients Taking Direct Oral FXa Inhibitors Using DOAC Filter.

Background: Direct oral factor Xa (FXa) inhibitors interfere with lupus anticoagulant (LA) assays challenging antiphospholipid syndrome diagnosis in treated patients. We evaluated a new device, called DOAC Filter, and its usefulness in this setting. It is a single-use filtration cartridge in which FXa inhibitor compounds are trapped by non-covalent binding while plasma is filtered through a solid phase. Patient samples were analyzed before and after filtration: 38 rivaroxaban, 41 apixaban, and 68 none. Anticoagulant plasma concentrations were measured using specific anti-Xa assays and HPLC-MS/MS. LA testing was performed using dilute Russell Viper Venom Time (dRVVT) and Silica Clotting Time (SCT). Baseline median [min–max] concentrations were 64.8 [17.6; 311.4] for rivaroxaban and 92.1 ng/mL [37.1; 390.7] for apixaban (HPLC-MS/MS). They were significantly correlated with anti-Xa assay results (r = 0.98 and r = 0.94, respectively). dRVVT was positive in 92% rivaroxaban and 72% apixaban and SCT in 28 and 41% of samples, respectively. Post-filtration, median % of neutralization was 100% with rivaroxaban and apixaban concentrations of, respectively, <2 [<2–2.4] and <2 ng/mL [<2–9.6] using HPLC-MS/MS. No significant effect of DOAC Filter was observed on LA testing in controls (n = 31) and LA-positive (n = 37) non-anticoagulated samples. dRVVT and SCT remained positive in, respectively, 16 and 8% of rivaroxaban and 41 and 18% of apixaban samples. DOAC Filter would be an easy-to-use device allowing FXa inhibitor removal from plasma samples, limiting their interference with LA testing in treated patients.

Applying index of circulating anticoagulant to mixing tests with lupus anticoagulant screen and confirm reagents can distinguish with high specificity between lupus anticoagulants and direct factor Xa inhibitors.

Lupus anticoagulants (LA) are detected by prolongation of clotting times for dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) screening tests. Direct oral anticoagulants (DOACs) can interfere with both screening and confirmatory tests. The present study aimed to investigate the influence of direct factor Xa inhibitors (DiXaIs) on screen, confirm and mixing tests and establish a method for differentiation from other sample types. A total of 257 samples including nonanticoagulated LA positive, LA positive with DiXaIs, factor deficiency, FVIII inhibitors, warfarin and non-APS DiXaIs were tested. APTT reagents Cephen LS/Cephen and dRVVT reagents LA1/LA2 were used, respectively, to screen/confirm the study group. Index of circulating anticoagulant (ICA) was calculated from clotting times based on the following formula as ICA screening and ICA confirmation. ICA= (1:1 Mix sample - Normal pooled plasma) / Screen patient x 100. An ICA matrix was established which suggested the presence of a DiXaI when both ICA screening and confirmation were above the cut-off. When only ICA screening is elevated, LA is suspected. Sensitivity and specificity of the ICA matrix were 52.2% and 92.8% for DiXaIs and 38.1% and 96.7% for LA in APTT, and 61.2% and 92.9% for DiXaIs and 22.2% and 88.4% for LA in dRVVT, respectively. The ICA matrix achieved high specificity with a lower apparent sensitivity for DiXaI samples comparatively to other devices but due only to less interferences: the matrix could contribute to differentiating DiXaIs from LA in samples where anticoagulation status is unknown.

Evaluation of the effect of apixaban using a viscoelastic coagulation assay with Russell\u2019s viper venom reagent

BackgroundConventional coagulation tests, such as prothrombin time and activated partial thromboplastin time, are not sensitive to anticoagulation by apixaban. We evaluated the antithrombotic effect of apixaban using a Russell viper venom (RVV) test for a patient who underwent posterior spine fusion surgery.Case presentationAn 84-year-old man was scheduled for percutaneous posterior spine fusion. He continued apixaban until the night before surgery and resumed it on the first day after surgery. We performed an RVV test as point-of-care coagulation monitoring in combination with chromogenic anti-activated factor X (anti-Xa) activity, prothrombin time, and activated partial thromboplastin time. Clotting time with the RVV test was prolonged according to the anti-Xa activity of apixaban, which was in the therapeutic range during surgery.ConclusionsAn RVV test might be useful as a point-of-care assay for estimation of the anti-Xa level induced by apixaban during the perioperative period.

28-Year-Old Man With Recurrent Vertigo, Syncope, and Progressive Memory Impairment

28-Year-Old Man With Recurrent Vertigo, Syncope, and Progressive Memory Impairment

Revisiting the effects of spectral interfering substances in optical end‐point coagulation assays

Hemolysis, icterus, and lipemia (HIL) are common pre-analytical variables in the clinical laboratory. Understanding their effects on coagulation laboratory results is essential. HIL effects on the prothrombin time (PT), activated partial thromboplastin time (APTT), dilute Russell's viper venom time (DRVVT), thrombin time (TT), and protein C chromogenic activity (CFx) were evaluated on the ACL TOP 750 optical analyzer and STA-R Evolution mechanical analyzer (PT and APTT only) by spiking normal donor, patient, and commercial control samples with varying concentrations of hemolysate, bilirubin, or a lipid emulsion. The relative difference or bias compared to the original results was determined. Hemolysis (H) indices up to 900mg/dL did not affect the APTT, PT, DRVVT Confirm, TT, and CFx; however, H indices above approximately 200mg/dL resulted in a false-negative DRVVT screen and screen/confirm ratio in samples with a lupus anticoagulant. There was an artifactual prolongation of the PT and APTT when conjugated bilirubin was dissolved in aqueous solvents and not when it was dissolved in dimethyl sulfoxide. Icterus (I) indices up to 45mg/dL did not result in significant (>15%) bias for all assays evaluated. The PT and APTT assays failed to produce a robust clot curve when the lipemia (L) index exceeded 6000 milliabsorbance units (mAbs), and the TT and DRVVT assays failed when the L index exceeded 3000 mAbs; the CFx assay was unaffected by lipemia. Verification of the manufacturer's recommended interference thresholds is important since it may avoid inappropriate instrument flagging and/ or sample rejection.

Microkinetic coagulation assays for human and zebrafish plasma.

Coagulation assays, prothrombin time (PT), and partial thromboplastin time (PTT) are tests to measure the clotting ability of plasma and used in evaluating patients suffering from bleeding disorders. These assays require 100 μl of human plasma. In zebrafish, dilute plasma with exogenously added human fibrinogen was used. Our objective is to create a microkinetic coagulation assay for human and zebrafish plasmas using 1 μl plasma under conditions similar to PT and PTTs. Here, we developed an assay using the Take3 plate with wells holding up to 6 μl, which can be loaded in a microplate reader for measuring the absorbance of fibrin formation. In this assay, we used 1 μl of citrated zebrafish or human plasma followed by the addition of either thromboplastin or Dade ACTIN or factor X activator from Russell viper venom as an activating agent and CaCl2. We found 4 or 3 μl of the final volume of reaction was optimal. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established curves using dilute plasma. This kinetic coagulation was inhibited by heparin and was reduced significantly in coagulation factor deficient plasmas. These results validated our microkinetic coagulation assays. Moreover, we derived clotting times from these kinetic curves, which were identical to human PT, PTT, and Russel's viper venom time. In conclusion, we established a microkinetic assay that could measure blood coagulation activity in models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.

How to Achieve Standardization? Diluted Russell Viper Venom Test for Lupus Anticoagulant Detection in a Chinese Female Population.

On an international scale, guidelines and proposals for lupus anticoagulant detection have been published over the last 20 years, but until now, standardization has not been completely realized. The aim of this study was to evaluate the different ways of interpreting the results of lupus anticoagulant detection for standardization. A retrospective review of 15 447 instances of lupus anticoagulant detection by the diluted Russell Viper Venom test for female patients presenting with problems relating to the areas of reproduction, gynecology and obstetrics was performed. Lupus anticoagulant data were compared between different departments, months, reagent lots and cutoffs. Significant differences were found in patient data between different reagent lots, especially between lots of screening reagents (monthly average: highest 37.96 s vs lowest 33.88 s) and in the positive rates of lupus anticoagulant by different detection cutoffs (47.58% by using LA1/LA2 > 1.20 without normalization as a cutoff in Lot 1 vs 1.52% by using LA1 > 44 s as a cutoff in Lot 3). Compared with the cutoff using the value above the 99th percentile of LA1 for the healthy donors per lot, the cutoff using integrated tests with normalization had the smaller deviation of positive rate between different reagent lots. Pregnant women had higher LA1/LA2 levels than nonpregnant women. Based on the results, normalization is needed because there are significant lot-to-lot variations. Integrated tests with normalization might be a better standard by which to confirm lupus anticoagulant. Pregnant women should have population-specific cutoffs because they have higher LA1/LA2 levels.

The mode of action of sugammadex on coagulation.

To explore the mode of action (MoA) by which sugammadex interferes with coagulation. The effect of sugammadex on various steps in the coagulation cascade including thrombin generation, factor Xa activity, and factor Xa generation was explored in human plasma. Sugammadex did not affect a conventional thrombin generation test (TGT), while it prolongs activated partial thromboplastin time (APTT) and prothrombin time (PT). However, a customized TGT with PT reagent revealed sugammadex effects. In addition, sugammadex prolonged a one-step prothrombinase-induced clotting time (PiCT) using human factor Xa. Furthermore, sugammadex interfered with factor Xa generation induced by an intrinsic and not by an extrinsic activator, nor by Russell's viper venom factor X (RVV-X). Adapted, rather than standard, experiments show that sugammadex is likely to decrease factor Xa activity in the common pathway and activation of factor X specifically in the intrinsic pathway.

The Forgotten Story of Vitamin C Deficiency

Vitamin C plays an essential role in the formation of collagen. A deficiency in vitamin C can lead to scurvy, manifested by blood vessel fragility, fatigue, and, rarely, death. Today, scurvy is rare in developed countries. Therefore, to diagnose scurvy requires a high index of suspicion. This will be illustrated by our patient of interest. A 66-year-old man presented to the emergency department (ED) with worsening bilateral leg swelling and bruising throughout his body. His past medical history was notable for a bowel resection with colostomy secondary to colorectal cancer, currently in remission. The bruising and swelling began two weeks prior without any inciting events. He denied taking blood thinners or non-steroidal-anti-inflammatory-drugs. He had no personal or family history of bleeding disorders. None of his previous surgeries were complicated by bleeding issues. Extremities showed large ecchymoses over left thigh and bilateral ankles, and hematoma over right patella. There were no perifollicular hemorrhages seen on skin examination. His hemoglobin was 13.5 g/dL and his platelet count was 145x109/L. A computed tomography angiography of his lower extremities revealed intramuscular hematomas in the calves, left adductor compartment and left sartorius. A venous ultrasound of bilateral lower extremities was unremarkable. He was advised to follow up with a hematologist outpatient. Ten days later, the patient reported worsening swelling and pain of his lower extremities and was advised to visit the ED (FIGURE 1A). His Hgb dropped to 10.8 g/dL. An extensive factor workup showed: factor VIII activity of 421.7% (ref range: 55-200), factor IX activity of 104% (ref range: 70-130), factor XI activity of 68% (ref range: 55-150), and von Willebrand factor activity of 355% (ref range: 55-200). Factor V, X and XIII were within normal limits. Other possible etiologies including vitamin K, HIV, hepatitis panel, antinuclear antibody and extractable nuclear antigen antibodies panel were normal. His activated partial thromboplastin time (aPTT) was prolonged at 44 seconds (ref range 25-37). The dilute Russel's viper venom time (dRVVT) was abnormal and his dRVVT/dRVVT-phospholipid ratio was greater than 1.3 or greater, indicative of a lupus anticoagulant. Beta-2 glycoprotein 1 antibodies and anticardiolipin antibodies were normal. Bleeding due to prothrombin (factor II) deficiency in the context of lupus anticoagulant has been reported (2). However, his factor II level was normal. Meanwhile, his Hgb fell to 6.9 g/dL, indicative of ongoing intramuscular bleeding. Upon further investigation by the consulting hematology team and registered dietitian, there was a concern for severe malnutrition, evident by substantial loss of subcutaneous fat and muscle mass. The patient revealed that he consumed six twelve-ounce cans of beers nightly. His diet was minimal in fruits and vegetables. One month prior to his admission, he had worsening fatigue, brittle nails and gum bleeding. His folate level was 2.0 ng/mL (ref range &amp;gt;3.9) and his albumin level dropped to 2.1 g/dL (ref range 3.5-5). His vitamin C level resulted &amp;lt; 0.1 mg/dL (ref range 0.4-2). He was started on three days of intravenous vitamin C, one gram per day. His hematoma and bruises dramatically improved (FIGURE 1B). As a result, a diagnosis of scurvy was made. On discharge, he was transitioned to oral vitamin C and advised to follow up with his hematologist outpatient. Scurvy is often viewed as a disease of the past. Yet, according to a national survey between 2003 and 2004, the prevalence of age-adjusted vitamin C deficiency is 7%. At risk patients include the elderly, institutionalized populations, alcoholics, and severe psychiatric illness leading to poor nutritional intake. Therefore, a dietary history of the patient should be obtained. Vitamin C contributes to the structure of blood vessels through its involvement in collagen synthesis. Characteristic signs and symptoms of scurvy feature fatigue, oral findings (spontaneous bleeding, gum retraction) and cutaneous abnormalities (petechiae and lesions). Rarely, it can lead to spontaneous intramuscular hematoma. The prognosis of scurvy is excellent, and the response to vitamin C is dramatic. This case illustrates the need to consider scurvy in diagnosing bleeding cases. A high index of suspicion remains integral in diagnosing scurvy to avoid expensive and lengthy workup. Disclosures No relevant conflicts of interest to declare.

Appropriate Laboratory Testing for Lupus Anticoagulants: An Observational Study at an Academic, Tertiary Care Hospital

Introduction: Lupus anticoagulants (LA) are a heterogeneous group of immunoglobulins that develop spontaneously, with autoimmune disorders or transiently in association with acute infectious or inflammatory conditions. Transient LA are generally presumed to be harmless, while LA that persist over time may be a risk factor for venous and arterial thrombosis. Accordingly, persistence of LA after a first thrombotic episode may signal a recommendation for long term thromboprophylaxis with an anticoagulant, thus, to guide therapy, correct interpretation of LA test results is paramount. LA test interpretation is complicated, however, and clinicians must understand the LA test characteristics, the importance of timing of testing and the implications of positive, negative and equivocal test results to use the information effectively for clinical decision making. We undertook this study to ascertain how clinicians at our institution approach LA testing and to identify knowledge gaps that may amenable to educational interventions aimed at improving patient care in this area. Methods: Our laboratory uses a combination of the dilute Russell viper venom time (dRVVT) and silica clotting time (SCT) for LA screening (Instrumentation Laboratories, Bedford MA, USA). A positive test for either dRVVT or SCT is considered positive for LA. Negative tests for both are required to exclude the presence of LA in a given patient. We provide cutoff values for negative, positive and indeterminate test results and recommend repeat testing after 12 weeks for any indeterminate or positive test to establish persistence in accordance with current guidelines. We recommend NOT testing patients who are currently anticoagulated with a direct oral anticoagulant (DOAC) due to the potential for a false positive test result. We retrospectively analyzed 715 completed LA tests between April 1, 2018 and April 30, 2019 to determine the proportion of positive, negative and indeterminate test results. We then reviewed individual patient records to identify patient and provider characteristics and document additional diagnostic actions taken based on the initial test results. We report our findings descriptively. Results: The average age of patients undergoing laboratory testing for LA was 46.9 years, (median, 47.0 years), with a preponderance of females (73%). The majority of tests were ordered by rheumatology or hematology providers (53%). Anticoagulation with a DOAC was present in 74 (10%). A negative initial test (dRVVT and SCT both negative) was documented in 413 patients (58%), and was repeated in nine individuals (2%) at a second time point. A positive test (dRVVT and/or SCT positive) was documented in 170 (24%), of which 53 (31%) were repeated subsequently and 18 (11%) were themselves repeat tests to follow up on a previous positive test, leaving 99 (58%) initial positive tests that were not repeated. The majority of repeat tests were conducted at a time point &amp;lt;12 weeks after the initial test (n = 37; 70%). Equivocal test results (dRVVT and/or SCT indeterminate) were obtained in 132 patients (18%) and repeated in 25 (19%). Conclusion: We discovered a surprisingly high rate of inappropriate LA test usage at our institution. The results of this investigation will guide our efforts to develop interventions aimed at informing our clinicians about best practices for laboratory testing for LA, and provide opportunities to leverage the features of the electronic health record to improve test ordering procedures. Disclosures No relevant conflicts of interest to declare.

Coagulation Testing Gone Awry; Effect of Direct Oral Anticoagulant (DOAC) Removal on Lupus Anticoagulant Assays

Abstract Background Direct oral anticoagulants (DOACs) such as apixaban and rivaroxaban are widely used to treat those with or at risk for thromboembolic disease. However, DOACs have been shown to interfere with coagulation assays making their interpretations challenging. Importantly, lupus anticoagulant (LA) assays using the dilute Russell Viper Venom Time (dRVVT) to screen for patients at increased risk of thrombosis are particularly affected by DOACs leading to inaccurate results. DOAC removal agents are available to improve dRVVT specificity for LA and eliminate assay interferences. Aims To evaluate the performance of DOAC-Remove to accurately measure LA in patient plasma. Methods 10 LA positive and 10 LA negative samples without DOAC as well as normal pool plasma (NPP) were used as controls. We assessed 64 patient plasma samples including 39 patients on apixaban and 25 patients on rivaroxaban (anticoagulation was confirmed by chart review). LA testing by dRVVT was performed using Staclot® DRVV Screen and Staclot® DRVV Confirm reagents on STA R Max analyzer. DRVVT testing was performed before and after DOAC removal (DOAC-Remove, Aniara). DOAC plasma concentrations were measured using STA Liquid Anti-Xa assay. Acceptable DOAC removal was defined as an anti Xa level &amp;lt;0.03 IU/mL. Positive LA was defined as a screen ratio and mix ratio greater than 1.15. Results Positive and negative samples without DOAC remained the same after DOAC removal treatment. Factor levels in NPP were not affected by DOAC removal treatment. DOAC removal resulted in shortening of the dRVVT in 91% of the samples. Surprisingly, 6 cases showed prolongation in the dRVVT after DOAC removal. Before DOAC removal, 47 cases (24 apixaban and 23 rivaroxaban) tested positive and 17 tested negative (15 apixaban and 2 rivaroxaban). Following DOAC removal, 28 cases tested positive and 36 tested negative. As expected, many samples of patients on DOAC were found to be falsely positive (27/64; 15 on apixaban and 12 on rivaroxaban). Interestingly, 8 of 64 tests were found to be falsely negative. Of the 8 samples that were falsely negative, 7 were on apixaban and 1 on rivaroxaban. 9 samples that were negative before DOAC removal remained negative after DOAC removal, and 20 tests that were positive before DOAC removal remained positive after DOAC removal for a total of 29 results (45%) that remained unchanged. It was found that DOACs caused inaccurate results in 55% of cases. Conclusions Our study was able to demonstrate the effectiveness of DOAC-Remove to eliminate DOACs from patient plasma and provide accurate LA results. DOAC interference on LA assays predictably creates many false positive results, however, presence of DOACs can produce false negative results as well, underlying the importance of pretest screening for presence of anticoagulants in plasma samples.

Intensity of immune/clotting assays relate to multiple antiphospholipid antibody positivity in thrombotic primary antiphospholipid syndrome.

The dual positivity (DP) and triple positivity (TP) concepts bypass the poor comparability of immune/clotting assay for the laboratory classification of antiphospholipid syndrome (APS). To evaluate intensity of immune/clotting assays and DP/TP through different clinical severity groups (CSG) as follows: (1) non-thrombotic asymptomatic carriers of aPL (N-THR), thrombotic primary APS (THR), deceased (D) for recurrent and fatal thrombosis. Activated partial thromboplastin time ratio (aPTTr), dilute Russell viper venom time ratio (DRVVTr), IgG/IgM anticardiolipin (aCL) and anti β-2-glycoprotein-I (aβ2GPI). Participants: 33N-THR, 64 THR and 11 D. The frequency of DP and TP (DRVVTr or aPTTr partnered with respective IgG aCL or aβ2GPI) increased across CSG (p = 0.006 and p = 0.003); mean DRVVTr and IgG aCL/aβ2GPI were always greater in TP versus non-TP within each CSG and progressively increased across the CSG. The intensity of individual lupus anticoagulants partnered with their corresponding IgG aPL related to the frequency of multiple positivity throughout CSG suggesting that of intensity of immune/clotting assays and multiple positivity are the different faces of the same diagnostic coin in our thrombotic PAPS cohort.

HEAVILY CALCIFIED CLOTS IN A PATIENT WITH CHRONIC THROMBOEMBOLIC PULMONARY HYPERTENSION PRESENTING WITH BACTEREMIA

SESSION TITLE: Medical Student/Resident Pulmonary Vascular Disease Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Antiphospholipid syndrome (APLS) is an autoimmune disorder that can lead to thrombotic disease. Here, we present a challenging case of catastrophic antiphospholipid syndrome with calcified clots, and chronic thromboembolic pulmonary hypertension (CTEPH) presenting with persistent bacteremia. CASE PRESENTATION: A 23-year-old female patient with a history of diabetes mellitus, methamphetamine and intravenous drug use disorder presented to an outside hospital with dry cough, shortness of breath, pleuritic chest pain, chills, and fatigue for 2 weeks. Blood cultures upon admission grew methicillin sensitive staphylococcus aureus (MSSA). Other labs were notable for thrombocytopenia and acute kidney injury. CT scan of chest and abdomen without contrast showed a large clot in her left pulmonary artery, inferior vena cava (IVC) extending into the right atrium (RA) with impressive calcifications (Figure 1). Transesophageal echocardiography showed no vegetations but revealed a large atrial thrombus extending from IVC into a dilated RA, dilated right ventricle, moderate tricuspid regurgitation with calculated right ventricular systolic pressure 55mmHg. Ventilation perfusion (VQ) scan showed a diffuse left lung perfusion defect without matched ventilation defect. She was transferred to our hospital for further care, her work-up revealed positive dilute Russell's viper venom time (dRVVT), cardiolipin antibodies and beta2-glycoprotein antibodies suggesting triple positive antiphospholipid syndrome. She was treated with intravenous heparin, however, developed hemoptysis requiring multiple blood transfusions and intubation, thus bronchial artery embolization was performed. Furthermore, she developed ST elevation myocardial infarction and splenic infarcts which were managed medically with resumption of heparin and antiplatelet therapy. She was eventually discharged on coumadin after completing six weeks of cefazolin and rifampin. DISCUSSION: This case illustrates an unusual presentation and calcification pattern of chronic clot, possibly due to the underlying infection, in a patient with CTEPH secondary to APLS. Mainstay of treatment for CTEPH remains anticoagulation followed by evaluation for pulmonary thromboendarterectomy (PTE) which is the only definite treatment (1). However, patients must tolerate anticoagulation, be clear of any infection and demonstrate adherence to therapy to undergo surgery. Furthermore, coumadin with an INR goal of 2.5 to 3.5 is recommended over direct oral anticoagulants in APLS with thromboembolism (2). CONCLUSIONS: In conclusion, CTEPH is associated with high morbidity and mortality, mainly from right heart failure (3). High level of suspicion is required to diagnose and promptly treat this rare disease with atypical presentation. Reference #1: Fedullo P, Kerr KM, Kim NH, Auger WR. Chronic thromboembolic pulmonary hypertension. Am J Respir Crit Care Med 2011; 183:1605. Reference #2: Pengo V, Denas G, Zoppellaro G, et al. Rivaroxaban vs warfarin in high-risk patients with antiphospholipid syndrome. Blood. 2018;132:1365–71. Reference #3: Hoeper MM, Mayer E, Simonneau G, Rubin LJ. Chronic thromboembolic pulmonary hypertension. Circulation 2006; 113:2011. DISCLOSURES: No relevant relationships by Timothy Fernandes, source=Web Response No relevant relationships by Abdurrahman Husain, source=Web Response No relevant relationships by Alexandra Rose, source=Web Response No relevant relationships by Qais Zawaydeh, source=Web Response

Lupus anti-coagulant (LA) screening &amp; confirmation testing by dilute Russell viper venom time (DRVVT) method

Background: Anti-phospholipid antibody syndrome is a life-threatening acquired disorder of the immune system which can lead to dysfunction of the multiple systems in the human body. Lupus anticoagulant (LA) detection by the normalized dilute Russell viper venom time (DRVVT) ratio provides a robust assay methodology with accurate results. The objective of this study was to evaluate the potential ability of diluted Russell viper-venom time (DRVVT) to identify Lupus anticoagulant (LAC) by screening and confirmation testing methods. Methods: Samples of the suspected auto-immune disorder patients with lengthened screening dRVVT (using LA Check) more than 2 standard deviations (SD) above the average level of normal donors undergo additional testing: a 1:1 blend with normal plasma which taken from various normal individuals and amalgamated together (using LA Check) and high phospholipid confirmatory testing (using LA Sure).Results: A total of 311 cases included in this study. Out of 311, 230 patients were females and 81 were males. Out of 230 females LAC positivity by DRVVT method was seen in 78 patients and 152 were Negative. Out of 81 males LAC positivity by DRVVT method was seen in 38 patients and 43 were Negative. Conclusions: Precise evaluation of lupus anticoagulant plays a major role in estimating the risk factors for thrombosis. A practically elevated lupus anticoagulant result may change the duration of anticoagulation medications usage for patients. The dRVVT is recognized and certified by all three expert panels as one of the preliminary assays to be included when screening for a LAC.

Loss of antiphospholipid antibody positivity post-thrombosis in SLE

Background/PurposeLoss of positivity of antiphospholipid antibodies has been observed in clinical practice post-thrombosis in patients with SLE with secondary antiphospholipid syndrome (APS). Our study defined the frequency of this loss...

Sodium oleate, arachidonate, and linoleate enhance fibrinogenolysis by Russell's viper venom proteinases and inhibit FXIIIa; a role for phospholipase A2 in venom induced consumption coagulopathy

Life-threatening symptoms produced by Russell's viper (RV, Daboia russelii) envenomation result largely from venom induced consumption coagulopathy (VICC). VICC is thought to be mediated to a large degree by venom serine and metalloproteinases, as well as by snake venom phospholipase A2 (svPLA2), the most abundant constituent of RV venom (RVV). The observation that the phenolic lipid anacardic acid markedly enhances proteolytic degradation of fibrinogen by RVV proteinases led us to characterize the chemical basis of this phenomenon with results indicating that svPLA2 products may be major contributors to VICC. ResultsOf the chemical analogs tested, the anionic detergents sodium dodecyl sulfate, sodium deoxycholate, N-lauryl sodium sarcosine, and the sodium salts of the fatty acids arachidonic, oleic and to a lesser extend linoleic acid were able to enhance fibrinogenolysis by RVV proteinases. Enhanced Fibrinogenolysis (EF) was observed with various venom size exclusion fractions containing different proteinases, and also with trypsin, indicating that conformational changes of the substrate and increased accessibility of otherwise cryptic cleavage sites are likely to be responsible for EF. In addition to enhancing fibrinogenolysis, sodium arachidonate and oleate were found to partially inhibit thrombin induced, factor XIIIa (FXIIIa) mediated ligation of fibrin chains. In clotting experiments with fresh blood RVV was found to disrupt normal coagulation, leading to small, partial clot formation, whereas RVV pretreated with the PLA2 inhibitor Varespladib induced rapid and complete clot formation (after 5 min) compared to blood alone. ConclusionThe observations that fatty acid anions and anionic detergents induce conformational changes that render fibrin(ogen) more susceptible to proteolysis by RVV proteinases and that RVV-PLA2 activity (which produces FFA) is required to render blood incoagulable in clotting experiments with RVV indicate a mechanism by which the activity of highly abundant RVV-PLA2 promotes degradation and depletion of fibrin(ogen) resulting in incoagulable blood seen following RVV envenomation (VICC).