Potential for False Negative Lupus Anticoagulant Testing in Patients Anticoagulated with Warfarin Using Dilute Russell's Viper Venom Time Assay

Introduction: Laboratory identification of a lupus anticoagulant (LAC) requires evidence of: a) prolongation of a phospholipid-dependent screening test, usually a LAC-responsive dilute Russell’s viper venom time (DRVVT) or activated partial thromboplastin time (APTT); b) inhibition on mixing with normal pooled plasma (NPP); c) confirmation of phospholipid-dependent inhibition by repeating the prolonged test with excess phospholipid. Despite decades of assay development and re-design and numerous attempts at standardization, diagnosis of a LAC remains challenging due to the heterogeneity of antiphospholipid antibodies, the marked variations in reagents, inconsistencies in post-analytic processes, and not least, the multitude of interferences, such as anticoagulant drugs, which influence test results. Guidelines recommend interruption of anticoagulation, if possible, prior to LAC testing to prevent obtaining false test results. While this is attempted in some instances for direct-acting anticoagulants, it is typically not considered for warfarin, and it is also not always possible. Our laboratory has observed a concerning pattern of potentially false-negative DRVVT screen/ confirm ratios in samples collected from patients on warfarin anticoagulation or with a factor deficiency (e.g. hypoprothrombinemia). The goal of this study is to replicate this observation using in vitro dilution experiments, mixing LAC-positive samples with either NPP or warfarin plasma.

Methods: DRVVT Screen, Mix and Confirm assays (Precision Biologic, Dartmouth, NS) were performed using archived LAC-positive plasmas from 5 unique patients not on anticoagulation, and titered with normal pooled plasma (NPP) (Precision Biologic, Dartmouth, NS) to a screen/confirm ratio of 1.20-1.35, near the assay cutoff for DRVVTC (<1.20) and analyzed on the ACL TOP 750 (Werfen, Bedford, MA). Warfarin donor plasma was then mixed in the same dilution scheme with each of the LAC-positive plasmas so that the final dilution factors for LAC-NPP and LAC-Warfarin contrived samples were the same. The DRVVT screen/ confirm ratios of the LAC-NPP and LAC-Warfarin plasma contrived samples were compared. Additionally, a DRVVT Mix Screen and Mix Confirm were performed after mixing in a 1:1 ratio with NPP.

Results: The average prothrombin time (PT) in the LAC-Warfarin samples was significantly longer than the LAC-NPP samples (20.7±8.5s vs 10.6±0.2s, p=0.03). All 5 LAC-NPP contrived samples had a positive DRVVT screen/ confirm ratio >1.20, while all of the LAC-Warfarin contrived samples had a negative DRVVT screen/ confirm ratio (1.26±0.04 vs 1.02±0.11, p<0.01). When the DRVVT screening time was divided by the DRVVT Mix Confirm time, 4 of the 5 LAC-Warfarin contrived samples reverted to positive using a cutoff of 1.20 (values of 1.30, 1.21, 1.21, 1.02 and 1.23 for LAC-Warfarin samples) and a similar pattern was observed with the LAC-NPP samples (values of 1.32, 1.29, 1.27, 1.14, 1.27, respectively, for LAC-NPP samples).

Conclusion: This study confirms our anecdotal observation that warfarin and/ or factor deficiencies in the common pathway can result in a false-negative DRVVT screen/ confirm ratio using the Precision Biologic LA Sure and LA Check reagents, similar to findings described for apixaban. The DRVVT assay is widely considered to be the most sensitive or responsive to LAC and this in vitro observation raises questions about whether negative DRVVT screen/ confirm ratios can reliably be used to exclude the presence of a LAC in patients on warfarin using this reagent system. Larger studies are required using pre-treatment and on-treatment paired specimens to confirm these findings, and to further explore the utility of the DRVVT Screen/Mix Confirm ratio in this patient population.