Does in vitro hemolysis affect measurements of plasma apixaban concentration by UPLC-MS and anti-Xa assay?

Abstract

Introduction: Hemolytic interference may impact various laboratory tests, including coagulation analyses. Apixaban is the most commonly used direct oral anticoagulant in Norway, and there is lacking knowledge on how apixaban concentration measure- ments might be influenced by hemolysis. Moreover, hemolysis-induced alterations in apixaban levels could potentially impact the risk of bleeding in specific clinical scenar- ios. We wanted to study whether hemolysis would increase apixaban concentration and investigate the impact of hemolytic interference on apixaban concentration measurements. Methods: Blood samples from 20 apixaban-treated patients and 8 healthy controls were hemolyzed in vitro by a freeze method. The degree of hemolysis was measured with plasma free hemoglobin (PfHb) at baseline and two levels of hemolysis. Apixa- ban concentration was measured in plasma using both the chromogenic anti-Xa method and the ultraperformance liquid chromatography mass spectrometry (UPLC-MS). Thrombin generation assay was performed to assess coagulability. Results: UPLC-MS measurements showed a mean concentration change of 1.66% (±3.2%, p = 0.005) and anti-Xa assay showed a mean concentration change of 3.37% (±6.5%, p = 0.09) with increasing hemolysis. Thrombin generation lagtime decreased, and endogenous thrombin potential and peak thrombin increased with increasing hemolysis in both the control group and the apixaban group. Conclusion: Apixaban concentration measurements by anti-Xa assay and UPLC-MS were not affected by hemolysis to a clinically relevant extent. Furthermore, hemolysis did not lead to hypocoagulability when assessed by thrombin generation.