A universal anti‐Xa assay for rivaroxaban, apixaban, and edoxaban measurements: method validation, diagnostic accuracy and external validation

Abstract

Summary A universal anti‐Xa assay for the determination of rivaroxaban, apixaban and edoxaban drug concentrations would simplify laboratory procedures and facilitate widespread implementation. Following two pilot studies analysing spiked samples and material from 698 patients, we conducted a prospective multicentre cross‐sectional study, including 867 patients treated with rivaroxaban, apixaban or edoxaban in clinical practice to comprehensively evaluate a simple, readily available anti‐Xa assay that would accurately measure drug concentrations and correctly predict relevant levels in clinical practice. Anti‐Xa activity was measured by an assay calibrated with low‐molecular‐weight heparin (LMWH) in addition to ultra‐high performance liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). As an external validation, LMWH‐calibrated anti‐Xa activity was also determined in nine external laboratories. The LMWH‐calibrated anti‐Xa activity correlated strongly with rivaroxaban, apixaban or edoxaban drug levels [rs = 0·98, 95% confidence interval (CI) 0·98–0·98]. The sensitivity for the clinically relevant cut‐off levels of 30, 50 and 100 µg/l was 96·2% (95% CI 94·4–97·4), 96·4% (95% CI 94·4–97·7) and 96·7% (95% CI 94·3–98·1) respectively. Concordant results were obtained in the external validation study. In conclusion, a universal, LMWH‐calibrated anti‐Xa assay accurately measured rivaroxaban, apixaban and edoxaban concentrations and correctly predicted relevant drug concentrations in clinical practice.

Accuracy of a Single, Heparin-Calibrated Anti-Xa Assay for the Measurement of Rivaroxaban, Apixaban, and Edoxaban Drug Concentrations: A Prospective Cross-Sectional Study

“…Samples were collected regardless of the time of last drug intake, covering the full range of drug levels observed in clinical practice. Ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) measuring rivaroxaban, apixaban, and edoxaban was used as a reference standard ( 16 , 21 ). The study was approved by the local ethics committees and all hospitals gave local feasibility approval.…”

“…Thus, implementing a single-calibration anti-Xa assay for unfractionated heparin, low molecular weight heparin, rivaroxaban, apixaban, and edoxaban would improve laboratory procedures but also the care of patients treated with DOAC. Recently, we demonstrated that a universal, LMWH-calibrated assay can accurately measure DOAC drug levels ( 21 ). However, it remains unclear if this can be achieved using a universal heparin-calibrator intended to detect unfractionated heparin and low molecular weight heparins.…”

Automated Thrombin Generation Assay for Rivaroxaban, Apixaban, and Edoxaban Measurements

“…This is a multicenter cross-sectional study conducted between 2018 and 2019 ( 20 ). Patients treated with rivaroxaban, apixaban, or edoxaban in routine clinical practice were included in nine specialized hemostasis laboratories affiliated to tertiary hospitals in Switzerland.…”

“…Rivaroxaban, apixaban, edoxaban, and edoxaban M4 were quantified by LC-MS/MS as previously described ( 20 ). Briefly, protein precipitation and analyte extraction were performed by adding to the plasma acetonitrile:water 1:1 (v/v), extraction buffer (MassTox TDM Series A, Chromsystems, Gräfelfing, Germany), and precipitation reagent (MassTox TDM Series, Chromsystems, Gräfelfing, Germany) containing the isotope labeled internal standards (13C6 rivaroxaban, 13CD3 apixaban, 13CD2 edoxaban) The samples were vortexed and then centrifuged at 14000 rcf and 20°C for 4 min.…”

“…The anti-Xa inhibitors rivaroxaban, apixaban, and edoxaban can be determined using chromogenic anti-Xa assays ( 19 ). These tests, which are successfully used to determine unfractionated and low molecular weight heparins, were further developed and successfully studied in various evaluation studies ( 15 , 20 ). Routine coagulation tests such as prothrombin time are not sensitive to detect clinically relevant concentrations of anti-Xa inhibitors ( 21 ).…”