Abstract Number: PB0336
Meeting: ISTH 2021 Congress
Background: Commercial DOAC assays for measuring FXa inhibitors (FXaI) apixaban and rivaroxaban on the ACL TOP/TOP50 analyzers are available in many countries. These assays utilize the anti-FXa reagent with drug specific calibrators and controls to determine the drug concentration in patient plasma. In the presence of andexanet alfa (AnXa), a specific reversal agent for FXaI, these assays show erroneous elevation of the measured anti-FXa activity. This is attributed to the large sample dilution in the assay setup (1:27), which causes dissociation of the FXaI from AnXa due to a reversible binding equilibrium.
Aims: The aim of this study was to verify the analytical performance of a modified user-defined setting for the apixaban and rivaroxaban tests on the ACL TOP/TOP50 analyzers with HemosIL Anti-Xa reagent and HemosIL Apixaban and Rivaroxaban calibrators.
Methods: The modified assay setup and calibration were optimized to minimize the sample dilution to 1:2.6 suitable for analyzing FXaI in AnXa-containing samples. Ten plasma samples containing different concentrations of apixaban (230, 460, 920 ng/mL, 0.5 – 2.0 μM) or rivaroxaban (218, 436, 872 ng/mL, 0.5 – 2.0 μM) in the presence of AnXa at different molar ratios were tested (Table 1). The optimized calibration curves reported in the 10-100 ng/mL linear range.
Molar Concentration | |||||
Sample ID | Total Apixaban (ng/mL) | Total Rivaroxaban (ng/mL) | FXaI (μM) | AnXa (μM) | Molar Ratio |
1 | 230 | 218 | 0.5 | 0 | 0 |
2 | 230 | 218 | 0.5 | 0.25 | 1:2 |
3 | 0.5 | 0.5 | 1:1 | ||
4 | 0.5 | 1.0 | 2:1 | ||
Data was obtained from ten samples each in the apixaban and rivaroxaban panel with different concentrations of FXaI at 0.5,1.0,2.0 μM and andexanet (AnXa at 0- 4.0μM). For clarity, only concentrations for Samples 1-4 are shown. Rivaroxaban MW=435.9 Da; Apixaban MW=459.5 Da; AnXa MW =41 kDa; FXaI- FXa inhibitor. |
Results: The data generated with minimized sample dilution on the ACL TOP/TOP50 is comparable to the 1:2 dilution in the modified 96-well plate assay previously used in AnXa clinical studies. Representative data shown in Figure 2 demonstrates a dose-dependent reversal of anti-FXa activity with increasing AnXa:FXaI molar ratio. Similar results were observed in samples with proportionally higher FXaI and AnXa concentrations (not shown).
Conclusions: The modified tests for apixaban and rivaroxaban on the ACL TOP/TOP50 analyzers demonstrate dose-dependent percent reversal comparable to the 96-well plate format. The modified user-defined setting could be used for measuring FXaI in AnXa-containing samples.
To cite this abstract in AMA style:
Khan A, Shishko K, Lu G, Nguyen V, Winkler A. Laboratory Assessment of DOAC Assays for FXa Inhibitors in the Presence of Reversal Agent Andexanet Alfa on the ACL TOP/TOP 50 Coagulation Analyzers [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/laboratory-assessment-of-doac-assays-for-fxa-inhibitors-in-the-presence-of-reversal-agent-andexanet-alfa-on-the-acl-top-top-50-coagulation-analyzers/. Accessed March 22, 2024.« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/laboratory-assessment-of-doac-assays-for-fxa-inhibitors-in-the-presence-of-reversal-agent-andexanet-alfa-on-the-acl-top-top-50-coagulation-analyzers/