Factor VIII assay discrepancies in post-infusion samples obtained from patients treated with efanesoctocog alfa

Introduction Efanesoctocog alfa is a B-domain-deleted, Fc-fusion factor VIII (FVIII) linked to the D'D3 domain of von Willebrand factor and 2 XTEN polypeptides. This design results in improved half-life but also in discrepancies in FVIII diagnostic tests. For example, it has been shown that FVIII chromogenic substrate assays (CSA) overestimate efa by a factor of 2 – 3, what led to the proposal of a correction factor (CF) of /2.5 (*0.4) when interpreting results obtained by FVIII CSAs [ 1 ] [ 2 ]. The aim of the present study was to evaluate FVIII assay discrepancies in post-infusion samples obtained from patients treated with efa. Method This retrospective analysis included 43 male patients on efa prophylaxis (26 on once-weekly-, 17 on twice-weekly treatment scheme) summoned for determination of incremental recoveries. FVIII activity was measured at trough (3.5 or 7 days post-infusion)- and peak levels (30 minutes post-infusion) using three diagnostic tests: two one-stage clotting assays (OSCAs) based on Actin FSL (AFSL) or Actin FS (AFS), and a chromogenic substrate assay (FVIII CSA). Results Incremental recoveries as determined by the recommended AFSL-based OSCA were found to be comparable between patient groups. At peak levels (64 to 214 IU/dL), comparable overestimation (1.9-fold) of plasma efa activity relative to Actin FSL-based OSCA was observed for both, the AFS-based OSCA and the FVIII CSA. In contrast, at trough levels (5 to 64 IU/dL), rate of overestimation relative to the AFSL-based OSCA results was found to be lower for the FVIII CSA (1.3-fold) when compared to the AFS-based OSCA (1.9-fold). Further analysis demonstrated different behavior of spiked- and post-infusion samples within the FVIII CSA. Conclusion This study revealed lower CFs than previously proposed (/1.9 and /1.3 versus /2.5). Discrepancies between spiked and post-infusion samples, particularly at low efa levels, may result from matrix effects or in vivo molecular modifications of efa that influence assay performance. Because bovine FVIII CSAs are essential for monitoring patients receiving emicizumab, product-specific calibration has been proposed to enhance measurement accuracy and comparability across settings [ 3 ] [ 4 ] [ 5 ]. While the present data support this approach, differences between sample types indicate that assay discrepancies may still persist, underscoring the need for further research into their biochemical mechanisms and for improved assay standardization. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany