Assay or astray? discrepancies in efanesoctocog alfa monitoring

Efanesoctocog alfa (EFA) is an ultraextended half-life factor VIII (FVIII) with a unique structure that may influence laboratory monitoring. Although assay discrepancies have been described in spiked samples, data in patient samples are limited.

To assess the performance of seven FVIII assays for measuring EFA in patient plasma and compare results with those from spiked samples.

FVIII activity was measured using four one-stage assays (OSAs) and three chromogenic assays (CSAs) on two analyzers. Assays were calibrated either with human plasma or an EFA-specific calibrator. Measurements were performed in FVIII-deficient plasma spiked with EFA and in samples from patients with hemophilia A treated with EFA. Results were compared with a reference assay (Actin FSL) and expressed as recovery percentages (acceptable range: 80%-120%).

In spiked samples, plasma-calibrated CSA overestimated FVIII activity (+37% to +94%), whereas EFA-calibrated CSA showed good agreement with the reference assay (+2% to +16%). OSA results were generally acceptable, with slight underestimation depending on the reagent. In patient samples, patterns differed markedly: most assays deviated significantly from the reference, and none remained within the acceptable recovery range across the full FVIII spectrum. Compared with spiked samples, discrepancies were more pronounced at later time points and trough levels, where OSA tended to overestimate FVIII activity, whereas EFA-calibrated CSA underestimated activity.

Assay performance for EFA differs between spiked and patient samples, underscoring the limitations of in vitro evaluations. No assay provided consistent agreement with the reference assay across the full FVIII range, highlighting the need for cautious interpretation and improved standardization of EFA monitoring in clinical practice.