Simultaneous detection of factor VIII and emicizumab

Introduction The factor VIIIa mimetic emicizumab has a profound impact on aPTT and aPTT-based assays leading to very short measurement times and, therefore, overestimation of factor levels. On the other hand, presence of factor VIII leads to an overestimation of emicizumab levels. Possible workarounds are the use of two-stage assays with or bovine coagulation factors, heat inactivation or neutralization of emicizumab. As the biochemical behavior of emicizumab and factor VIII are varying significantly, it is possible to distinguish them based on reaction kinetics. In our study, we use clot waveform analysis on optical data of standard factor VIII one-stage assays to derive emicizumab and factor VIII levels simultaneously. This might be especially helpful in inhibitor testing, where emicizumab from the patient sample interferes with the factor VIII applied in the Bethesda assay. Method Emicizumab and factor VIII were calibrated using spiked factor VIII deficient plasma with varying concentrations of both entities. Inhibitor positive as well as negative samples, with or without emicizumab were investigated to determined factor VIII levels as well as inhibitor potency. Optical data from factor VIII assays (e.g. Roche aPTT, Siemens Actin FS/FSL, Werfen SynthasIL) on various platforms (Siemens Atellica COAG, Roche T711, Sysmex CN6000, ACLTOP 300) were extracted and processed using clot waveform analysis. Lag time and kinetic data from fibrin formation were used to calculate factor VIII and emicizumab levels. Results The approach allowed to obtain emicizumab as well as factor VIII levels from samples simultaneously. The dilution of emicizumab in the Bethesda assay could be retraced as well as the inhibition of factor VIII, or lack thereof, in samples with and without inhibitors to factor VIII. Inhibitor potencies could be calculated and are comparable to the expected values in the absence of emicizumab. Conclusion Simultaneous detection of factor VIII and emicizumab is possible based on standard one-stage factor VIII assays. The only requirement is the application of clot waveform analysis on the optical data obtained during the measurement. This approach makes the application of two-stage assays, heat inactivation or neutralization of emicizumab obsolete. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany