Functional determination of emicizumab in presence of factor VIII activity

• Published in: Journal of thrombosis and haemostasis 2023-12, Vol.21 (12), p.3490-3500
• Main Authors: Hamedani, Nasim Shahidi, Donners, Anouk Anna Marie Therese, van Luin, Matthijs, Gasper, Simone, Rühl, Heiko, Klein, Claudia, Albert, Thilo, EL Amrani, Mohsin, Pötzsch, Bernd, Oldenburg, Johannes, Müller, Jens
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Bispecific - pharmacology; Antibodies, Bispecific - therapeutic use; Cattle; emicizumab; factor VIII; Factor VIII - therapeutic use; hemophilia A; Hemophilia A - diagnosis; Hemophilia A - drug therapy; Hemostatics - therapeutic use; Humans; neutralizing antibody; Partial Thromboplastin Time; preanalytical phase
• ISSN: 1538-7836; 1538-7836
• DOI: 10.1016/j.jtha.2023.09.011

Accurate measurement of emicizumab in the presence of factor (F) VIII is required in patients with severe hemophilia A treated with emicizumab, as well as additional need for FVIII substitution or emicizumab prophylaxis in patients with acquired or moderate to mild hemophilia A. However, the presence of FVIII potentially biases the results. To assess the impact of plasma FVIII activity on determined emicizumab levels and evaluate different strategies for correction for or preanalytical inhibition of FVIII. Evaluated strategies comprised of the following: (1) calculation of actual emicizumab plasma levels based on measured FVIII activities and FVIII-affected emicizumab values, (2) preanalytical heat treatment (56 °C for 40 minutes), and (3) neutralization of FVIII activity using FVIII inhibitors. Emicizumab levels and FVIII activities were measured using a modified FVIII one-stage clotting assay and a chromogenic FVIII assay based on bovine factors, respectively. Spiking experiments revealed a consistent linear association between FVIII activities and determined (FVIII-affected) emicizumab results at different emicizumab input levels (∼0.12 μg/mL per IU/dL of FVIII). This principally allowed for mathematical correction of measured emicizumab levels in the presence of FVIII. While a 40% to 50% activity loss of intrinsic plasma emicizumab through heat treatment was observed in patient samples, emicizumab spiked into FVIII-deficient plasma was not or only marginally affected. Application of inhibitor-based FVIII neutralization led to good agreement of results when compared with direct quantification of emicizumab by liquid chromatography-tandem mass spectrometry. Inhibitor-based FVIII neutralization appears to be a feasible strategy for accurate measurement of plasma emicizumab levels in the presence of FVIII activity. •Plasma factor (F) VIII influenced emicizumab results in a modified FVIII one-stage clotting assay.•Cross-titration experiments enabled mathematical correction of FVIII-affected emicizumab values.•Heat inactivation significantly decreased emicizumab levels in patient plasma measured by a modified FVIII one-stage clotting assay.•Preanalytical FVIII neutralization allows accurate emicizumab measurements in the presence of FVIII.