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Functional determination of emicizumab in presence of Factor VIII activity.

Journal of thrombosis and haemostasis : JTH

Authors: Nasim Shahidi Hamedani, Anouk Anna Marie Therese Donners, Matthijs van Luin, Simone Gasper, Heiko Rühl, Claudia Klein, Thilo Albert, Mohsin El Amrani, Bernd Pötzsch, Johannes Oldenburg, Jens Müller

BACKGROUND: Accurate measurement of emicizumab in the presence of FVIII is required in severe HA patients treated with emicizumab and additional need for FVIII substitution or in emicizumab prophylaxis in patients with acquired or moderate to mild HA. However, the presence of FVIII potentially biases the results.

OBJECTIVE: To assess the impact of plasma FVIII activity on determined emicizumab levels and to evaluate different strategies for correction for or pre-analytical inhibition of FVIII.

METHODS: Evaluated strategies comprised (1) calculation of actual emicizumab plasma levels based on measured FVIII activities and FVIII-affected emicizumab values, (2) pre-analytical heat-treatment (56°C for 40 min), and (3) neutralization of FVIII activity using FVIII-inhibitors. Emicizumab levels and FVIII activities were measured using a modified FVIII one-stage clotting-assay (mOSA) and a chromogenic FVIII assay based on bovine factors, respectively.

RESULTS: Spiking experiments revealed 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%-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 to direct quantification of emicizumab by liquid chromatography-tandem mass spectrometry.

CONCLUSION: Inhibitor-based FVIII neutralization appears to be a feasible strategy for accurate measurement of plasma emicizumab levels in presence of FVIII activity.

Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.

PMID: 37741510

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