Preanalytical quenching of FVIII using FVIII-inhibitors improves the specificity of functional emicizumab testing

Introduction While the measurement of FVIII-activity in haemophilia A (HA) patients treated with Emicizumab (Emi) is easily possible using chromogenic FVIII-assays based on bovine factors, functional measurement of plasma Emi is potentially falsified by the presence of FVIII [1]. However, the latter may be of interest in severe HA patients treated with Emi and additional need for FVIII or in Emi prophylaxis in patients with moderate or mild HA [2]. The aim of the present study was to assess different strategies for quantification of plasma Emi levels in the presence of FVIII-activity.

Method Evaluated strategies comprised (1) calculation of actual Emi plasma levels based on measured FVIII-activity and FVIII-affected Emi values, (2) silencing of FVIII-activity by FVIII-inhibitors or (3) heat-inactivation (HI, 56°C for 40 min) of patient plasma samples in order to extinguish FVIII-activity before Emi analysis. FVIII-deficient plasma was spiked at different ratios with Emi and FVIII to initially evaluate strategies 1 and 2. For FVIII-inhibitor treatment, samples were mixed 1+1 with a commercially available FVIII-inhibitor plasma preparation (approx. 15 BU/ml) before analysis. Plasma samples obtained from patients treated with Emi were used for further assessment and to determine the impact of HI on Emi measurements (strategy 3). Emi plasma levels were measured using a modified FVIII one-stage clotting-assay calibrated against Emi. Plasma FVIII-activities were determined using a chromogenic FVIII-assay based on bovine factors. Multiple linear regression (MLR) analysis was used to allow for calculation of actual Emi plasma levels according to strategy 1.

Results Spiking experiments at different Emi concentrations (20 to 80 µg/ml) revealed a linear relationship between increasing plasma FVIII-activity (up to 2 IU/ml) and determined (FVIII-affected) Emi levels. MLR analysis showed an adjusted multiple R-squared value of >0.99 and allowed for calculation of actual Emi levels in patient plasma samples (relative error [RE] <10%) based on Emi- and FVIII-activity measurements. Pre-analytical mixing of plasma samples with FVIII-inhibitor plasma (1+1) demonstrated efficient inhibition of FVIII-activity and yielded good recovery (RE <10%) of Emi plasma levels as measured in the absence FVIII-activity. In contrast to the successfully applied strategies 1 and 2, HI of patient plasma samples according to strategy 3 resulted in a significant decrease of determined functional Emi levels (mean loss: 48%, range: 35% to 58%).

Conclusion The presence of FVIII activity impairs the specificity of functional Emi-testing in a concentration-dependent manner. Quenching of FVIII activity through addition of FVIII-inhibitors increases the specificity of Emi-testing, allowing accurate measurement of Emi plasma levels. MLR analysis may be used for results correction. HI significantly decreased the sensitivity / accuracy of functional Emi-testing.

Conflict of Interest

JM received honoraria from Roche / Chugai, Siemens Healthineers and Stago. JO has received research funding from Bayer, Biotest, CSL Behring, Octapharma, Pfizer, Swedish Orphan Biovitrum, and Takeda; consultancy, speakers bureau, honoraria, scientific advisory board, and travel expenses from Bayer, Biogen Idec, BioMarin, Biotest, Chugai Pharmaceutical Co., Ltd., CSL Behring, Freeline, Grifols, LFB, Novo Nordisk, Octapharma, Pfizer, F. Hoffmann-La Roche Ltd., Sanofi, Spark Therapeutics, Swedish Orphan Biovitrum, and Takeda. NM has received honoraria for speaking/consulting, funds for research and travel support from Bayer, NovoNordisk, Chugai, CSL Behring, Octapharma, Pfizer, Roche, Shire/Takeda.

• References

• 1 Müller J. et al Laboratory Monitoring in Emicizumab-Treated Persons with Hemophilia A. Thromb Haemost 2019; 119: 1384-1393
• 2 Negrier. et al Emicizumab Prophylaxis in Persons with Mild or Moderate Hemophilia A: Results from the Interim Analysis of the HAVEN 6 Study. Blood 2021; 138 (Suppl. 01) 343

Publication History

Article published online:
20 February 2023