PB0158 - Preanalytical Quenching of FVIII Using FVIII-Inhibitors Improves the Specificity of Functional Emicizumab Testing

Sunday, June 25, 2023

18:30 – 19:30 ET

Room: Exhibition

Presenting Author(s)

Nasim Shahidi Hamedani, PharmD PhD (she/her/hers)

Scientific Staff Member
University Hospital Bonn; Institute of Experimental Haematologie and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

CK

Claudia Klein

Physician
University Hospital Bonn; Institute of Experimental Haematologie and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany
GG

Georg Goldmann

Senior Physician
University Hospital Bonn; Institute of Experimental Haematologie and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany
Natascha Marquardt, MD

Senior Physician
Institute for Experimental Hematology and Transfusion Medicine, University Clinic Bonn
Bonn, Nordrhein-Westfalen, Germany
HR

Heiko Rühl

Senior researcher
Institute of Experimental Haematology and Transfusion Medicine, University Hospital Bonn
Bonn, Nordrhein-Westfalen, Germany
BP

Bernd Pötzsch

Senior Physician
University Hospital Bonn; Institute of Experimental Haematologie and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg, MD (he/him/his)

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany
JM

Jens Mueller

Scientific Staff Member
University Hospital Bonn; Institute of Experimental Haematologie and Transfusion Medicine
Bonn, Nordrhein-Westfalen, Germany

Background: 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. 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 .

Aims: The aim of the present study was to assess different strategies for quantification of plasma Emi levels in the presence of FVIII-activity.

Methods: Evaluated strategies comprised (1) calculation of actual Emi plasma levels by Multiple linear regression (MLR) analysis based on measured FVIII-activity and FVIII-affected Emi values, (2) silencing of FVIII-activity by FVIII-inhibitors (using commercially available FVIII-inhibitor plasma) or (3) heat-inactivation of patient plasma samples in order to extinguish FVIII-activity before Emi analysis. 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.

Results: Spiking experiments at different Emi concentrations revealed a linear relationship between increasing plasma FVIII-activity 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 based on Emi- and FVIII-activity measurements. Pre-analytical mixing of plasma samples with FVIII-inhibitor plasma demonstrated efficient inhibition of FVIII-activity and yielded good recovery of Emi plasma levels as measured in the absence FVIII-activity. HI of patient plasma samples according to strategy 3 resulted in a significant decrease of determined functional Emi levels (mean loss: 48%).

Conclusion(s): 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 accuracy of functional Emi-testing.