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Thank you for attending THSNA 2026. The virtual meeting is now closed.
Thank you for attending THSNA 2026. The virtual meeting is now closed.
Presentation Details
| Assay Performance and Heat-Inactivation Challenges of Efanesoctocog Alfa in Factor VIII Activity and Inhibitor Testing Erin L Aakre, Melissa S Stuart, Julie I Tange, Juliana Perez Botero, Rajiv K Pruthi. Division of Hematopathology, Department of Laboratory Medicine, Mayo Clinic, Rochester, MN, USA |
Abstract
Background: Accurate assessment of factor VIII (FVIII) inhibitor titers in the Nijmegen Bethesda inhibitor assay (NBA) requires heat inactivation (HI) of FVIII down to below 10% FVIII within the test sample. Efanesoctocog alfa (Efa) is an extended half-life FVIII concentrate used in management of hemophilia A (HA). Efa has been shown to be resistant to standard HI conditions used in the NBA, which may lead to inaccurate calculation of inhibitor titers. In addition, Chromogenic FVIII (CH8) and the most commonly used one-stage FVIII (FVIII:C) assays are known to overestimate by 2-2.5-fold and underestimate Efa-treated samples by approximately 30-40% respectively. Objectives: To determine the optimal HI conditions of samples containing Efa as measured by the one-stage and chromogenic FVIII assays. Methods: Commercial pooled donor HA plasma and positive inhibitor donor plasma (George King Biomedical, Overland Park, KS) were spiked with varying concentrations of Efa (Sanofi, Morristown, NJ). FVIII activity was measured using FVIII:C (SynthASil, Werfen, Bedford, MA) and CH8 (Precision BioLogic Inc., Dartmouth, Nova Scotia) assays following manufacturer instructions. HI times were varied (30, 60, 90, &120 min) to determine the optimal time to fully heat-inactivate Efa. The positive inhibitor donor plasma was spiked with varying levels of Efa and subjected to 90 min HI inactivation before performing the Bethesda assay. Results: As seen in Figure 1, FVIII activity varied by assay: the CH8 assay overestimated about 2-fold and SynthASil underestimated about 30-40% of spiked Efa levels. Standard HI (30 min) was not sufficient to inactivate Efa, whereas an optimized HI protocol (90 min) was more than adequate. Standard HI for Donor 1 resulted in a Bethesda titer of 0.7BU CH8 and 1.4BU FVIII:C. Standard HI for Donor 2 resulted in a Bethesda titer of 1.0BU CH8 and 1.4BU FVIII:C. The optimized heat-inactivation time did not significantly alter the Bethesda unit (BU) titers across various Efa concentrations, Figure 2. However, concentrations of Efa above 100 IU/mL yielded potential false-negative Bethesda titers. Conclusions: This study confirms the variability in Efa measurement with FVIII:C and CH8 assays. Efa causes assay-dependent variability in Factor VIII activity measurements and interferes with inhibitor testing. Optimized heat-inactivation guidelines are needed for accurate titers in patients receiving Efa. Immediate post-Efa infusion sample collection for inhibitor testing should be avoided.
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No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, including photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author.