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Thank you for attending THSNA 2026. The virtual meeting is now closed.
Presentation Details
| Heat inactivation of Efanesoctocog alfa (Altuviiio) in patient’s plasma to accurately determine Factor VIII Inhibitor titer by Bethesda assay. Andrea Staum, Andrew Podd, Tina Agostini, Geoffrey Wool, Bailey Hutchison, Sandra Haberichter, Kenneth Friedman. Versiti, Milwaukee, WI, USA |
Abstract
Background: The presence of factor VIII (FVIII) activity in a patient sample, either endogenous or due to FVIII replacement therapy, can lead to underestimation of FVIII inhibitor titer. Therefore, heat treatment of patient samples at 56⁰C is recommended by the International Society on Thrombosis and Haemostasis (ISTH) and the World Federation of Hemophilia (WFH) as a preanalytical procedure to inactivate residual FVIII activity prior to performing a Bethesda assay. Our lab currently performs a 60-minute incubation for this purpose. With the increased use of Efanesoctocog alfa (Efa, Altuviiio) for treatment of hemophilia A (HA), we have observed that a subset of samples from patients treated with Efa demonstrate high percent residual FVIII activity (measured as residual FVIII activity in patient sample/residual FVIII activity in control sample) in the Bethesda assay, suggesting failure of heat treatment to completely inactivate Efa in the sample. For example, one sample demonstrated a chromogenic FVIII activity of 332 and 16.4 IU/dL before and after heat treatment, respectively. This sample showed 147% residual activity, with a final result of 87% residual activity after subtracting out the remaining FVIII activity after heat treatment. A sample from another patient being treated with Efa demonstrated chromogenic FVIII activity of 251 and 8.2 IU/dL before and after heat treatment and similarly revealed a final result of 80% residual activity. This residual FVIII activity following heat inactivation could lead to falsely low titers or false negative FVIII inhibitor results in patients on Efa, preventing proper management of these patients. Objectives: To determine a heat inactivation protocol (temperature and time of incubation) which completely inactivates Efa without degradation of any inhibitor present in patient samples. Methods: Plasma samples from patients with severe HA were spiked with known concentrations of Efa, incubated at increasing temperatures (56-62⁰C) for varying times (15-120 min), and assessed by a chromogenic FVIII assay to determine the extent of FVIII inactivation following heat treatment. HA patient samples with previously proven inhibitors were similarly treated, and inhibitor titer following heat inactivation was measured via a Bethesda assay. Results: Low-titer inhibitor activity was degraded at temperatures above 56°C (Table 1), while complete inactivation of Efa at 56⁰C required 120 minutes (Table 2). Conclusions: An extended 120-minute heat treatment at 56°C appears sufficient to inactivate endogenous and therapeutic FVIII activity prior to Bethesda assay in samples collected from HA patients receiving Efa, thus allowing for accurate measurement of inhibitor titer.
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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.