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Two-center validation of assays for the detection of binding and neutralizing anti-factor VIII antibodies.

Haemophilia : the official journal of the World Federation of Hemophilia

Authors: Jens Müller, Sonja Neimanis, Jörg Kahle, Thilo Albert, Stephan Schultze Strasser, Bonita Rup, Bernd Pötzsch, Christoph Königs, Johannes Oldenburg

INTRODUCTION: Patients with hemophilia A treated with coagulation Factor VIII (FVIII) products are at risk for developing anti-FVIII antibodies. The ABIRISK Consortium aimed to provide knowledge on the formation and detection of anti-drug antibodies against biopharmaceutical products, including FVIII. Accordingly, standardized and validated assays for the detection of binding (total) and neutralizing antibodies are needed.

AIM: Two-center validation of an ELISA for the detection of total FVIII-binding IgG-antibodies and Nijmegen-Bethesda assays for the quantification of FVIII-neutralizing antibodies according to consensus validation guidelines.

METHODS: Validation of assays at both sites was done according to published recommendations and included preanalytics, the determination of key assay parameters, including cut-points, assay sensitivity, precision, and FVIII interference.

RESULTS: The validated assays reproducibly detected FVIII-binding and -neutralizing antibodies with comparable performance in both laboratories. Floating screening cut-points were established for both assays. Determined mass-based sensitivity of both assays (all values ≤66 ng/mL) complied with the minimum sensitivity for the detection of anti-drug antibodies as recommended by the FDA (<100 ng/mL). Intra- and inter-assay coefficients of variation did not exceed 25%. Assay validation further revealed that pre-analytical heat treatment led to potentially false-positive ELISA results, while up to 0.15 IU/mL, residual FVIII showed no significant impact. Overall, good agreement of results was found for patient samples analyzed at both study sites.

CONCLUSION: Comprehensive validation of different anti-FVIII-antibody assays in two laboratories gave novel insights into the impact of pre-analytical sample treatment as well as the comparability of test results generated by the use of methodically different assays.

© 2023 The Authors. Haemophilia published by John Wiley & Sons Ltd.

PMID: 37824540

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