Methods for anti-factor VIII antibody levels in haemophilia A patients - validation of a multiplex immunoassay and comparability with assays measuring non-neutralising and neutralising antibodies (inhibitors).

Abstract

The development of neutralising (inhibitors) and non-neutralising antibodies (NNAs) is a complication to factor replacement therapy in haemophilia. The diagnostic methods available lack standardisation, have high inter-laboratory variation, and false-negative as well as false-positive results may affect treatment. Both functional inhibitors and NNAs may be detected with higher reproducibility, sensitivity and specificity using the immunological Luminex xMAP-based fluorescence-immunoassay (xFLI). Validation of our xFLI and comparability with enzyme-linked immunosorbent assay (ELISA) and chromogenic Nijmegen-Bethesda assay (CBA) for anti-FVIII antibodies in haemophilia A (HA) patients. The xFLI method was developed with full-length and B-domain deleted factor coupled to magnetic beads, optimised and validated for performance characteristics. Comparability with ELISA and CBA was evaluated in HA patient samples (n=112), serial samples in six inhibitor patients and reference interval and decision-limits in healthy donors (n=44). The intra- and inter-assay precision (CV%) for the xFLI method was below 6% and detection limit (LLOQ) .084ng/mL (NovoEight). All ELISA-positive samples were positive with either Advate or NovoEight. Additionally, 10.7%-14.3% were xFLI-positive and ELISA-negative. All but one CBA-positive sample was above 3SD with xFLI; one was between 2 and 3SD. 29.1% were xFLI-positive and CBA negative. The overall concordance between xFLI and ELISA was 82.1% and xFLI and CBA 77.9%. The anti-FVIII antibody xFLI method is adaptable to clinical practice and more sensitive and reproducible than ELISA and CBA. Actual NNA titers are determined to both full-length and B-domain deleted FVIII. The xFLI is thus valuable for confirmation of all anti-FVIII antibodies.