Characterization of inhibitors to FVIII with an ELISA in congenital and acquired haemophilia A.

Abstract

Different methods can be used for the detection and quantification of inhibitors or antibodies to coagulation factor VIII (FVIII). Traditionally, clotting assays have been used, in particular the Bethesda assay. These assays have, however, several shortcomings, due to the complex reaction kinetics of some inhibitors and a low sensitivity to low-titre antibodies. In addition, a universal FVIII inhibitor standard is lacking. Furthermore, clotting assays do not detect noninhibitory antibodies. Use of ELISAs has been described and FVIII from various commercially available FVIII concentrates has been used as target antigen in the assays. In the present study, we systematically explored the influence of different FVIII concentrates on the performance of an ELISA for detection of FVIII antibodies. The described ELISA was also used for further characterization of FVIII inhibitors in patients with acquired and congenital haemophilia A. We found that the source of FVIII had a substantial impact on the frequency of antibody detection. Albumin-free recombinant FVIII as target antigen gave the highest sensitivity for the assay, whereas plasma-derived concentrates containing a high level of von Willebrand factor (vWF) gave the lowest sensitivity. Presumably vWF interferes with the binding of antibodies to FVIII. We suggest that albumin-free recombinant FVIII should be used as target antigen when ELISAs are used for detection of FVIII antibodies.