Detection and Prediction of Factor VIII Antibody Formation in Congenital and Acquired Haemophilia A

Abstract

Factor VIII (FVIII) is a co-factor in the haemostatic system required for fibrin-rich clot formation. Inherited F8 gene defects result in haemophilia A (HA), one of the commonest inherited bleeding disorders. Acquired FVIII defects (acquired haemophilia A, AHA) occur through auto-antibody formation. FVIII antibodies (allo and auto-antibodies) are the greatest challenges facing the haemophilia treating physician. Prediction of risk of antibody formation is based on genetic and environmental factors. There is incomplete understanding of the total immune response to FVIII due to limitations in current laboratory methodology used for FVIII antibody testing. The aim of this this work was to assess clinical practices, laboratory methodology and high-throughput approaches to further characterise the immune response to FVIII. The key findings are as follows: 1) Sub-optimal compliance with targeted inhibitor screening following FVIII treatment was seen in non-severe HA in London haemophilia centres. 2) A national survey of AHA demonstrated heterogeneity in the management of immunosuppression and testing. 3) A FVIII ELISA was specific with a high negative predictive values for FVIII antibody detection in routine practice. 4) Pre-analytical heat treatment prior to antibody testing, improved sensitivity for auto-antibody detection and a systematic evaluation optimised incubation conditions for this modification. 5) A novel re-usable high-throughput peptide microarray, characterised B-cell epitopes of monoclonal and polyclonal FVIII antibodies, demonstrating immunodominant epitopes in regions of functional or structural importance. 6) A modified low volume RNA sample tube demonstrated feasibility for transcriptome analysis in patients with severe haemophilia A, providing a repository of transcriptome data for developing understanding of the allo-immune response to FVIII. Heterogeneity in clinical and laboratory practices limits interpretation of data from observational studies of FVIII antibodies. Improvements in detection and characterisation of FVIII antibodies, may further advance understanding of the total immune response to FVIII