A novel, rapid test for factor VIII antibodies in patients with haemophilia A

Background and objectives About 30% of patients with haemophilia A develop inhibitors to factor VIII (FVIII), subsequent to which treatment with FVIII concentrates becomes ineffective. The standard test for diagnosis of FVIII inhibitors is Nijmegen modified Bethesda assay (NBA). This test is not easily accessible and not widely available. We report development of a simple, rapid, equipment-free, latex agglutination test (LAT) for detecting antibodies to FVIII. Methods A novel, rapid semiquantitative point of care (POC) test using recombinant FVIII, covalently coupled to carboxylated latex beads and stabilised in an optimised buffer was established for detection of FVIII antibodies. The sensitisation conditions such as ionic strength of the incubation buffer, pH, antigen concentration, the covalent linking conditions and composition of storage buffer were optimised. The test was validated using approximately 200 samples from haemophilia patients and 47 samples from normal healthy volunteers. All samples from patients with haemophilia were tested for FVIII antibodies by NBA and FVIII antibody ELISA. Results The test was conducted by mixing antigen-sensitised beads with samples and observing agglutination within 5 min. A clear visual agglutination was observed in samples containing anti-FVIII antibodies. Compared to NBA, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the POC test were 92%, 96%, 85%, and 98%, respectively. Working cost of per test is approximately 100 INR. Interpretation and conclusions This is a novel rapid POC assay for FVIII antibodies that can support immediate clinical decision-making in settings without access to specialised coagulation laboratories.