Aim: Inhibitors to factor VIII (FVIII) are common in inherited haemophilia A patients receiving factor replacement therapy. Acquired haemophilia A is a rare condition and inhibitors occur mostly in elderly and postpartum patients. Detecting and quantitating inhibitors in these patients is important for managing haemorrhage and monitoring inhibitor elimination. The Classical Bethesda Assay (CBA) was described as a uniform measurement of inhibitors to FVIII using pooled normal plasma (PNP) and imidazole buffer (Kasper et al., 1975). In 1995 Verbruggen et al. showed that PNP buffered with imidazole at pH 7.4 and FVIII deficient plasma as a patient diluent improved FVIII stability by maintaining constant protein concentration and pH dependant loss of FVIII activity. In 2014 Miller et al. introduced a 56°C 30 minute pre incubation procedure to denature endogenous and exogenous FVIII increasing the inhibitor sensitivity. We aim to evaluate these modifications to the CBA.
Method: From 2017 to 2019, 47 samples that were requested for inhibitor assays were tested in parallel using the CBA and the CBA with the Nijmegen/CDC modifications. The CBA involves performing doubling dilutions of patient plasma with imidazole buffer. The patient dilutions are then mixed 1:1 with PNP and incubated at 37°C for 2 hours. Following incubation, FVIII activity is determined for each dilution. The dilution which generates a residual FVIII activity closest to 50% represents one Bethesda Unit/mL and is then multiplied by the dilution titre. The Nijmegen/CDC modifications mentioned previously were introduced to the CBA.
Results: Increased FVIII inhibitor levels were detected in all 47 samples (from 5.70 BU/mL to 8.78 BU/mL, p = 0.09) using the CBA with the Nijmegen/CDC modifications. 9 samples that were negative using the CBA were positive using the CBA with the Nijmegen/CDC modifications.
Conclusion: The Nijmegen and CDC modifications to the CBA show increased sensitivity to FVIII inhibitor detection which is important in the management of haemorrhage in patients who develop inhibitors.