PB0666 - A new FVIII chromogenic assay with bovine FX is sensitive enough for the diagnosis of severe haemophilia A.

Background: The development of extended half-life recombinant factor VIII (rFVIII) and of the bispecific monoclonal antibody that binds to both factor IXa and factor X (emicizumab) has considerably increased the complexity of the monitoring of FVIII replacement therapy in patients with haemophilia A. To allow the monitoring of FVIII substitution therapy in patients treated with emicizumab, a novel FVIII chromogenic assay with bovine Factor X and human Factor IXa has recently been developed by Hyphen Biomed.

Aims: We determined whether the sensitivity of this novel assay was sufficient for the diagnosis of severe haemophilia A.

Methods: The tests were carried out with the BIOPHEN FVIII variant (emicizumab insensitive) chromogenic assay (Hyphen BioMed, Neuville-Sur-Oise) according to the manufacturer’s instructions. Three calibration curves were generated by diluting the calibrator plasma in diluent buffer provided with the kit, in FVIII deficient plasma and in pooled normal plasma inactivated at 58°C for 30 min.

Results: There was a significant difference between the calibration curve generated with different diluents (Figure). The optical density recorded for 0% FVIII when using diluent buffer only corresponds to more than 1% FVIII when it is read on the calibration curves generated by dilution in severe haemophilia A plasma or inactivated plasma. Accordingly, the use of the calibration curves generated with diluent buffer is not appropriate to measure low FVIII levels as the bias would result in the measurement of more than 1% FVIII in plasma of patients with severe haemophilia A.

Conclusion(s): The assay can be used for the diagnosis of severe haemophilia A when the calibration curve is generated in FVIII-deficient plasma.