PB0173 - Coagulation assay discrepancies for factor IX activity in hemophilia B

Background: In non-severe hemophilia A, the assay discrepancies of factor VIII activity are well known. However, little is known about the discrepancies in factor IX activity (FIX:C) in hemophilia B.

Aims: The present study aimed to measure FIX:C in Japanese patients with hemophilia B by various assay methods and to clarify the status and characteristics of the FIX:C discrepancies.

Methods: Plasma was collected from eight patients whose etiology genetic variant was already known and for whom at least 7 days had elapsed since the last administration of the concentrated formulation. FIX:C was measured by the one-stage clotting assay (FIX:C_OSA) using five different APTT reagents, including Actin FS (FS) and Actin FSL (FSL), and the chromogenic substrate assay (FIX:C_CSA) using Revohem FIX chromogenic kit, which is identical to Hyphen kit.

Results: The results of this study observed that FIX:C_OSA tends to show higher values than FIX:C_CSA, and the tendency is more pronounced for the lower values. In 7 of the 8 cases, at least one of the 5 FIX:C_OSA values was measured at greater than two-fold the FIX:C_CSA value (Figure). A very marked discrepancy in measurements was observed in two cases of the same family with the p.Ala74Pro variant. Furthermore, they exhibited different values even though they had the same variant. Their FIX:C_OSA measurements, obtained using FS and FSL, showed values more than eight and five-fold greater than the other four measurements, respectively. Since the p.Ala74Pro variant is located within the Gla domain, it was predicted that alterations in interactions with calcium ions and/or phospholipids might be involved in this discrepancy.

Conclusion(s): Our analysis implied that FIX:C also shows a definite, notably in p.Ala74Pro, discrepancy between assays. The mechanism underlying the discrepancy needs to be further elucidated.