Comparison of factor VIII assays using physiological triggers

G. J. Praefcke, M. Kusch, C. Grundmann, S. Keitel, A. Hunfeld, R. Seitz (Langen, Germany)

Laboratory tests
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: The determination factor VIII activity of is a critical method in haemophilia care. It is widely used for diagnosis of patients and for monitoring of their treatment with clotting factor concentrates. Furthermore, the method is used during the manufacturing process and batch release of plasma-derived and recombinant FVIII concentrates. Several assays have been established for the determination of FVIII activity but the one-stage clotting assay and the chromogenic method are predominantly used. For potency measurements of FVIII concentrates, the European Medicines Agency (EMA) requires the chromogenic method in accordance with the European Pharmacopoeia, while the US FDA requires the one-stage clotting assay. However, modified recombinant FVIII products such as B-domain deleted as well as fusion proteins, yield different potencies with these two assays and also with different reagent kits.

Methods: We have developed a fluorogenic assay for the determination of FVIII activity which uses physiological amounts of FIXa as trigger and in which spontaneous activation of the intrinsic pathway is inhibited by addition of corn trypsin inhibitor. Recently, other groups have shown that also FXIa can be used as a trigger and that the addition of small amount of tissue factor enhances the performance under certain conditions.

Results: Here we present a comparison of FIXa and FXIa as triggers for the reaction and also of different inhibitors of the intrinsic pathway. Furthermore, we have simplified our assay by using commercially available FVIII-deficient plasma and optical monitoring of clotting instead of a fluorogenic detection of FXa and thrombin activity. Finally, we used this assays to compare the potencies of different FVIII concentrates.

Conclusion: The principle of using a physiological trigger may help to resolve assay discrepancies between different FVIII concentrates.