Investigations of the thrombin generation test for the measurement of factor VIII
Haemophilia A is a genetic bleeding disorder in which the plasma level of the clotting protein FVHI is reduced or absent Treatment of haemophilia is by replacement of the missing FVTII with FVIII concentrates made from human plasma, or by recombinant technology. The two widely used assays for FVIII measurement (one-stage APTT and chromogenic assay) have disagreements in potency of FVIII concentrates and in post-infusion plasma samples these discrepancies are largest for the recombinant products. The aim of this project was to use a modified thrombin generation test (TGT) for a more physiological approach to FVQI measurement. Two different TGTs were used, a clotting based and a fluorogenic method. Thrombin generation was carried out in commercial FVH1 deficient plasma to which a FWI concentrate was added, the reaction was triggered with the addition of phospholipid, calcium and FEXa. A range of FVQI concentrates were investigated, four plasma-derived, three recombinant products (two full-length, one B-domain deleted). There were no substantial differences between the concentrates under the various conditions tested. Platelets were added to the system to investigate if the different amounts of von Willebrand factor (VWF) in the concentrates affected the thrombin generation, but no substantial differences were observed between low purity plasma-derived and recombinant products containing no VWF. It was also found that a large amount of thrombin could be generated at low levels of FVTQ, and both assays were sensitive to FVIII levels of 0.001 IU/ml. However, it was dependent upon the high amount of FIXa used to trigger the reaction. A wide variation in the amount of thrombin generated by several commercial FVIII deficient plasmas was also observed, both between different manufacturers, and between batches of the same plasma. The ability to detect low levels of FVTII could be beneficial for monitoring patients during prophylaxis, gene therapy and in phenotyping patients with severe haemophilia.