A modified thrombin generation test for investigating very low levels of factor VIII activity in hemophilia A

Discrepancies between low levels of FVIII:C and clinical symptoms in severe hemophilia A are well-known. We have recently demonstrated that levels of FVIII:C < 0.2 IU/dl were consistent with clinical phenotype by clot waveform analysis, suggesting that precise measurement of very low levels of FVIII:C was clinically important. Thrombin generation tests (TGTs) triggered by tissue factor (TF) have been recently utilized to monitor coagulation function in hemophilia A. We examined whether TGT was useful for evaluating hemophilia A patients with very low levels of FVIII:C. TGTs in 40 hemophilia A plasmas with FVIII:C < 0.2-17 IU/dl (measured by clot waveform analysis using MDA-II) were performed using TF and/or ellagic acid (ELG). The lagtime in ELG-TGT at very low levels of FVIII:C was shortened dose-dependently, whilst this parameter in TF-TGT was not significantly affected. Other parameters (endogenous thrombin potential, peak thrombin, time to peak) correlated with FVIII:C levels to some extent in both assays (r = 0.4-0.7). Using a TF/ELG mixture in TGT, however, the correlation coefficients increased to ~0.85. TGT parameters correlated well with levels of FVIII:C > 0.2 IU/dl, although the lagtime was not especially informative. We conclude that modified TGT, using a TF/ELG mixture as the trigger, is useful for monitoring coagulation function at very low levels of FVIII:C in hemophilia A.