In vitro kinetics of factor VIII activity in patients with mild haemophilia A and a discrepancy between one-stage and two-stage factor VIII assay results

Br J Haematol. 2007 Jan;136(1):138-45. doi: 10.1111/j.1365-2141.2006.06402.x.


In some mild haemophilia A patients (discrepant haemophilia), factor VIII coagulant activity (FVIII:C) levels, by one-stage assay are more than double than those by two-stage assay. This may be due to the longer incubation times (10-12 min) in the two-stage assay. This study aimed to determine the time course of the activation phase of the two-stage assay, using both classical coagulation and chromogenic detection methods. In both systems, for equivalent patients (equivalent FVIII:C levels by one-stage and two-stage assays, n = 6, all different mutations), similar FVIII:C results were obtained with short- or long-incubation times. In contrast, plasma from discrepant patients (n = 8, five different mutations) showed higher FVIII:C at shorter incubation times than after longer incubation times. In the chromogenic assay, FVIII:C levels were higher after incubation for 2 min (23-56%, mean 41%) than after 10 min (19-41%, mean 29%). In the classical coagulation assay, FVIII:C levels were higher at shorter incubation times (21-64%, mean 37%) than with the longer incubation times usually used (13-29%, mean 23%). These time-course experiments have verified that the longer incubation time used in the two-stage assay is at least partly responsible for the lower FVIII:C measured by that assay in discrepant haemophilia.

MeSH terms

  • Blood Coagulation Tests*
  • Chromogenic Compounds / pharmacology
  • Factor VIII / genetics
  • Factor VIII / pharmacokinetics*
  • Hemophilia A / blood*
  • Hemophilia A / genetics*
  • Humans
  • Mutation
  • Predictive Value of Tests
  • Thromboplastin / metabolism
  • Time Factors


  • Chromogenic Compounds
  • Factor VIII
  • Thromboplastin