• FVIII assay;
  • one-stage clotting method;
  • hemophilia A;
  • FVIII concentrates

Summary. Background: The basic prerequisite of factor (F) VIII clotting assay (FVIII:C) by one-stage method is that all clotting factors other than FVIII are present in constant concentration in each dilution of both standard reference and patient plasma curves. Indeed, the plasma content of each dilution should decrease as the dilution factor increases. Objectives and methods: To keep the plasma content exactly constant in each mixture, we performed all dilutions of both standard reference and patient plasma with FVIII-deficient plasma and with a fixed amount of buffer (method B). To show the discrepancies between this method and regular method A, using buffer to make dilutions, a comparative study was conducted on FVIII:C assay on samples at known FVIII concentration and in patient plasma. Imidazole or Owren's buffers and five different activated partial thromboplastin time reagents were employed, both in method A and B. Results: A discrepancy between FVIII:C assays obtained by methods A and B was observed, mainly when Pathrontin SL and Imidazole buffer were used. The assays derived from method B always fitted better with the expected, calculated, values of FVIII:C concentrations. Furthermore, FVIII:C was assayed in 60 patients: the outcome of method A was always higher than values of method B. The discrepancy between the two methods was higher at FVIII concentrations below 50 U dL−1 but nil at 100 U dL−1. The A slope was steeper than B slope and the difference was statistically significant starting from 1/10 dilution. Accordingly, FVIII:C of patients’ plasma obtained by method A was always higher than those obtained by method B, even two or three times for FVIII level ≤10 U dL−1 or 1.4–1.6 times for FVIII levels between 10 and 25 U dL−1. Conclusions: Only method B is able to give FVIII:C assays in agreement with the expected values. The dilution of reference standards and samples with FVIII-deficient plasma is crucial to accurately evaluate the postinfusion FVIII concentrations in pharmacokinetic studies or the trough level during prophylactic therapy and to investigate the discrepancy among different FVIII:C assays. In addition, the assessment of severity and classification of hemophilia should be reviewed.