Impact of variation in reagent combinations for one-stage clotting assay on assay discrepancy in nonsevere haemophilia A

Introduction: Factor VIII activity (FVIII:C) is measured by one-stage clotting assay (OSA) or chromogenic substrate assay (CSA). Significant differences in FVIII:C between OSA (FVIII:C_1st ) and CSA (FVIII:C_Chr ) are described as assay discrepancy in nonsevere haemophilia A (HA). A large number of reagent combinations (APTT reagent and FVIII-deficient plasma) are used for OSA, but the impact of variations in reagent combinations on assay discrepancy has not been fully characterized.

Aim: To clarify the variations in FVIII:C_1st /FVIII:C_Chr ratios according to OSA reagent combination in HA subjects with/without assay discrepancy.

Methods: Thirty-nine patients previously diagnosed with nonsevere HA were enrolled, and their FVIII genes were investigated and FVIII:C levels were assessed by a single CSA reagent and 11 OSA reagent combinations. Receiver operating characteristic (ROC) curve analysis was used to predict possible cut-off values of the FVIII:C_1st /FVIII:C_Chr ratio to define FVIII assay discrepancy for each reagent combination.

Results: Patients were categorized into nondiscrepant (n = 25), discrepant (n = 5) and unclassified (n = 9) groups according to their genotypes and information in the database. The FVIII:C_1st /FVIII:C_Chr ratio in nondiscrepant HA varied widely, depending on the APTT reagents and FVIII-deficient plasma used. The ratio in discrepant HA patients differed with respect to their genotype and the reagent combination used. ROC curve analyses revealed that cut-off values to distinguish the assay discrepancy differed depending on the reagents used, but revealed two novel genotype variants, p.Cys573Gly and p.Gly582Arg, associated with FVIII assay discrepancy.

Conclusion: Our findings showed that the FVIII:C_1st /FVIII:C_Chr ratio is dependent on the reagent combination used for OSA.