Discrepancies between the one-stage clotting assay and the chromogenic assay in haemophilia B

Assay discrepancy in factor VIII activity between the one-stage and the chromogenic assays has been described in approximately one third of patients with non-severe haemophilia A. Whether assay discrepancy may also occur in patients with haemophilia B remains unknown.

This study compared the results from the one-stage and the chromogenic assays in patients with haemophilia B.

Plasma samples from patients with haemophilia B attending the haemophilia centre in Malmö, Sweden, were collected after a wash-out period of more than 7 days and analysed with both assays.

Fifty samples from 36 patients were analysed. No discrepancy was found in patients with severe haemophilia B. Among the 44 plasma samples from patients with non-severe disease, 15 showed a twofold or greater difference between the results of the two methods, with the chromogenic method presenting the higher value (mean FIX:C_one-stage 0.02 vs. FIX:C_chromo 0.06 IU mL^-1 ). Of these 15 samples, 14 were from seven individuals from five families with the same mutated amino acid at the N-terminal cleaving site of the activation peptide (FIX: c.572G>A; p.Arg191His or FIX: c.571C>T; p.Arg191Cys). These mutations were not observed in any patients with non-discrepant results. The reported bleeding frequency for these patients was low and indicative of a mild bleeding phenotype.

Our findings imply that assay discrepancy occurs for factor IX activity and that both type of assays are needed for a correct diagnosis and classification of haemophilia B. The underlying mechanism by which the mutation influences the assays remains to be determined.