Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B

Abstract

Background Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management.

Aim To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype.

Methods Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories.

Results FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories.

Conclusion FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

Keywords

hemophilia B - factor IX - diagnostics - assays - genotype

Authors' Contribution

D.E.S. analyzed data and wrote the manuscript. Å.T., A.S., and N.S. performed experiments. J.A.H., A.L., and R.L. contributed data and discussed the findings. A.B., J.A., S.R., and M.H. contributed data and discussed the findings. M.B. designed and supervised the study.

Note

This study was performed in the Special Coagulation Laboratory L7:04, Clinical Chemistry, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.

Publication History

Received: 21 March 2023

Accepted: 25 July 2023

Accepted Manuscript online:
26 July 2023

Article published online:
18 September 2023

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