The use of the new ReFacto AF Laboratory Standard allows reliable measurement of FVIII:C levels in ReFacto AF mock plasma samples by a one‐stage clotting assay

Abstract

Factor VIII coagulant (FVIII:C) levels measured in patients receiving ReFacto® (B-domain-deleted recombinant FVIII) using chromogenic substrate assay (CSA) and one-stage clotting assay (OSA) have frequently shown discrepancies, and the use of the ReFacto Laboratory Standard (RLS) has therefore been recommended to minimize these differences. The potency of ReFacto AF®, the albumin-free successor of ReFacto®, is determined using CSA for the titration of vials, and a new standard (RLS-AF) was developed to measure its biological efficacy using OSA. This multicentre study therefore evaluated the efficacy of this new RLS in minimizing differences between OSA and CSA when measuring FVIII:C levels in plasma. Mock plasma samples were prepared by diluting ReFacto AF® in FVIII-deficient plasma to obtain four concentrations ranging from 15 to 90 IU dL⁻¹ . FVIII:C levels were then measured in six laboratories on four separate days using three different procedures, i.e. OSA with a plasma standard (PS) as reference, OSA with RLS-AF and CSA with PS. The inter-centre standard deviation ranged from 1.4 to 5.5 IU dL⁻¹. However, FVIII:C levels measured with OSA were closer to the expected values when RLS-AF was used. In addition, the uncertainty of measurement, reflecting the inter-method discrepancy was greatly reduced when RLS-AF was employed in OSA (15%) in place of PS (33%). This study demonstrates that the OSA performed with RLS-AF to establish calibration curves provides a valuable alternative to CSA to measure FVIII:C in ReFacto-AF-treated patients.