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.

Factor VIII assay variability in postinfusion samples containing full length and B‐domain deleted FVIII

“…This may relate to the level of FVIII present in the test material but may also be partly a consequence of the pattern of reagents/kits used by the centres in our study since we noted one stage assay results were around 40% higher for users of Instrumentation laboratory reagents compared to results obtained by There are a number of studies reporting higher results for chromogenic assay compared to one stage assay in samples containing ReFacto ( 8,10,12,16) and ReFacto AF (14,15). This relationship is affected by the nature of the laboratory reagents used in one stage assays.…”

“…For the 2 FVIII deficient plasmas the most obvious difference An alternative and more widely used approach to restoring agreement between chromogenic and one stage assay in samples containing ReFactois to calibrate one stage assays with a product specific standard and it has been proposed that concentrate specific standards for post infusion monitoring should be used when recommended by the concentrate manufacturer ( 25). This approach has been used to successfully abolish differences between one stage and chromogenic assay results of around 30-40% (lower by one stage) in some studies (8,(11)(12)(13)(14)(15). Recalibration of both ReFacto and the RLS reduced the differences between one stage and chromogenic assay results (13), though discrepancies have persisted following the reformulation of this particular BDD FVIII as ReFacto AF (15).…”

“…Data related to its impact on results of one stage FVIII assays involve only a few types of assay reagent in a small number of centres (14,15). We therefore decided to survey UK Haemophilia centres on their current practice in relation to monitoring infusions of recombinant FVIII and at the same time centres were invited to assay FVIII in 3 samples which had been obtained from patients infused with one of 2 different widely used full length recombinant FVIII products (Kogenate FS, Bayer or Advate, Baxter) or a B-domain deleted FVIII (ReFacto AF), using either a plasma standard or the ReFacto AF laboratory standard (RAFLS) for assay calibration.…”

Clotting and chromogenic factor VIII assay variability in post‐infusion and spiked samples containing full‐length recombinant FVIII or recombinant factor VIII Fc fusion protein (rFVIIIFc)

“…This can be abolished by use of a concentrate standard for assay calibration . In earlier studies, results of chromogenic assays have been up to 50% higher than those obtained by one‐stage assay for some B‐domain deleted (BDD) FVIII products with standard half‐lives . The laboratory reagents used in the assay have an impact on these differences, and use of the ReFacto AF laboratory standard (RLS) for one‐stage assay calibration has been shown to deliver agreement with chromogenic results .…”

Chromogenic assay for BAY 81‐8973 potency assignment has no impact on clinical outcome or monitoring in patient samples

“…The discrepancy for postinfusion samples was substantially reduced when BDDrFVIII was recalibrated to contain ~ 20% more protein with the labeled potency unchanged . The results were found to be comparable with both assays when a product‐specific standard was used ; similarly, a recombinant standard reduced the discrepancy between the one‐stage assay and the chromogenic assay observed with the BDDrFVIII ReFacto AF .…”