A Multi-centre Assessment of the Chromogenic Method for Factor VIII:C Estimation in Plasma-derived and Recombinant Concentrates

Abstract number: P1246

Hubbard¹ AR, Barrowcliffe¹ TW, Dodt² J, Grancha³ S, Helgeson⁴ E, Jorquera³ JI, Metzner⁵ H, Mikaelsson⁶ M, Oswaldsson⁶ U, Smith⁷ G, Turecek⁸ PL, Seitz² R

¹¹National Institute for Biological Standards & Control, Potters Bar, UK ¹¹National Institute for Biological Standards & Control, Potters Bar, UK ²²Paul Ehrlich Institut, Langen, Germany ³³Instituto Grifols SA, Barcelona, Spain ⁴⁴Bayer HealthCare LLC, Berkeley CA, USA ⁵⁵ZLB Behring, Marburg, Germany ⁶⁶Biovitrum, Stockholm, Sweden ⁷⁷Therapeutic Goods Administration, Woden, Australia ⁸⁸Baxter AG, Vienna, Austria

Under the auspices of the European Directorate for the Quality of Medicines, this study evaluated the Ph Eur chromogenic method for Factor VIII:C (FVIII:C) estimation in plasma-derived and recombinant concentrates. Three kit methods (Chromogenix Coatest 4 labs; Chromogenix Coamatic 2 labs; Baxter Immuno Immunochrom 2 labs) were included. Participants initially followed the time course of Factor X (FX) activation in two plasma-derived concentrates, Replenate (method-M) and EU BRP FVIII Concentrate (EP) standard #3 (gel filtration), and in two recombinant concentrates, WHO 6th International Standard (full-length) and ReFacto (B-domain-deleted, BDD) in order to determine the time for the plateau of FXa to be reached. The time to plateau was similar for all concentrates within each laboratory but ranged from 5–20 minutes between laboratories. Potencies were estimated using 1st stage activation times based on the pre-plateau (initial rate) and plateau phases of FX activation relative to the EP standard. Estimates for the method-M and the full length recombinant FVIII concentrate using the pre-plateau and plateau FX activation times differed by only 2%. However, estimates for the BDD concentrate were significantly greater by a mean of 20% when the plateau FX activation times were used. Similar results were obtained with all kit methods. These results indicate that differences in the FX activation time rather than the kit can have a marked effect on the potency of the BDD concentrate. The Working Group has therefore proposed a revision to the monograph whereby ‘the activation of FX in the 1st stage be terminated when the FXa concentration has reached approximately 50% of the maximal (plateau) level’. This will ensure that estimates are based on the initial rate of FX activation with all kit methods and should thereby minimize discrepancies between laboratories. The proposal is open to public discussion and has yet to be formally adopted.