*Significant dierence between 1-stage and chromogenic potencies at 5% level or less. Mean potency ratios were calculated from the results of two independent assays on each batch of product. n, number of batches tested.

We have investigated the possibility that differences in the profile of factor VIII (FVIII) activation, by thrombin, may help to explain the one-stage/chromogenic potency discrepancies in two therapeutic concentrates. A Method M concentrate and a recombinant B-domain-deleted (B-DD) concentrate were found to have one-stage/chromogenic ratios of approximately 1.15 and 0.70, respectively, relative to the World Health Organization (WHO) 6th International Standard (IS) FVIII concentrate, whether pre-diluted in FVIII-deficient plasma or buffer (+/- von Willebrand factor, VWF). The activation of FVIII, by thrombin, was followed in a buffer medium (+/- VWF) and all three concentrates showed similar times to reach peak FVIII coagulation (FVIII:C) activity. However, despite the use of equivalent amounts of FVIII:C for all three concentrates, the B-DD concentrate reached a higher peak level and maintained higher FVIII:C compared with the WHO 6th IS throughout the incubation period, whereas the Method M concentrate reached a lower peak level and maintained lower FVIII:C throughout the incubation period. We propose that the higher levels of FVIII:C found with the B-DD concentrate and the lower levels with the Method M concentrate, following activation, may be reflected in the potencies obtained by the chromogenic method and may be consistent with one-stage/chromogenic ratios of < 1.0 and > 1.0 respectively.

Summary. Large potency discrepancies between the chromogenic and one-stage clotting methods have been reported for patients' plasma samples following the infusion of recombinant factor VIII (rFVIII) concentrates. We have investigated the potency estimation of two different full-length rFVIII concentrates using both assay methods relative to both plasma and concentrate standards. Potencies by the chromogenic method were significantly higher (53% and 45%) than potencies by the one-stage clotting method when a plasma standard was used. In contrast, there was no significant potency difference between methods when a concentrate standard was used. Time-course studies into thrombin and activated factor X (FXa) generation, in modified clotting and chromogenic methods, respectively, revealed that the two rFVIII concentrates behaved very similarly to the concentrate standard, whereas the plasma standard showed slightly more rapid thrombin generation and markedly slower FXa generation. The different behaviour of rFVIII and plasma FVIII in the chromogenic method is proposed as the main cause of the methods-based potency discrepancy. The results support the use of a concentrate standard to measure rFVIII in post-infusion plasma.

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To cite this article: Raut S., Bevan S., Hubbard A. R., Sands D., Barrowcliffe T. W. A collaborative study to establish the 7th International Standard for Factor VIII Concentrate. J Thromb Haemost 2005; 3: 119-26.Summary. A candidate concentrate, preparation N (99/678), was assayed and calibrated, as a potential replacement, against four established factor (F) VIII concentrate standards: the current WHO 6th International Standard (IS) (97/616), the previous 5th IS (88/640), the Mega 1 standard and Ph. Eur. BRP Batch 2 standard, in a collaborative study involving 38 laboratories. All laboratories were instructed to use the ISTH/ SSC recommendations, including predilution of concentrates in FVIII-deficient plasma. Several laboratories performed more than one assay method and altogether there were 27 sets of assays with the one-stage method, 31 with the chromogenic method, and 18 with both methods. There was good agreement between laboratories using each of the two methods for comparison of preparation N against the four established standards, with overall potencies by one-stage and chromogenic methods differing only by less than 2%. However, there were significant differences in potencies relative to the different standards, ranging from 10.1 IU per ampoule against the Ph. Eur.BRP2 to 11.4 against the WHO 6th IS. Accelerated degradation studies showed that the proposed standard is very stable, with a predicted loss of activity per year of less than 0.001% at the recommended storage temperature of )20°C. Various options for potency of preparation N were considered by the participants and by members of the ISTH/SSC FVIII/ FIX Subcommittee. In November 2003, preparation N (NIBSC 99/678) was proposed to and accepted by the Expert Committee on Biological Standardization of the World Health Organization to be the 7th International Standard for Factor VIII Concentrate with an assigned potency of 11.0 IU per ampoule.