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INTRODUCTION: Although the variability in factor VIII (FVIII):C measurement is well recognized, this has not been widely reported for post-FVIII infusion samples. AIM/METHODS: Three samples from haemophilia A patients were distributed in a UK National External Quality Assessment Scheme survey, each after treatment with either ReFacto AF, Kogenate FS or Advate. Fifty-two UK haemophilia centres performed FVIII assays using one-stage (n = 46) and chromogenic (n = 10) assays. Centres calibrated assays with the local plasma standard and with ReFacto AF laboratory standard for the ReFacto AF sample. RESULTS/CONCLUSIONS: Chromogenic assays gave significantly higher results than one-stage assays (P < 0.0001, 32% difference) in the post-Kogenate sample but not in the post-ReFacto AF (11% higher by chromogenic assay, ns) or post-Advate samples (3% lower by chromogenic, ns) when assays were calibrated with plasma standards. Twenty centres used all Instrumentation Laboratory (IL)-activated partial thromboplastin time reagents (Synthasil)/IL deficient plasma/reference plasma) in the one-stage assay and 15 used all Siemens reagents (Actin FS/Siemens deficient plasma/reference plasma); this made a significant difference to results post-ReFacto AF (41% higher by IL reagents, P < 0.0001) and Advate (39% higher by IL reagents, P < 0.0001), but not Kogenate (7% higher by IL, ns) when calibrated with plasma standards. Differences between results obtained with different one-stage assay reagents for monitoring Advate have implications for dosing patients. Furthermore, there was considerable inter-laboratory variation as indicated by CVs in the range 15-26% for chromogenic assay and 12-19% for one-stage assay results. This study suggests that external quality assessment schemes should offer participation in post-FVIII infusion schemes where haemophilic patients are monitored.

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Factor-Eight-Inhibitor-Bypassing-Activity (FEIBA) is a bypassing-agent used to control spontaneous bleeding or cover surgical interventions in Haemophiliacs who develop neutralizing antibodies against FVIII/FIX. The market lot-release of FEIBA is dependent on specific clot-based assays, carried out by both the manufacturer and regulatory authorities, relative to manufacturer's in-house standards, which are produced on a small-scale and are replaced frequently. We sought to standardize the FEIBA assay by developing a FEIBA primary standard which would be internationally available in sufficiently large quantities, with a predicted lifetime of many years. A collaborative study involving the manufacturer and three regulatory authorities, was carried out in which a candidate material, sample B (06/172), was calibrated by assays relative to the manufacturer's in-house FEIBA standards (C and D). All laboratories used their routine validated methods (16 APTT-assays, 8 ACTIN-FS-assays and 27 DAPTTIN-assays). Intra-laboratory geometric coefficients of variation (GCVs) for candidate B ranged from 3% to 29% (GCVs <9% from majority of labs). Assessment of inter-laboratory variability gave overall GCV values of 6.9% and 4.4% relative to standards C and D, respectively, for all methods. There was good agreement in potency estimation between laboratories using each of the three methods, with the overall potencies by the three methods differing by less than 10% of the overall mean, giving an overall combined potency of 28.0 units per ampoule. All participants agreed that candidate B (06/172) be established as the 1st NIBSC Working Standard for FEIBA with an assigned potency of 28.0 units per ampoule, based on combined results for both methods, relative to either standard C or D.

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The potency assay of human coagulation factor VII concentrate preparations as described in the European Pharmacopoeia (Ph. Eur.) requires a reference preparation calibrated in International Units (IU). The current Ph. Eur. Biological Reference Preparation (BRP) batch 1 was established in 2005 during an international collaborative study. It has an assigned potency of 8.2 IU/vial for the chromogenic assay method. Stocks of this BRP are dwindling and a replacement batch needs to be established. A candidate material was produced by a manufacturer from a plasma-derived concentrate preparation, with the same formulation and approximately the same potency, in the interest of continuity. The candidate material fulfilled the requirements of a BRP with regard to precision and homogeneity of fill, residual water content and stability. The potency of the candidate BRP (cBRP) was determined using chromogenic assays as required by the Ph. Eur. and in-house clotting assays in an attempt to assign a potency for both methods, as is the case for the current batch. The statistical model used for most laboratories was the maximum likelihood of the parallel line model using a logarithmic transformation of the responses. In the chromogenic assay, a potency of 9.9 IU/vial (+/- 1.8 %) was obtained for the cBRP with a very good consistency between laboratories. The results from the clotting assay, however, were less homogenous and yielded consistently higher results (13 IU/vial +/- 12 %), probably due to a higher activated factor VII (FVIIa) content than in the current BRP (3 % as compared to 0.3 %). Due to the large difference between the values obtained with the 2 different methods, it was not possible to reconcile the outcomes with each other. On the other hand, the uncertainty observed with the clotting assay method was quite large and seemed questionable for a reference preparation. Therefore the use of BRP batch 2 as a reference for the clotting assay method is not recommended. Nevertheless, the results of the study showed that the candidate BRP (cBRP) is suitable as a reference standard for the chromogenic assay according to the Ph. Eur. general chapter 2.7.10 Assay of human coagulation factor VII. It was adopted by the Ph. Eur. Commission in December 2009 as an official Ph. Eur. BRP for human coagulation factor VII concentrate with an assigned potency of 9.9 IU/vial.

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Plasma-derived factor VIII (FVIII) and von Willebrand Factor (VWF)/FVIII concentrates have been successfully used to treat haemophilia since the late 1960s. These products are derived from pools of plasma donations that may contain viral contaminants - including hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) - and may therefore present a transmission risk to recipients. To ensure the safety of Haemate P/Humate-P, a plasma-derived VWF/FVIII concentrate, donors of plasma are carefully selected and all donations are screened for viral antigens (HBV), virus-specific antibodies (HIV-1/2, HCV) and genomic material [hepatitis A virus, HBV, HCV, HIV-1 and high titres of human parvovirus B19 (B19V)]. As a quality control measure, plasma pools for fractionation are only released for further processing when non-reactivity has been demonstrated in serological and genome amplification assays. The manufacturing process for plasma-derived products, especially the fundamental procedure of pasteurization, is effective in inactivating and/or removing a wide variety of viruses that may potentially be present despite the screening process. This has been demonstrated in virus validation studies using a range of different viruses. New emerging infectious agents, including prions, which potentially pose a threat to recipients of plasma derivatives, are also the subject of safety evaluations. The multiple precautionary measures that are inherent in the overall production process of Haemate P/Humate-P have resulted in an excellent safety record, documented during 25 years of clinical use, and will help to maintain the high safety margin in the future.

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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.

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