D. Sands

International collaborative study to establish the 3rd International Standard for Streptokinase

Summary.  An international collaborative study was organized to calibrate a replacement for the current (2nd) International Standard (IS) for Streptokinase, stocks of which are almost exhausted. Two candidate preparations were assayed against the 2nd IS in a study involving 16 laboratories in 12 countries: preparation 88/824 (coded B), and preparation 00/464 (C and D, coded duplicates). Laboratories could use two methods provided, either a fibrin clot lysis assay or a solution chromogenic method, or an in‐house method. Laboratories were encouraged to perform more than one method if possible. With the exception of one laboratory which gave outlying results for preparation 00/464, there was good agreement within and between laboratories and no significant differences between potencies using the different methods employed. This study demonstrates that a solution chromogenic assay is an acceptable format for potency determination of the streptokinase preparations in this study and fibrin is not necessary. It has now been agreed that a solution chromogenic plasminogen activation assay replace the current euglobulin reference method for streptokinase activity determination in the European Pharmacopoeia. Study participants, SSC of the International Society on Thrombosis and Haemostasis and the Expert Committee on Biological Standardization (ECBS) at the World Health Organization approved preparation 00/464 (C,D in the study) as the 3rd IS for Streptokinase with a potency of 1030 IU per ampoule.

Variability in factor VIII concentrate measurement: results from SSC field collaborative studies

Summary.  Seven ‘field’ collaborative studies on factor (F)VIII concentrate potency measurements were carried out, using local routine methodology, standards and calculation of results. Data from five of the 12 different concentrates studied are described in detail. These studies revealed that, for the intermediate‐purity and the recombinant FVIII concentrates, one‐stage potencies were significantly lower than chromogenic potencies, whilst for the two high‐purity FVIII concentrates one‐stage potencies were significantly greater than chromogenic potencies. On comparing predilution methods for the intermediate‐purity concentrate, equivalent potencies were obtained using either buffer or FVIII‐deficient plasma as prediluent. For the two high‐purity and the recombinant concentrates, potencies obtained using buffer as prediluent were significantly greater and lower, respectively, than potencies obtained using FVIII‐deficient plasma as prediluent. Interlaboratory variabilities were compared over all 12 concentrates studied and coefficients of variation (CVs) for one‐stage assays were found to be much greater than for chromogenic assays. This was true for all concentrates except for the intermediate‐purity concentrate and samples A and B from the first study, where the reverse was true. Furthermore, much better CVs were obtained when using FVIII‐deficient plasma than when using buffer as prediluent, for all FVIII concentrates except for the intermediate‐purity concentrate where the reverse was true, and sample B where CVs were equivalent. Overall, CVs were far worse than those obtained in controlled collaborative studies. Generally, however, CVs were better with chromogenic assays and predilution in FVIII‐deficient plasma, as is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee, particularly for higher purity and recombinant concentrates.

Collaborative studies to establish the first WHO Reference Reagent for detection of human antibody against human platelet antigen-5b.

Background and Objectives This report describes the production of a freeze‐dried preparation of pooled human plasma, coded 99/666, containing immunoglobulin G (IgG) antibodies against human platelet antigen 5b (HPA‐5b).

A collaborative study to establish the 7th International Standard for Factor VIII Concentrate

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.

A competitive enzyme-linked immunoassay using erythrocytes fixed to microtitre plates for anti-D quantitation in immunoglobulin products.

Background and Objectives: The batch control of anti-D immunoglobulin for prevention of haemolytic disease of the newborn necessitates assessment of its potency. Anti-D quantitation is usually performed using automated haemagglutination methodology although this can only be carried out in specialist centres. The aim of this study was to develop a simple and robust assay for anti-D quantitation. Methods: We developed a competitive enzyme-linked immunoassay (EIA) in which unlabelled anti-D immunoglobulin and a biotinylated monoclonal anti-D compete for red cell binding. Binding of biotinylated anti-D is detected using an alkaline-phosphatase-labelled avidin preparation. The assay is conveniently carried out using erythrocytes fixed to microtitre plates. Results: The competitive EIA was specific for anti-D activity, highly reproducible and showed good correlation with manufacturers’ potency estimates using automated haemagglutination. The assay was quick and simple to perform using freshly prepared or stored plates, and the biotinylated monoclonal anti-D could be lyophilized in ampoules for distribution as a standardized reagent. Conclusions: The competitive EIA described can be used for the specific quantitation of anti-D and provides a robust alternative method to automated haemagglutination.

International collaborative study to evaluate methods for quantification of anti-D in immunoglobulin preparations

Background and Objectives The disadvantages of autoanalyser methodology for anti‐D potency estimation have prompted the search for an alternative reference method. The aim of this study was to carry out a direct comparison of autoanalyser methodology, competitive enzyme‐linked immunoassay (EIA) and flow cytometry.

A global standard for anti-D immunoglobulin: international collaborative study to evaluate a candidate preparation.

Background and Objectives  The aim of the study was to evaluate a lyophilized anti‐D immunoglobulin preparation to serve as a global standard for potency assays of anti‐D immunoglobulin products.

National Institute for Biological Standards and control/UK Blood Transfusion service working standards for HBsAg, anti-HCV and anti-HIV-1 ('go/no-go' controls)

The positive controls supplied with the commercial kits used in the testing of blood donations for markers of viral infection are usually of high reactivity and do not give users a reliable indication of optimal performance of the kit on a day‐to‐day basis.