Thus, in the first stage of their method dilutions of the FVIII containing sample were incubated with a source of FIXa, FX,

phospholipid and Ca2+ ions, in the absence of prothrombin. Subsamples were then taken and added to a source of prothrombin and fibrinogen (usually normal plasma), and clotting times were measured after addition of Ca2+ ions. In the original method, published in 1955 [8], platelets were used as the source of phospholipid, Dinaciclib mw but as with the one-stage method this was soon replaced by a stable freeze-dried phospholipid reagent. Other technical variations were introduced over time, and these are reviewed elsewhere [9]. Detailed comparisons of the one-stage and two-stage methods have LY294002 been published elsewhere [10]. Briefly, the one-stage method is simpler and easier to automate, but has a large variety of reagents (FVIII-deficient plasma and APTT reagents), which perhaps accounts for the fact that it is less precise. The two-stage method has less variation in reagents, which may explain why it is generally more precise, but is technically more complex and more difficult to automate. The latter problem has been circumvented by the introduction of chromogenic

substrates to measure the FXa produced in the first stage, and the chromogenic version has now largely replaced the original clotting method. Unlike the one-stage assay, the two-stage method does not MCE require a source of FVIII-deficient

plasma, a distinct advantage to control laboratories such as NIBSC and to manufacturers of concentrates. The one-stage assay remains the most popular in clinical laboratories, but the chromogenic method is used extensively by manufacturers and control laboratories, and is the official method of the European Pharmacopoeia (EP). When I joined NIBSC in 1974 my remit was to establish a laboratory for testing clotting factor concentrates and other coagulation-related products such as heparin, as well as to organize the development of national and international standards for these products. Initially, I worked in the Division of Hormones and Blood Products under Dr Derek Bangham, but a few years later Blood Products became a separate Division with Dr Duncan Thomas as Head. The procedure for establishment of international standards had been in place for many years under Dr Bangham as Head of the Biological Standards Division at the National Institute of Medical Research (NIMR), Mill Hill, before NIBSC was formed in the early 1970s. In fact this procedure started as far back as the 1920s when the NIMR was first formed – curiously enough this was initially in the same building at Hampstead where NIBSC was established, so it could be said that the Standards work eventually came back home.