Development of an International Standard for Thrombin with a Unified Unit. An international collaborative study was organized to identify and calibrate a replacement standard for the current US Standard Thrombin, Lot J, and the 1st International Standard (IS) for alpha-Thrombin (89/588). The study involved 25 laboratories from 15 countries. Laboratories were asked to assay two candidate replacement standards, C and D, relative to both the US Standard and the IS using either fibrinogen clotting or plasma clotting methods, and/or chromogenic assays. Data analysis indicated that candidate D is the more suitable replacement for the US Standard and IS. From clotting assays, the potency of candidate D was 106.4 US units/ampoule against the US Standard, and 114.5 IU/ampoule against the IS. Averaging provided 110 Units/ampoule (both US units and IU). The ratios between the potencies derived from the chromogenic and clotting assays indicated that candidate D had a higher proportion of alpha-Thrombin than candidate C. Initial stability studies after 6 months of storage at elevated temperatures showed that both candidates were equally stable with a projected loss of activity of <0.1% per year at -20 ?C. With the concurrence of participating laboratories, CBER/NIBSC recommended that candidate D be adopted as the 2nd International Standard for Thrombin with a potency of 110 IU/ampoule. The report and recommendation of CBER/NIBSC received approval from the Expert Committee on Biological Standardization at the World Health organization in February 2003. Since then, the design of the label was completed and agreed upon by representatives from NIBSC and CBER. At this moment, both parties are working together on the Instructions For Use for this new international standard for thrombin, which will soon be available for distribution from the two agencies. Development of a Collagen Binding Assay for von Willebrand Factor (VWF:CBA). von Willebrand disease (VWD) is the most common inherited bleeding disorder. Individuals with VWD have defects in, or reduced levels of von Willebrand Factor (VWF), an adhesive plasma protein with two primary hemostatic roles (permits adhesion of platelets to sites of vascular damage and acts to stabilize Factor VIII). VWD is a heterogeneous disorder, and patients are subtyped according to their pathophysiology, using different clinical and laboratory criteria. Discrimination of Type 1 and 2 VWD is important because patients may then be managed with different therapeutic regimens. VWF:CBA is reported to be effective in discriminating Type 2 VWD because of its ability to deferentially bind high molecular weight VWF, which is absent or deficient in Type 2A and 2B VWD. Thus, VWF:CBE is a pertinent test to assess the quality of various preparations of VWF. We have so far tested one protocol of VWF:CBA using human placenta-derived, pepsin-digested type III collagen covalently immobilized on microtiter plates as the capture for VWF. The bound VWF is then detected with a peroxidase conjugated rabbit anti-human VWF antibody. Initial results indicate that there is dose dependence between signal and [VWF] but the conditions of the reaction need to be further optimized.
