1. Development of an International Standard for Thrombin with a Unified Unit Dr. Andrew Chang initiated this project in collaboration with the WHO and NIBSC. The purposes of which are (a) to replenish the much depleted inventory of Lot J, the current U.S. national thrombin standard; and (b) to unify the unit for thrombin to eliminate the inconvenience of having to convert between the U.S. unit and the international unit. Two suppliers donated the thrombin concentrates for the NIBSC to make into two thrombin preparations (candidates C and D) with 10,000 ampoules each and approximately 100 units per ampoule. Twenty-five laboratories worldwide participated in the collaborative study to determine the potencies of these two preparations against both the U.S. thrombin standard (Lot J) and the current international standard for alpha-thrombin (89/588). Preliminary results showed that the overall variability in clotting assay is 8.8%, and in chromogenic assay, 6.5%. No differences concerning activity were observed when either human or bovine fibrinogen was used as substrate. However, the activity was slightly lower when plasma was used as substrate. Chromogenic assays displayed a trend towards higher levels of thrombin activity. Candidate C seems to be similar to the U.S. standard, whereas candidate D is close to the international standard with regard to thrombin activity in clotting and chromogenic assays. At this moment, candidate D seems to be a better reference for determining the potency of thrombin and unifying the two units. Its geometric mean is 110 units per ampoule against both the U.S. and international thrombin standards, using plasma as well as human and bovine fibrinogen as substrate. Stability study is now in progress at the NIBSC. Assays and SDS-PAGE analysis of thrombin were performed by Eleanor Koo. 2. 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: It permits the adhesion of platelets to sites of vascular damage, and it acts to stabilize Factor VIII. VWD is a heterogeneous disorder, and patients are subtyped according to pathophysiology, and using different clinical and laboratory criteria. Discrimination of Type 1 and 2 VWD is important because patients may then be differentially managed with regard to their therapy. VWF:CBA is reported to be effective in discriminating Type 2 VWD because of its ability to preferentially bind high molecular weight VWF, which is absent or deficient in Type 2A and 2B VWD. Thus, VWF:CBA is a pertinent test to assess the quality of various preparations of VWF because of this characteristics. 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 optimized. This project is done with the technical assistance of Donald Lebel. During the 18th Scientific and Standardization Committee Meeting of the International Society on Thrombosis and Haemostasis, we learned that there continued to be difficulties with agreement on a standard approach to the collagen-binding assay. A working party will be formed to address problems such as the choice of collagen reagents and methodologies of assay. We will follow closely and proceed with the findings of this working party on this assay.
