AN INTERNATIONAL COLLABORATIVE STUDY TO INVESTIGATE STANDARDIZATION OF HIRUDIN POTENCY

An international collaborative study has been carried out to investiga te the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple ass ay procedure was proposed involving the titration of alpha-thrombin wi th inhibitor and measurement of residual activity using a chromogenic substrate. A standard alpha-thrombin preparation was supplied to ensur e that this reagent was of uniform quality throughout the study. The m ethod appeared to present no difficulties and laboratories reported si milar potencies for the 5 hirudin samples, in line with expected value s. This gave 200-222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) o f lyophilised hirudin, with geometric coefficient of variation (gcv) v alues ranging from 10.15-15.97%. This corresponds to specific activiti es of approximately 14,300-15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We co nclude that the precision of this determination compared with the wide r range of values in the literature (8,000-16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard a-thromb in by all laboratories. This study has important implications for hiru din standardisation.