Comparison of antithrombin III assays using biological and chromogenic substrates

Antithrombin III (At III) levels in plasma samples were determined by incubation of diluted plasma with thrombin, either with or without heparin. Followed by measurement of residual thrombin using clotting and amidolytic methods. All assays were of multidose bioassay design suitable for parallel line analysis. Assays without heparin showed only a small difference between amidolytic and clotting methods, which was not significant at the 95% level. Assays with heparin showed a much larger difference between clotting and amidolytic methods which is shown to be attributable to the heat defibrination step used in the clotting assay. Heat defibrination and ancrod defibrination methods are compared using the amidolytic heparin cofactor assay and it is shown that heat defibrination can cause the loss of nearly 50% of the functional At III in a reconstituted freeze-dried plasma which was used as a standard. No loss occurs when ancrod is used for defibrination. It is an advantage of the amidolytic heparin cofactor assay that defibrination is not required.