Defibrinogenation Procedures for Antithrombin III (AT-III) Biological Assay

Abstract

Since fibrinogen has itself an antithrombin activity, defibrinogenation of the sample is a prerequisite for AT-III determination. Five different defibrinogenation procedures were tested: heat precipitation for either 3 or 10 minutes, ristocetin precipitation, clotting with either Reptilase-R or Botropase-R. AT-III was measured as biological activity according to Howie et al. (Br. J. Haemat. 25, 101, 1973) and assayed immunologically by radial immunodiffusion. Defibrinogenation was complete with all the procedures used (< 16 μg/ml F. R.-antigen being left). In the samples defibrinogenated by ristocetin, the biological assay could not be performed, since some ristocetin remained in the supernatant and provoked a rapid precipitation of the substrate fibrinogen used for the assay. The possibility that Botropase-R or Reptilase-R had themselves some antithrombin activity was preliminarly ruled out. In samples definbriogenated by heating (3 min), Reptilase-R or Botropase-R the same biological AT-III activity was found; in addition, no differences in immunological reactivity of the protein were observed as compared with the corresponding-plasma before defibrinogenation. In contrast, a statistically significant (P < 0.001) reduction of both biological and immunological AT-III activity was found in the samples defibrinogenated by heating (10 min), as compared with the other 3 procedures. These results indicate that Reptilase-R and Boropase-R are suitable reagents for defibrinogenation of plasma prior to AT-III assay; heat precipitation can also be used, provided the duration of the incubation period is accurately controlled.(Supported by Grant N. 73.00400.04 of the Italian Research Council (C. N. R.).)