The Effects Of Defibrination Procedures In Plasma At III Assays

Abstract

Defibrination procedures using heat or ancrod (a coagulant fraction from Agkistrodon rhodostoma venom) are commonly used in heparin co-factor AT III assays which use a clotting time endpoint to measure residual thrombin. The test plasma is compared to a freeze-dried standard plasma which has been defined to contain a particular number of AT III units. This study examines the effects of both heat and ancrod defibrination of fresh and freeze-dried plasmas using a chromogenic-based AT III assay system in which no defibrination is necessary.The mean potency of the heat (56°C × 15 min) defibrinated freeze-dried standard plasma compared to the untreated standard was 0.51 (mean of 9 assays using a multidose bioassay design) whereas the corresponding figure for the frozen test plasmas was 0.72. Thus a test plasma would give falsely elevated readings for AT III compared to a freeze- dried plasma standard. Assays of ancrod (0.55 iu/ml) defibrinated plasma against untreated plasma gave mean potencies not significantly different from 1.0 for both the freeze-dried and frozen plasmas. Thus this form of defibrination allows a more valid comparison of fresh and freeze- dried plasmas.Analysis of these samples by crossed immunoelectrophoresis in the presence of heparin confirms that the ancrod defibrinated samples are essentially identical to the untreated ones whereas the heat defibrinated samples are altered, the freeze-dried plasmas markedly so.The data suggest that, should a defibrination step be used in the assay of AT III, the use of ancrod is preferred to heat. The heparin co-factor AT III assay using the chromogenic substrate, S-2238, to measure residual thrombin would seem the preferred method of assay in order to avoid any defibrination step.