The amidolytic method using Xa, purified antithrombin III(At-III) and the chromogenic substrate Bz-Ile-Glu-Gly-Arg-pNA(S-2222 from KABI, Stockholm, Sweden) measures heparin down to a concentration of 0.010 U/ml plasma. The accuracy of the method was evaluated by adding heparin to plasma samples from 10 normals and 10 patients, to concentrations of 0.05 and 0.5 U/ml. The results indicated that the content was assayed more accurately than by existing clotting assays. The precision was 5 and 2 per cent of the mean value at the two heparin concentrations, respectively. The concentrations of platelet factor 4 and At-III in test plasma had some influence on the result at low heparin concentrations. The “error” due to At-III, however, may be corrected by a simple formula. Heparinized plasma(containing less than about 15 000 platelets/μl) could be kept at 0 – 4 °c for 24 hours before assay, kept frozen and thawed repeatedly without significant loss of heparin activity.
In plasma from patients on heparin prophylaxis with 5 000 U twice daily, heparin was detected in 98 per cent of the samples (range 0.010 – 0.241 U/ml).
The present assay method reflects heparin concentration more directly than the activated partial thromboplastin time test and the thrombin clotting time test. In order to investigate to what extent these three tests mirror the clinical effects of heparin (therapeutic response and bleeding complications) a collaborative study involving 14 hospitals has been started.
