Automated Coagulation Factor Assays Using A Thrombin Specific Chromogenic Substrate

Abstract

An automated method for specific factor assays has been developed using a novel thrombin-specific chromogenic substrate (developed by A. Aiyappa, Ph. D., Abbott Labs). For factors VIII, IX, XI and XII, the test system includes an ellagic acid activator and brain phospholipid in a calcium containing tris buffer pH 7.4. These are mixed with a plasma sample consisting of a mixture of specific factor deficient substrate and test plasma for 5 minutes and then the chromogenic substrate is added. After another 5 minutes the increase in optical density ΔOD is recorded. Normal plasma serially diluted in specific factor deficient substrate is used to construct a standard graph and test plasma results are interpolated from this.Fifty-four comparative assays have been done for factors VIII and IX and correlated with the results obtained from conventional clotting assays. For factor VIII the correlation coefficient (r)=0.935 (N=35) and for factor IX r=0.941 (N=19). While insufficient data exists to date for factors XI and XII, these assays appear to be comparable to the VIII and IX results.The assays for factors V, VII and X use a simplified system in that the chromogenic substrate and phospholipid are added together. The ΔOD is again recorded after 5 minutes. Test plasma results are interpolated from a standard graph constructed using serially diluted normal plasma as before. To date only the factor V assay has enough data to do a correlation with the clotting assays and r=0.953 (N=13) which is similar to the r value for the VIII and IX assays.Because of the widespread availability of the automated analyzer used in the assay and the standardization of end point determination it provides, specific factor assays might be made more widely available outside specialized coagulation centers.