Automated prothrombin-time test with use of a chromogenic peptide substrate and a centrifugal analyzer

We modified a test for prothrombin time, to automate its performance by centrifugal analysis. The sample is activated with calcium-thromboplastin in the presence of a thrombin-specific chromogenic peptide substrate, Tos-Gly-Pro-Arg-p-nitroaniline (Chromozym TH). The endpoint of the reaction is set as the time after onset until a defined amount of substrate has been cleaved, as reflected in a fixed change in absorbance. The assay is as sensitive as clotting-type methods are to the clotting factors of the extrinsic pathway--except for fibrinogen, which can be estimated from the maximum absorbance difference. Substrate concentration has little effect on the result, and varying it does not significantly change the sensitivity to factor X, which indicates little interference with the enzymes of the coagulation cascade. The method was applied to the Cobas Bio centrifugal analyzer. An external calculator must be used to convert absorbance readings into reaction times, which can be expressed as percent-of-normal values or as "ratios" by use of reagent-specific factors. CVs were less than 2% within run, less than 6% day to day. Overall correlation with the clotting test (r = 0.95) was good. Using the Cobas Bio, we achieved a throughput rate of 200 samples per hour.