CAT Assay: Principle, Protocol, Instrumentation
Thrombin generation (TG) works by introducing a thrombin-specific chromogenic or fluorescent substrate to clotting plasma, permitting measurement of the activity of thrombin as a function of time. The Calibrated Automated Thrombogram (CAT) assay developed by Thrombinoscope/Diagnostica Stago is a research use only semi-automated method for running TG assays.
Testing on the CAT begins by first placing normal and treated plasmas of interest into six wells of a 96-well plate, followed by introduction of TF or the thrombin calibrator to three of the six total wells. The patented thrombin calibrator allows for correction of substrate consumption and nonlinearity of the test as well as donor-to-donor color differences of the plasma, turbidity, or hemolysis.
After the plate is filled with all platelet-poor or platelet-rich plasmas of interest, it is placed into a Thermo Fluoroskan Ascent 96- well fluorescent plate reader from Thermo Scientific (Figure 1). A dispenser inside the instrument then dispenses the fluorescent substrate/Calcium mixture into each well and the fluorescence from the plate read for up to 60 minutes.
After the run is finished, software specific to this application will then calculate all pertinent parameters in the thrombogram curve in order to assess the relative activity of the plasma being tested. The parameters include the lag time, time to peak, area under the curve (also known as the endogenous thrombin potential, ETP), slope of thrombin formation (also known as the velocity index, VI), and the time to peak completion (Figure 2).
Past standardization studies have shown that when the ETP of the plasma being tested is compared to normal plasma run on the same plate, the researcher can fully understand how the bulk coagulation activity of the plasmatic coagulation system has been affected.