Thrombinoscope software

Frozen samples were thawed at 37 °C and thrombin generation was measured using the fluorometric CAT method³¹ as previously described.¹³ Briefly, 80 µL of PPP was added to round‐bottom 96‐well plates (Immulon 2HB, Thermofisher) containing 20 µL of calibrator or PPP reagent (Stago). Thrombin generation was initiated by the addition of 20 µL of CaCl₂ and fluorogenic thrombin substrate (Z‐Gly‐Gly‐Arg‐AMC, Merck). Fluorescence was continuously measured over 60 minutes on a Fluoroskan Ascent plate reader (Thermofisher). All samples were run in triplicate with curves and results automatically generated by Thrombinoscope software (Stago). Variables derived from the thrombin generation curve include the time‐to‐curve onset (lag time), initial slope and overall ascending slope of the curve (velocity index), peak thrombin absorbance (peak), time taken to reach the peak (time to peak), area under the thrombin curve (endogenous thrombin potential [ETP]), and the time taken to reach the thrombin curve tail (time to tail) (Figure 1B). α₂‐Macroglobulin levels were also calculated.

Thrombin generation triggered by 5 pM tissue factor was measured in platelet-poor plasma using calibrated automated thrombogram (CAT) method.¹⁴ (link) The main output parameter of this assay was the area under the thrombin generation curve, which represents the endogenous thrombin potential (ETP). ETP was calculated in nM.min by the Thrombinoscope software (Stago, Valencia, Spain).