Data management in Thrombin Generation

To obtain a thrombin generation (TG) curve from the conversion of added fluorogenic substrate, thrombin concentrations are to be derived from the observed velocity of increase of fluorescence (dF/dt). The relation between velocity and thrombin concentration varies during the experiment because substrate is consumed and because fluorescence is not linear with the concentration of product.Here we review the techniques that we developed to:A: Transform the fluorescence trace into the “ideal” trace that would be seen if substrate consumption and non-linearity of fluorescence would not play a role.B: Subtract the contribution of α2M-thrombin so as to obtain the course of free thrombin.C: Calculate the velocity of prothrombin conversion from the course of free thrombin.D: Fit a smooth curve through the points obtained in a TG experiment