Method for determining thrombin activity

A method for determining the course of thrombin activity in time in a plasma sample, the method comprising the following steps:

• a) adding a signal substrate to a reaction mixture that contains a predetermined amount of thrombin activity, said signal substrate causing a detectable signal related to a conversion product formed upon reaction with said thrombin activity, and measuring the detectable signal;
• b) providing a curve showing the time course of signal development in the reaction mixture, and determining an initial slope of the curve, the curve's end-level and the curve's curvature;
• c) adding a signal substrate to a plasma sample in which thrombin generation takes place, said signal substrate causing a detectable signal related to a conversion product formed upon reaction with thrombin, and measuring the detectable signal to provide a curve showing an intensity of fluorescence in time;
• d1) adding a fluorescent compound to the same sample of step c), or another sample of the same plasma in which thrombin generation takes place, providing a detectable signal to be used as a measure for the influence of turbidity and/or color caused by the plasma on the detectable signal;
• or
• d2) measuring an optical density of the same sample in step c);
• or another sample of the same plasma in which thrombin generation takes place, providing a measure for the influence of turbidity and/or color caused by the plasma on the detectable signal;
• or
• d3) providing a fluorescent source and positioning the fluorescent source such that excitation light passes through the plasma sample to reach the fluorescent source and/or emission light passes through the sample to reach a detector, and measuring the detectable signal, providing a measure for the influence of turbidity and/or color caused by the plasma on the signal;
• e) comparing the measurement of detectable signal in any one of steps d1), d2) or d3) with the measurement of detectable signal in step a) to provide a correction factor for turbidity and color of the plasma in which thrombin generation takes place;
• and
• f) using the curvature of step b) to correct for non-linearities in the curve of step c), using the correction factor of step e) to correct the curve of step c) for turbidity and color of the plasma, and using the initial slope of step b) to calculate the concentration of thrombin in time from the curve of step c) to determine the course of thrombin activity.