Method for automatically determining the endogenous thrombin potential

1. A method for determining the endogenous thrombin potential (ETP) of a sample of coagulating blood or plasma, comprising

a) contacting a thrombin substrate with the sample of coagulating blood or plasma,

b) allowing cleavage of the thrombin substrate to occur,

c) measuring turnover kinetics of the thrombin substrate as a function of time, wherein the turnover kinetics comprise a saturation phase, and wherein the saturation phase comprises a linear region, and

d) determining the ETP;

wherein the ETP is determined by:

i) determining the linear region of the saturation phase of the turnover kinetics within a time window,

ii) calculating the slope of the linear region of part (i), which slope corresponds to the slope of α 2 MT kinetics;

iii) iteratively determining the binding constant of thrombin to α 2 macroglobulin using slope and kinetics values, wherein the slope consists of said slope of the linear region of the saturation phase;

iv) determining values of the kinetics of free thrombin by

(1) determining values of the α 2 MT kinetics from the binding constant of thrombin to α 2 macroglobulin, and

(2) subtracting the values of the α 2 MT kinetics from the corresponding values of the turnover kinetics; and

iv) determining the raw ETP value by averaging the values of the kinetics of free thrombin in the linear region of the saturation phase of the turnover kinetics.

2. The method as claimed in claim 1 , wherein the time window encompasses at least 3 measurement points within the saturation phase of the turnover kinetics.

3. The method as claimed in claim 1 , wherein the linear region of the saturation phase of the turnover kinetics is determined using a regression method.

4. The method as claimed in claim 1 , wherein the slope of the linear region of the saturation phase of the turnover kinetics is calculated using a linear regression.

5. The method as claimed in claim 4 , further comprising minimizing the deviations of the calculated slope of α 2 MT from the actual slope of the linear region of the saturation phase of the turnover kinetics.

6. The method as claimed in claim 5 , wherein the method of least squares is used to minimize deviations of the calculated slope of α 2 MT from the actual slope of the linear region of the saturation phase of the turnover kinetics.

7. The method as claimed in claim 1 , wherein a time series of the values of the α 2 MT kinetics is calculated using the method of finite differences.

8. The method as claimed in claim 1 , further comprising verifying the quality of the raw ETP value by a method comprising calculating the coefficient of variation of the values of the kinetics of the free thrombin.

9. The method as claimed in claim 1 , further comprising comparing the raw ETP value of the sample to the raw ETP value of a normal standard sample.

10. The method as claimed in claim 3 , wherein the linear region of the saturation phase of the turnover kinetics is determined by a method comprising obtaining time windows comprising one end point located in the saturation phase of the turnover kinetics and variable starting points located downstream of the lag phase of the turnover kinetics, and calculating the square of the Pearson's correlation coefficient for each said time window.

11. The method as claimed in claim 10 , further comprising determining the maximum value from the squares of each said Pearson's correlation coefficient, and selecting the time window corresponding to said maximum value as the linear region of the saturation phase of the turnover kinetics.

12. The method as claimed in claim 11 , wherein, if the linear region of the saturation phase of the turnover kinetics falls below a defined minimum width, a window of a previously defined minimum width is selected as the linear region of the saturation phase of the turnover kinetics.

13. The method as claimed in claim 1 , wherein the method does not require the input of pre-determined substrate-specific kinetic constants.

14. The method as claimed in claim 1 , wherein the method is an automatic method.

15. A method for determining the endogenous thrombin potential (ETP) of a sample of coagulating blood or plasma, comprising

a) contacting a thrombin substrate with the sample of coagulating blood or plasma,

b) allowing cleavage of the thrombin substrate to occur,

c) measuring turnover kinetics of the thrombin substrate as a function of time, wherein the turnover kinetics comprise a saturation phase, and wherein the saturation phase comprises a linear region, and

d) determining the ETP;

wherein the ETP is determined by:

i) determining the linear region of the saturation phase of the turnover kinetics within a time window,

ii) calculating the slope of the linear region of part (i), which slope corresponds to the slope of α 2 MT kinetics;

iii) iteratively determining the binding constant of thrombin to α 2 -macroglobulin using slope and kinetics values, wherein the slope consists of said slope of the linear region of the saturation phase, by the following equation:

C

j

+

1

=

A

·

∑

⁢

⁢

T

i

⁡

(

C

j

)

∑

⁢

⁢

T

i

2

⁡

(

C

j

)

in which C 0 =0, and wherein

C is the binding constant,

A is the slope,

T i is the thrombin kinetics value at time t i ,

i is the numeric index for the thrombin kinetics values T(t i ),

j is the numeric index for the iterations;

iv) determining values of the kinetics of free thrombin by

(1) determining values of the α 2 MT kinetics from the binding constant of thrombin to α 2 -microglobulin, and

(2) subtracting the values of the α 2 MT kinetics from the corresponding values of the turnover kinetics; and

iv) determining the raw ETP value by averaging the values of the kinetics of free thrombin in the linear region of the saturation phase of the turnover kinetics.

16. The method as claimed in claim 15 , wherein the method comprises, in advance of step (iii), selecting kinetics values from within the linear region of the saturation phase from the turnover kinetics of part (c).