Determination of the thrombogenic power of human immunoglobulins

1. A process for determining the thrombogenic power of human immunoglobulins for therapeutic use contained in a sample to be tested, with a kit comprising the following components:

a platelet-poor human blood plasma deficient in Factor XI (FXI),

phospholipids,

CaCl 2 ,

human tissue factor, and

a dilution buffer,

said process comprising the following stages:

mixing the components of the kit and the sample to be tested in order to form a reaction medium, wherein the final concentration of said human tissue factor in the reaction medium is 0.3 pM; and

obtaining a thrombinogram by carrying out a thrombin generation test on said reaction medium.

2. A process for determining the thrombogenic power of human immunoglobulins, linked to the presence of Factor VII, Factor XI, Factor IX, Factor X and/or of their activated forms in a sample to be tested, with a kit comprising the following components:

a platelet-poor human blood plasma deficient in Factor XI,

phospholipids,

CaCl 2 ,

human tissue factor, and

a dilution buffer,

said process comprising the following stages:

mixing the components of the kit and the sample to be tested in order to form a reaction medium, wherein the final concentration of said human tissue factor in the reaction medium is 0.3 pM; and

obtaining a thrombinogram by carrying out a thrombin generation test on said reaction medium.

3. The process according to claim 1 , wherein the dilution buffer of said kit is identical to that of the sample to be tested.

4. The process according to claim 1 , wherein said kit further comprises a fluorogenic substrate.

5. The process according to claim 1 , wherein said kit comprises:

the platelet-poor human blood plasma deficient in FXI, the volume of which represents from 80% to 40% of the volume of the reaction medium,

human phospholipids, the final concentration of which in the reaction medium is from 1 μM to 10 μM,

the human tissue factor, the final concentration of which in the reaction medium is 0.3 pM, and

CaCl 2 .

6. The process according to claim 1 , wherein a ratio between the volume of the human blood plasma and that of the sample to be tested is from 8:1 to 2:1.

7. The process according to claim 1 , wherein the human tissue factor of the kit is of plasma origin, of recombinant origin, or of transgenic origin.

8. The process according to claim 1 , wherein the human blood plasma is a pool of fresh or frozen human blood plasmas or a calibrated commercial blood plasma.

9. A process for measuring the thrombogenic power of human immunoglobulins contained in a sample to be tested, comprising the following stages:

a) mixing a dilution buffer with a platelet-poor human blood plasma deficient in Factor XI (FXI) in order to form an intermediate negative control,

b) mixing a sample to be tested with the platelet-poor human blood plasma in order to form an intermediate reaction medium,

c) adding to the intermediate reaction medium obtained in stage b) and to the intermediate negative control obtained in stage a), a mixture comprising human phospholipids, CaCl 2 , and 0.3 pM final concentration of human tissue factor, in order to form a reaction medium and a negative control;

d) obtaining a first thrombinogram by carrying out a thrombin generation test on the reaction medium obtained in stage c) and a second thrombinogram by carrying out a thrombin generation test on the negative control obtained in stage c);

e) comparing at least one of the parameters of each of the thrombinograms obtained in stage d) with a homologous parameter obtained from standard thrombinograms established on the basis of a series of calibrators the thrombogenic power of which linked to the presence of activated Factor VII, and/or activated Factor XI, and/or activated Factor IX, and/or activated Factor XII, and/or activated Factor X is known and varies between each calibrator; and

f) determining from stage e), the thrombogenic power of human immunoglobulins contained in the sample to be tested.

10. The process according to claim 9 , wherein the standard thrombinogram is obtained by carrying out a thrombin generation test on a reaction medium comprising: i) a calibrator, the thrombogenic power of which linked to the presence of activated Factor VII, and/or activated Factor XI, and/or activated Factor IX, and/or activated Factor XII, and/or activated Factor X is known, ii) a human blood plasma, iii) a reaction mixture comprising human phospholipids and human tissue factor.

11. The process according to claim 9 , wherein the final concentration of the human phospholipids in the reaction medium is from 1 μM to 10 μM.

12. The process according to claim 9 , wherein the volume of the human blood plasma represents from 80% to 40%, of the volume of the reaction medium.

13. The process according to claim 9 , wherein a ratio between the volume of the human blood plasma and that of the sample to be tested is from 8:1 to 2:1.

14. Process according to claim 9 , wherein the thrombinogram is obtained by the thromboelastography method or the thrombinography method.

15. A process for measuring the thrombogenic power of human immunoglobulins contained in a sample to be tested, comprising the following stages:

a) mixing a dilution buffer of a sample to be tested with a platelet-poor human blood plasma deficient in Factor XI in order to form an intermediate negative control;

b) mixing the sample to be tested with the platelet-poor human blood plasma deficient in Factor XI representing 53% of the volume of the reaction medium, in which the ratio between the volume of the blood plasma and that of the sample is from 8:1 to 2:1, in order to form an intermediate reaction medium;

c) adding to the intermediate reaction medium obtained in stage b) and to the intermediate negative control obtained in stage a), a mixture comprising 4 μM of phospholipids, and 0.3 pM of human tissue factor and CaCl 2 , in order to form a reaction medium and a negative control;

d) obtaining two thrombinograms by the implementation of thrombinography, by carrying out a thrombin generation test on the reaction medium and on the negative control obtained in stage c);

e) comparing at least one of the parameters of each of the thrombinograms obtained in stage d) with a homologous parameter obtained from standard thrombinograms established on the basis of a series of calibrators the thrombogenic power of which linked to the presence of activated Factor VII, and/or activated Factor XI, and/or activated Factor IX, and/or activated Factor X is known and varies between each calibrator; and

f) determining from stage e), the thrombogenic power linked to the presence of Factor VII, Factor XI, Factor IX, Factor X and/or of their activated form in the sample.

16. The process according to claim 1 , wherein the sample to be tested is a biologically acceptable product.

17. The process according to claim 1 , wherein said dilution buffer is a Tris NaCl buffer.

18. The process according to claim 5 , wherein said kit comprises:

the platelet-poor human blood plasma deficient in FXI, the volume of which represents from 75% to 50% of the volume of the reaction medium,

the human phospholipids, the final concentration of which in the reaction medium is from 1 μM to 10 μM,

the human tissue factor, the final concentration of which in the reaction medium is 0.3 pM, and

CaCl 2 .

19. The process according to claim 5 , wherein said kit comprises:

the platelet-poor human blood plasma deficient in FXI, the volume of which represents 53% of the volume of the reaction medium,

the human phospholipids, the final concentration of which in the reaction medium is from 1 μM to 10 μM,

the human tissue factor, the final concentration of which in the reaction medium is 0.3 pM, and

CaCl 2 .

20. The process according to claim 5 , wherein said kit comprises:

the platelet-poor human blood plasma deficient in FXI, the volume of which represents from 80% to 40% of the volume of the reaction medium,

the human phospholipids, the final concentration of which in the reaction medium is 4 μM,

the human tissue factor, the final concentration of which in the reaction medium is 0.3 pM, and

CaCl 2 .

21. The process according to claim 6 , wherein the ratio between the volume of the human blood plasma and that of the sample to be tested is 4:1.