METHOD FOR DETERMINING THE STRUCTURAL PROFILE OF A FIBRIN CLOT REFLECTING THE STABILITY THEREOF, IN ORDER TO PREDICT THE RISK OF BLEEDING, THROMBOSIS OR RETHROMBOSIS

Abstract

The present invention relates to a method for dynamically determining the structural profile of a fibrin clot, reflecting the stability thereof in a biological sample of a patient. The method preferably includes a step that makes it possible to predict the risk of bleeding, thrombosis or rethrombosis and to select the anticoagulant that is best suited to the clinical situation of a patient.

Claims

1-17. (canceled)

18. A method for determining the structural profile of a fibrin clot, reflecting the stability thereof in a biological sample from a patient, said method comprising steps of: a) mixing the undiluted biological sample with tissue factor and phospholipids; b) incubating the mixture obtained in step a), then adding calcium ions to the mixture obtained, in order to initiate the formation of a clot; c) measuring the turbidity or the optical density of the clot being formed in step b), at least two wavelengths of between 450 nm and 850 nm, and for a time of between 1 and 35 minutes; and d) determining the structural profile of the clot analyzed in c) expressed as number of protofibrils, density and radius, and calculated by means of the formula
τ.λ.sup.5=A[Fg].(λ.sup.2−B), wherein τ is the turbidity of the clot or the expression of the optical density in turbidity, at a given wavelength λ, [Fg] is the initial weight concentration of fibrinogen, and A and B are coefficients proportional to the density and to the radius of the fibers constituting the clot, respectively.

19. The method according to claim 18, wherein in step a), the tissue factor and the phospholipids are premixed with a solution of plasminogen activator, then the whole mixture is added to the undiluted biological sample.

20. The method according to claim 19, wherein the plasminogen activator is tissue plasminogen activator.

21. The method according to claim 19, wherein the tissue factor of the mixture of step a) is present in an amount such that its final concentration in the mixture with the undiluted biological sample is between 0.01 and 20 pM, or between 0.1 and 5 pM, or between 1 and 5 pM.

22. The method according to claim 19, wherein mixing the tissue factor with the tissue plasminogen activator is carried out in a [t-PA/TF] respective weight ratio of between 800 and 1700, or in a ratio of 75-150 ng/ml of t-PA for 0.1-5 pM of TF.

23. The method according to claim 22, wherein mixing the tissue factor with the tissue plasminogen activator is carried out in a ratio of 75-150 ng/ml of t-PA for 1-5 pM of TF.

24. The method according to claim 18, wherein the mixture of step a) comprises at least one divalent cation.

25. The method according to claim 24, wherein the at least one divalent cation is calcium ions.

26. The method according to claim 18, wherein the biological sample is a blood sample, a plasma sample, a sample of platelet-rich plasma or of platelet-poor plasma, or a sample of plasma containing platelet microparticles, erythrocytes or any other cell.

27. The method according to claim 26, wherein the biological sample is a sample of platelet-poor plasma.

28. The method according to claim 18, wherein the biological sample has a volume of between 5 μl and 500 μl, or of between 50 μl and 400 μl, or of between 50 μl and 300 μl, or of between 100 μl and 300 μl, or of approximately 200 μl.

29. The method according to claim 19, wherein the plasminogen activator has an affinity for fibrin.

30. The method according to claim 18, wherein step b) comprises incubating the mixture obtained in step a) for a time of between 60 seconds and 400 seconds, or of between 200 seconds and 350 seconds, at a temperature of between 30° C. and 40° C.

31. The method according to claim 18, wherein step b) initiates the thrombin generation and the formation of a clot.

32. The method according to claim 18, wherein measuring the turbidity or the optical density of step c) is carried out at at least two wavelengths closest to the extremes.

33. The method according to claim 32, wherein the two wavelengths closest to the extremes are 540 nm and 760 nm.

34. The method according to claim 32, wherein measuring the turbidity or the optical density of step c) is carried out at at least two wavelengths closest to the extremes simultaneously

35. The method according to claim 18, wherein measuring the turbidity or the optical density of step c) is carried out for a time of between 1 minute and 35 minutes, or of approximately 15 minutes with tissue factor alone, or of approximately 30 minutes with a mixture of tissue factor and tissue plasminogen activator.

36. The method according to claim 18, wherein at least steps c) and d) are carried out on an automated diagnostic device.

37. The method according to claim 36, wherein the automated diagnostic device is a coagulation analyzer.

38. A method for predicting the risk of bleeding, thrombosis or rethrombosis using a biological sample from a patient, said method comprising following steps of: a) mixing the undiluted biological sample with tissue factor and phospholipids; b) incubating the mixture obtained in step a), then adding calcium ions to the mixture obtained, in order to initiate the formation of a clot; c) measuring the turbidity or the optical density of the clot being formed in step b), at at least two wavelengths of between 450 nm and 850 nm, and for a time of between 1 and 35 minutes; and d) determining the profile of the clot analyzed in c) by means of the formula
τ.λ.sup.5=A[Fg].(λ.sup.2−B), wherein τ is the turbidity of the clot at a given wavelength A, [Fg] is the initial weight concentration of fibrinogen, and A and B are coefficients proportional to the density and to the radius of the fibers constituting the clot, respectively, and e) comparing the profile obtained in d) with a control.

39. The method according to claim 38, further comprising a step f), after step e), of selecting the anticoagulant most suitable for the clinical situation of said patient, said clinical situation being chosen from atrial fibrillation or another cardiac impairment, a cancer or another malignant condition or precancerous state, and a risk of venous or arterial thrombosis.

40. The method according to claim 38, wherein the control of step e) is chosen from: a biological sample from one or more healthy individuals, preferably a reference plasma, a biological sample from one or more quality controls for mimicking the conditions of patients, a biological sample from a hemorrhagic patient, a biological sample from a thrombotic patient, and a biological sample from a patient having suffered rethrombosis.

Description

[0097] The figure legends are given below.

[0098] FIG. 1: Measurement of the number of protofibrils as a function of time for a normal plasma, for a hypocoagulant plasma (heparinized control plasma 0.2 IU/ml) and for a hypercoagulant plasma (control plasma deficient in protein S).

[0099] FIG. 2: Measurement of the number of protofibrils as a function of time for normal plasmas (control plasma CCN and normal pool PN), hypocoagulant plasma (plasma congenitally deficient in FVIII:C DEF VIII) and hypercoagulant plasma (plasma deficient in protein S DPS).

[0100] FIG. 3: Measurement of the number of protofibrils as a function of time for normal plasmas (control plasma CCN and normal pool PN), hypocoagulant plasma (plasma congenitally deficient in FVIII:C DEF VIII) and hypercoagulant plasma (plasma deficient in protein S DPS). [0101] A: t-PA at 100 ng/ml; [0102] B: t-PA at 150 ng/ml; [0103] C: t-PA at 175 ng/ml.

[0104] FIG. 4: Measurement of the number of protofibrils as a function of time for 4 normal control plasmas comprising 2.52 g/l, 2.32 g/l, 3.34 g/l and 2.50 g/l of fibrinogen, respectively, with t-PA at 150 ng/ml.

[0105] FIG. 5: Measurement of the number of protofibrils as a function of time for 9 control plasmas (plasmas from N-CCN 5587 to N-CCN 5594), a normal control plasma (C-CCN) and a hypercoagulant control plasma (C-DPS) with t-PA at 150 ng/ml.

[0106] FIG. 6: [0107] 6a) Measurement of the number of protofibrils for a normal plasma (Normal), a hypocoagulant plasma (heparinized control plasma 0.2 IU/ml) and a hypercoagulant plasma (plasma deficient in protein S) at 2, 3, 7 or 20 wavelengths as a function of time, [0108] 6b) Measurement of the number of protofibrils for a normal control plasma as a function of time, at the wavelength of 540 nm, and at the two optical wavelengths of 540 nm and 780 nm and in continuous spectrum.

[0109] FIG. 7: Measurement of the number of protofibrils at the time of arrest, for a normal control plasma (STA COAG CONTROL N), a normal pool (NORMAL POOL), a hypocoagulant control plasma (STA HEPARIN CONTROL 2) and a hypercoagulant control plasma (STA DEFICIENT PS), as a function of the variations in percentage fibrinogen of the patient over the determination of the profile thereof

[0110] FIG. 8: Measurement of the density of protofibrils at the time of arrest, for a normal control plasma (STA COAG CONTROL N), a normal pool (NORMAL POOL), a hypocoagulant control plasma (STA HEPARIN CONTROL 2) and a hypercoagulant control plasma (STA DEFICIENT PS), as a function of the variations in percentage of fibrinogen of the patient over the determination of the profile thereof.

[0111] FIG. 9: Measurement of the density of protofibrils at the time of arrest, for a normal control plasma (STA COAG CONTROL N), a normal pool (NORMAL POOL), a hypocoagulant control plasma (STA HEPARIN CONTROL 2) and a hypercoagulant control plasma (STA DEFICIENT PS), as a function of the variations in level of fibrinogen of the patient over the determination of the profile thereof.

[0112] FIG. 10: Measurement of the density of protofibrils at the time of arrest, for a normal control plasma (STA COAG CONTROL N), a normal pool (NORMAL POOL), a hypocoagulant control plasma (STA HEPARIN CONTROL 2) and a hypercoagulant control plasma (STA DEFICIENT PS), as a function of the variations in level of fibrinogen of the patient over the determination of the profile thereof.

[0113] FIG. 11: Measurement of the number of protofibrils as a function of time for 9 normal plasmas (plasmas from N-CCN 5587 to N-CCN 5594), a normal control plasma (C-CCN) and a hypercoagulant control plasma (C-DPS), without t-PA.

[0114] FIG. 12: Measurement of the number of protofibrils as a function of time for plasmas at 0, 20 and 40 AU/ml of PAI-1 (P-PAI 4A, 4B and 4C respectively), a plasma deficient in PAI-1 (P-DPAI), a normal control plasma (C-CCN) and a hypercoagulant control plasma (C-DPS), without t-PA.

[0115] FIG. 13: Measurement of the number of protofibrils as a function of time for plasmas with an overload of rivaroxaban at 0, 100 and 200 ng/ml (P-R0, P-R100 and P-R200 respectively), a plasma deficient in TFPI (P-DTFPI), a normal control plasma (C-CCN) and a hypercoagulant control plasma (C-DPS), without t-PA.

[0116] FIG. 14: Measurement of the number of protofibrils as a function of time for 9 normal plasmas (plasmas from N-CCN 5587 to N-CCN 5594), a normal control plasma (C-CCN) and a hypercoagulant control plasma (C-DPS), with t-PA at 150 ng/ml.

[0117] FIG. 15: Measurement of the number of protofibrils as a function of time for plasmas at 0, 20 and 40 AU/ml of PAI-1 (P-PAI 4A, 4B and 4C respectively), a plasma deficient in PAI-1 (P-DPAI), a normal control plasma (C-CCN) and a hypercoagulant control plasma (C-DPS), with t-PA at 150 ng/ml.

[0118] FIG. 16: Measurement of the number of protofibrils as a function of time for plasmas with an overload of rivaroxaban at 0, 100 and 200 ng/ml (P-R0, P-R100 and P-R200 respectively), a plasma deficient in TFPI (P-DTFPI), a normal control plasma (C-CCN) and a hypercoagulant control plasma (C-DPS), with t-PA at 150 ng/ml.

EXAMPLES

Implementation of the Method According to the Invention

Experimental Protocols

[0119] The following protocols are used in the examples which follow:

[0120] Protocol A (Comparative, Manual Method):

[0121] The following are added manually to a 1 ml spectrophotometer cuvette: [0122] 667 μl of a sample of pure plasma, [0123] 167 μl of a [TF 1 to 5 pM final concentration+PL 4 μM final concentration] mixture.

[0124] This is then agitated manually.

[0125] It is incubated for 600 seconds at 37° C.

[0126] 167 μl of CaCl.sub.2 at a final concentration of 16.7 mM are added manually.

[0127] This is agitated manually.

[0128] Finally, the number of protofibrils is measured at 20 wavelengths of between 450 nm and 850 nm as a function of time for 90 minutes.

[0129] Protocol B (Without t-PA, Method on STA-R® with Tissue Factor):

[0130] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0131] 200 μl of a sample of pure plasma, [0132] 50 μl of a [TF 2 to 5 pM final concentration+PL 4 μM final concentration] mixture.

[0133] The automated device agitates by means of the arm, and carries out an incubation for 300 seconds at 37° C.

[0134] It then adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle of the initiating reagent.

[0135] Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 15 minutes.

[0136] Protocol C (With t-PA, Method on STA-R® with Tissue Factor and Plasminogen Activator):

[0137] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0138] 200 μl of a sample of pure plasma, [0139] 50 μl of a [TF 2 to 5 pM final concentration+PL 4 μM final concentration+tPA 0.1 to 0.2 μg/ml final concentration] mixture.

[0140] The automated device agitates by means of the arm, and carries out an incubation for 300 seconds at 37° C.

[0141] It then adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle of the initiating reagent.

[0142] Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 30 minutes.

Example 1

Structural Profile of Fibrin with 3 Normal, Hypocoagulant and Hypercoagulant Control Plasmas:

1a) Protocol A: Manual Method

[0143] The following are added manually to a 1 ml spectrophotometer cuvette: [0144] 667 μl of a sample of pure plasma: normal (frozen normal pool), hypocoagulant (heparinized control plasma 0.2 IU/ml) or hypercoagulant (protein S-depleted control plasma), [0145] 167 μl of a [TF 2 pM final concentration+PL 4 μM final concentration] mixture, followed by manual agitation.

[0146] It is incubated for 600 seconds at 37° C.

[0147] 167 μl of CaCl.sub.2 at a final concentration of 16.7 mM are added manually and the mixture is agitated manually.

[0148] Finally, the number of protofibrils is measured at 20 wavelengths of between 450 nm and 850 nm, as a function of time for 90 minutes.

[0149] The results are presented in FIG. 1.

[0150] The number of protofibrils measured as a function of time for the various normal, hypocoagulant and hypercoagulant plasmas makes it possible to distinguish these plasmas during thrombin generation and clot formation.

1b) Protocol B: Method on STA-R® with Tissue Factor TF

[0151] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0152] 200 μl of a sample of pure: normal (Coag Control N control plasma and frozen normal pool), hypocoagulant (heparinized control plasma 0.2 IU/ml) and hypercoagulant (protein S-depleted plasma and plasma congenitally deficient in FVIII:C), [0153] 50 μl of a [TF 2 pM final concentration+PL 4 μM final concentration] mixture.

[0154] After agitation, and incubation for 300 seconds at 37° C., the automated device adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle. Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 15 minutes.

[0155] The results are presented in FIG. 2.

[0156] The number of protofibrils of the various normal, hypocoagulant and hypercoagulant plasmas, as a function of time, makes it possible to distinguish these plasmas in less than 6 minutes in this example, during thrombin generation and clot formation.

[0157] The distinction is based on the number of protofibrils, the time to reach the plateau and the rate at which the plateau is reached.

1c) Protocol C: Method on STA-R® with Tissue Factor and Plasminogen Activator TF+t-PA—Influence of t-PA Concentration

[0158] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0159] 200 μl of a sample of pure plasma: normal (Coag Control N control plasma and frozen normal pool), hypocoagulant (heparinized control plasma 0.2 IU/ml) and hypercoagulant (protein S-depleted plasma and plasma congenitally deficient in FVIII: C), [0160] 50 μl of a [TF 2 pM final concentration+PL 4 μM final concentration+t-PA 100, 150 and 175 ng/ml final concentration] mixture.

[0161] After agitation and incubation for 300 seconds at 37° C., the instrument adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle.

[0162] Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 30 minutes. [0163] a) [t-PA]=100 ng/ml

[0164] The results are presented in FIG. 3A. [0165] b) [t-PA]=150 ng/ml

[0166] The results are presented in FIG. 3B. [0167] c) [t-PA]=175 ng/ml

[0168] The results are presented in FIG. 3C.

[0169] The number of protofibrils of the various normal, hypocoagulant and hypercoagulant plasmas makes it possible to distinguish the plasmas in a time which depends on the t-PA concentration contained in the tissue factor. The distinction is based on the number of protofibrils, the time to reach the plateau and the rate at which the plateau is reached, and also the duration of the plateau and its slope.

[0170] The duration of the protofibril plateau, reflecting the stability of the clot, is reached in 30 minutes for the various profiles only starting from the concentration of 175 ng/ml of t-PA in this example.

[0171] The descending slope of the protofibril plateau is greater the faster the lysis; it is zero or small when the clot is resistant to lysis (deficient in factor FVIII:C, and protein S-depleted plasma respectively).

[0172] The disappearance of the protofibrils, corresponding to total lysis, is reached at 175 ng/ml oft-PA in this example.

Example 2

Structural Profile of Fibrin with the Normal Plasmas from Healthy Donors or from Patients with Normal Hemostasis Results

2a) Protocol A: Manual Method

[0173] The following are added manually to a 1 ml spectrophotometer cuvette: [0174] 667 μl of pure plasma sample of 4 frozen normal plasmas from healthy donors, [0175] 167 μl of a [TF 2 pM final concentration+PL 4 μM final concentration+t-PA 150 ng/ml] mixture, followed by manual agitation. It is incubated for 600 seconds at 37° C.

[0176] 167 μl of CaCl.sub.2 at a final concentration of 16.7 mM are added manually and agitation is carried out manually.

[0177] Finally, the number of protofibrils is measured at 20 wavelengths as a function of time for 90 minutes.

[0178] The results are presented in FIG. 4.

[0179] The number of protofibrils of the various normal plasmas, the time to reach the plateau and the rate at which the plateau is reached are very close. On the other hand, the duration of the plateau and its slope vary a great deal from one plasma to the other at the t-PA concentration of 150 ng/ml. The disappearance of the protofibrils is not achieved in 50 min for all the plasmas.

2b) Protocol C: Method on STA-R® with Tissue Factor and Plasminogen Activator TF+t-PA

[0180] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0181] 200 μl of a sample of fresh pure plasma from patients deemed to be normal by means of routine hemostatis tests (quick time, partial thromboplastin time, fibrinogen), [0182] 50 μl of a [TF 2 pM final concentration+PL 4 μM final concentration+t-PA 150 ng/ml final concentration] optimized mixture.

[0183] After agitation and incubation for 300 seconds at 37° C., the instrument adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle.

[0184] Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 30 minutes.

[0185] Hemostatis Results for the Fresh Plasmas from Patients “Deemed to be Normal by Means of Routine Hemostatis Tests”

TABLE-US-00001 Plasmas from Prothrombin Partial thromboplastin normal patients level (%) time (sec) Fibrinogen (g/l) N-CCN 5586 100 40.5 3.80 N-CCN 5587 99 34.4 2.30 N-CCN 5588 100 31.1 4.00 N-CCN 5589 96 35.7 3.60 N-CCN 5590 94 32.7 3.30 N-CCN 5591 100 37.9 2.90 N-CCN 5592 100 37.8 3.00 N-CCN 5593 99 31.4 3.70 N-CCN 5594 92 32.6 3.50

[0186] The results are also presented in FIG. 5.

[0187] Number of Protofibrils

[0188] The number of protofibrils of the various fresh normal plasmas varies from 70 to 120;

[0189] the time to reach the plateau and the rate at which the plateau is reached vary more than for the healthy donors, in connection with the hemorrhagic or thrombotic risk associated with their hospitalization.

[0190] The duration of the plateau and its slope vary less than for the healthy donors, in connection with the optimized t-PA concentration in this example.

[0191] The disappearance of the protofibrils is reached in 33 min at the t-PA concentration used for all the plasmas. All these plasmas are distinguished from the protein S-depleted hypercoagulant plasma on the basis of the protofibril plateau.

Example 3

Structural Profile of Fibrin with 3 Normal, Hypocoagulant and Hypercoagulant Control Plasmas: Influence of the Wavelength

[0192] Protocol A was carried out as follows:

[0193] The following are added manually to a 1 ml spectrophotometer cuvette: [0194] 667 μl of pure plasma sample of the normal plasma (frozen normal pool), hypocoagulant plasma (heparinized control plasma 0.2 IU/ml) and hypercoagulant plasma (protein S-depleted plasma), [0195] 167 μl of a [TF 2 pM final concentration+PL 4 μM final concentration] mixture, followed by manual agitation.

[0196] It is incubated for 600 seconds at 37° C.

[0197] 167 μl of CaCl.sub.2 are added manually to a final concentration of 16.7 mM and manual agitation is carried out.

[0198] Finally, the number of protofibrils is measured at 2, 3, 7 or 20 wavelengths as a function of time for 90 minutes.

[0199] The results obtained at various numbers of wavelengths are then compared.

[0200] The results are presented in FIG. 6.

[0201] The curves are superimposable for each of the plasmas regardless of the number of wavelengths. The number of a minimum of 2 wavelengths was thus selected for the automated method on the routine instrument.

Example 4

Structural Profile of Fibrin with 3 Normal, Hypocoagulant and Hypercoagulant Control Plasmas: Repeatability and Reproducibility of the Automated Method on STA-R®

[0202] Protocol B on STA-R® with tissue factor TF was used as follows:

[0203] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0204] 200 μl of normal (frozen normal plasma), hypocoagulant (heparinized control plasma 0.2 IU/ml, STA HEP C2) and hypercoagulant (protein S-depleted plasma, STA DEF PS) pure plasma sample, [0205] 50 μl of a [TF 2 pM final concentration+PL 4 μM final concentration] mixture.

[0206] After agitation, and incubation for 300 seconds at 37° C., the automated device adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle. Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 15 minutes.

4a) Repeatability of the Method

[0207] 24 tests were carried out in 3 consecutive assay series:

[0208] The results are in the table hereinafter.

[0209] The coefficients of variation (CV) obtained on the structural parameters of fibrin: number of protofibrils, radius and slope, are less than 2.5% for the normal plasma and less than or equal to 5% for the pathological plasmas, and therefore much lower than those obtained by means of the thrombin generation test. The CV on the time parameters (coagulation time, gel time and arrest time) are higher, in particular the arrest time, but they are not used for determining the structure of the clot, or for the interpretation, in contrast to the turbidimetric methods used to measure fibrin formation or lysis time.

4b) Reproducibility of the Method

[0210] 20 measurements were carried out in 10 series of 2 measurements, for 5 consecutive days.

TABLE-US-00002 Reproducibility of the method: NUMBER OF n = 10 series of 2 DOD DOD PROTOFIBRILS measurements 540 nm 780 nm TCOAG TGEL TARREST TARREST TMAX Normal Mean 0.617 0.301 80.6 103 372 77.0 81.9 Standard 0.009 0.006 2.439 4.646 64.806 2.212 2.359 deviation CV 1.5% 2.1% 3.0% 4.5% 17.4% 2.9% 2.9% Hypocoagulant Mean 0.684 0.394 150.3 219 745 103.4 104.6 Standard 0.010 0.011 4.961 11.803 57.846 3.158 3.133 deviation CV 1.5% 2.7% 3.3% 5.4% 7.8% 3.1% 3.0% Hypercoagulant Mean 0.398 0.181 76.9 97 157 48.0 49.7 Standard 0.005 0.004 1.719 3.358 5.749 1.208 1.461 deviation CV 1.3% 2.0% 2.2% 3.5% 3.7% 2.5% 2.9% Reproducibility of the method: SLOPE n = 10 series of 2 MEAN RADIUS DENSITY AROUND measurements TARREST TMAX TARREST TMAX TGEL Normal Mean 83.3 83.5 0.051 0.054 1.052 Standard 0.702 0.899 0.001 0.001 0.044 deviation CV 0.8% 1.1% 2.2% 1.9% 4.2% Hypocoagulant Mean 91.7 91.8 0.056 0.057 0.492 Standard 0.608 0.606 0.001 0.001 0.014 deviation CV 0.7% 0.7% 2.1% 1.9% 2.8% Hypercoagulant Mean 79.3 79.1 0.035 0.036 1.077 Standard 0.713 1.034 0.001 0.000 0.040 deviation CV 0.9% 1.3% 1.5% 1.4% 3.7% Np: Number of protofibrils Gel time: curve inflection point Coagulation time: extrapolation on the X-axis or tangent to the inflection point Arrest time: time at which the reaction rate corresponds to 1% of the maximum rate

[0211] The coefficients of variation (CV) obtained on the structural parameters of fibrin: number of protofibrils, radius and slope, are all less than 5% regardless of the plasma. The CVs on the time parameters (coagulation time, gel time and arrest time) are higher, in particular the arrest time, but they are not used for determining the structure of the clot, or for the interpretation, in contrast to the turbidimetric methods used to measure fibrin formation or lysis time.

Example 5

Structural Profile of Fibrin with 4 Normal, Hypocoagulant and Hypercoagulant Plasmas: Influence of Variations in Fibrinogen of the Patient on the Determination of the Profile Thereof

[0212] Protocol B on STA-R® with tissue factor TF was used as follows:

[0213] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0214] 200 μl of normal (frozen normal plasma and frozen normal pool), hypocoagulant (heparinized control plasma 0.2 IU/ml) and hypercoagulant (protein-S depleted plasmas) pure plasma sample, [0215] 50 μl of a [TF 2 pM final concentration+PL 4 μM final concentration] mixture.

[0216] After agitation, and incubation for 300 seconds at 37° C., the automated device adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle. Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 15 minutes.

[0217] The calculations are carried out for each plasma with variations in fibrinogen level of plus or minus 20% in order to study the influence of these variations on the determination of the profile of each plasma.

[0218] The results are presented in FIGS. 7 to 10.

[0219] The optical density and also the structure of the fibrin depend on the intrinsic fibrinogen level of the sample. The optical density and also the number of protofibrils and the density increase all the more as the fibrinogen increases according to a linear relationship y=ax−b.

Example 6

Structural Profile of Fibrin with Normal, Hypocoagulant, Hypercoagulant, Hypofibrinolytic and Hyperfibrinolytic Plasmas: Distinction Between Normal Samples and Pathological Samples

[0220] 6a) Protocol B on STAR® with Tissue Factor TF:

[0221] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0222] 200 μl of a sample of pure plasma from 9 patients with normal hemostatis results, of a frozen plasma deficient in PAI-1, of 3 control plasmas at 0, 20 and 40 AU/ml of PAI-1, of a protein S-deficient plasma, of a TFPI-depleted plasma and of 3 plasmas with an overload of rivaroxaban at 0, 100 and 200 ng/ml, [0223] 50 μl of a [TF 2 to 5 pM final concentration+PL 4 μM final concentration] mixture.

[0224] The automated device agitates by means of the arm, and carries out an incubation for 300 seconds at 37° C.

[0225] It then adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle.

[0226] Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 15 minutes.

[0227] The results are presented in FIGS. 11 to 13.

[0228] The number of protofibrils of the various normal, hypocoagulant and hypercoagulant plasmas makes it possible to distinguish these plasmas in 15 minutes; on the other hand, it does not make it possible to distinguish the hypofibrinolytic and hyperfibrinolytic plasmas from the other plasmas.

[0229] The distinction of the hypercoagulant, normal and hypocoagulant profiles is based on the number of protofibrils, the time to reach the plateau and the rate at which the plateau is reached: [0230] these parameters increase in parallel to the concentration of anticoagulant, attesting to a looser structure of the clot; [0231] they are lower for the hypercoagulant plasmas than for all the normal plasmas, attesting to a more compact structure of the clot; [0232] these parameters are included in the range of the normal samples for the hypofibrinolytic plasmas with an overload of PAI-1. The information obtained during the thrombin generation is insufficient and does not make it possible to distinguish this type of plasma.
6b) Protocol C on STA-R® with Tissue Factor and Plasminogen Activator TF+t-PA:

[0233] To 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago), are added, simultaneously by the instrument: [0234] 200 μl of sample of pure plasma from 9 patients with normal hemostatis results, of a frozen plasma deficient in PAI-1, of 3 control plasmas at 0, 20 and 40 AU/ml of PAI-1, of a protein S-depleted plasma, of a TFPI-depleted plasma and of 3 plasmas with an overload of rivaroxaban at 0, 100 and 200 ng/ml, [0235] 50 μl of a [TF 2 to 5 pM final concentration+PL 4 μM final concentration+t-PA 150 ng/ml] mixture.

[0236] The automated device agitates by means of the arm, and carries out an incubation for 300 seconds at 37° C.

[0237] It then adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and agitates by means of the needle.

[0238] Finally, the automated device measures the number of protofibrils at the 2 wavelengths of 540 nm and 780 nm as a function of time for 30 minutes.

[0239] The results are presented in FIGS. 14 to 16.

[0240] The number of protofibrils of the various normal, hypocoagulant, hypercoagulant, hypofibrinolytic and hyperfibrinolytic plasmas makes it possible to distinguish all these plasmas in 30 minutes on the basis of their structural profile during thrombin generation, fibrin formation and lysis.

[0241] The distinction of all the profiles is obtained on the basis of the number of protofibrils, the time to reach the plateau and the rate at which the plateau is reached, the duration of the plateau and its slope.

[0242] The protofibril plateau, reflecting the stability of the clot, lasts for more than 20 min, 10 to 20 min and less than 10 min for a hypercoagulant profile, a normal profile and a hypocoagulant profile, respectively: [0243] the protofibril plateau is shortened proportionally to the concentration of anti-FXa anticoagulant (rivaroxaban) of the hypocoagulant plasma, in connection with the hemorrhagic risk due to the anticoagulant; [0244] it is extended proportionally to the concentration of plasminogen activator inhibitor (PAI-1) of the hypofibrinolytic plasma, and with the deficiencies in protein S and TFPI, in connection with the thrombotic risk associated with these hemostasis disorders.

[0245] The downward slope of the protofibril plateau is all the greater the faster the lysis, this is the case with the hyperfibrinolytic plasmas (deficient in PAI-1 or containing rivaroxaban); it is zero when the clot is resistant to lysis, which is the case with the hypercoagulant plasmas (deficient in protein S or in tissue factor inhibitor TFPI).

[0246] The disappearance of the protofibrils, corresponding to total lysis, is reached in 33 min for all the samples, except for one of the hypercoagulant plasmas, deficient in protein S.