METHOD FOR ASSAYING D-DIMERS SPECIFIC TO VENOUS THROMBOEMBOLISM AND USE THEREOF FOR DIAGNOSING PULMONARY EMBOLISM AND DEEP VENOUS THROMBOSIS

Abstract

The present invention relates to a method for assaying D-dimers that are specific to venous thromboembolism, resulting from the degradation of intravascular fibrin in a blood sample, including routine assay of D-dimers contained in the sample and dynamic measurement of fibrin formation in same. This method may be used to rule out pulmonary embolism or deep venous thrombosis in patients and/or to diagnose thrombosis or coagulation activation or thrombophilia in patients.

Claims

1. A method for assaying D-dimers specific for venous thromboembolism in a blood sample from a patient, said method comprising, on the one hand, the assaying of D-dimers in the sample in order to obtain the level of D-dimers in the sample (Ddi.sub.S), and on the other hand, the dynamic measurement of fibrin formation in this same sample, said dynamic measurement comprising steps of: a) initiating the activation of coagulation in the sample without triggering it; b) incubating the mixture obtained in step a), and triggering, in the incubated sample, the generation of thrombin and the formation of a fibrin clot; c) measuring the time variation of at least one property of the sample obtained in b), in which the fibrin clot forms; d) establishing the formation profile of the fibrin clot analyzed in c), and extracting from said profile the fibrin formation time (FFT) measured at the point of inflection of the tangent of the profile curve, the value of the property (Vp.sub.(TA)) of the sample measured at the time to reach (TA) the fibrin polymerization plateau; e) calculating the level of D-dimers resulting from intravascular fibrin degradation (R): e1) by adjusting the level of D-dimers of the sample (Ddi.sub.S) as a function of the level of D-dimers generated by hypercoagulation using FFT determined in d), in order to obtain the level of D-dimers adjusted as a function of the hypercoagulation (Ddi.sub.S/HC); and e2) by adjusting the level of adjusted D-dimers Ddi.sub.S/HC obtained in e1) as a function of the level of D-dimers generated by inflammation using Vp(T.sub.A) determined in d), in order to obtain R; f) comparing the level of D-dimers resulting from the fibrin degradation R obtained in e), with respect to a threshold, preferably to the threshold of 0.5 μg/ml; g) determining the level of fibrinogen degradation products generated by hyperfibrinolysis (FgDP.sub.(HF)) present in the coagulation activation states, using the level of D-dimers resulting from the fibrin degradation, R, obtained in e) and the level of D-dimers of the sample (Ddi.sub.S); h) comparing the FgDP.sub.(HF) level obtained in g) with respect to a threshold, and comparing the fibrin formation time (FFT) obtained in d) with respect to a threshold.

2. The method according to claim 1, wherein, in step e), calculating the level R expressed in initial fibrinogen units (FEUs) is carried out: e1) by calculating the level of D-dimers adjusted as a function of hypercoagulation (Ddi.sub.S/HC) using the following equation: ${\mathrm{Ddi}}_{S/\mathrm{HC}}={\mathrm{Ddi}}_S\times \frac{\mathrm{FFT}}{\mathrm{Control}\mathrm{Time}}$ wherein the Control Time is the average fibrin clot formation time of samples from healthy patients with no suspicion of thrombosis, measured according to steps a)-d), and e2) by calculating the level R using the following equation: $R=\frac{{\mathrm{Ddi}}_{S/\mathrm{HC}}}{{\left[\mathrm{Fib}\right]}_{\left(\mathrm{Vp}\left(\mathrm{TA}\right)\right)}}$ wherein [Fib].sub.(Vp(TA)) is the fibrinogen concentration deduced for the value of the property Vp.sub.(TA) on a standard curve having the equation:
y=a ln(x)−b, wherein: y is the value of the property measured at the time to reach (TA) the fibrin polymerization plateau, x is the fibrinogen concentration, a and b are the constants of the logarithmic equation which links the level of the fibrin plateau, and the fibrinogen concentration, the standard curve having been established for blood samples, the fibrinogen concentration of which has been determined and the value of the property (Vp.sub.(TA)) of which has been determined by steps a)-d).

3. The method according to claim 1, wherein, in step f), a level R obtained in e), below the threshold, preferably below the threshold of 0.5 μg/ml, makes it possible to exclude a thrombosis in the patient, and a level R obtained in e), above the threshold, preferably above the threshold of 0.5 μg/ml is indicative of the possibility of a thrombosis in the patient.

4. The method according to claim 1, wherein step g) comprises steps of: g1) determining the level of FgDP corresponding to the level of D-dimers in the sample (Ddi.sub.S) on a standard curve established using blood samples with known levels of FgDP and known levels of D-dimers, g2) determining the level of FgDP corresponding to the adjusted level of D-dimers (R) obtained in step e) on the standard curve used in g1), and g3) determining the level of FgDP generated by hyperfibrinolysis (FgDP.sub.(HF)) by subtracting the level of FgDP obtained in g2) from the level of FgDP obtained in g1).

5. The method according to claim 4, wherein step h) comprises steps of: h1) comparing FgDP.sub.(HF) to a threshold, in particular a threshold of 1 μg/ml, wherein FgDP.sub.(HF) below the threshold or negative makes it possible to exclude thrombosis in the patient, and FgDP.sub.(HF) above the threshold is indicative of a possibility of thrombosis in the patient, h2) comparing FFT to a threshold, in particular to a threshold equal to [Control Time of e1)−1 standard deviation], for example a threshold of 120 seconds for a Control Time of 135 seconds, where FFT below the threshold is indicative of a patient without thrombosis but having an acute coagulation activation state, and a FFT above the threshold is indicative of a thrombosis in the patient.

6. The method according to claim 5, wherein: when a thrombosis has been diagnosed in step h2), the level of D-dimers specific for venous thromboembolism (Ddi.sub.VTE) is the level of D-dimers, R, obtained in step e), when a thrombosis has been excluded in steps h1) and h2) but the level R obtained in step e) is above a threshold, preferably above the threshold of 0.5 μg/ml, the method also comprises a step consisting of calculating the level of D-dimers specific for venous thromboembolism (Ddi.sub.VTE) using the following equation: ${\mathrm{Ddi}}_{\mathrm{VTE}}=0.5\times \frac{R}{{\mathrm{Ddi}}_S}$ wherein Ddi.sub.S is the level of D-dimers in the sample.

7. A method for assaying D-dimers specific for venous thromboembolism in a blood sample from patient, said method comprising, on the one hand, the assaying of D-dimers in the sample in order to obtain the level of D-dimers in the sample (Ddi.sub.S), and on the other hand, the dynamic measurement of the fibrin formation of this same sample, said dynamic measurement comprising steps of: a) initiating the activation of coagulation in the sample without triggering it; b) incubating the mixture obtained in step a), and triggering, in the incubated sample, the generation of thrombin and the formation of a fibrin clot; c) measuring the time variation of at least one property of the sample obtained in b), in which the fibrin clot forms; d) establishing the formation profile of the fibrin clot analyzed in c), and extracting from this profile the fibrin formation time (FFT) measured at the inflection point of the tangent of the curve of the profile, and the value of the property (Vp.sub.(TA)) of the sample measured at the time to reach (TA) the fibrin polymerization plateau; e′) calculating the level of D-dimers resulting from intravascular fibrin degradation (R); e′ 1) by adjusting the level of D-dimers of the sample, Ddi.sub.S, as a function of the level of D-dimers generated by inflammation using Vp.sub.(TA) determined in d), in order to obtain the level of D-dimers adjusted for inflammation (Ddi.sub.S/I); and e′2) by correcting the level of D-dimers adjusted for inflammation (Ddi.sub.S/I) obtained in e′1) for the low D-dimer levels; and classifying the sample from the patient as a function of inflammation; f) comparing the level of D-dimers resulting from the degradation of the fibrin R obtained in e), with respect to a threshold, preferably to the threshold of 0.5 μg/ml; g′) determining the level of D-dimers generated by hyperfibrinolysis (Ddi.sub.(HF)) using the level of D-dimers in the sample (Ddi.sub.S) and the level R obtained in e′) or using the level of D-dimers adjusted as a function of inflammation, Ddi.sub.S/I, obtained in e′1) and the level R obtained in e′); and h′) comparing the level Ddi.sub.(HF) obtained in g′) with respect to a threshold, and comparing the TA/FFT ratio with respect to a threshold, wherein TA is the time to reach the fibrin polymerization plateau obtained in d), and FFT is the fibrin formation time obtained in d).

8. The method according to claim 7, wherein, in step e′), the level R expressed in initial fibrinogen equivalent units (FEUs) is obtained: e′1) by calculating the level of D-dimers adjusted as a function of inflammation (Ddi.sub.S/I) by the following equation: ${\mathrm{Ddi}}_{S/I}=\frac{{\mathrm{Ddi}}_S}{{\left[\mathrm{Fib}\right]}_{\left(\mathrm{Vp}\left(\mathrm{TA}\right)\right)}}$ wherein [Fib].sub.(Vp(TA)) is the fibrinogen concentration deduced for the value of the property Vp.sub.(TA) on a standard curve having the equation:
y=a ln(x)−b, wherein: y is the value of the property measured at the time to reach (TA) the fibrin polymerization plateau, x is the fibrinogen concentration, a and b are the constants of the logarithmic equation which links the level of the fibrin plateau and the fibrinogen concentration, the standard curve having been established for blood samples, the fibrinogen concentration of which has been determined and the value of the property Vp.sub.(TA) of which has been determined by steps a)-d); e′2) by correcting the level Ddi.sub.S/1 obtained in e′ 1) by the following equation:
R=Ddi.sub.S/I+[0.5−F.sub.Ddi-S] wherein F.sub.Ddi-S is a correction factor for the low D-dimer levels (<4 μg/ml), the value of which corresponds to the value of the correction factor for the level of D-dimers of the sample (Ddi.sub.S) on the standard curve having the equation:
y=ax.sup.2+bx+c wherein y is the correction factor, F, x is the level of D-dimers, a, b and c are the constants of the polynomial equation which links the correction factor and the level of D-dimers, the standard curve having been established for blood samples, the level of D-dimers of which has been determined and for which the correction factor has been determined empirically so that the level Ddi.sub.S/I is related back to the threshold of 0.5 μg/ml FEUs (fibrinogen equivalent units).

9. The method according to claim 7, wherein, in step e′), classifying the sample from the patient as a function of inflammation comprises: calculating the ratio 1/[Fib].sub.(Vp(TA)), wherein [Fib].sub.(Vp(TA)) is the fibrinogen concentration determined in e1′); and classifying the sample from the patient in group I of patients without inflammation if the ratio 1/[Fib].sub.(Vp(TA))>0.20, or classifying the sample from the patient in group II of patients with inflammation if the ratio 1/[Fib].sub.(Vp(TA))≤0.20.

10. The method according to claim 7, wherein, in step f), a level R obtained in e′), below the threshold, preferably below the threshold of 0.5 μg/ml, makes it possible to exclude a thrombosis in the patient, and a level R obtained in e′), above the threshold, preferably above the threshold of 0.5 μg/ml, is indicative of the possibility of a thrombosis in the patient.

11. The method according to claim 9, wherein, in step g′), the level of D-dimers generated by hyperfibrinolysis (Ddi.sub.HF) is calculated: g′1) as the ratio between the level R determined in step e′) and the level of D-dimers of the sample, Ddi.sub.S, using the following equation: ${\mathrm{Ddi}}_{\mathrm{HF}}={\left(R/{\mathrm{Ddi}}_S\right)}_{\mathrm{patient}}=\frac{R}{{\mathrm{Ddi}}_S}$ g′2) as the ratio between the level R determined in step e′) and the level Ddi.sub.S/I, obtained in e′ 1), using the following equation: ${\mathrm{Ddi}}_{\mathrm{HF}}={\left(R/{\mathrm{Ddi}}_{S/I}\right)}_{\mathrm{patient}}=\frac{R}{{\mathrm{Ddi}}_{S/I}}.$

12. The method according to claim 11, wherein, in step h′): the level (R/Ddi.sub.S).sub.patient obtained in g′1) is compared to a threshold: h′ 1) by determining, for the level Ddi.sub.S of the sample, the value of the ratio (R/Ddi.sub.S) standard on a standard curve having the equation:
y=ax.sup.−b wherein x is the level of D-dimers, y is the ratio between the level of D-dimers which result from intravascular fibrin degradation and the level of D-dimers, (R/Ddi.sub.S), a and b are the constants of the equation which links the ratio R/Ddi.sub.S and the level of D-dimers, the standard curve having been established: using, if the sample from the patient has been classified in group I: blood samples classified in group I by step e′) and the level of D-dimers of which is known or has been determined and the level R of which has been obtained by steps a)-e′2); and using, if the sample from the patient has been classified in group II: blood samples classified in group II by step e′) and the level of D-dimers of which is known or has been determined and the level R of which has been obtained by steps a)-e′2); and h″1) by comparing the value of the level of D-dimers generated by hyperfibrinolysis (R/Ddi.sub.S).sub.patient obtained in g′1) with the value of the ratio (R/Ddi.sub.S).sub.standard obtained in h′1), wherein: (R/Ddi.sub.S).sub.patient less than the ratio (R/Ddi.sub.S).sub.standard makes it possible to exclude thrombosis in the patient, and (R/Ddi.sub.S).sub.patient greater than or equal to the ratio (R/Ddi.sub.S) is indicative of a possibility of thrombosis in the patient; the level (R/Ddi.sub.S/I).sub.patient obtained in g′2) is compared to a threshold: h′2) by determining, for the level Ddi.sub.S of the sample, the value of the ratio (R/Ddi.sub.S/I).sub.standard on a standard curve having the equation:
y=ax.sup.−b wherein x is the level of D-dimers, y is the ratio between the level of D-dimers which result from intravascular fibrin degradation and the level of D-dimers adjusted as a function of inflammation, (R/Ddi.sub.S/I), a and b are the constants of the equation which links the ratio R/Ddi.sub.S/I and the level of D-dimers, the standard curve having been established: using, if the sample from the patient has been classified in group I: blood samples classified in group I by step e′) and the level of D-dimers of which is known or has been determined, the level Ddi.sub.S/I of which has been obtained by steps a)-e′1), and the level R of which has been obtained by steps a)-e′2); and using, if the sample from the patient has been classified in group II: blood samples classified in group II by step e′) and the level of D-dimers of which is known or has been determined, the level Ddi.sub.S/I of which has been obtained by steps a)-e′1), and the level R of which has been obtained by steps a)-e′2); and h″2) by comparing the value of the level of D-dimers generated by hyperfibrinolysis (R/Ddi.sub.S/I).sub.patient obtained in g′2) with the value of the ratio (R/Ddi.sub.S/I).sub.standard obtained in h′2), wherein: (R/Ddi.sub.S/I).sub.patient less than the ratio (R/Ddi.sub.S/I).sub.standard makes it possible to exclude thrombosis in the patient, and wherein (R/Ddi.sub.S/I).sub.patient greater than or equal to the ratio (R/Ddi.sub.S/I).sub.standard is indicative of a possibility of thrombosis in the patient.

13. The method according to claim 12, wherein, in step h′), comparing the ratio TA/FFT with respect to a threshold comprises: h′3) calculating the ratio TA/FFT wherein TA is the time to reach the fibrin polymerization plateau determined in step d) and FFT is the fibrin clot formation time determined in step d); and h″3) if the sample from the patient has been classified in group I: comparing the ratio TA/FFT with respect to a first threshold, in particular to a first threshold of 1.75, wherein: a ratio TA/FFT above the first threshold makes it possible to exclude thrombosis and to diagnose thrombophilia or a coagulation activation state in the patient classified in group I, and a ratio TA/FFT below or equal to the threshold is indicative of a thrombosis in the patient classified in group I; if the sample from the patient has been classified in group II: comparing the ratio TA/FFT with respect to a first threshold, in particular a first threshold of 1.65, wherein: a ratio TA/FFT above the first threshold makes it possible to exclude thrombosis and to diagnose thrombophilia or a coagulation activation state in the patient classified in group II, and a ratio TA/FFT below or equal to the threshold is indicative of a thrombosis in the patient classified in group II.

14. The method according to claim 13, wherein: when a thrombosis has been diagnosed in step h″3), the level of D-dimers specific for venous thromboembolism (Ddi.sub.VTE) is the level of D-dimers, R, obtained in step e′), when a thrombosis has been excluded in steps h″1), h″2) and h″3), but the level R obtained in step e′) is above the threshold, preferably above the threshold of 0.5 μg/ml, the method also comprises a step consisting in calculating the level of D-dimers specific for venous thromboembolism (Ddi.sub.VTE) using the following equation: ${\mathrm{Ddi}}_{\mathrm{VTE}}=0.5\times \frac{R}{{\mathrm{Ddi}}_S}$ wherein Ddi.sub.S is the level of D-dimers in the sample.

15. The method according to claim 1, wherein that the blood sample has a volume of between 1 μl and 300 μl, preferably between 50 μl and 200 μl.

16. The method according to claim 1, wherein that the blood sample is undiluted.

17. The method according to claim 1, wherein the assaying of D-dimers of the sample is carried out according to an immunoturbidimetric or immunoenzymatic method.

18. The method according to claim 1, wherein step a) is carried out by mixing the blood sample from the patient with tissue factor and optionally phospholipids, preferably by mixing the blood sample from the patient with tissue factor and phospholipids.

19. The method according to claim 18, wherein the tissue factor of step a) is present in a concentration of between 0.5 and 5 pM, preferably 2 pM.

20. The method according to claim 18, wherein the mixture of step a) comprises calcium ions.

21. The method according to claim 1, wherein step b) comprises incubating the mixture obtained in step a) for a time of between 20 seconds and 400 seconds, preferably between 60 seconds and 300 seconds, at a temperature between 30° C. and 40° C.

22. The method according to claim 1, wherein, in step b), triggering thrombin generation and fibrin clot formation is carried out by adding calcium ions to the sample incubated.

23. The method according to claim 1, wherein the blood sample from a patient is a plasma sample.

24. The method according to claim 23, wherein the plasma sample is a platelet-poor plasma sample.

25. The method according to claim 23, wherein, in step c), measuring the time variation of at least one property of the sample obtained in b) is carried out by measuring the time variation of the optical density (DOD) at a wavelength of between 350 and 800 nm, preferably at the wavelength of 540 nm.

26. The method according to claim 25, wherein the measurement of the optical density of step c) is carried out at the same wavelength as that used for assaying the D-dimers, 540 nm.

27. The method according to claim 1, wherein the blood sample from a patient is a whole-blood sample.

28. The method according to claim 27, wherein the whole-blood sample is a citrated whole-blood sample.

29. The method according to claim 27, wherein measuring the time variation of at least one property of the sample obtained in b) is carried out by thromboelastography, by rheometry or by image analysis.

30. The method according to claim 1, wherein at least steps c) and d) are carried out on an automated diagnostic device or on a remote biology analyzer, preferably on a coagulation analyzer.

31. An in vitro method for diagnosing venous thromboembolism (VTE) in a patient, comprising steps of: carrying out an assay of D-dimers specific for VTE in a blood sample from the patient using a method according to claim 1; and providing a diagnosis regarding the patient.

32. The in vitro method of diagnosis according to claim 31, wherein the diagnosis regarding the patient is (i) exclusion of thrombosis, (ii) acute coagulation activation state, or (iii) thrombosis.

33. The in vitro method of diagnosis according to claim 31, wherein, if the patient is an elderly individual, a patient suffering from cancer, a patient suffering from an infection or a patient suffering from thrombophilia, the assaying of the D-dimers specific for VTE is carried out using a method according to claim 7.

34. The in vitro method of diagnosis according to claim 32, wherein the diagnosis regarding the patient is (i) exclusion of thrombosis, (ii) acute coagulation activation state, (iii) thrombophilia, or (iv) thrombosis.

35. The in vitro method of diagnosis according to claim 34, wherein the diagnosis regarding the patient is a non-serious thrombosis or thrombosis in the case of pulmonary infarction.

36. The method according to claim 6, wherein a high level of D-dimers specific for venous thromboembolism, Ddi.sub.VTE, is representative of the extent of the pulmonary embolism or of the deep vein thrombosis.

Description

FIGURE LEGENDS

[0311] FIG. 1: Diagram of coagulation activation. FpA-FpB=fibrinopeptides A and B; TAT=thrombin-antithrombin complex; and F1+2=prothrombin fragments.

[0312] FIG. 2: Dynamics of D-dimer formation by hypercoagulation, inflammation, and hyperfibrinolysis in coagulation activation states and venous thromboembolism (VTE).

[0313] FIG. 3: Principle of the method for assaying D-dimers specific for VTE according to the invention, in a plasma or whole-blood sample, comprising the usual assaying of D-dimers of the sample and the dynamic measurement of the fibrin formation of this sample.

[0314] FIG. 4: Interpretation of the method for assaying D-dimers specific for VTE, in a biological sample, on the basis of the D-dimers adjusted as a function of the D-dimers generated by hypercoagulation, inflammation and hyperfibrinolysis in said sample from the patient according to the first version of the method.

[0315] FIG. 5: Fibrin clot formation profile (step d), (A) in the case of a plasma from a normal patient (PN) in which the fibrinogen is 3.5 g/l and of a sample of plasma from a patient with a hypercoagulation state (P Hyper), in which the fibrinogen is 3.0 g/l, by measurement of the optical density (OD) as a function of time, according to the method of the invention; and (B) in the case of a whole-blood sample from a normal patient (PN) in which the fibrinogen level is 2.0 g/l and of a whole-blood sample from a hyperfibrinogenemic patient (P Hyperfib) in which the fibrinogen level is 5.0 g/1, by thromboelastometric measurement of the amplitude at the time to reach the plateau as a function of time, according to a variant of the method of the invention.

[0316] FIG. 6: Variation in optical density (DOD) at the time to reach the fibrin polymerization plateau, by the method according to the invention, for the 51 plasma samples from patients with a suspicion of thrombosis, as a function of inflammation, represented (A) by the level of C-reactive protein (CRP); and (B) by the level of fibrinogen (Fib).

[0317] FIG. 7: Time to reach the plateau as a function of fibrin formation time, by the method according to the invention (A) for 219 samples from patients with or without known cancer, or with a suspicion of thrombosis; and (B) for 34 samples from patients suffering from cancer, with a suspicion of thrombosis.

[0318] FIG. 8: Level of D-dimers generated by hypercoagulation as a function of D-dimers of the sample (step e)) (A) for 87 samples from patients with a suspicion of thrombosis; and (B) for 28 samples from patients with an active cancer.

[0319] FIG. 9: Variations in optical density (DOD) at the time to reach the fibrin polymerization plateau, measured according to the method of the invention, as a function of the level of fibrinogen determined by the reference method (Clauss) (A) in 219 samples from patients with or without cancer, with a suspicion of thrombosis; and (B) in 23 samples from patients suffering from active cancer, with a suspicion of thrombosis. (C) Amplitude at the time to reach the fibrin polymerization plateau measured according to the variant of the method according to the invention in 7 whole-blood samples from patients with a suspicion of thrombosis, as a function of the level of fibrinogen determined in plasma by the Clauss method.

[0320] FIG. 10: Correlation in the samples from 14 patients with and without venous thrombosis, between the level of fibrinogen degradation products (FgDPs) measured by the STA Liatest® FDP latex method (Stago) and the level (A) of the usual D-dimers (Stago method); (B) of the D-dimers (Stago method) adjusted as a function of the D-dimers generated by hypercoagulation and inflammation, according to the method of the invention carried out on plasma; and (C) of the D-dimers (Stago method) adjusted as a function of the D-dimers generated by hypercoagulation and inflammation, according to a variant of the method of the invention carried out on whole-blood samples.

[0321] FIG. 11: Correlation in the samples from 14 patients with and without venous thrombosis, between the level of fibrinogen degradation products (FgDPs) measured by the STA Liatest® FDP latex method (Stago) and the level (A) of the D-dimers (method of the Instrumentation Laboratory IL); and (B) of the D-dimers (IL method) adjusted as a function of the D-dimers generated by hypercoagulation and inflammation, according to the method of the invention carried out on plasma samples.

[0322] FIG. 12: Comparison of the adjustment of D-dimers (Stago method), by the method of the invention and by the ratio [D-dimers/Fibrinogen] (A) in the plasmas from 215 patients without venous thrombosis in imaging; and (B) in the plasmas from 21 patients with PE and/or DVT in imaging. (I: inflammation, F: hyperfibrinolysis, Ddi: D-dimers, Fib: fibrinogen).

[0323] FIG. 13: Comparison of the adjustment of D-dimers (IL method), by the method of the invention and by the ratio [D-dimers/Fibrinogen] (A) in the plasmas from 199 patients without venous thrombosis in imaging; and (B) in the plasmas from 15 patients with PE and/or DVT in imaging. (I: inflammation, F: hyperfibrinolysis, Ddi: D-dimers, Fib: fibrinogen).

[0324] FIG. 14: Correlation between the correction factor F.sub.Ddi-S for the low levels of D-dimers (<4 μg/ml) (A) and the level of D-dimers, R, resulting from intravascular fibrin degradation (B) obtained after adjustment as a function of inflammation (Ddi.sub.S/I) and correction for the low levels of D-dimers (<4 μg/ml) by the equation R=Ddi.sub.S/I+[0.5−F.sub.Ddi-s] as a function of the level of D-dimers of the sample (Ddi.sub.S), according to the method of the invention carried out on plasma samples.

[0325] FIG. 15: Comparison of the percentage exclusion of thrombosis as a function of age, by i) the assaying of D-dimers, ii) the assaying of D-dimers adjusted with respect to age, iii) the method of the invention (A) for 796 patients with a suspicion of venous thrombosis; and (B) for the 58 patients suffering from cancer among the 796 patients with a suspicions of venous thrombosis.

EXPERIMENTAL PROTOCOLS

[0326] The protocols used in the examples are the following:

Protocol A

[0327] Protocol A is a method carried out on the STAR® Evolution Expert Series automatic coagulation analyzer (Stago).

[0328] The following are simultaneously added, by the instrument, to 8 cuvettes of the STAR® Evolution automated device (step a)): [0329] 200 μl of pure plasma sample, obtained after centrifugation of the citrated tube intended for usual assaying of D-dimers, for 15 minutes, at a speed of 2500 g, [0330] 50 μl of a mixture of tissue factor and of phospholipids [TF+PL], that has been freeze-dried and taken up with water; at final concentrations of tissue factor (TF) of 2 to 5 pM and of phospholipids (PL) of 4 μM.

[0331] The automated device stirs by means of the arm and carries out an incubation for 300 seconds at 37° C. It then adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and stirs, by means of the needle, the triggering reagent (step b)). The automated device then measures the optical density (OD) at 540 nm as a function of the time for 10 minutes (step c)) and plots the fibrin formation curve.

[0332] The algorithm for “post-processing” of the measurement makes it possible to calculate the fibrin formation time (FFT) from the tangent to the curve, and the variation in OD (DOD) at the time to reach (TA) the plateau, from the OD at the time TA of the plateau and the OD at the time T0 to 10 seconds, as described in FIG. 5a (step d)). The level of D-dimers adjusted as a function of the D-dimers generated by hypercoagulation is calculated by the formula [(X)×(FFT/Control Time)], wherein X is the level of D-dimers measured, as shown in FIG. 8 and example 3.

[0333] The level of D-dimers adjusted as a function of the D-dimers generated by inflammation is calculated by the ratio [adjusted D-dimers/inflammation]. The level of inflammation is determined from the equation y=a ln(x)−b, wherein y is the DOD, x is the fibrinogen concentration, and a and b are the constants for the logarithmic equation which links the level of the fibrin plateau and the fibrinogen concentration, as shown in FIG. 9 and example 4. The D-dimers are thus expressed in fibrinogen equivalent units (FEUs) (step e)).

[0334] The time to reach the clot polymerization is calculated by the ratio TA/FFT, wherein TA is the time to reach the fibrin polymerization plateau determined in step d) and FFT is the fibrin clot formation time determined in step d).

Protocol B

[0335] Protocol B is a method carried out on the Rotem® delta automatic coagulation analyzer.

[0336] 300 μl of non-diluted whole blood (steps a) and b) of the variant of the method according to the invention), taken from the citrated tube intended for the usual assaying of D-dimers, preheated to 37° C., are added to a cuvette of the instrument, preheated beforehand, containing 40 μl of a mixture of tissue factor and of phospholipids [TF+PL], that is freeze-dried and taken up with water; at final concentrations of tissue factor (TF) of 2 to 5 pM and of phospholipids (PL) of 4 μM, and of CaCl.sub.2 at a final concentration of 16.7 mM. The thromboelastomeric measurement is initiated (step c) of the variant of the method according to the invention) then 300 μl of the mixture of blood and reagent are analyzed by the instrument which continuously records, for 30 minutes, the coagulation activation, the formation, the polymerization and the stability of the fibrin clot, and provides the usual parameters (CT, CFT, angle α, A5, A10, MCF, MCF-t).

[0337] The fibrin formation time (FFT) from the tangent and the amplitude at the time to reach (TA) the plateau of the fibrin formation curve are calculated from the temporal change in the amplitude as a function of time, as described in FIG. 5(B) (step d) of the variant of the method according to the invention). The level of D-dimers adjusted as a function of the D-dimers generated by hypercoagulation is calculated by the formula [(X)×(FFT/Control Time)], wherein X is the level of D-dimers measured, as shown in example 3.

[0338] The level of D-dimers adjusted as a function of the D-dimers generated by inflammation is calculated by the ratio [adjusted D-dimers/level of inflammation]. This level is determined from the linear equation which links the amplitude at the time to reach the plateau (mm) and the fibrinogen concentration, as shown in FIG. 9 and example 4. The D-dimers are thus expressed in initial fibrinogen equivalent units (FEUs) (step e) of the variant of the method according to the invention).

Protocol C

[0339] Protocol C is a method carried out on the STAR® Evolution Expert Series automatic coagulation analyzer (Stago).

[0340] The following are simultaneously added to the cuvettes of the STAR® Evolution automated device, by the instrument (step a)): [0341] 200 μl of sample of each of the negative control or positive control plasmas, freeze-dried and reconstituted with water, [0342] 50 μl of a mixture of tissue factor and of phospholipids [TF+PL], that has been freeze-dried and taken up with water; at final concentrations of tissue factor (TF) of 2 pM and of phospholipids (PL) of 4 μM.

[0343] The automated device stirs by means of the arm, carries out an incubation for 300 seconds at 37° C. then adds 50 μl of CaCl.sub.2 at a final concentration of 16.7 mM, and stirs, by means of the needle, the triggering reagent (step b)). The automated device then measures the optical density (OD) at 540 nm as a function of the time for 10 minutes (step c)) and the fibrin formation curve is plotted for each of the control plasmas.

[0344] The algorithm for “post-processing” of the measurement makes it possible to calculate the fibrin formation time (FFT) from the tangent to the curve, and the variation in OD (DOD) at the time to reach (TA) the plateau, from the OD at the time T of the plateau and from the OD at the time T0 to 10 seconds for each of the control plasmas, as described in FIG. 5(A) (step d))).

[0345] The level of D-dimers adjusted as a function of the D-dimers generated by hypercoagulation is calculated by the formula [(X)×(FFT/Control Time)], wherein X is the level of D-dimers measured, as shown in FIG. 8 and example 3.

[0346] The level of D-dimers adjusted as a function of the D-dimers generated by inflammation is calculated by the ratio [adjusted D-dimers/level of inflammation]. This level is determined from the equation y=a ln(x)−b, wherein y is the DOD, x is the fibrinogen concentration, a and b of the constants for the logarithmic equation which links the level of the fibrin plateau and the fibrinogen concentration, as shown in FIGS. 9(A) and (B) and in example 4. The D-dimers are thus expressed in fibrinogen equivalent units (FEUs) (step e)).

Example 1: Fibrin Formation Time of Various Plasmas from Normal Patients and from Patients with a Suspicion of Thrombosis (with or without Thrombosis, and with or without Cancer)

[0347] The protocol used in this example is protocol A. Step d) of the method used in this example is without implied distinction that of the first or the second version of the method.

[0348] Among the samples from patients with a suspicion of PE and/or of DVT that are tested, the samples from patients with a coagulation activation state are differentiated from the normal patients (normal healthy subjects, with no suspicion of thrombosis), from those with an exclusion of thrombosis, and from those with a positive diagnosis of venous thrombosis in imaging, with regard to the fibrin formation time (FFT), representing hypercoagulation, with regard to the time to reach (TA) the plateau, and with regard the time to reach clot polymerization (ratio TA/FFT), as shown in FIG. 5 and table 1 below.

[0349] The fibrin formation time (FFT) is shorter for the patients exhibiting a hypercoagulation state; but surprisingly, there is no hypercoagulation state in the patients with a diagnosis of PE and/of DVT, contrary to the patients with an exclusion of thrombosis, including in cancer. Advantage is taken of this in the first version of the method of the invention in order to discriminate the patients with and without thrombosis.

[0350] The time to reach the plateau does not exceed 8 minutes, all patients included, except for the patients receiving anticoagulant treatment, for whom the rendering of the result is limited to the fibrin formation time.

[0351] The time to reach clot polymerization is shorter (ratio TA/FFT lower) in the patients with a diagnosis of PE and/or of DVT, than in the normal patients, or the patients with a coagulation activation state, or with an exclusion of thrombosis, including in cancer. Advantage is taken of this in the second version of the method of the invention in order to discriminate the patients with and without thrombosis.

TABLE-US-00001 TABLE 1 Discrimination of the plasmas from patients with a hypercoagulation state among the normal patients and the patients with a suspicion of thrombosis, whether or not they have a thrombosis or a cancer, with regard to the fibrin formation time (FFT) and with regard to the time to reach (TA) the plateau and with regard to the time to reach clot polymerization (TA/FFT). Diagnosis D-dimers (threshold Plasmas 0.5 μg/ml) and/or FFT (sec) TA (sec) TA/FFT from imaging N Mean Range Mean Range Mean Normal Ddi negative Negative 150 133  88-206 1.82 172-324 1.82 patients Patients Ddi negative Negative 127 141  84-304 1.62 154-440 1.62 with Ddi positive Negative 88 154  88-258 1.55 154-384 1.55 suspicion of without thrombosis thrombosis without Ddi positive Positive 21 166 116-218 1.45 176-298 1.45 cancer with PE thrombosis and/or DVT Patients Imaging Negative 16 155 116-210 1.56 190-304 1.56 with without suspicion of thrombosis thrombosis Imaging Positive 6 176 126-212 1.46 204-298 1.46 with cancer with PE thrombosis and/or DVT Under anticoagulants 10 313 150-694 447  220-1122 /

Example 2: Generation of D-Dimers by Hypercoagulation in Various Plasmas from Patients with a Suspicion of Thrombosis (with or without Thrombosis, and with or without Cancer)

[0352] The protocol used in this example is protocol A. Step e) of the method used in this example is that of the first version of the method.

[0353] Among the samples from patients with a suspicion of PE and/or of DVT that are tested, the D-dimers generated as a function of the fibrin formation time correlate well with the initial D-dimers of the sample, whether for the patients with a positive diagnosis of thrombosis in imaging or with an exclusion of thrombosis, with or without cancer, as shown by FIGS. 8 (A, B).

[0354] Advantage is taken of the linear function to adjust the D-dimers of the sample as a function of the D-dimers generated by hypercoagulation, by calculating the adjusted D-dimers Y, from the level of D-dimers of the sample X, by the following formula:

[00015]$Y=X\times \frac{\mathrm{Fibrin}\mathrm{formation}\mathrm{time}}{\mathrm{Control}\mathrm{Time}}$

[0355] This makes it possible to amplify the D-dimers proportionally to the elongation of the fibrin formation time FFT in the patients with thrombosis, and to subtract them proportionally to the shortening of the fibrin formation time FFT in the patients with hypercoagulation.

[0356] The ratio varies as a function of the type and batch of D-dimer reagent used for the assay; and as a function of the batch of tissue factor-phospholipid reagent used for measuring the fibrin formation. The batches of reagents can then advantageously be calibrated according to this formula.

Example 3: Adjustment of the D-Dimers as a Function of the D-Dimers Generated by Hypercoagulation, for Various Plasmas from Patients with a Suspicion of Thrombosis (with and without Thrombosis, and with or without Cancer)

[0357] The protocol used in this example is protocol A. Step e) of the method used in this example is that of a first version of the method.

[0358] Advantage is taken of the linear correlation y=ax+b of example 2 in the method according to the invention, in order to adjust the D-dimers as a function of the D-dimers generated by hypercoagulation, as described in particular in example 2 and table 2 below. The patients with and without thrombosis are especially discriminated on the basis of a high level of D-dimers 3.6 times higher for the patients with thrombosis than for the patients without thrombosis and 2.3 times higher for the cancer patients with a thrombosis than for the patients without thrombosis, as shown in table 2 below.

[0359] The adjustment of the D-dimers as a function of the D-dimers generated by hypercoagulation makes it possible to subtract the D-dimers generated by hypercoagulation in coagulation activation states.

TABLE-US-00002 TABLE 2 Adjustment of the D-dimers as a function of the D-dimers generated by hypercoagulation in the plasmas from patients with a suspicion of thrombosis (with or without thrombosis, and with or without cancer). Level of adjusted Diagnosis Level of initial D-dimers D-dimers/hypercoag D-dimers and/or (μg/ml) (μg/ml) Plasmas from imaging Number Mean Range Mean Range Normal Ddi negative 150 0.28 0.27-0.47 0.28 0.27-0.54 patients Patients with Ddi negative 127 0.31 0.27-0.49 0.33 0.19-0.66 suspicion of Ddi positive 88 1.60 0.51-11.6 2.00 0.43-19.0 thrombosis without with or thrombosis without cancer Ddi positive 21 5.72 1.33-30.0 7.19 1.41-41.0 with thrombosis Variation +/− thrombosis ×3.6 ×3.6 Patients with Imaging 16 2.52 0.27-11.6 2.94 0.59-19.0 cancer with without suspicion of thrombosis thrombosis Imaging 6 5.21 2.73-10.9 6.89 2.59-15.5 with cancer with thrombosis Variation +/− thrombosis ×2.1 ×2.3

Example 4: Level of the Fibrin Plateau, of Various Plasmas from Normal Patients and from Patients with a Suspicion of Thrombosis (with or without Thrombosis, and with or without Cancer), by the Method of the Invention

Plasma Samples

[0360] The protocol used in the first part of this example is protocol A. Step d) of the method used in this example is without implied distinction that of the first or the second version of the method.

[0361] Among the samples from patients with a suspicion of PE and/or DVT that are tested, the samples from patients with a positive diagnosis of thrombosis in imaging or with an exclusion of thrombosis are differentiated from the patients with a coagulation activation state and the normal patients (normal healthy subjects with no suspicion of thrombosis) with regard to the level of the fibrin formation plateau, representative of inflammation, as shown in FIGS. 6 and 9, and table 3 below. The level of the DOD plateau by the method of the invention is higher the greater the inflammation, represented by C-reactive protein (CRP) and fibrinogen in FIGS. 6(A, B), independently of the presence or of the exclusion of thrombosis.

[0362] The level of the fibrin formation plateau, which represents the maximum clot polymerization, correlates well with the level of fibrinogen of the sample, regardless of the patients, according to a logarithmic equation y=a ln x+b, wherein y is the variation in optical density at the plateau start time, and ln x is the Naperian log of x which represents the level of fibrinogen, as described in FIGS. 9(A, B). The variable x is calculated from the equation, by the exponential function of the Naperian log of x. The factor linking the level of fibrinogen and the DOD is constant regardless of the patients, as shown in table 3 below.

[0363] The patients with and without thrombosis, including in the case of patients who have a cancer, are discriminated on the basis of the inflammation, represented by the amount of fibrinogen and/or of DOD, as shown in table 3 below.

TABLE-US-00003 TABLE 3 Level of the fibrin plateau of plasmas from patients with a suspicion of thrombosis (with or without thrombosis, and with or without cancer) determined by the DOD at the time to reach the fibrin formation plateau. Diagnosis Level of fibrinogen D-di and/or (μg/ml) DOD 540 nm Delta Plasmas from imaging Number Mean Range Mean Range DOD/Fib Normal Ddi negative 150 2.93 2.12-4.56 0.937 0.618-1.423 0.32 patients Patients with Ddi negative 127 3.79 1.93-8.40 1.225 0.581-2.237 0.32 suspicion of Ddi positive 88 4.50 1.67-8.27 1.437 0.458-2.119 0.32 thrombosis without with or thrombosis without Ddi positive 21 4.83 3.00-7.54 1.509 1.007-2.059 0.31 cancer with thrombosis Patients with Imaging 16 4.15 2.63-7.23 1.424 0.716-2.051 0.34 suspicion of without thrombosis thrombosis with cancer Imaging 6 4.91 3.43-7.54 1.598 1.134-2.059 0.33 with thrombosis

Whole-Blood Samples

[0364] The protocol used in the second part of this example is protocol B. This method is carried out in the presence of an anti-platelet inhibitor, in the case in point cytochalasin D.

[0365] The level of inflammation is determined from the linear equation, which links the amplitude at the time to reach the plateau, according to the alternative method in whole blood as shown in FIG. 5(B), and the level of fibrinogen in Clauss, as shown in FIG. 9(C). The patients tested with the variant of the method according to the invention are discriminated both with the method based on the amplitude at the time to reach the plateau, and with the method of the invention based on the determination of DOD.

Example 5: Adjustment of the D-Dimers as a Function of the D-Dimers Generated by Inflammation, for Various Plasmas from Normal Patients and from Patients with a Suspicion of Thrombosis (with or without Thrombosis, and with or without Cancer), by the Method of the Invention

[0366] The protocol used in this example is protocol A. Steps e) and f) of the method used in this example are without distinction those of the first or of the second version of the method.

[0367] Advantage is taken of the logarithmic function of example 4 in the method according to the invention, in order to adjust the D-dimers as a function of the D-dimers generated by inflammation, by the ratio R:

[00016]$R=\frac{\begin{array}{c}D\text{-}\mathrm{dimers}\mathrm{adjusted}\mathrm{as}a\mathrm{function}\mathrm{of}\mathrm{the}\\ D\text{-}\mathrm{dimers}\mathrm{generated}\mathrm{by}\mathrm{hypercoagulation}\end{array}}{\mathrm{Level}\mathrm{of}\mathrm{inflammation}}$

and to express them in fibrinogen equivalent units FEUs.

[0368] Since the amount of D-dimers generated by plasmin is approximately 50% of the FEU unit with the antibodies 8D2 and 2.1.16 of the latex reagent used, the positivity threshold of the measurement is thus 0.5 μg/ml.

[0369] The level of adjusted D-dimers is determined with respect to a threshold, preferably a threshold of 0.5 μg/ml, in order to determine the probability of a pulmonary embolism (PE) or of a deep vein thrombosis (DVT) in the sample from the patient, as shown by FIGS. 12(A, B) and table 4 below.

[0370] The 127 patients excluded with regard to D-dimers all have a negative adjusted D-dimer level. Among the 88 patients without thrombosis, the D-dimers of whom are falsely positive, 80% have a negative adjusted D-dimer level and avoid imaging. More than 90% of the patients without thrombosis thus avoid imaging with the method of the invention.

[0371] 100% of the patients with thrombosis have a positive adjusted D-dimer level ranging from 0.52 μg/ml to 10.5 μg/ml, as shown in table 4 below.

[0372] Among the samples from patients without thrombosis, found to be falsely positive in terms of adjusted D-dimers with a level of 0.50 μg/ml to 2.73 μg/ml, ⅓ have a level <0.60 μg/ml, ⅓ have a cancer, and ⅓ have a coagulation activation state.

TABLE-US-00004 TABLE 4 Discrimination of plasmas from patients with a suspicion of thrombosis (with or without thrombosis, and with or without cancer), with an adjustment of D-dimers as a function of the D-dimers generated by hypercoagulation and as a function of the D-dimers adjusted for inflammation. Level of adjusted Diagnosis Level of adjusted D- D-dimers/ Ddi and/or dimers/hypercoag H inflammation I Result Plasmas from imaging N Mean Range Mean Range rendered Patients with a Ddi negative 127 0.33 0.19-0.66 0.09 0.04-0.21  0 False+ suspicion of Ddi positive 88 2.00 0.43-19.0 0.42 0.11-2.73 18 False+ thrombosis without with or thrombosis without Ddi positive 21 7.19 1.41-41.0 1.65 0.52-10.5  0 False− cancer with thrombosis Patients Imaging 16 4.24 0.59-19.0 0.72 0.12-2.73  7 False+ with a without suspicion of thrombosis thrombosis Imaging 6 7.20 2.59-15.5 1.44 0.58-3.65  0 False− with cancer with thrombosis

Example 6: Differentiation of D-Dimers Generated by Intravascular Fibrin, from Those Generated in High Coagulation Activation States with Regard to Hyperfibrinolysis

[0373] The protocol used in this example is protocol A. Step g) of the method used in this example is that of the first version of the method.

[0374] The differentiation between the D-dimers present in these activation states and those of thrombosis is carried out with regard to hyperfibrinolysis. The generation of fibrinogen degradation products FgDPs by plasmin in acute activation states enables their exclusion. The hyperfibrinolysis is calculated from the linear equations of FIG. 10(A, B) for the Stago reagent, and FIG. 11(A, B) for the IL reagent (step g)).

[0375] In the presence of hyperfibrinolysis (FgDP>7 μg/ml), the FgDPs generated with intravascular fibrin are systematically lower in the samples originating from patients with thrombosis than in those from patients without thrombosis; this being whatever the D-dimer reagent used. This makes it possible to discriminate the patients with thrombosis from those without thrombosis, on the basis of a difference between FgDPs before and after D-dimer adjustment which is positive, at the threshold of 1 μg/ml, as shown in table 5 below. Conversely, a difference which is negative or <1.0 in the patients without thrombosis makes it possible to turn attention to an acute coagulation activation state.

TABLE-US-00005 TABLE 5 Discrimination of the plasmas from patients with a suspicion of thrombosis (with or without thrombosis, and with or without cancer), as a function of hyperfibrinolysis with deduction of FgDPs, by the optical method on STAR ® (Stago). Difference Diagnosis FgDP FgDP Plasmas Ddi positive Adjusted Adjusted threshold ≥1 Result from and imaging N Ddi FgDP/Ddi Ddi μg/ml rendered Patients with Ddi neg 127 0.09 Neg Neg / Negative suspicion of Without 69 0.26 Neg Neg / Negative thrombosis thrombosis 4 0.51 Neg Neg / 4 False+ with or 9 1.13 11.2 10.9 +0.3 Negative without 6 0.91 15.3  9.2 +6.1 6 False+ cancer (+2.6 to +20.3) With 2 0.55 Neg Neg / Positive thrombosis 19 1.77 18.8 14.5 +4.3 Positive (0.58-10.5) (8.3-83.3) (7.1-68.7) (+1.2 to +14.6) Patients with Without 10 0.26 Neg Neg / Negative suspicion of thrombosis 2 1.97 16.1 15.8 +0.3 Negative thrombosis 1 0.51 Neg Neg / 1 False+ with cancer 3 0.97 18.8  9.5 +9.2 3 False+ With 6 1.44 16.2 12.4 +3.8 Positive thrombosis (0.58-3.65) (9.5-31.7) (7.1-26.2) (+1.3 to +6.7)  Positive

[0376] Thus, 95% of the patients avoid imaging on the basis of a negative level of D-dimers adjusted as a function of the D-dimers generated equally by hypercoagulation (H), inflammation (I) and hyperfibrinolysis (F).

[0377] Among the false positives, 3 are at the threshold limit, 4 are cancers and 3 have a coagulation activation cause and can avoid imaging on the basis of a considerable shortening of the fibrin formation time, without generating false negatives, as shown in example 7.

Example 7: Adjustment of D-Dimers by the Method of the Invention Compared with the Ratio [Ddi/Fibrinogen by the Clauss Method]

[0378] The protocol used in this example is protocol A. Step h) of the method used in this example is that of the first version of the method.

[0379] The adjustment of the D-dimers as a function of the D-dimers generated by hypercoagulation (H), inflammation (I) and hyperfibrinolysis (F) according to the first version of the method of the invention (step h)) is more effective than the ratio [D-dimers/fibrinogen], as shown by FIG. 12 and table 6 below. The patients with thrombosis are discriminated from those without thrombosis on the basis of a high level of adjusted D-dimers, which are multiplied by a factor of 5.3, as shown in table 6 below. Thus, 92% of the patients with a falsely positive level of D-dimers, that is to say 97% of the patients without thrombosis, are rendered negative with the adjustment of D-dimers according to step h), as shown by FIG. 12. Among the 7 false +, 4 are cancers and 3 are at the threshold limit.

TABLE-US-00006 TABLE 6 Adjustment of D-dimers by the optical method of the invention, compared with adjustment by the ration [D-dimers/fibrinogen by the Clauss method], in the plasmas from patients with a suspicion of thrombosis (with or without thrombosis, and with or without cancer) (H: hypercoagulation, I: inflammation, F: hyperfibrinolysis, Ddi: D-dimers, Fib: fibrinogen) Diagnosis Adjusted Ddi Ratio Plasmas Ddi and/or [Ddi/(H + I + F)] [Ddi/Fib] Clauss from imaging N Mean Range Result Mean Range Result Patients with Ddi negative 127 0.09 0.04-0.21 Neg 0.09 0.04-0.16 Neg suspicion of Ddi positive 88 0.31 0.11-1.56 7 False+ 0.40 0.09-3.60 17 False+ PE and/or Without DVT with or thrombosis without Ddi positive 21 1.65 0.52-10.5 0 False− 1.35 0.38-7.71  2 False− cancer With thrombosis Variation +/− thrombosis ×5.3 ×3.4 Patients with Without 16 0.45 0.12-1.56 4 False+ 0.75 0.09-3.60  7 False+ suspicion of thrombosis thrombosis With 6 1.44 0.58-3.65 0 False− 1.18 0.38-3.0   1 False− with cancer thrombosis Variation +/− thrombosis ×3.2 ×1.6

[0380] The false negatives with the ratio [D-dimers/fibrinogen] are detected by the method of the invention, which makes it possible not to miss a thrombosis.

[0381] There are fewer false positives by the method of the invention than with the ratio [D-dimers/fibrinogen], which makes it possible to reduce the number of patients diagnosed by imaging.

Example 8: Influence of the Level of D-Dimers Adjusted According to the Method of the Invention, with Regard to the Probability of PE and/or of DVT

[0382] The protocol used in this step is protocol A. Step h) of the method used in this example is that of the first version of the method.

[0383] The threshold of the D-dimers adjusted according to the method of the invention was advantageously not adjusted as a function of age. This is because the level of D-dimers, the coagulation activation and the inflammation gradually increase as a function of age after the age of 50. As a result, the ratio is not adjusted as a function of age.

[0384] The higher the levels of D-dimers adjusted according to the method of the invention, the higher the denominator of the formula with the fibrin formation time and of the ratio with the DOD, the higher the probability of PE or of DVT.

[0385] The greater the extent of the PE and the more proximal rather than distal or superficial the DVT, the higher the adjusted D-dimers, as shown in table 7 below.

TABLE-US-00007 TABLE 7 Probability of PE and of DVT as a function of the level of D-dimers that have been adjusted for hypercoagulation (H), inflammation (I) and hyperfibrinolysis (F), (FFT: fibrin formation time, T: mean Control Time − 1 standard deviation = 120 sec), FgDPs: fibrinogen degradation products, Ddi: D-dimers, Act: coagulation activation state). Probability Adjusted Ddi Delta FFT ≤ PE and/or Plasmas from patients Number Ddi [Ddi/C + I + F] FgDPs T DVT with PE and/or DVT 1 1.56 0.58 / 190 Medium Subsegmental PE 3 3.60 0.92 +2.6 157 High Segmental PE 1 7.0 1.75 +6.7 212 High Bilateral pulmonary artery PE 1 10.9 3.65 +5.5 188 High Proximal and distal PE 1 30.0 10.47 +14.6  182 Very high Massive PE 1 0.72 0.25 / 88 Activation Peroneal vein inflammation 2 1.44 0.49 / 162 Low Superficial DVT 1 2.28 0.58 +1.2 162 Medium Distal DVT 1 3.60 0.91 +2.7 218 High Proximal DVT 1 7.70 1.62 +9.3 192 High 1 12.1 4.19 +5.3 144 High Large jugular DVT

Example 9: Inter-Instrument Reproducibility of the Fibrin Formation Measurement

[0386] The protocol used in this example is protocol C on STA-R®, which was used as follows, according to steps a), b), c) and d) of the first or of the second version of the method.

[0387] The following are simultaneously added, by the instrument, to 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago): [0388] 200 μl of control-plasma sample: normal (STA® CCN); hypercoagulant (plasma depleted of protein S, STA DEF PS) and of hypofibrinolytic control plasma (plasma containing plasminogen activator inhibitor PAI-I, PAI 4C), [0389] 50 μl of a mixture of tissue factor and of phospholipids [TF 2 pM final+PL 4 μM final].

[0390] After stirring, 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 stirs by means of the needle. Finally, the automated device measures the fibrin formation time and the variation in optical density DOD at the time to reach the plateau (TA); at the wavelength of 540 nm, as a function of time for 10 minutes.

[0391] The inter-instrument reproducibility of the method was determined on three STAR® instruments, as follows: 12 series of 2 measurements, that is to say 24 measurements, were carried out on each of the 3 instruments, for 5 consecutive days, using 2 bottles of each of the normal control plasmas (CCN) and pathological control plasmas (DPS:

[0392] control plasma depleted of protein S, hypercoagulant, PAI: hypofibrinolytic control plasma), that is to say 48 measurements for each control plasma. The aberrant values were eliminated according to Rosner.

[0393] The coefficients of variation (CV) obtained on the fibrin formation time FFT are all less than 6%; those obtained on the OD at the time of the plateau and on the maximum OD are all less than 3%, whatever the control plasma, as shown in table 8 below. The inter-STAR® variations are less than 2.5% whatever the parameter and the control plasma.

TABLE-US-00008 TABLE 8 Inter-instrument reproducibility of the measurement of fibrin formation (CCN: normal control plasma, DPS: control plasma depleted of protein S, hypercoagulant, PAI: hypofibrinolytic control plasma). N = 48 measurements Fibrin formation time FFT OD at the time of the (sec) plateau Maximum OD Control plasma CCN DPS PAI CCN DPS PAI CCN DPS PAI STAR ® 1 mean 100.4 82.4 117.8 2.200 1.977 2.280 2.223 2.004 2.306 min 86.0 78.0 102 2.157 1.751 2.217 2.174 1.768 2.239 max 110.0 86.0 126 2.278 2.053 2.347 2.310 2.071 2.384 CV 5.8% 2.8% 4.5% 1.4% 2.8% 1.4% 1.5% 2.9% 1.5% STAR ® 2 mean 99.7 83.1 118.2 2.222 2.015 2.309 2.337 2.043 2.337 min 88.0 78.0 100.0 2.073 1.788 2.248 2.265 1.804 2.265 max 116.0 90.0 138.0 2.280 2.114 2.381 2.422 2.154 2.422 CV 5.6% 3.3% 5.7% 1.9% 2.7% 1.3% 1.5% 2.8% 1.5% STAR ® 3 mean 100.1 84.2 118.7 2.245 2.030 2.328 2.265 2.057 2.352 min 90.0 76.0 106.0 2.192 1.879 2.261 2.213 1.904 2.279 max 110.0 90.0 134.0 2.323 2.112 2.374 2.365 2.129 2.395 CV 4.4% 3.5% 4.6% 1.2% 2.6% 1.2% 1.3% 2.4% 1.1% Inter-STAR ® variations mean 99.9 83.2 118.2 2.222 2.007 2.306 2.275 2.035 2.332 standard 0.35 0.90 0.42 0.02 0.03 0.02 0.06 0.03 0.02 deviation CV 0.3% 1.1% 0.4% 1.0% 1.4% 1.1% 2.5% 1.3% 1.0%

Example 10: Repeatability of the Fibrin Formation Measurement

[0394] The protocol used in this example is protocol A on STA-R®, which was used as follows, according to steps a), b), c) and d) of the first or of the second version of the method.

[0395] The following are simultaneously added, by the instrument, to 8 cuvettes of the STA-R® Evolution Expert Series automated device (Stago): [0396] 200 μl of sample of citrated plasma from normal patients, or from patients with or without a suspicion of venous thrombosis, [0397] 50 μl of a mixture of tissue factor and of phospholipids [TF 2 pM final+PL 4 μM final].

[0398] After stirring, and incubation for 300 seconds at 37° C., the automated device adds 50 μl of CaCl.sub.2 at 16.7 mM final concentration, and stirs by means of the needle. Finally, the automated device measures the fibrin formation time and the variation in optical density DOD at the time to reach the plateau (TA); at the wavelength of 540 nm, as a function of time, for 10 minutes.

[0399] The repeatability of the measurement duplicates was determined on 4 STAR® instruments, for each parameter of the method, and for each of the samples. The 154 plasmas from normal patients were tested with a first batch of reagents on one of the STAR® instruments, the 83 samples from patients with a suspicion of thrombosis were tested with a second batch of reagent on the other 3 STAR® instruments for the D-dimers, the fibrin formation time, the DOD at the plateau and the fibrinogen.

[0400] The mean and the standard deviation of the variations obtained on each of the parameters are reported in tables 9 and 10 below.

[0401] The mean of the variations obtained on the fibrin formation measurements is less than 2% for the 154 normal patients; it is less than 1% for the 83 patients with and without venous thrombosis, as shown in tables 9 and 10.

[0402] The measurements of the parameters of fibrin formation are as repeatable as those of fibrinogen in the Clauss method.

[0403] The measurements of the specific D-dimers are as repeatable as those of the D-dimers and of the ratio [D-dimers/fibrinogen]; the means of the variations is less than 2.3%.

[0404] Among the 5 samples rendered positive by specific D-dimers and negative by the ratio [Ddi/Fib] on the duplicates, 2 are from patients with a pulmonary embolism confirmed in imaging, 2 have coagulation hyperactivation and 1 is from a patient with a cardiopathy.

[0405] Thus, 2 patients with a PE, found to be falsely negative by the ratio [Ddi/Fib], can be diagnosed by the method of the invention; while a single patient without PE requires imaging by the method of the invention.

TABLE-US-00009 TABLE 9 Repeatability of the duplicates of fibrin formation measurement in the samples from normal patients. Fibrin formation measurements Measurement variations Samples from normal patients Standard Standard Number of duplicates Mean deviation Mean deviation Fibrin formation time FFT 150   134 24 −1.1% 7.1% (seconds)  82-206 DOD at the plateau 150 0.937 0.20 −0.5% 1.8% (540 nm) 0.618-1.423

TABLE-US-00010 TABLE 10 Repeatability of the duplicates of measurement of fibrin formation, of D-dimers and D- dimers adjusted according to the method of the invention, and also of the ratio [D- dimers/fibrinogen] in the samples from patients with and without thrombosis in imaging. Samples from patients with and Fibrin formation measurements Measurement variations without thrombosis in imaging Standard Standard Number of duplicates Mean deviation Mean deviation Fibrinogen (g/l) 83 3.97 1.92-8.40 1.10 0.4% 3.0% Fibrin formation time FFT 83 151.6 88-382 41.4 0.9% 5.0% (seconds) DOD at the plateau (540 nm) 83 1.262 0.559-2.241 335 0.2% 2.0% Positive D-dimers >0.5 30 2.20 0.52-12.13 3.15 2.1% .sup. 7% μg/ml Ratio [D-dimers/fibrinogen] 30 0.67 0.12-4.92 1.14 2.1% 6.7% Negative ratio 25 0.28 0.12-0.53 0.13 2.4% 7.3% Positive ratio 5 2.61 0.60-4.92 1.85 0.2% 1.8% D-dimers specific for 30 0.71 0.14-4.28 1.00 2.3% 6.8% thrombosis (μg/ml) Negative specific Ddi 21 0.30 0.14-0.50 0.11 3.6% 7.7% Positive specific Ddi 9 1.66 0.51-4.28 1.44 −0.6% 1.8%

Example 11: Adjustment of D-Dimers According to the Method of the Invention with Another Reagent for Assaying D-Dimers

[0406] The protocol used in this example is protocol A. Steps e) to h) of the method used in this example are those of the first version of the method.

[0407] The D-dimers are adjusted as a function of the D-dimers generated by hypercoagulation (H) by the formula of example 2, of the inflammation (I) by the logarithmic function of example 4 and the ratio R of example 5, and of the hyperfibrinolysis (F) by the linear equations of example 6, with the Instrumentation Laboratory (IL) method for assaying D-dimers.

[0408] The adjustment of the D-dimers as a function of the D-dimers generated by hypercoagulation makes it possible to subtract the D-dimers generated by hypercoagulation in coagulation activation states, but does not make it possible to discriminate the patients with and without thrombosis, as shown in table 11.

TABLE-US-00011 TABLE 11 Adjustment of the D-dimers with the IL method for assaying D- dimers as a function of the D-dimers generated by hypercoagulation in plasmas from patients with a suspicion of thrombosis. Plasmas Diagnosis Level of initial D- Level of adjusted D- from D-dimers dimers (μg/ml) dimers/hypercoag (μg/ml) patients and/or imaging Number Mean Range Mean Range With a Ddi negative 120 0.43 0.11-3.32 0.46 0.09-3.35 suspicion of Ddi positive 79 1.75 0.20-7.34 2.12 0.21-16.7 thrombosis without thrombosis Ddi positive 15 8.42 1.39-25.4 10.9 1.82-34.7 with thrombosis

[0409] The adjustment of the D-dimers as a function of the D-dimers generated by inflammation makes it possible to discriminate the patients with and without thrombosis, as shown in FIG. 13 and table 12. Thus, approximately 90% of the patients can avoid imaging with one or other of the 2 Stago and IL reagents, on a negative adjusted D-dimer level.

TABLE-US-00012 TABLE 12 Adjustment of the D-dimers with the IL method for assaying D-dimers as a function of the D-dimers generated by hypercoagulation and by inflammation in plasmas from patients with a suspicion of thrombosis. Level of adjusted Level of adjusted Plasmas Diagnosis D-dimers (μg/ml)/ D-dimers (μg/ml)/ Result from Ddi and/or hypercoag H inflammation I False+ patients imaging N Mean Range Mean Range False− Plasmas from Ddi negative 120 0.46 0.09-3.35 0.12 0.02-1.04  2 F+ patients Ddi positive 79 2.12 0.21-16.7 0.45 0.06-2.47 22 F+ with a without suspicion of thrombosis thrombosis Ddi positive 15 10.9 1.82-34.7 2.45 0.49-8.84 1 lim with thrombosis

[0410] The adjustment of the D-dimers as a function of the D-dimers generated by hyperfibrinolysis (F), in addition to hypercoagulation (H) and inflammation (I), makes it possible to discriminate more patients, as shown in FIG. 13 and tables 13 and 14 below. Thus, more than 95% of the patients can avoid imaging, on the basis of a negative level of D-dimers adjusted according to the method of the invention. Among the 9 false +, 3 are cancers with metastases, 3 are at the threshold limit (≤0.60) and 3 are elderly patients (>80 years old).

TABLE-US-00013 TABLE 13 Discrimination of the plasmas from patients with a suspicion of thrombosis (with or without thrombosis), by the method of the invention, with the IL method for assaying D-dimers, as a function of the D-dimers generated by hyperfibrinolysis with deduction of the FgDPs. Plasmas Diagnosis FgDP FgDP from Ddi positive Adjusted FgDP Adjusted Threshold ≥ patients and imaging N Ddi Ddi Ddi 1.0 Result With Ddi negative 120 0.12 / / / Neg suspicion of Without 79 0.45 5.67 5.52 / 11 F+ thrombosis thrombosis With 15 2.45 23.4 17.7 +5.66 Positives thrombosis 1 limit

[0411] This adjustment according to the method of the invention is more effective than the ratio [D-dimers/fibrinogen], as shown in FIG. 13 and table 14 below. The false negatives with the ratio [D-dimers/fibrinogen] are detected by the method of the invention, which makes it possible not to miss a thrombosis. There are fewer false positives by the method of the invention than with the ratio [D-dimers/fibrinogen], which makes it possible to reduce the number of patients diagnosed using imaging.

TABLE-US-00014 TABLE 14 Comparison of the adjustment of D-dimers by the optical method of the invention with the IL reagent for assaying D-dimers and by the ratio [D-dimers/fibrinogen], in the plasmas from patients with or without thrombosis (H: hypercoagulation, I: inflammation, F: hyperfibrinolysis, Ddi: D-dimers, Fib: fibrinogen). Plasmas Diagnosis Adjusted Ddi Ratio [Ddi/Fib] Result from Ddi and/or [Ddi/(C+ I + F)] (Clauss) False+ patients imaging Number Mean Range Result Mean Range False− With a Ddi negative 120 0.12 0.02-0.41 0 F+ 0.12 0.01-1.01 2 F+ suspicion of Ddi positive 79 0.37 0.06-2.23 9 F+ 0.41 0.05-2.26 17F+ PE and/or Without DVT thrombosis Ddi positive 15 2.45 0.49-8.84 1 lim 2.01 0.37-6.52 3 F− With thrombosis

Example 12: Diagnostic Performance Levels of the Method According to the Invention for Patients with a Suspicion of Thrombosis (with or without Thrombosis, with or without Cancer) as a Function of Age

[0412] The protocol used in this example is protocol A. Steps e) to h) of the method used in this example are those of the second version of the method.

[0413] The level of D-dimers which result from intravascular fibrin degradation, R, was calculated according to step e′) by adjusting the level of D-dimers of the sample as a function of the level of D-dimers generated by inflammation, and by correcting this adjusted level for the low levels of D-dimers (<4 μg/ml). The level of D-dimers generated by hyperfibrinolysis was calculated according to step g′), for each of the samples classified as a function of inflammation. The ratio TA/FFT was then compared with respect to a threshold, in order to exclude or to diagnose a thrombosis in the patient, or to turn attention to a coagulation activation state in the patient.

[0414] A total of 218 patients with a suspicion of thrombosis (with or without thrombosis, with or without cancer) were tested. Among these 218 patients tested, 47% were more than 50 years old, 17% were more than 75 years old and 15% had a cancer. The diagnosis of PE and/or of DVT was excluded on the basis of an imaging test or of a negative assay of D-dimers according to clinical probability, in 199 patients (91.3%). The diagnosis of PE and/or of DVT was confirmed on the basis of a positive imaging test in 19 patients, that is to say 8.7%. The exclusion and/or the diagnosis of a thrombosis or of a coagulation activation state was carried out for the 218 patients using the second version of the method of the invention. The diagnostic performance levels of the method were compared to those of the usual assaying of D-dimers and to those of the age-adjusted D-dimers.

[0415] The diagnostic performance levels of the method of the invention were then determined on a larger number of patients with a suspicion of thrombosis (with or without thrombosis, with or without cancer). On a total of 796 patients tested, 49% were more than 50 years old, 19% were more than 75 years old and 7.3% had a cancer. The diagnosis of PE and/or of DVT was excluded in 730 patients (91.7%). The diagnosis of PE and/or of DVT was confirmed in 66 patients, that is to say 8.3%.

[0416] The clinical usefulness of the determination of the level of D-dimers is limited in elderly patients. This is because the threshold of D-dimers must be adjusted with age in order to exclude pulmonary embolism (Righini et al., JAMA, 2014, 311: 1117-1124). This adjusted threshold corresponds to the age multiplied by 10 for patients 50 years old and older. The combination of the D-dimer threshold adjusted for age and of the pretest evaluation of the clinical probability is combined with a large number of patients in whom pulmonary embolism might have been excluded (Righini et al., JAMA, 2014, 311: 1117-1124). Methods for pretest evaluation of the clinical probability of pulmonary embolism (PE) and of deep vein thrombosis (DVT) are known in the art (see Kolok et al., Arch. Intern. Med., 2008, 168: 21-31, and Wells et al., NEJM, 2003, 349: 1227-1235, respectively).

[0417] The threshold of the D-dimers adjusted according to the method of the invention was advantageously not adjusted as a function of age. This is because the levels of D-dimers, the coagulation activation and the inflammation gradually increase as a function of age after the age of 50. As a result, the ratio is not adjusted as a function of age. The higher the levels of D-dimers adjusted according to the method of the invention, the higher the denominator of the ratio with the DOD, the higher the probability of PE or of DVT.

[0418] For the 218 patients with a suspicion of thrombosis, the method of the invention makes it possible to further exclude more patients (approximately 20% more) than the usual assaying of D-dimers or of age-adjusted D-dimers, as shown in table 15 below. Thus, more than 75% of the patients, tested by the method of the invention, do not need supplementary imaging tests.

[0419] The performance levels in table 15 are given at the usual threshold of 0.50 μg/ml for the D-dimers and the age-adjusted D-dimers, and at the threshold of 0.51 μg/ml in this example with the method of the invention. The sensitivity of the three methods is 100% given that there was no falsely negative diagnosis; on the other hand, the specificity of the method of the invention is much higher than that of the other two methods, as shown in table 1.5. The positive likelihood ratio is higher than that of the other two methods, confirming that the patient has three times more chance of having a thrombosis when the result is positive, than with the other methods.

[0420] Similar diagnostic performance levels are found when the method according to the invention is applied to a larger number of patients. Among the 796 patients with a suspicion of thrombosis, the method makes it possible to exclude 79% of patients (more than 20% more than the other methods) as shown in table 16, and regardless of age, as shown in table 17 and FIG. 15(A). The method according to the invention also makes it possible to exclude more patients (<75 years of age) suffering from a cancer (see FIG. 15(B)). Finally, the method according to the invention allows a better exclusion not only of elderly patients and patients suffering from a cancer, but also of elderly patients suffering from a cancer (see FIG. 15(B)). The results on a larger number of patients demonstrate a greater specificity of the method according to the invention compared with the reference method (see table 16). The positive predictive value is approximately two times higher than that of the reference method.

TABLE-US-00015 TABLE 15 Comparison of the diagnostic performance levels of the method of the invention with those of the assays of D-dimers and of age-adjusted D-dimers, for the 218 patients with a suspicion of thrombosis (19 with thrombosis and 199 without thrombosis). Diagnostic performance levels N = 218 patients Number of patients excluded for the Positive Negative diagnosis of PE Sensitivity Specificity likelihood ratio likelihood ratio or of DVT % % (LR+) (LR−) Method of 166/218 100% 83.2% 6.03 0.00 the invention (76.2%) D-dimers 115/218 100% 57.8% 2.37 0.00 (52.8%) Age-adjusted 123/218 100% 61.8% 2.62 0.00 D-dimers (56.4%)

TABLE-US-00016 TABLE 16 Comparison of the diagnostic performance levels of the method of the invention with those of the assays of D-dimers and of age-adjusted D-dimers, for the 796 patients with a suspicion of thrombosis (66 with thrombosis and 730 without thrombosis). Diagnostic performance levels N = 796 patients % Speci- PPV (positive Exclusion Sensitivity ficity predictive value) Method of the 79% (+28%) 99.8%  86% 39% invention (X2) D-dimers 51% 100% 56% 17% Age-adjusted  57% (+6%) 100% 62% 17% D-dimers

TABLE-US-00017 TABLE 17 Level of D-dimers (D-di) resulting from intravascular fibrin degradation according to the method of the invention, for the 386 patients under the age of 50 and the 379 patients over the age of 50 in whom the level of D-dimers (D-di) is <5.1 μg/ml, among the 796 patients with a suspicion of thrombosis. Method of the Diagnosis D-di invention Age PE and/or DVT n μg/ml D-di μg/ml Average age <50A Positive 13 2.23 0.69 38 years old N = 386 PE or DVT 0.52-1.62 Negative 375 0.55 0.51 35 years old 0.45-1.39 >=50A Positive 36 2.25 0.65 72 years old N = 379 PE or DVT 0.55-0.95 Negative 343 1.04 0.56 68 years old 0.46-1.76

[0421] The levels of D-dimers resulting from intravascular fibrin degradation according to the method of the invention are similar for the patients with a diagnosis of thrombosis regardless of their age (average age 38 or 72 years old). On the other hand, in the patients without thrombosis, the D-dimers are higher as a function of age, as expected.

Example 13: Diagnostic Performance Levels of the Method According to the Invention by Patient Group

[0422] The protocol used in this example is protocol A. Steps e) and h) of the method used in this example are those of the second version of the method.

[0423] The level of D-dimers which result from intravascular fibrin degradation, R, was calculated according to step e′) by adjusting the level of D-dimers of the sample as a function of the level of D-dimers generated by inflammation, and by correcting this adjusting level for the low levels of D-dimers (<4 μgimp. The level of D-dimers generated by hyperfibrinolysis was calculated according to step g′), for each of the samples classified as a function of inflammation. The ratio TA/FFT was then compared with respect to a threshold, in order to exclude or to diagnose a thrombosis in the patient, or to direct attention to a coagulation activation state in the patient.

[0424] The diagnostic performance levels of the method of the invention were determined on a total of 796 patients with a suspicion of thrombosis using the second version of the method of the invention. The results obtained by means of the method of the invention on the 8.3% of patients with a thrombosis, the 7.3% of patients with a cancer, on the patients with a predisposition to thrombosis, those with a hypercoagulation state and those with a coagulation activation state are presented in tables 18 to 21 below.

Diagnosis of Pulmonary Embolism (PE) and of Deep Vein Thrombosis (DVT)

[0425] The diagnostic performance levels of the method of the invention, obtained on 46 patients with a pulmonary embolism (PE), 16 patients with a deep vein thrombosis (DVT), 4 patients with both a PE and a DVT, 15 patients of whom have a history (HIST) of venous thrombosis, are described in table 18.

TABLE-US-00018 TABLE 18 Diagnostic performance levels of the method of the invention, obtained on the 66 patients with a diagnosis of thrombosis among the 796 patients with a suspicion of thrombosis. Method of the Diagnosis of Ddi Inflammation invention Ddi True positive (TP) thrombosis n (μg/ml) (DOD) (μg/ml) performance levels PE+ positive 46 5.2 4.8 1.43 97.8% TP 0.68-30 2.6-9.9 0.52-7.64 1 non-serious SSPE* DVT+ positive 16 4.1 4.4 1.28 100% TP 0.77-13 2.6-6.6 0.55-5.05 PE+ DVT+ positive 4 5.0 4.3 1.36 100% TP 1.15-10 3.3-5.4 0.61-3.03 HIST PE or DVT 15 5.8 4.5 1.83 100% TP 0.68-20 2.6-8.4 0.54-7.24 Negative superficial 3 1.1  4.0 0.60 2 false positive VT Negative venous 2 0.52 4.2 0.50 2 true negatives insufficiency *SSPE: subsegmental pulmonary embolism.

[0426] The results obtained show that the method according to the invention makes it possible to diagnose close to 100% of patients suffering from venous thromboembolism, including in the event of pulmonary infarction, the level of inflammation of which is high, and in the event of non-serious, isolated subsegmental pulmonary embolism, as shown in table 19. It should be noted that the levels of D-dimer which result from intravascular fibrin degradation that are determined are higher in the case of a history (HIST) of venous thromboembolism (pulmonary embolism or deep vein thrombosis), as shown in table 18.

TABLE-US-00019 TABLE 19 Diagnostic performance levels of the method of the invention, obtained on the 66 patients with a diagnosis of thrombosis among the 796 patients with a suspicion of thrombosis. Inflammation Method of the invention (DOD) Ddi (μg/ml) Diagnosis of thrombosis N D-dimers N: 2 to 4.5 g/l TP = True positives Massive, bilateral or 24 7.1 1.2-30.0 4.8 1.91 segmental PE 2.8-8.4 0.57-7.52 (100% TP) Distal PE 7 1.7 0.89-2.77 5.4 0.60 3.7-9.9 0.55-0.65 (100% TP) Pulmonary infarction 7 2.5 1.1-5.1 5.8 0.73 3.1-8.4 0.52-1.62 (100% TP) SSPE associated with PE 6 3.3 1.15-5.1 5.0 0.80 3.1-7.5 0.52-1.62 (100% TP) Isolated SSPE 6 1.9 0.68-3.8 4.2 0.62 2.6-6.9 0.54-0.58 (1 False negative)

Thrombosis Predisposition States

[0427] The 113 patients presenting a predisposition to thrombosis who were tested in this study were patients suffering from a known cancer (58 patients, including 47 diagnosed negative for VTE, and 11 diagnosed positive), patients suffering from an unknown cancer (21 patients, including 17 diagnosed negative and 4 diagnosed positive), pregnant women (14 patients, all diagnosed negative), women having given birth (4 patients, all diagnosed negative), patients in post-operative phase (2 patients, all diagnosed negative), and patients who had just taken a lengthy trip (14 patients, all diagnosed negative, except 1 diagnosed positive).

TABLE-US-00020 TABLE 20 Diagnostic performance levels of the method of the invention, obtained on 113 patients with a thrombosis predisposition state, 16 of whom have a diagnosis of thrombosis, among the 796 patients with a suspicion of thrombosis. Patients with a Method of thrombosis Diagnosis the predisposition of Inflammation invention Performance state thrombosis n Ddi (DOD) Ddi (μg/ml) levels Known cancer Positive 11 5.0 4.7 1.50 100% true (n = 58) positives Negative 47 1.7 4.7 0.68 53% exclusion Unknown Positive 4 6.4 4.2 2.0 100% true cancer (n = 21) positives Negative 17 2.0 4.8 0.67 48% exclusion Pregnancy Negative 14 0.78 3.8 0.56 93% exclusion Childbirth Negative 4 0.67 3.5 0.54 100% exclusion Post-operative Negative 2 1.40 3.9 0.61 100% exclusion Trip Positive 1 2.23 8.4 0.58 100% true N = 14 positives Negative 13 0.76 3.7 0.54 93% exclusion

[0428] The results obtained in the patients with a thrombosis predisposition state clearly show that the method according to the invention makes it possible to diagnose 100% of the patients suffering from venous thromboembolism. The method of the invention also allows the exclusion of more than 93% of the non-cancer patients, as shown in table 20. In the patients suffering from cancer, the method of the invention allows the exclusion of 50% of the patients, compared with 22% with the usual method for determining D-dimers.

Hypercoagulation State

[0429] The 436 patients with a hypercoagulation state who were tested in this study were patients suffering from thrombophilia (4 patients, all diagnosed negative for VTE, except 1 diagnosed positive), patients suffering from renal insufficiency (26 patients, all diagnosed negative, except 1 diagnosed positive), patients having suffered a trauma, a fall or a fracture (13 patients, all diagnosed negative), and a group of subjects aged 50 or over (393 subjects, including 357 diagnosed negative and 36 diagnosed positive).

TABLE-US-00021 TABLE 21 Diagnostic performance levels of the method of the invention, obtained on 436 patients with a hypercoagulation state, 38 of whom have a diagnosis of thrombosis, among 796 patients with a suspicion of thrombosis. Patients with a Method of hyper- Diagnosis the coagulation of Inflammation invention Performance state thrombosis n Ddi (DOD) Ddi (μg/ml) levels Thrombophilia Positive 1 4.0 4.8 0.82 True positive Negative 3 0.37 3.8 0.48 100% exclusion Renal Positive 1 2.2 8.4 0.58 True positive insufficiency Negative 25 3.0 5.1 0.78 50% exclusion (RI) nephropathy Trauma, fall, Negative 13 1.86 3.9 0.70 77% exclusion fractures Age ≥ 50 Positive 36 2.25 5.0 0.65 100% true Average age: positives 69 Negative 357 1.21 4.4 0.58 77% exclusion compared with 94% for < 50 years old

[0430] The results obtained in the patients with a hypercoagulability state clearly show that the method according to the invention makes it possible to diagnose 100% of the patients suffering from venous thromboembolism. The method of the invention also allows the exclusion of more than 75% of the patients, including in the elderly patients, as shown in table 21. Among the elderly patients not excluded by the method of the invention, ⅓ have a cancer and ⅔ are suffering from renal insufficiency or from coagulation activation.

Coagulation Activation States

[0431] The 144 patients with a coagulation activation state who were tested in this study were patients suffering from infections or from sepsis (5 patients, all diagnosed negative for VTE), patients suffering from pneumopathy, bronchitis or respiratory insufficiency (71 patients, all diagnosed negative, except 1 diagnosed positive), patients suffering from inflammatory diseases (6 patients, all diagnosed negative), patients suffering from gastritis (7 patients, all diagnosed negative), patients suffering from cardiomyopathies (45 patients, all diagnosed negative, except 1 diagnosed positive), and patients with a history of stroke (10 patients, all diagnosed negative).

TABLE-US-00022 TABLE 22 Diagnostic performance levels of the method according to the invention, obtained on 144 patients with a coagulation activation state, 2 of whom have a diagnosis of thrombosis, among the 796 patients with a suspicion of thrombosis. Patients with a Method of hyper- Diagnosis the coagulation of Inflammation invention Performance state thrombosis n Ddi (DOD) Ddi (μg/ml) levels Infections, Negative 5 2.88 5.3 0.80 100% exclusion sepsis Pneumopathy, Positive 1 2.76 9.9 0.56 True positive bronchitis, Negative 70 0.86 5.0 0.53 83% exclusion respiratory insufficiency Inflammatory Negative 6 2.16 4.3 0.85 100% exclusion diseases Gastritis Negative 7 0.72 4.3 0.51 86% exclusion Cardio- Positive 1 2.77 4.1 0.65 True positive myopathies Negative 44 1.19 4.3 0.57 66% exclusion (SCA, heart failure, etc.) HIST stroke Negative 10 0.74 4.0 0.53 90% exclusion

[0432] The results obtained in the 144 patients with a coagulation activation state clearly show that the method according to the invention makes it possible to diagnose 100% of the patients suffering from venous thromboembolism and also to exclude 80% of the negative patients (with the exception of the elderly patients who have a cardiomyopathy, of average age: 80), as shown in table 22.