In vitro measurement of fibrin clot lysis

Abstract

The invention relates to an in vitro method for measuring fibrin clot degradation based on a fibrin clot lysis curve over time in a blood or plasma sample previously obtained from a patient suspected of having a deficiency in at least one coagulation factor. This method includes determining a baseline fibrin clot level at time t1 and determining a degraded fibrin clot level at a subsequent time t2. The method may include an additional step of classifying the tested sample into a group reflecting a fibrin clot lysis profile. The invention also relates to a method for monitoring therapeutic treatment administered to a patient suspected of having, or who has, a deficiency in at least one coagulation factor. This method may further include a step of adjusting the patient's therapeutic treatment based on the classification obtained.The invention relates to enclosing the use of tranexamic acid (TXA) or a composition comprising it for use in a therapeutic treatment, said use comprising the implementation of a method according to the invention, and means for the implementation of the invention.

Description

Title of the invention: In vitro measurement of fibrin clot lysis. Technical field

[0001] The present disclosure falls within the field of hemostasis. More particularly, it relates to hemophilia and concerns a method for performing an in vitro measurement of the lysis curve of a fibrin clot over time from a blood or plasma sample, to enable the determination of patient profiles likely to be hemophiliac, to be diagnosed with hemophilia, or to have hemophilia, based on the lysis kinetics of said clots. In another aspect, the invention relates to the possibility of adapting treatment or choosing a treatment based on the physiological parameter observed according to the invention, for these patients.

[0002] The invention relates more specifically to an in vitro method for measuring the degradation of the fibrin clot from a lysis curve of a fibrin clot over time (kinetic curve) in a blood or plasma sample previously obtained from a patient likely to have a deficiency in at least one coagulation factor, the method comprising the following steps: a. mixing the previously obtained sample from said patient with a reagent composition comprising tissue factor, phospholipids, t-PA, and one or more activators of the intrinsic coagulation pathway, including one or more of these activators selected from: ellagic acid, silica, FIXa, FXIa, and optionally one or more coagulation factors lacking in the patient, including FVIII, FIX, FXI; then b. incubation of the mixture obtained in a., then c. triggering coagulation by adding calcium ions to the mixture incubated in step b., to allow the formation of a fibrin clot in the mixture, then lysis of the clot formed, and d. measurement of the kinetics of fibrin clot degradation during lysis in step c., and e. determination of a baseline level value of the fibrin clot at a time tl, the time tl being chosen to be located between the Tmax and the TL of the fibrin clot lysis curve, and of a degraded level value of the fibrin clot at a later time t2, the time t2 being chosen between 300 and 900 seconds after tl. This method includes determining a baseline level value of the fibrin clot at a time t1, and determining a degraded level value of the fibrin clot at a later time t2.

[0003] According to one aspect, the method may include an additional step f. of classifying the tested sample into a group reflecting a lysis profile of a fibrin clot, said lysis profile being determined on the basis of the values ​​measured at t1 and t2 in step e., with regard to a predefined classification model based on classification parameters obtained with training data processed under the same experimental conditions as those of the analyzed sample.

[0004] The invention also relates to a method for monitoring therapeutic treatment administered to a patient who may have or has a deficiency in at least one coagulation factor, involving the implementation of a method for carrying out an in vitro measurement of a lysis curve of a fibrin clot over time according to the invention, on a blood or plasma sample obtained from said patient, at at least one given time and possibly at another or more subsequent time(s), said method including a step f.The classification of the tested sample into a group reflecting a fibrin clot lysis profile, and based on the classification obtained for the analyzed sample within a group, a conclusion regarding the coagulation factor(s) deficiency of the analyzed patient observed by the classification method or regarding the patient's health status, and possibly a conclusion regarding the evolution of said coagulation factor(s) deficiency of the analyzed patient or the patient's health status if several classifications performed at separate and successive times are available. Such a method may also further include a step of adjusting the therapeutic treatment followed by said patient, according to the classification obtained.

[0005] The invention relates to enclosing the use of tranexamic acid (TXA) or a composition comprising it for use in the therapeutic treatment of a patient who may have or has a deficiency in at least one coagulation factor, in particular a patient diagnosed as hemophiliac, said use comprising the implementation of a method according to the invention for in vitro measurement of the degradation of the fibrin clot from a lysis curve of a fibrin clot over time (kinetic curve) in a blood or plasma sample previously obtained from a patient, where appropriate for classification of the sample, monitoring or adjustment of the therapeutic treatment of said patient.

[0006] The invention further relates to means for implementing the invention, i.e., a data processing system or a device comprising particular means for implementing a method according to the invention, or a computer program, or a non-transient recording medium readable by a computer on which is stored a computer program for implementing, when this program is executed by a processor, the method according to the invention for in vitro measurement of fibrin clot degradation from a lysis curve of a fibrin clot over time (kinetic curve) in a blood or plasma sample previously obtained from a patient, or a kit. A data processing system or device includes means for implementing at least step e of the in vitro method for measuring fibrin clot degradation from a lysis curve of a fibrin clot over time (kinetic curve) according to the invention, or step f.classifying the tested sample into a group reflecting a lysis profile of a fibrin clot according to the invention, and optionally also at least one other step of one of the method claims according to the invention, and optionally includes means for providing input and / or output the variables generated during these steps, to make the classification result taking into account the variables provided as input, and optionally also includes a device for measuring the kinetics of degradation of a fibrin clot, or uses a device for measuring the kinetics of degradation of a fibrin clot, in particular remote, and optionally also includes a processor adapted to implement said steps.A computer program includes program code instructions for performing the steps of the method according to the invention for in vitro measurement of fibrin clot degradation from a fibrin clot lysis curve over time (kinetic curve) in a blood or plasma sample previously obtained from a patient, when said program is run on a computer, in particular it includes code instructions that cause a data processing system or a device according to the invention, in particular a device including a kinetic measuring apparatus or a data processing system or a device using such a measuring apparatus, in particular remote, to perform at least step e.of the method according to the invention for in vitro measurement of fibrin clot degradation from a lysis curve of a fibrin clot over time (kinetic curve) in a blood or plasma sample previously obtained from a patient, or step f. of classifying the tested sample into a group reflecting a lysis profile of a fibrin clot according to the invention, and optionally also at least one other of the steps of the method according to the invention for in vitro measurement of fibrin clot degradation from a lysis curve of a fibrin clot over time (kinetic curve) in a blood or plasma sample previously obtained from a patient, for example step d. of this method. A kit adapted for the implementation of a method according to the invention for in vitro measurement of the degradation of the fibrin clot from a lysis curve of a fibrin clot over time. (kinetic curve) in a previously obtained blood or plasma sample from a patient includes: a. one or more of the following reagents: tissue factor, phospholipids, t-PA, an intrinsic coagulation pathway activator selected from elagic acid, silica, FIXa, FXIa, or several of these, calcium ions, b. Optionally, a coagulation factor chosen from factor VIII, factor IX, factor XI, or several of these, c. Optionally, one or more suitable stamps, d. Optionally, instructions for performing one or more fibrin clot degradation kinetics, and e. Optionally, a computer system and / or device and / or program and / or computer-readable data storage medium according to the invention, f. Optionally, instructions for implementing the in vitro method of measuring fibrin clot degradation from a fibrin clot lysis curve over time (kinetic curve) in a blood or plasma sample previously obtained from a patient according to the invention, g. Optionally, instructions relating to the use of a signal from a data carrier, for the implementation of an in vitro method for measuring the degradation of the fibrin clot from a lysis curve of a fibrin clot over time (kinetic curve) in a blood or plasma sample previously obtained from a patient according to the invention. Previous technique

[0007] Hemophilia is a congenital recessive disorder characterized by spontaneous or prolonged bleeding due to a deficiency in one of the coagulation factors (VIII, IX, or XI, also referred to as "FVIII," "FIX," and "FXI" in this description, for "factor VIII," "factor IX," and "factor XI," respectively). There are three types of hemophilia: Hemophilia A, Hemophilia B, or Hemophilia C. Hemophiliacs are generally classified into three classes based on their factor VIII level (FVIII - for hemophilia A, FIX - for hemophilia B, FXI - for hemophilia C): • Severe hemophilia (<1% of factor - 1% of factor is equivalent to 0.01 IU / mL by convention. The unit IU / mL is also used in this description instead of percentages.) • Moderate hemophilia (between 1 and 5% of factors); • Minor hemophilia (between 5% and 40% of factors).

[0008] In particular, FVIII deficiency can lead to insufficient fibrin production during coagulation. As a result, the clot is less resistant and forms with difficulty. The coagulolytic balance is compromised compared to a healthy individual. A person with hemophilia A will have a higher risk of bleeding in the event of trauma (injury) or spontaneously in the joints and muscles. The more severe the disease, the more frequent the bleeding. The frequency of bleeding is assessed by the Annual Bleeding Rate (ABR), which corresponds to the number of bleeding episodes per year.

[0009] However, the bleeding risk in hemophiliacs is highly heterogeneous: some patients with very low FVIII levels will have a low ABR, while patients diagnosed as moderate hemophilia A may bleed more. With FVIII replacement therapy, which is the treatment of choice to date, the goal is to raise the plasma concentration of FVIII to a level that minimizes the time spent below a threshold level (through level). However, this optimal FVIII threshold for suppressing bleeding is uncertain and is not correlated with the patients' clinical phenotype.

[0010] It has been suggested that hemostatic balance, and particularly the clot lysis process, can be altered in hemophilia patients. The association of low FVIII levels with hyperstimulation of the fibrinolytic system may predispose hemophilia A patients to bleeding. Pro- / anti-thrombotic proteins also have an influence (TFPI, Antithrombin, Proteins C and S). These multiple effects cause a difference between the clinical phenotype and FVIII concentration. Leong et al. in 2017 (Leong et al. 2017 Research and practice in thrombosis and haemostasis) indicate that the ability to form an effective hemostatic clot depends on more than just the FVIII level. They raise the possibility that the clinical variation in patient response to FVIII replacement therapy can be reduced, among other things, by modulating the contribution of fibrin, platelets, and erythrocytes to hemostasis.The formation of the fibrin clot and its fibrinolysis, particularly in hemophiliac patients, could influence the severity of the bleeding tendency.

[0011] Clinical studies suggesting dose adjustment of FVIII treatment based on adapted pharmacokinetic profiles have not achieved zero bleeding. The mean ABR during prophylaxis (1% FVIII) is 6.3 bleedings / patient / year. The wide range of the ABR [4.4–9.9] suggests that some patients require a higher dose of FVIII to prevent spontaneous bleeding. It is recognized that a level between 12 and 15% FVIII would prevent the maximum number of bleedings. This level is difficult to achieve in prophylaxis due to the short half-life and the cost of treatment.

[0012] The Thrombin Generation Test (TGT), the Close Waveform Analysis (CWA) test in aPTT, or viscoelastic tests (TEG / ROTEM) have been used in hemophilia A applications and are recognized as sensitive to FVIII variations (Chitlur 2012 Thrombosis Research, Challenges in the laboratory analyses of bleeding disorders). The through level based on the patient's ETP may be more reliable than the FVIII level at 1% (Dargaud 2017), but the latter is not optimal for monitoring hemophilia A patients (Tarandowsky 2013). It should be noted that Dargaud et al. found that 4 of the 11 patients (36%) with the lowest ETP did not have a clinical bleeding phenotype (Dargaud 2017).

[0013] The inventors note that the ideal hemostatic test should correlate the patients' biological phenotype with the clinical bleeding phenotype and allow for the determination of bleeding severity. Such characteristics would make it possible to tailor the treatment dosage to individual patients. To date, no test has been approved for monitoring hemophilia A patients and for predicting the predisposition of hemophilia patients to bleed (Tripodi 2019, Tarandowsky 2013). Indeed, a wide inter-patient difference exists in the response to FVIII therapy, and current global tests (TGT, CWA, and TEG / ROTEM) are not sufficiently sensitive to small variations in FVIII, particularly in the low range (Matsumoto 2009, Aghighi 2019).Tissue factor-activated TGT alone has relatively low sensitivity to deficiencies in intrinsic coagulation factors; it is necessary to modify it (diluted tissue factor, CTI, PRP) to achieve sufficient sensitivity. Furthermore, other factors, not explored in these tests, contribute to the severity of the phenotype (Aghighi 2019, Leong 2017, He 2018).

[0014] In 2020, three Novo Nordisk (Guardian) and three Bayer (Leopold) studies showed that inter-patient variability in the bleeding phenotype remains significant and unexplained, that the risk of bleeding can change over time, and that it is influenced by factors independent of FVIII pharmacokinetics and the threshold level. The need for a treatment individualization tool that takes into account personalized bleeding risk remains to this day (Tiede 2020, Abrantes 2019).

[0015] In addition to this scientific observation, clinical and economic considerations highlight the value of a lysis test addressing the issues outlined above in hemophiliacs. In France, the prevalence of hemophilia A is approximately 1 in 5,000 (with 50% of cases being severe hemophilia). Treatment with anti-hemophilic factors accounts for approximately 98% of the cost of care for a severe hemophiliac. Furthermore, in France (in 2003), 7,000 hospitalizations related to hemophilia A were recorded annually. Ultimately, today, the total costs incurred by a hemophiliac are significant. severe is €800,000 per year on average (Poster P190 ECTH 2018 B. Polack et al.) in France.

[0016] In France, according to the 2019 HAS study, 1944 patients are treated prophylactically: for the Advate® type molecule: 20 to 40 IU (International Units) of FVIII / kg every 2 to 3 days / Novoeight®: 20 to 40 IU of FVIII / kg every 2 days up to 50 IU 3 times / week; in case of bleeding, every 8 hours to every 2 days. For Hemlibra®, the target population is 1410 to 1880 patients, once / week or every 2 weeks for 4 weeks or every 4 weeks (HAS 2019 website and National Diagnostic and Treatment Protocol for Hemophilia V3 of 2019). By convention, 1 IU = 100% of FVIII (or FIX or FXI) as described in

[0007] of this description (IU corresponds to the same thing, in English).

[0017] In India, there are an estimated 170,000 hemophiliacs, of whom 100,000 have the severe form of the disease. With a prophylaxis of 3 IU / person, this would require a volume of 4 billion IU at $0.25 / IU. The total cost would amount to $1 billion / year (GFHT CoMETH 2019 - Symposium “Hemophilia in the World” “Epidemiological Data” by Alok Srivastava).

[0018] In 2018, the global market for the treatment of hemophilia A was $9.8 billion. This market is constantly growing, with an estimated $15.8 billion in 2026 (EAHAD 2020 - Symposium “Ethical aspect of gene therapy in children” by Rieke van der Graaf and Fortune Business Insights website “Hemophilia Drug Market”).

[0019] These examples illustrate that improving the cost-effectiveness balance is a real international challenge for the treatment of hemophiliacs and in particular hemophiliacs A.

[0020] The present invention relates, according to a particular embodiment, to a classification of hemophilia patients based on the lysis kinetics of the fibrin clot forming in a sample obtained from these patients, thus enabling the selection or personalization of the maintenance treatment followed by the patients. It follows that the present invention paves the way, at the laboratory level, for predicting the treatment best suited to each patient. For clinicians, the expected advantages are a better characterization of patients by their predisposition to bleeding, incorporating the variable of their low, moderate, or high bleeding risk, and a practical tool for tailoring treatment. Among the overall advantages enabled by the present invention are reduced treatment costs, improved quality of life for patients, and potentially a reduction in the annual bleeding rate.

[0021] A method for determining the structural profile of a fibrin clot reflecting its stability, for predicting the risk of WO 2016 / 012729, is known. bleeding, thrombosis, or re-thrombosis. However, the disclosed protocol does not provide for the possibility of classifying patients, particularly hemophiliacs, using a statistical or machine learning method, and lysis must be interpreted using two wavelengths during the turbidimetric test, which adds to the complexity of implementing the method. Finally, the starting reagents differ.

[0022] Furthermore, the prior art does not report any methodology for considering the degradation of a fibrin clot due to its lysis in hemophiliacs as the sole tool for personalizing missing factor therapy administered to a hemophiliac patient, whether or not associated with antifibrinolytic treatment. In contrast, the present invention enables such an implementation. Detailed description

[0023] This disclosure is intended to address the aforementioned issues.

[0024] A method is proposed for the in vitro measurement of fibrin clot degradation from a lysis curve of a fibrin clot over time in a blood or plasma sample previously obtained from a patient likely to have or having a deficiency in at least one coagulation factor, the method comprising the following steps: a. Mixing of the previously obtained sample from said patient with a reagent composition comprising tissue factor, phospholipids, t-PA (tissue plasminogen activator), and one or more activator(s) of the intrinsic coagulation pathway, including one or more of these activator(s) selected from: ellagic acid, silica, FIXa, FXIa, and optionally one or more coagulation factor(s) missing from the analyzed patient, including selected from: FVIII, FIX, FXI or a mixture of two or more of these factors; b. Incubation of the mixture obtained in a., then c. Initiation of coagulation by adding calcium ions to the mixture incubated in step b. to allow the formation of a fibrin clot in the mixture, then allowing lysis of the clot formed, and d. measurement of the degradation kinetics of the fibrin clot formed and degraded during lysis following step c., and e. determination of a baseline level value of the fibrin clot at a time tl, the time tl being chosen to be located between the Tmax and the TL of the fibrin clot lysis curve, and of a degraded level value of the fibrin clot at a later time t2, the time t2 being chosen to be from 300 to 900 seconds after tl.

[0025] The characteristics of the means employed in this method will be specified below.

[0026] The features described in the following paragraphs may optionally be implemented independently of each other or in combination with each other.

[0027] The Tmax of a fibrin clot lysis curve is a value easily determined by a person skilled in the art, and by conventional means. During the formation of a fibrin clot in a sample, which is naturally followed by fibrinolysis (the degradation process of the formed clot), a curve reflecting the state of the fibrin clot in the sample necessarily passes through a maximum (the point at which the measured value, for example, the maximum change in Optical Density (ODD), is reached during the entire measurement period). The time to reach this maximum is denoted Tmax. In this application, the term "fibrin clot lysis curve" means the curve measured and / or plotted at the times indicated in this description, according to any embodiment, including the area corresponding to the formation of the fibrin clot and the area corresponding to the degradation of the fibrin clot.The "fibrin clot lysis curve" is thus synonymous, unless a different interpretation arises from the context, with the "generation and lysis curve" or the "measured curve" or the "plot curve", in its entirety.

[0028] The TL of a fibrin clot lysis curve is also a value easily determined by a person skilled in the art, and in a conventional manner. It is the time value corresponding to 50% of the maximum value (measured at Tmax), determinable once the return to the initial amplitude of the fibrin clot value has been observed in the tested sample, that is to say, the amplitude observable before the generation of the fibrin clot.

[0029] Thus, the time tl is a time located between the Tmax and the TL of the fibrin clot lysis curve, which can therefore be located either in the plateau (between Tmax and T90% of the max) of the curve, or after the plateau (between T90% of the max and TL).

[0030] According to the invention, the time t2 is chosen within a fixed range of values ​​after t1. Conventionally, a generation and lysis curve of a fibrin clot starts after the triggering of the reaction by the addition of calcium ions (which corresponds to the time t0).

[0031] The optimal choice of times t1 and t2 within the aforementioned ranges can be made by a person skilled in the art by determining the times that yield the best quality results in quadratic discriminant analysis. For example, the inventors determined the percentage of correct classification in quadratic discriminant analysis initially using a single parameter, as described in [Fig. 15]. Subsequently, the inventors worked with a combination of parameters, as described in [Fig. 16]. We can see that the observed variation in correct classification percentages allows a person skilled in the art to easily verify whether the times t1 and t2 used are optimally chosen.

[0032] According to the invention, the method can be performed on the basis of a blood or plasma sample previously obtained from a patient who is likely to have, or has, a deficiency in at least one coagulation factor. In particular embodiments, the missing coagulation factor(s) is / are chosen from: factor VIII, factor IX, factor XI. In other aspects, the method can be performed on a sample: i. Whose patient is undergoing therapy with supplementation containing at least one coagulation factor, recombinant or non-recombinant, chosen from: factor VIII (recombinant or non-recombinant), factor IX (including factor IX concentrate) (recombinant or non-recombinant), factor XI (recombinant or non-recombinant), and / or ii. Whose patient is undergoing therapy with bispecific antibody treatment, for example, emicizumab or Hemlibra® (Roche) or iii. Whose patient is undergoing therapy with factor VIIa, recombinant or not, for example, NovoSeven® (NovoNordisk), or iv. Whose patient is undergoing therapy with a treatment directed against both hemophilia A and hemophilia B, such as Fitusiran® ​​(Sanofi) or Concizumab® (Novo Nordisk) or Marstacimab® (Pfizer), or v. Whose patient is under therapy with an anti-fibrinolytic treatment, for example Exacyl® or tranexamic acid (Cheplapharm).

[0033] Fitusiran® ​​is an AT complementary RNAi, a coagulation inhibitor.

[0034] Marstacimab® and Concizumab® are TFPI-specific antibodies, also coagulation inhibitors.

[0035] In the case of a patient undergoing antifibrinolytic therapy, this type of treatment is generally considered today for hemophiliacs undergoing dental extractions in severe, moderate, and mild hemophilia without inhibitors (HAS 2019, Steve Chaplin 2016 DOI 10.17225 / jhp00085). This treatment is also known to prevent bleeding in polytrauma patients. The literature reports the use of tranexamic acid in clinical practice at doses > 1 g three times daily (Forbes et al. 1972 DOI 10.1136 / bmj.2.5809.311), which can be considered equivalent to a dose > 15 mg / kg in a hemophiliac patient. Pharmacokinetic data for tranexamic acid show that at 15 mg / kg, the plasma concentration is 2 pg / mL (Steve Chaplin 2016 DOI 10.17225 / jhp00085).

[0036] If it is anticipated that at this plasma concentration of 15 mg / kg of tranexamic acid in a hemophiliac patient, a lysis curve of a fibrin clot will remain usable, according to a particular embodiment, it is preferred that the The plasma concentration of tranexamic acid in the tested sample does not exceed 5 pg / mL. It can be predicted that TXA treatment resulting in a plasma concentration of 5 pg / mL of tranexamic acid in the sample would lead to an optical density change (ODC) at time t2 that is identical to the ODC at time t1. While a lysis curve can still be obtained, this situation would make interpreting the curve for the purpose of drawing conclusions difficult. However, monitoring remains possible, particularly for dosages resulting in low plasma concentrations of TXA, as the method also allows for a significant variation in plasma TXA concentration given the dosages generally used.

[0037] In one embodiment, the analyzed samples come from patients diagnosed with hemophilia. In specific embodiments, the patients are hemophiliac A (FVIII deficiency), hemophiliac B (FIX deficiency), or hemophiliac C (FXI deficiency). The reference method for diagnosing hemophilia is the measurement of Factor VIII or Factor IX levels, which confirms severe hemophilia when the level is less than 1% of the expected normal level, moderate hemophilia when it is between 1% and 5%, and mild hemophilia when it is between 5% and 40% (missing factor levels presented above). However, as previously stated, the missing factor level does not predict the risk of bleeding. Two types of conventional methods exist.The "one-stage clotting assay" method, which is derived from the measurement of TCA, or the "chromogenic assay" method, which is based on the indirect measurement of FVIII by generation of FXa and cleavage of a specific substrate. Reference is made to the literature: Srivastava et al. 2013 DOI 10.1111 / hae. 14046 and Srivastava et al. 2020 DOI 10.1111 / hae. 14046 - § Recommendation 3.2.9.

[0038] According to a particular embodiment, in the case where the analyzed sample comes from a patient who is under therapy with an antifibrinolytic treatment which is tranexamic acid, and in particular, but not necessarily, that this patient has been diagnosed as hemophiliac, the sample analyzed in the method described here is a sample in which the plasma concentration of tranexamic acid in the tested sample does not exceed 5 pg / mL.

[0039] According to one embodiment, the blood or plasma sample is an undiluted sample of whole blood or plasma, in particular platelet-rich plasma or platelet-poor plasma, plasma containing platelet microparticles, erythrocytes or any other cell, preferably is a platelet-poor plasma sample.

[0040] According to a particular embodiment, if the sample analyzed is a whole blood sample, t2 is chosen 650 seconds after t1.

[0041] According to a particular embodiment, if the sample analyzed is a plasma sample, t2 is chosen 600 seconds after t1.

[0042] According to a particular embodiment, in step a) of the method, tissue factor, phospholipids, intrinsic coagulation pathway activator, t-PA (tissue plasminogen activator), and optionally coagulation factor missing in the analyzed patient are premixed, and then the whole is added to the blood or plasma sample to be analyzed, which is undiluted.

[0043] According to a particular embodiment, the sample is added in a volume of 200 pL, for a final reaction mixture volume of 300 pL.

[0044] According to a particular embodiment, in step a., the tissue factor is present in the reagent composition in such quantity that the final concentration of tissue factor in the mixture on which the kinetic measurement is carried out in step d. is between 0.01 and 5.0 pM.

[0045] According to a particular embodiment in which the analyzed sample is a whole blood sample, the tissue factor is, in step a., present in the reagent composition in such quantity that the final concentration of tissue factor in the mixture on which the kinetic measurement is carried out in step d. is between 0.01 and 5.0 pM, or between 2.0 and 5.0 pM, in particular is 2.0 pM.

[0046] According to a particular embodiment in which the analyzed sample is a plasma sample, the tissue factor is, in step a., present in the reagent composition in such quantity that the final concentration of tissue factor in the mixture on which the kinetic measurement is carried out in step d. is between 0.01 and 1.0 pM, or between 0.1 and 0.7 pM, more preferably between 0.3 and 0.6 pM, in particular is 0.5 pM.

[0047] According to a particular embodiment, in step a., the phospholipids are present in the composition of reactants in such quantity that the final concentration of phospholipids in the mixture on which the kinetic measurement is carried out in step d. is between 1 and 10 pM, or between 3 and 7 pM, more preferably between 3 and 5 pM, in particular is 4 pM.

[0048] According to a particular embodiment, in step a., the activator of the intrinsic coagulation pathway, in particular chosen from one or more of: ellagic acid, silica, FIXa, FXIa is present in the reagent composition in such quantity that the final concentration of activator in the mixture on which the kinetic measurement is carried out in step d. is between 1 and 1000 pM, or between 10 and 200 pM, more preferably between 75 and 200 pM, in particular is 100 pM, in particular the activator is FXIa at 100 pM.

[0049] According to a particular embodiment, in step a, the activator of the intrinsic coagulation pathway, when it is ellagic acid or silica, is present in the reagent composition in such quantity that the final concentration of activator in the mixture on which the kinetic measurement is carried out in step d. is between 1 and 1000 pM, or between 10 and 900 pM, or between 20 and 800 pM, or between 30 and 700 pM, or between 40 and 600 pM, or between 50 and 500 pM, or between 60 and 400 pM, or between 70 and 300 pM, or between 80 and 200 pM, or between 90 and 100 pM, in particular is 1000 pM.

[0050] According to a particular embodiment, in step a., the activator of the intrinsic coagulation pathway, when it is FIXa or FXIa, is present in the reagent composition in such quantity that the final concentration of activator in the mixture on which the kinetic measurement is carried out in step d. is between 1 and 200 pM, or between 10 and 200 pM, more preferably between 75 and 200 pM, in particular is 100 pM, in particular the activator is FXIa at 100 pM.

[0051] According to a particular embodiment, in step a., t-PA (tissue plasminogen activator) is present in the reagent composition in such quantity that the final concentration of t-PA (tissue plasminogen activator) in the mixture on which the kinetic measurement is carried out in step d. is between 0.01 and 5 pg / mL, or between 0.1 and 2 pg / mL, in particular is 0.13 pg / mL.

[0052] According to a particular embodiment, in step a., the coagulation factor missing in the analyzed patient, in particular chosen from one or more of: FVIII, FIX, FXI, is present in the mixture on which the kinetic measurement is carried out in step d., in a concentration between 0 and 0.4 IU / mL, or between 0 and 0.2 IU / mL, or between 0 and 0.1 IU / mL, or between 0 and 0.05 IU / mL, or between 0 and 0.025 IU / mL, in particular in a concentration of 0.015 IU / mL.

[0053] It is understood that the missing factor may or may not be present. Indeed, although a "missing factor" may be deficient in a patient, it will be understood that a patient diagnosed with hemophilia is generally treated to compensate for this absence by administering the missing factor, so it is expected that the analyzed sample will contain a concentration as mentioned above without any particular procedure being taken to achieve this. Conversely, it is also possible, according to a particular embodiment, to provide a quantity of the missing factor by external addition to the mixture to be analyzed, or to systematically supplement the samples to be analyzed by external addition to the mixture to be analyzed. While not essential to the implementation of the method described here, homogeneous supplementation can indeed help to normalize the results.

[0054] According to a particular embodiment, in step b, the incubation of the mixture obtained in step a is carried out between 20 and 39°C, preferably at 37°C for 2 to 10 minutes, in particular 5 minutes, and then the addition of calcium ions to the incubated mixture is carried out in an amount allowing a final concentration of calcium ions between 5 and 25 mM, preferably 17 mM.

[0055] According to a particular embodiment, the blood or plasma sample obtained from the patient and used for the method has a volume between 5qL and 500 qL, preferably between 50 qL and 400 qL, preferably between 50 qL and 300 qL, preferably between 100 qL and 300 qL, preferably about 200 qL.

[0056] According to distinct particular embodiments, the degradation kinetics of the fibrin clot due to its lysis in step d of the method is obtained by any method for measuring the degradation kinetics of said fibrin clot due to its lysis, in particular a method chosen from: a viscoelastic method, a rheometric method, an acoustic method, an optical method, a waveform analysis method, a fluorometric method, a magnetic resonance method, a turbidimetric method, in particular is carried out by turbidimetry (absorbance and transmittance) or by a viscoelastic method (for example ROTEM, or Quantra).

[0057] The evolution of a fibrin clot formed following thrombin generation, as implemented in the present method, is independent of the type of measurement method used to track its evolution kinetics. This evolution necessarily involves a stage of fibrin clot degradation due to lysis. The method described here is based on tracking the degradation kinetics of the fibrin clot during lysis. Generally, a lysis curve of a fibrin clot is followed over time until it returns to its initial amplitude, that is, the amplitude observed before the fibrin clot was generated. It follows from the above that any method that allows tracking the evolution of fibrin clot lysis, particularly over a sufficiently long period to observe a return to the initial amplitude, can be suitable for implementing the invention described herein.The literature includes examples of the implementation of various methods in a conventional manner. Examples are cited here. This application presents examples employing a turbidimetric method for measuring the optical density (also called absorbance) of the analyzed sample over time. Results obtained using a viscoelastic measurement method (where a signal amplitude in millimeters is measured) in whole blood are also presented. These embodiments are not, however, limiting, since the principle on which the present invention is based—namely, observing the lysis kinetics by means of such a curve and subsequently processing the values ​​extracted from it—is independent of the measurement method used. Originally, the measurement method of the invention takes into account not only the rate of degradation as relevant parameters. of a fibrin clot in an analyzed sample, but also the basal level of said clot.

[0058] As indicated above, clot lysis is conventionally observed over a period that includes the return to the initial amplitude, at which point the clot is completely degraded. The return time to the initial amplitude can vary depending on the measurement method used and is therefore not a limiting factor. Observation will be continued for as long as necessary.

[0059] According to a particular embodiment involving a turbidimetric measurement method, specifically by measuring the change in Optical Density (OD) of the analyzed sample (also called Absorbance in the literature, with arbitrary values ​​because they are dimensionless, related to the type of instrument used and a control sample for calibration), the measurement of the degradation kinetics is carried out at a wavelength between 350 and 800 nm, preferably at 540 nm, and for a duration between 1400 and 3600 seconds, or between 1400 and 2400 seconds, from the onset of coagulation by the addition of calcium ions. It should be noted that in this description, the term "OD" is used to signify this measured "change in optical density." This requires measuring an optical density (OD) at a (lysis) curve in order to determine its value on the curve.The expressions "DDO parameter" or "two-time DDO parameter" are also used and mean the same thing, namely the measurement of an Optical Density value. The invention is based on measuring two Optical Density (OD) values ​​at two different times on a lysis curve as measured in the present invention. Therefore, measuring an optical density parameter at a given time corresponds to measuring a "DDO" parameter in this description.

[0060] According to a particular embodiment involving a measurement method by viscoelastometry, in particular by measuring a curve amplitude in millimeters, the measurement of the degradation kinetics is carried out over a period of between 1400 and 3600 seconds from the triggering of coagulation by the addition of calcium ions.

[0061] According to the invention, in step e of the method, two values ​​are determined during this observation period: a basal level value of the fibrin clot at a time t1 of lysis and a degraded level value of the fibrin clot at a time t2, the time t2 being subsequent to the time t1.

[0062] The measurement taken at time tl corresponds to a value reflecting the end of clot polymerization (after its stabilization and before or at the beginning of its lysis), or the initial phase of fibrin clot lysis, tl being chosen in the plateau portion or at the beginning of the lysis curve. Indeed, tl is a time located between Tmax and TL of the fibrin clot lysis curve, which can therefore be located either in the plateau (between Tmax and T90% of the max) of the curve, or after the plateau (between T90% of the max and TL).

[0063] The measurement taken at time t2 corresponds to a value reflecting the degraded or partially degraded fibrin clot.

[0064] Reference is made to the description above regarding a more precise determination of the t1 and t2 times that can be retained. Those skilled in the art can easily verify whether the chosen t1 and t2 times are appropriate. t1 and t2 may also vary depending on the concentration of the t-PA activator (tissue plasminogen activator) used in vitro in the method and possibly on the type of method employed (e.g., turbidimetric or viscoelastic method). The t1 and t2 times, however, remain within the measurement ranges described here and can be optimized by verifying the values ​​that give the best classification percentages in quadratic discriminant analysis, particularly under the following sample measurement conditions:

[0065] A) For a turbidimetric method: FT (tissue factor) at 0.5 pM and PPL (phospholipids) at 4 pM are mixed with FXIa at 100 pM, and t-PA (tissue plasminogen activator) at 0.13 pg / mL (final plasma test values),

[0066] B) For a viscoelastic method: FT (tissue factor) at 2.2 pM and PPL (phospholipids) at 8 pM are mixed with FXIa at 200 pM, and t-PA (tissue plasminogen activator) at 0.07 pg / mL (final values ​​of the whole blood test).

[0067] According to an embodiment where the measurement is made by turbidimetry, the time t1 is chosen in the range between 700 and 900 seconds from the triggering of coagulation by the addition of calcium ions and the time t2 is chosen in the range between 1100 and 1400 seconds from the triggering of coagulation by the addition of calcium ions.

[0068] According to a particular embodiment, preferred in the case of turbidimetry, t1 is defined between 700 and 800 seconds, preferably is defined at 750 seconds and t2 is defined between 1300 and 1500 seconds, preferably is defined at 1400 seconds.

[0069] In a particular embodiment where the measurement is made by turbidimetry, t1 is defined at 750 seconds from the triggering of coagulation by the addition of calcium ions and t2 is defined at 1400 seconds from the triggering of coagulation by the addition of calcium ions.

[0070] It should be noted that the choice of times t1 and t2 can be made, in a viscoelastic measurement method, as in a turbidimetric method as previously described, based on the correct classification in quadratic discriminant analysis. Indeed, as shown in [Fig. 17], the number of correct classifications obtained is 100% when using the same time values ​​as those used in the turbidimetric method (750 sec and 1400 sec). Therefore, the times indicated above for a method where the measurement is made by turbidimetry can also be transposed to a viscoelastic measurement method.

[0071] According to a particular embodiment where the measurement is made by a viscoelastic measurement method, the time t1 is chosen in the range of 900 to 1200 seconds from the triggering of coagulation by the addition of calcium ions and the time t2 is chosen in the range of 1500 to 1800 seconds from the triggering of coagulation by the addition of calcium ions.

[0072] According to a particular embodiment where the measurement is made by a viscoelastic measurement method, t1 is defined at 900 seconds from the triggering of coagulation by the addition of calcium ions and t2 is defined at 1500 seconds from the triggering of coagulation by the addition of calcium ions.

[0073] According to a particular embodiment where the measurement is made by a viscoelastic measurement method, t1 is defined at 1200 seconds from the triggering of coagulation by the addition of calcium ions and t2 is defined at 1800 seconds from the triggering of coagulation by the addition of calcium ions.

[0074] The values ​​obtained (measured on the curve, or measured at t1 and t2) to quantify the basal level of the fibrin clot and the degraded level of the fibrin clot constitute parameters of the kinetics of fibrin clot degradation, measured at two time points. The measurements performed at t1 and t2, regardless of their expression (for example, absorbance for a turbidimetric method or amplitude in millimeters for a viscoelastic method), provide information concerning both the rate of lysis and the basal level of the fibrin clot, measured at t1, which "reflects the end of clot polymerization" in the initial state. Indeed, as shown in [Fig. 2], the experimentally observed lysis kinetics allow for the distinction of several different profiles.

[0075] The group bearing the number 3 (Group 3) on [Fig.2] and in the present description, has a low basal level of fibrin clot and shortened lysis.

[0076] The group bearing the number 2 (Group 2) on [Fig.2] and in the present description, has a basal level of intermediate fibrin clot and normal lysis.

[0077] The group bearing the number 1 (Group 1) on [Fig.2] and in the present description, has a high basal level of fibrin clot and delayed lysis.

[0078] In parallel with the observation of the aforementioned differences in lysis profile, the inventors were able to statistically identify three groups of samples among those tested, based on the two 2-time optical density (OD) parameters reported herein, using a centered and normalized hierarchical analysis, as also described in the experimental section. Finally, a classification into three patient groups was confirmed on another dataset from newly selected patients at a different site.

[0079] However, it is anticipated that this number of 3 groups retained in the present application, with regard to the sets of samples analyzed, the results of which are set out, This may vary depending on the samples selected. It is indeed possible, for example when samples with a 0% missing factor rate are included, to have more groups in order to allow for the specific discrimination of untreated plasmas.

[0080] For example, using the database employed for the results described here, and a 90% similarity criterion employed in a standardized hierarchical analysis, a total of 8 groups were identified. Conversely, implementing a full standardized hierarchical analysis with 2 groups allowed for the separation of group 3 (with the 0% concentrations of group 2) from the other 2 groups.

[0081] The inventors then obtained a correct classification rate of 100% in quadratic discriminant analysis and 99% (3 errors) by the normalized K-means method (analyses carried out on Minitab).

[0082] Thus, in addition to characterizing the behavior (profile) of a fibrin clot in a patient, the data pairs consisting of the values ​​obtained at times t1 and t2, and the times t1 and t2, can also, according to another aspect of the invention, serve as classifiers to distinguish the different profiles mentioned above. The parameters determined in the present invention can therefore, according to another aspect of the invention, classify an analyzed sample based on the behavior of the clot during lysis, and in particular based on the rate of lysis but also on the baseline level of the fibrin clot, measured at t1, which "reflects the end of clot polymerization," relative to a training group constructed under the same conditions.

[0083] In this text, the terms “classification” and “classification” may be used interchangeably and are synonymous.

[0084] According to the invention, any unsupervised analysis method can be used to classify into groups. Such a method can optionally be supplemented by any supervised method to verify the classification. Similarly, any supervised analysis method can be used to predict a classification.

[0085] In this regard, when an unsupervised statistical learning method is used, the number of groups is an input for implementing the method, allowing a person skilled in the art to adapt the implementation of the method described herein to their investigative needs. The elements presented herein enable a person skilled in the art to choose the most appropriate embodiment for their investigative needs.

[0086] According to different independent embodiments, a classification model can be used, where appropriate, to define groups.

[0087] A classification model is obtained by an unsupervised or supervised learning method trained on training data processed in the same experimental conditions as those applied to the blood or plasma sample whose analysis is sought by the method described here.

[0088] According to the invention, an unsupervised analysis method can be chosen from: a hierarchical analysis, in particular a centered and normalized hierarchical analysis or a K-means method.

[0089] The relevant parameters for implementing such unsupervised methods are: a. The number of groups or similarity b. The bonding method c. Measuring the distance d. Standardization

[0090] In the experimental part of this application, the parameters used were: a: 3 groups, b: centered correlation, c: Euclidean distance, and d: normalized values. Those skilled in the art can refer to the literature, or even simply to the Minitab software documentation (Group Observations - General Information), for the implementation of such a method with its various parameters.

[0091] According to the invention, a supervised analysis method can be chosen from: a discriminant (quadratic) analysis, logistic regression or a “random forest” method.

[0092] The relevant parameters for implementing such supervised methods are: a. The discriminant function: Linear or quadratic b. Random forest method: the number of factors per tree / number of trees c. Logistic regression: multinomial (logistic regression) / reference grouping

[0093] In the experimental part of this application, the parameters that were used are: a: quadratic discriminant analysis (bibliographic reference: Minitab software documentation (General)), b: two factors per tree, 3000 trees (bibliographic reference: Leo Breiman 2001 Random Forest, Machine Learning 45, 5-32), c: Multinomial logistic regression with group no. 2 chosen as the reference group chosen no. 2 (similar analysis obtained with reference group no. 1 or no. 3) (bibliographic reference: R software: Fit Multinomial Log-linear Models (r-project.org) and Venables and Ripley 2002 Modern Applied Statistics with S. Fourth edition).

[0094] A person skilled in the art can refer to the aforementioned literature for the implementation of these methods, with their various parameters.

[0095] The groups obtained by learning reflect different degradation behaviors due to the lysis of a fibrin clot in patients as represented in the learning database.

[0096] According to a particular embodiment, the supervised learning method used is a discriminant analysis method, more specifically, a quadratic discriminant analysis method. The implementation characteristics of such a method are known in the literature. It can be implemented using numerous statistical tools or software programs, for example Minitab 18 and R.

[0097] The statistical analysis of all the samples is carried out using statistical software, preferably Minitab 18 and R software.

[0098] The training data can be conventionally defined by a person skilled in the art. In this case, the experimental part provides complete guidelines for the sampling chosen to constitute the test database.

[0099] The unsupervised or supervised statistical learning method used results in the determination of a model or classification parameters that are thus defined and therefore predetermined. In particular, the two parameters at t1 and t2 are used for the statistical analysis. These predefined models or classification parameters then allow, if necessary, during the classification step of a method as described here, the analyzed sample to be classified into a group, with regard to the values ​​measured at t1 and t2 in step e for the analyzed sample (which serve as classifiers).

[0100] The experimental section shows that, in a model-based prediction context, it was possible to reliably allocate each analyzed sample to a group reflecting a particular behavior or lysis profile of the fibrin clot. In particular, [Fig. 4] shows that it was possible to define three groups with no overlap between them.

[0101] The three groups, labeled 1 to 3, defined in the experimental section using a turbidimetric measurement method under the aforementioned t1 and t2 time definition conditions for plasma from hemophilia A patients, correspond to three typical profiles of fibrin degradation in this patient group, as shown in [Fig. 2]. Each group has its own fibrinogen level ([Fig. 5] and [Fig. 9]). Group 3, defined here, is the group with a shortened lysis time.

[0102] According to another aspect, it is also envisaged to have a method for determining a patient's clinical phenotype, based on their fibrin degradation profile, in particular to have a clinical bleeding phenotype, which indicates the bleeding tendency of said patient. Indeed, it is anticipated that a patient's predisposition to bleed differs according to the different profiles analyzed here. According to this aspect, the determination of the clinical phenotype, in particular the phenotype Clinical hemorrhagic phenotype determination takes advantage of a method such as the one described herein, involving at least one step (e) of determining values ​​at times t1 and t2, as detailed in this application. In one aspect, the determination of the clinical phenotype, particularly the clinical hemorrhagic phenotype, also benefits from a classification, as described and further developed in this application.

[0103] In fact, based on the classification that can be carried out according to their fibrin clot degradation profile group following the lysis of said clots, it is anticipated that the plasmas of the analyzed patients will react differently to a therapeutic treatment (supplementation with a missing factor or administration of an antifibrinolytic) to which the patient will be subjected. Reference is made in particular to the experimental data corresponding to Figures 7A and 7B.

[0104] Thus, according to another aspect of the invention, a method is proposed for monitoring therapeutic treatment administered to a patient who may have or has a deficiency in at least one coagulation factor, comprising the following steps: a. Implementation of a method for performing an in vitro measurement of a fibrin clot lysis curve over time according to any of the embodiments described herein, including one involving a measurement by tubidimetry, on a blood or plasma sample obtained from said patient, at at least one given time and possibly at one or more subsequent time points, said method including a step f. of classifying the tested sample into a group reflecting a fibrin clot lysis profile; and b. Based on the classification obtained for the analyzed sample in a group reflecting a lysis profile of a fibrin clot in said patient, conclusion concerning the deficiency in coagulation factor(s) of the analyzed patient observed by the classification method or concerning the state of health of said patient, and possibly conclusion concerning the evolution of said deficiency in coagulation factor(s) of the analyzed patient or of the state of health of said patient if several classifications carried out at separate and successive times are available.

[0105] The definitions and descriptions provided above in the context of the previously described method are used. The relevant descriptions also apply to this aspect, and the features set forth throughout this description may be incorporated. independently of each other or in combination with each other, in the present aspect.

[0106] Hemophilia patients are generally treated for their condition and therefore follow a maintenance therapy, the dosage of which is not necessarily always optimized, particularly with a view to bringing them to a clinical profile that limits risks such as, for example, the annual bleeding rate, or simply to ensure an improvement in their quality of life. It is here that the classification enabled by the present invention makes it possible to personalize either the threshold level of the maintenance therapy, or to consider complementary treatment with an antifibrinolytic agent, or to adjust both types of treatment, for these purposes. As indicated in the experimental part for hemophilia A patients.

[0107] The possible types of disease-modifying therapies for hemophilia A, B, or C patients consist of the administration of factor VIII, IX, or XI, respectively. Known commercial formulations of factor VIII include, in particular, the drugs Advate® (Shire), Elocta® (Sanofi), and Jivi® (Bayer).

[0108] Antifibrinolytic treatments are also known, notably the administration of tranexamic acid (TXA), a synthetic derivative of lysine (medicines Lysteda® or Cyklokapron® (USA), Transamin® or Transcam® in Asia, Espercil® in South America, and Exacyl® or Spotof® in Europe), prescribed in cases of excessive bleeding, by inhibiting the fibrinolysis system. Tranexamic acid blocks the binding of the lysine residue of fibrin by plasmin, the enzyme responsible for fibrinolysis. The normal action of plasmin, namely dissolving the clot (fibrinolysis), is thus blocked. It is prescribed in moderate to mild hemophilia without inhibitors or in cases of surgery (HAS 2019).

[0109] The experimental section (Figure 7) shows that the addition of TXA to background therapy modifies the lysis kinetics of a fibrin clot. In particular, the lysis kinetics following the addition of TXA to background therapy become slower. Therefore, in this specific case, it is group 3 of patients described here, with a shortened lysis time, who would benefit most from adjusting their treatment with the addition of TXA. It becomes possible to move these patients to group 2 following a treatment adjustment (Figure 7). Similarly, the invention allows for the personalization of the threshold level of background therapy, based on an initial classification or at a given time in a therapeutic protocol.

[0110] By "conclusion concerning the state of health of said patient" is thus referred to the conclusions made possible from a classification into a group reflecting the lysis profile of a fibrin clot in a patient, in particular concerning the question of whether the clot lysis behavior is abnormal.

[0111] Alternatively, the method described here simply allows the lysis profile of a fibrin clot in a patient to be qualified by classification into a group as a physiological parameter, allowing, where appropriate, for adjustments to treatment dosage to vary this physiological parameter, in particular in relation to pre-established criteria.

[0112] The term "conclusion concerning the evolution of the patient's health status if several classifications carried out at separate times are available" refers to the reiteration of the classification at one or more time intervals for the same patient, in order to assess, by comparison with previously available data, the evolution of the patient's situation, taking into account, where applicable, a change in their treatment, or the absence of a change in their treatment. For example, following treatment, a patient may or may not change group. Similarly, following the absence of treatment, a patient may or may not change group.

[0113] According to a particular embodiment, the monitoring method further includes a step of adjusting the therapeutic treatment followed by said patient, according to the classification obtained.

[0114] By way of example, administration of antifibrinolytic therapy may be offered to a patient whose sample has been classified as group 3, with the aim of prolonging the fibrin clot lysis time in that patient. In the case of patients in group 1 or 2, an adjustment of the background therapy (dosage of the missing factor) may be offered to optimize treatment based on the effects obtained on fibrin clot lysis. As another example, both antifibrinolytic therapy and an adjustment of the background therapy may be offered.

[0115] According to specific embodiments, the adjustment of the therapeutic treatment followed by said patient, based on the classification obtained, may consist of: a. If the classification reveals a high risk of bleeding: assessment of the opportunity for or switch to a background treatment supplemented by an antifibrinolytic treatment, for example tranexamic acid (TXA); b. If the classification reveals a low risk of bleeding: assessment of the opportunity to or adjustment of the background treatment to reduce the dosage; c. If the classification reveals a moderate risk of bleeding: assessment of the opportunity to or adjustment of the background treatment to increase the dosage.

[0116] A particular example presenting specific adjustment proposals is included in the experimental part (Table 3): the proposed treatments can be combined with the general presentation given above.

[0117] According to another aspect, the use of tranexamic acid (TXA) or a composition comprising tranexamic acid (TXA) is proposed for use in the treatment of a patient who may have or has a deficiency in at least one coagulation factor, in particular a patient diagnosed as hemophiliac, said use including the performance of a measurement method on a sample of said patient according to any of the embodiments described herein, where appropriate with a classification of the sample, or monitoring or adjustment of the therapeutic treatment of said patient.

[0118] The aforementioned definitions apply identically in the context of this aspect.

[0119] According to a particular embodiment, the use of tranexamic acid (TXA) or a composition comprising tranexamic acid (TXA) in the treatment of a patient on the blood or plasma sample of which a classification or monitoring method according to any of the embodiments described herein has been implemented, applies in the case where said blood or plasma sample tested has been classified in a group indicating a reduced fibrin clot lysis time, of type group 3 described herein (determination of a high risk of bleeding - see experimental part, combinable with the general presentation given herein).

[0120] Furthermore, it is noted that the step involving the lysis kinetics of a fibrin clot in the methods described herein can advantageously be carried out on an automated diagnostic device, preferably a coagulation analyzer. Such a device can also include computer means for data processing, thus making it possible to carry out, with a single automated device or via suitable means, possibly remote, at least one other step of the methods described herein that can be implemented through a computer program or processor.

[0121] According to another aspect, a data processing system or device is thus proposed comprising means for implementing at least step e of the method described herein or step f of the classification method described herein, according to any of the embodiments described, and where appropriate also at least one other of the steps described in an embodiment of this description, and optionally comprising means for providing input and / or output the variables generated during these steps, for returning the classification result taking into account the variables provided as input, and optionally also comprising a device for measuring the kinetics of degradation of a fibrin clot, or using a device for measuring the kinetics of degradation of a fibrin clot, in particular remote, and optionally also comprising a processor adapted to implement said steps.

[0122] According to another aspect, a computer program is proposed comprising program code instructions for executing the steps of a method according to any of the embodiments described herein when said program is executed on a computer. According to a particular embodiment, the computer program allows the implementation of the method steps to be controlled and / or at least partially performed the calculation operations for a classification or conclusion related to the monitoring.

[0123] According to one embodiment, the computer program includes instructions which, when the program is executed by a computer, cause the computer to implement at least step e of the method described herein or step f of the sorting method described herein, according to any embodiment, and where appropriate at least one other step of the method described herein, and optionally instructions also enabling the input to be retrieved and / or the output of the variables generated during these steps.

[0124] According to one embodiment, the computer program includes instructions that lead a data processing system or a device as described herein, in particular a device including a kinetic measuring apparatus as defined in any of the embodiments described herein or a data processing system or a device using such a measuring apparatus, in particular remote, to execute at least step e. of the method described herein or step f. of the classification method described herein, and where appropriate also at least one other step of the method described herein, according to any embodiment, in particular step d. of the method described herein, according to any embodiment.

[0125] According to another aspect, a computer-readable recording medium is proposed comprising instructions which, when executed by a computer, cause the computer to implement at least step e of the method described herein or step f of the classification method described herein, and optionally also at least one other step of the method described herein, according to any embodiment, and optionally comprising means for providing as input and / or output the variables generated during these steps, and / or for returning the classification result taking into account the variables provided as input, and optionally also comprising means for giving instructions to a kinetic measuring device, in particular a remote one, for the implementation of a method described herein, according to any embodiment

[0126] According to another aspect, a computer-readable data carrier is proposed on which the computer program described herein is recorded, or a signal from a data carrier carrying the computer program described herein.

[0127] According to another aspect, a non-transient recording medium readable by a computer is proposed on which a program is recorded for the implementation of the method described herein, according to any embodiment, when this program is executed by a processor.

[0128] According to another aspect, a kit is proposed, particularly adapted for the implementation of a measurement, classification or monitoring method described herein, according to any embodiment, comprising: • a. one or more of the following reagents: tissue factor, phospholipids, an activator of the intrinsic coagulation pathway chosen from: ellagic acid, silica, FIXa, FXIa, or several of these, t-PA (tissue plasminogen activator), calcium ions, • b. Optionally, a coagulation factor chosen from factor VIII, factor IX, factor XI, or several of these, • c. Optionally, one or more suitable stamps, • d. Optionally, instructions for performing one or more fibrin clot degradation kinetics, and • e. Optionally, a computer system and / or device and / or program and / or computer-readable data storage medium according to any of the embodiments described herein, • f. Optionally, instructions for implementing the measurement, classification, or monitoring method according to any of the embodiments described herein, • g. Optionally, instructions relating to the use of a signal from a data carrier, for the implementation of a measurement, classification or monitoring method according to any of the embodiments described herein.

[0129] Within the framework of such a kit, all the features disclosed in other parts of this description may, individually or in combination, be integrated. Brief description of the drawings

[0130] Other features, details and advantages will become apparent from the detailed description below and from the analysis of the accompanying drawings, in which: Fig. 1

[0131] [Fig. 1] shows an explanatory diagram of the Coag-Lyse test method according to the invention, as described in the Examples. 1. 200 pL pure plasma. 2. 50 pL FT t-PA FXIa reagent. 3. Incubation at 37°C. 4. 50 pL calcium reagent; T0 of the test. 5. OD measurements. 6. Post-processing software and statistical analysis. Fig. 2

[0132] [Fig.2] shows typical fibrin degradation profiles from three plasmas different types of hemophilia A. Fig. 3

[0133] [Fig.3] shows a scheme of sample analysis for the study reported in the Examples. Legend: 1. 26 ranges of 10 levels of FVIII [0-100%]. 2. Unsupervised and supervised analysis. 3. Patient samples. 4. Supervised analysis. Fig. 4

[0134] [Fig.4] shows a classification of patients according to the DDO parameter (variation optical density) in two steps (Results by grouping). Fig. 5

[0135] [Fig.5] shows the fibrinogen level according to each of the groups. Fig. 6

[0136] [Fig.6] shows the two-time turbidity difference in the presence of hemophilic plasmas Severe. Fig. 7A and Fig. 7B

[0137] [Fig.7A] and [Fig.7B] show the combined effect of TXA 1 pg / mL in association with the missing factor for hemophilic plasma A with shortened lysis. Fig. 8

[0138] [Fig.8] shows the two-time turbidity difference (phenotypic study). Fig. 9

[0139] [Fig.9] shows the fibrinogen level by grouping (phenotypic study). Fig. 10

[0140] [Fig. 10] shows the fibrinogen level versus the DDO value (density variation) optics) at time 1 (phenotypic study). Fig. 11

[0141] [Fig. 11] shows the lysis time per grouping (phenotypic study). Fig. 12

[0142] [Fig. 12] shows the rate of lysis per grouping (phenotypic study). Fig. 13A, Fig. 13B and Fig. 13C

[0143] [Fig.13A], [Fig.13B] and [Fig.13C] show a test on the ROTEM analyzer (viscoelastic method) at a concentration of 20% of FVIII (0.2 IU / mL) (Groups 3, 2, and 1, respectively). Fig. 14A

[0144] [Fig.l4A] shows the two-time amplitude deviation with ROTEM parameters A15 and A25. Fig. 14B

[0145] [Fig. 14B] shows the amplitude difference at two times at 20 minutes and 30 minutes (viscoelastic method). Fig. 15

[0146] [Fig. 15] shows the percentage of correct classification in quadratic discriminant analysis with a single parameter (Optical Density measured at a single time with a turbidimetric measurement method). Fig. 16

[0147] [Fig. 16] shows the percentages of correct classification in quadratic discriminant analysis with two parameters (Optical Densities measured at two times with a turbidimetric measurement method), compared to a single parameter. Fig. 17

[0148] [Fig. 17] shows the percentage of correct classification in quadratic discriminant analysis with a single parameter (Optical Density measured at a single time with a viscoelastic measurement method). Examples

[0149] A. The Coag-Lyse test

[0150] The dynamic measurement of fibrin clot formation and degradation is performed on a set of samples. The test, called the "Coag-Lyse" test, is a turbidimetric, automated, and comprehensive test for measuring fibrin clot formation and lysis. The invention takes advantage of the "lysis" portion of the fibrin clot formation and lysis curve for analysis. The test is performed by mixing the undiluted plasma sample with an intermediate reagent containing a very low concentration of tissue factor (TF), phospholipids (PPL), a plasminogen activator (t-PA), and an intrinsic pathway activator, preferably factor Xla (FXIa). After incubation of the mixture, the calcium reagent triggers thrombin generation and fibrin clot formation, which is rapidly degraded by t-PA (tissue plasminogen activator).

[0151] The test can be performed using any existing instrument, and in particular a turbidimeter or spectrophotometer. In one advantageous embodiment, the steps of the method are carried out on an automated STA-R Max or STA-R type analyzer. In one embodiment, the blood or plasma sample is a platelet-poor plasma sample. It is obtained, in particular, by centrifuging a citrated tube containing the patient's blood sample for 15 minutes at a speed of 2000 to 2500 g, at a temperature between 18 and 22°C. The sample can also undergo a second centrifugation for 15 minutes, at a speed 2000 to 2500 g, at a temperature between 18 and 22°C. This is a conventional treatment.

[0152] As shown in [Fig. 1], FT (tissue factor) and PPL (phospholipids) are mixed with an activator of the intrinsic pathway, preferably FXIa, and t-PA (tissue plasminogen activator). According to one embodiment, FT (tissue factor), PPL (phospholipids), FXIa, and t-PA (tissue plasminogen activator) are mixed at the following concentrations: 0.5 pM, 4 pM, 100 pM, and 0.13 pg / mL final test.

[0153] A quantity of the missing factor can be added to achieve a final test concentration between 1.5 and 200%. To this end, depending on the initial quantity of factor in the sample (which can be determined), the missing factor can be added via the intermediate reagent.

[0154] The plasma sample, preferably 200 pL, undiluted, is mixed with the intermediate reagent, preferably 50 pL.

[0155] This is followed by an incubation step at 37°C of the mixture and then the addition of the calcium ion-based triggering reagent to the mixture ([Fig.1]), preferably 50pL at 102 mM final reagent (17 mM final test), to initiate thrombin generation and fibrin clot formation ([Fig.1]).

[0156] Then, the optical density variation is read ([Fig.1]) at a single wavelength between 350 and 800 nm, dynamically (every 2 seconds), preferably at 540nm and for a duration of preferably 1986 seconds (33 minutes).

[0157] The fibrinogram is the curve that shows the evolution of the physical properties of the clot, preferably the evolution of the optical properties of the clot, during fibrin formation until its lysis. Only the lysis of the clot is necessary to analyze a profile within the scope of the present invention.

[0158] The lysis is monitored in terms of optical density change (ODC) over time until it returns to the initial amplitude. The kinetics of ODC over time thus obtained allows the calculation of ODC parameters at both times.

[0159] In the context of this experimental part, the parameters or steps that were specifically used or carried out to obtain the results shown are as follows: a. FT (tissue factor) and PPL (phospholipids) are mixed with an intrinsic pathway activator, FXIa, and t-PA (tissue plasminogen activator), at the following concentrations: 0.5 pM, 4 pM, 100 pM and 0.13 pg / mL final test. b. Incubate the mixture at 37°C and then add the calcium ion-based triggering reagent to the mixture, 50pL at 102 mM final reagent (17 mM final test), to initiate the formation of the fibrin clot. c. The optical density variation is read at a single wavelength dynamically (every 2 seconds), at 540nm and for a duration of 1986 seconds (33 minutes). d. The database consists of all the optical density deltas at the different measurement times and for each of the method parameters (Tmax; T09D - 90% of the max DDO; TL - 50% of the max DDO - max DDO is an optical density measurement). e. These results are analyzed using quadratic discriminant analysis to determine the % of correct classification at t1 and t2. f. This database is used as a training set on at least 70% of the overloaded samples taken randomly, and as a test set on the remaining overloaded samples (30%) and then as a validation set on patient samples.

[0160] Figure 2 shows what typical fibrin degradation profiles look like in three different hemophilia A plasmas. This figure illustrates that the DDO (optical density change) parameters at times 1 and 2 vary according to the hyper / hypofibrinolysis of the hemophilia patient plasma.

[0161] B. Study of the entire sample set and training set

[0162] The sample set may include hemophilia plasma, and in particular hemophilia A plasma with or without inhibitors, and samples from patients treated on demand or prophylactically. The method is possible with all commercial treatments, and preferably with Advate®, Elocta®, and Jivi®. The reasons why these different treatments do not introduce any bias in the analysis with regard to the samples used are related to the marketing authorizations of said drugs with different mechanisms of action (rFVIII and long-acting FVIII).

[0163] In particular: a. Advate® (Takeda) is a market-leading recombinant molecule to date; https: / / www.fortunebusinessinsights.com / industry-reports / hemophilia-drugs-market-100068 b. Elocta® (Sanofi) and Jivi® (Bayer) are "Long-Acting FVIII" hemoglobin inhibitors. Elocta® is the only long-acting product authorized in France and features a fusion with an FC fragment of immunoglobulin; it holds approximately 20% of the market: https: / / www.lesechos.fr / 2018 / 01 / sanofi-fait-son-entree-sur-le-marche-de-lhemophilie-982460. Jivi® is a pegylated molecule (polyethylene glycol). from Bayer; a molecule which is considered representative of the 3 molecules authorized on the market.

[0164] The data reported here are based on the use of 9 severely hemophilia A patients overloaded with 3 FVIII molecules (Advate®, Elocta® and Jivi®) ([Fig. 3]). Samples from hemophilia A patients before and after treatment with Jivi® or Elocta® (the type of treatment was performed blinded) were analyzed.

[0165] The training set was created using severe hemophilia A plasma (<1%) spiked with molecules at different concentrations. A total of 250 samples (and results) from non-splattered and spiked patients were analyzed as the training and test groups. Then, 47 additional patient samples were analyzed as the validation group. Typically, a training sample comprises between 50% and 80% of the data, and a test sample comprises between 20% and 40% of the data, which may be or are randomly separated. Thus, for the present study, a total of 250 spiked and non-splattered patient samples were analyzed as the training (70%) and test (30%) groups, and then 47 additional patient samples were used for model validation.

[0166] Plasmas from overloaded patients were obtained following the protocol: Several µL aliquots of patient plasma are thawed for 5 minutes at 37°C and mixed together. A 100% FVIII loading is achieved while respecting the maximum 2% plasma dilution. The FVIII level obtained after plasma loading is assessed using an automated chromogenic assay sensitive to FVIII. • After adjusting the level to 100%, the plasma ranges are obtained by diluting the 100% level in the 0% low-level plasma to obtain the following levels: 0%; 1%; 2%; 5%; 7%; 10%; 15%; 20%; 60%; 80%; 100% of FVIII. Note: some intermediate levels (e.g., 15%; 80%) were not manufactured for all overload ranges.

[0167] After a minimum of 24 hours of freezing at -80°C, each range is tested with the Coag-Lyse test.

[0168] Plasmas from hemophilia A patients tested for the invention were collected according to two pharmacokinetic profiles: pre-dose and 0.25h, 0.5h, 1h post-dose. Each sample was tested using an automated chromogenic assay, sensitive to FVIII, and with the Coag-Lyse assay.

[0169] The statistical analysis of all samples is carried out using statistical software, preferably Minitab 18 and R.

[0170] C. Statistical analysis on the two-time DDO (optical density variation) parameters

[0171] The statistical analysis is performed on the DDO (optical density variation) parameter at two times.

[0172] Initially, differences in lysis profile were observed (Figures 2).

[0173] Then, three groups were statistically identified on the two DDO (optical density variation) parameters at 2 time points using centered and normalized hierarchical analysis. Hierarchical analysis (centered and normalized) – or group observation – was thus also used to classify the training data, which were confirmed by quadratic discriminant analysis.

[0174] Finally, the inventors achieved a 100% correct classification rate using quadratic discriminant analysis as the learning model and 99% (3 errors) using the normalized K-means method (analyses performed on Minitab). The results obtained using the normalized K-means method are not shown in the Figures of this application; only the results obtained using quadratic discriminant analysis are shown. Furthermore, the 100% correct classification rate obtained using quadratic discriminant analysis was achieved with samples where Factor FVIII was greater than or equal to 1.5% (10 errors otherwise, if rates of 0% are included), when all the data (250 results + 47 patients) are considered.

[0175] The group membership is determined using the two parameters DDO (optical density variation). For the turbidimetric method employed, the times are defined in the ranges [700–900] sec for time t1 and [1100–1400] sec for time t2. Preferably, time t1 was defined at 750 sec and time t2 at 1400 sec. More precisely, the results shown in the Figures were obtained with t1 = 750 sec and t2 = 1400 sec. The choice of these parameters was made in accordance with the method for optimal selection or verification of these parameters, described herein. In fact, the range of [700–900] sec is the range of results that showed the best discrimination when all results were considered with a single parameter. The range for time t2 was defined in combination with the results obtained at time t1.

[0176] Each patient is classified with these parameters measured at two times according to the lysis, relative to the training group, constructed in quadratic discriminant analysis under the same conditions ([Fig.4]: Results by grouping).

[0177] Note: Patient #5 did not show the same classification based on their pharmacokinetic profiles with Jivi® or Elocta®. They moved from group #1 to group #2. This difference is related to the baseline level of the fibrin clot (DDO at time tl). In this regard, it should be noted that group assignment can initially be done using an unsupervised learning method. A visual inspection of the results may prove useful in certain specific cases (as in the case of patient #5). The [Fig. 4] shows that patient no. 5 is indeed classified in both groups no. 2 and 3 (left-hand squares). In fact, patient no. 5 was sampled at two different times. In the first instance, he had a fibrinogen level of 3 g / L and a low bleeding risk. In the second instance, he likely experienced a bleeding event that increased his inflammation, hence the increase in fibrinogen to 4.2 g / L, which explains his change in classification. This particular case, however, does not interfere with the general presentation of this application, given the inherent risks of error associated with the type of method described here, which can, moreover, be calculated, as detailed here.

[0178] Patients in group 1 ([Fig. 4]) have a high baseline fibrin clot level (DDO at time t1 > 1.1). This was verified by measuring the fibrinogen level, which is higher than the normal range (4.0 to 5.0 g / L). In addition, patients in group 1 have a significantly faster lysis rate (from -1.2 mDDO / sec to -2.5 mDDO / sec).

[0179] Patients in group 2 ([Fig. 4]) have a baseline level of fibrin clot and moderately degraded fibrin level. This group constitutes the reference group. It has a lysis time between 1100 sec and 1900 sec and a lysis slope between -2.2 mDDO / sec and -0.7 mDDO / sec.

[0180] Patients in group 3 ([Fig. 4]) have a reduced baseline level of fibrin clot and a reduced level of degraded fibrin (a reduced DDO at t1 and t2 times). This was verified by measuring the lysis time, which was significantly reduced (from 840 sec to 1300 sec).

[0181] Additional analysis: Random Forest

[0182] We performed the analysis with 2 parameters per tree and 3000 trees.

[0183] On the training data, we obtained a correct ranking rate The accuracy rates were 100% and 68% (13 errors) for the validation data (patients) compared to the reference classification. These values ​​correspond to FVIII levels > 1.5. The first value represents the database of results with an overload, and the second represents the patient results. It is also possible to combine these values ​​into a single figure of 95% correct classifications.

[0184] Additional analysis: Logistic regression

[0185] We performed the analysis using multinomial logistic regression, taking group 2 as the reference (the analysis is similar when taking groups 1 or 3 as the reference).

[0186] On the training data, we obtained a correct classification rate of 100% and 93% (3 errors) on the validation data (patients) compared to the reference classification. These values ​​correspond to FVIII rates > 1.5. The first value corresponds to the results database with overloaded results and the second to patient results. It is also possible to group these values ​​into a single figure representing 99% correct classifications.

[0187] D. Validity of the learning model

[0188] According to one aspect, a minimum quantity of FVIII in the analyzed sample is necessary to sensitize the Coag-Lyse test method. However, the proposed classification is then, in itself, independent of the FVIII level and the molecule used. Indeed, in the training database used, the same plasmas were spiked at different concentrations, and the "classification" is patient-dependent; it is therefore possible to predict the patient's classification for all tested concentrations, and in particular the 0% concentration.

[0189] It should be noted that since hemophilia patients are generally treated for their condition by administering the missing factor, the case where a tested sample does not meet the minimum required FVIII level would be the exception rather than the rule in the field of the invention. If necessary, an exogenous supply of the missing factor can be provided during the implementation of the method according to the invention, at the level of the sample to be tested.

[0190] In the presence of all samples (including levels < 1.5% and levels > 1.5%, i.e., the NxO population), an overlap of groups 3 and 2 was observed ([Fig. 6]). The level of overlap, using quadratic discriminant analysis on the entire database (when 0% levels are included), was determined to be 97% (10 classification errors) compared to the reference classification, which was the initial classification performed with concentrations > 1.5%. Overlap is evaluated here in terms of the number of correct classifications compared to the initial classification performed with concentrations > 1.5%. In this regard, the initial classification was performed using an unsupervised learning method with a Factor FVIII level > 1.5%. By extension, level 0 samples from the same patients were classified into the same groups.

[0191] It can be seen that while increased awareness (achieved by studying levels > 1.5%) made it possible to achieve 100% correct classifications and eliminate the 10 misclassifications (false positives), observation of the results including levels with 0% Factor FVIII may show that patients in group #2 could be classified as #3 when they have a very low amount of Factor FVIII found in the corresponding samples. This remains useful for identifying a threshold or "through level" Factor FVIII level for group #2 and potentially adjusting FVIII replacement therapy accordingly. Therefore, the present invention is not limited to implementation on samples with a Factor FVIII level greater than or equal to 1.5%.

[0192] Furthermore, a limit to be set on the error rate depends primarily on the severity of the phenomenon being studied. For example, in a particular embodiment, the objective may be to limit the number of patients belonging to group 3 who end up in group 2. Indeed, patients belonging to group 3 have insufficient treatment: this may therefore be a group of interest to be identified as a priority.

[0193] According to one aspect of the invention, a result given by a discriminant analysis or another type of analysis can be evaluated by the "Number of correct classifications", a target of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of correct classification being a criterion for success of the test.

[0194] In this case, the number of correct classifications can conventionally be obtained from the sensitivity / specificity measure derived from a contingency table, as in the case of ROC curves. Here, the number of correct classifications was obtained from the sensitivity / specificity measure derived from the contingency table shown in Table 1.

[0195] Based on the database including 0% levels of Factor FVIII, the contingency table is as follows (Table 1):

[0196] [Tables 1] Ranking summary Place in group True group 1 3 2 1 80 0 0 3 0 59 6 2 1 3 148 Total number 81 62 154 Correct number 80 59 148 Sensitivity 0.988 0.952 0.961 Specificity 1.000 0.974 0.972

[0197] In this contingency table (Table 1): 6 results classified in Group No. 2 are found in Group No. 3.

[0198] According to a specific but non-limiting embodiment of the invention, in particular in which a Group 3 is apparent from the results as described herein, it is possible to evaluate a result given by a discriminant analysis or another type of analysis, by the "Number of correct classifications", a target of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% correct classification of patients in Group 3 being a criterion for success of the test.

[0199] E. The benefit of grouping for samples

[0200] According to one aspect, the invention allows the samples to be classified according to the clinically observed groupings ([Fig.7A] and [Fig.7B]). • 1 - Depending on their classification and grouping, plasmas Patients with hemophilia A will react differently to the presence of FVIII and TXA. This allows for the study of patients individually and within the groups to which they belong, in order to identify abnormal lysis behavior and choose the appropriate treatment. The test highlights the effect of TXA in reducing lysis. In the example presented here, patients in group 3, with a reduced lysis time, require this treatment in combination with the missing FVIII factor (Figure 7). • 2 - Classification can also allow for personalization the threshold level of treatment for missing FVIII and / or TXA (“optimal through level”) depending on the group belonging.

[0201] Patient groups No. 1 and No. 2 (Figure 7) do not require TXA but require treatment adjustment to the missing factor alone, with two adjustment subgroups.

[0202] More specifically, it can be deduced that groups no. 1 and 2 would not need TXA, unlike group no. 3, as shown in the table below (Table 2):

[0203] [Tables2] Patient Group Treatment* Comment Group 3 Patient at high risk of bleeding Replacement therapy + TXA TXA avoids increasing replacement therapy doses Group 2 Patient at low risk of bleeding Comfort Potential reduction in replacement therapy Group 1 Patient at moderate risk of bleeding Replacement therapy Potential increase in already prescribed doses

[0204] * Prophylactic: Advate® type: 20 to 40U of FVIII / kg every 2 to 3 days / Novoeight®: 20 to 40 units of FVIII / kg every 2 days up to 50 units 3 times / week; in case of bleeding, every 8 hours to every 2 days / Hemlibra®: target population 1410 to 1880 patients, once a week or every 2 weeks for 4 weeks or every 4 weeks (HAS 2019).

[0205] F. Study of phenotyped patients

[0206] Based on phenotyped patient data, results from samples of patients other than the 47 additional patient samples mentioned above, which were analyzed, are also presented.

[0207] 1. Characteristics of the samples a. 15 samples from 12 patients with severe (8), moderate (2) and mild (2) hemophilia A b. Treated on demand (3), as prophylaxis (7) or not treated (2) c. Distribution of commercial molecules by sample: • Recombinant FVIII: Advate® (2), Novoeight® (5), Kogenate® (2); • Plasma-derived FVIII: Factane® (2); • Recombinant FVIII with extended half-life: Elocta® (2) d. The annual bleeding rate (ABR) for these patients is equal to 0 (5), 1 (2), 3 (1), 5 (2), 12 (1), 24 (1) e. One patient was excluded from the study because they were out of analytical specification (turbid).

[0208] 2. Patient study

[0209] As with the 47 patient samples previously mentioned above, the 14 new samples were tested according to the method described herein.

[0210] This made it possible to classify the samples according to the DDO (variation in optical density) at times t1 750 seconds and t2 1400 seconds.

[0211] It is observed that samples taken from the same patient are classified in the same group ([Fig.8] and Table 3)

[0212] [Tables3] Group No. 1 N ABR prophylactic treatment 3 7(+ / -5) on demand 0 NA none 1 24 Total 4 11(+ / -9) Group #2 N ABR prophylactic treatment 3 0 on demand 3 2 (+ / - 2) none 1 0 Total 7 1(+ / -2)

[0213] No patient is classified in group 3, considered the high-risk bleeding group. This can be explained by the fact that this group represents only 10% of patients (3 / 27) and is therefore not representative of 11 patients.

[0214] For patients classified in group 1, 4 out of 4 have an ABR > 3 (Table 3). This group is therefore at moderate risk of bleeding despite prophylactic treatment. These patients experience regular bleeding, the key factor of which is inflammation (Vulpen et al. 2018 DOI 10.1111 / hae.13449). This inflammation is reflected biologically by an increase in fibrinogen, as found in these patients (4.21 g / L group 1 vs. 2.83 and 2.79 g / L groups 2 and 3 respectively, see [Fig. 9]) and also in the DDO at time 1 ([Fig. 10]). Although the fibrinogen level is much higher than in the other groups and increases the lysis time (TL: 1668 sec vs. 1460 and 1100 sec respectively), meaning that lysis occurs later, this does not protect them against bleeding because they lyse faster (slope at lysis time - PTL: -1.92 mDDO / sec vs. -1.40 and -1.13 mDDO / sec respectively) (Figures 11 and 12).

[0215] For patients classified in group 2, 5 out of 7 have an ABR = 0 (Table 3). This means that these patients have a low risk of bleeding. The 2 patients with an ABR > 0 are treated on demand, i.e., with curative treatment. These patients would benefit from a lower ABR by switching to personalized prophylactic treatment.

[0216] 3. Conclusion

[0217] The previous results are confirmed as the classification is found in these new patients. The moderate bleeding in group 1 compared to the low bleeding in group 2 is explained. Group 3 remains the priority group at high risk of bleeding because it presents both the lowest DDO at time 1 and a very shortened lysis time (1100 sec vs 1460 and 1668 sec).

[0218] G. Implementation of a viscoelastic measurement method

[0219] Using a ROTEM analyzer on whole blood, the following results were obtained.

[0220] By passing into whole blood, the concentrations of the different activators were modified as follows: • The TF reagent for Coag. Lyse was concentrated (2.2 pM TF and 8 pM Phospholipid) • FXIa was concentrated x2 (200 pM of FXIa) • The tPA reagent from Coag. Lyse was diluted x2 (0.07 pg / mL of t-PA) • The calcium buffer has not been modified • The proportions (2 / 3) between samples and reagents were respected for a larger final cuvette volume (340 pL).

[0221] The inventors tested a plasma from each group 1, 2, and 3 at different concentrations of FVIII under these conditions. The results of a test on the ROTEM analyzer at a concentration of 20% FVIII (0.2 IU / mL) are shown in Figures 13A (Group 3), 13B (Group 2), and 13C (Group 1). In Figure 13C, the grayscale change occurs automatically at an amplitude of 10 mm; this allows the calculation of the ROTEM CFT (Close Formation Time) parameter, which is not used in the present invention.

[0222] To do this, the inventors used the parameters returned by the ROTEM A15 and A25 (15 minutes or 25 minutes after the coagulation time - CT), which correspond to parameters comparable to those used in the method of the invention previously described using an optical density measurement method ([Fig. 14A]). The inventors also reprocessed the results by setting the t1 and t2 values ​​fixed at 20 minutes and 30 minutes ([Fig. 14B]). This reprocessing of the results was carried out using image processing software for the returned curve (software developed in-house or http: / / www.graphreader.com) because the ROTEM does not provide raw data for each time point. It only provides the parameters A5, A10, A15, A20, A25, LY30, and LY45, corresponding to 5 to 45 minutes after the coagulation time, respectively.According to the method described here, the result is processed at a fixed time after the calcium triggering. During this reprocessing, the inventors ensured beforehand that the results obtained were the same as those of ROTEM (+ / - 1 mm).

[0223] It follows that the method according to the invention is also implementable using a viscoelastic measurement method in whole blood, in particular on a ROTEM automated system. Industrial application

[0224] These technical solutions may be applicable in particular in the field of hemostasis monitoring via dedicated devices, or in clinical practice with regard to patient monitoring.

[0225] This disclosure is not limited to the examples described above only by way of example, but encompasses all the variants that a person skilled in the art may consider in the context of the protection sought. List of documents cited Patent documents

[0226] For all intents and purposes, the following patent document is cited: - patcitl: WO 2016 / 012729 (publication number). Non-patent literature

[0227] For the avoidance of doubt, the following non-patent elements are cited: - nplcitl: Matsumoto et al. 2009 Int J Hematol, A modified thrombin generation test for investigating very low levels of factor Vil 1 activity in hemophilia A, 10.10071-12185-009-0450;

[0228] - nplcit2: Tarandovskiy et al. 2013 Thrombosis Research, Investigation of the phenotype heterogeneity in severe hemophilia A using thromboelastography, thrombin generation, and thrombodynamics, 10.1016 / j.thromres.2013.04.004;

[0229] - nplcite3: Leong et al. 2017 Research and practice in thrombosis and haemostasis, Clôt stability as a determinant of effective factor VIII replacement in hemophilia A, 10.1002 / rth2.12034;

[0230] - nplcit4 : He et al. 2018 Thrombosis Research, A ROTEM method using APTT reagent and tissue factor as the clotting activators may better define bleeding heterogeneity in moderate or severe haemophilia A, 10.1016 / j.thromres.2018.09.041;

[0231] - nplcit5 : Dargaud et al. 2017 Haemophilia, Individual thrombin génération and spontaneous bleeding rate during personalized prophylaxis with Nuwiq® (human-cl rhFVIII) in previously treated patients with severe haemophilia A, 10.1111 / hae. 13493;

[0232] - nplcitô : Chitlur 2012 Thrombosis Research, Challenges in the laboratory analyses of bleeding disorders, 10.1016 / j.thromres.2012.03.011;

[0233] - nplcit7 : Tripodi et al. 2019 Clinical Chemistry, Advances in the Treatment of Hemophilia: Implications for Laboratory Testing, 10.1373 / clinchem.2O17.284356;

[0234] - nplcit8 : Aghighi et al. 2019 Research and practice in thrombosis and haemostasis, Global coagulation assays in hemophilia A: A comparison to conventional assays, 10.1002 / rth2.12295;

[0235] - nplcit9 : Tiede et al. 2020 Haematologica, Factor VIII activity and bleeding risk during prophylaxis for severe hemophilia A: a population pharmacokinetic model, 10.3324 / haematol.2019.241554;

[0236] - nplcitlO : Abrantes et al. 2019 Haematologica, Relationship between factor VIII activity, bleeds and individual characteristics in severe hemophilia A patients, 10.3324 / haematol. 2019.217133

Claims

Demands

1. Method for measuring in vitro fibrin clot degradation from a fibrin clot lysis curve over time in a blood or plasma sample previously obtained from a patient likely to have a deficiency in at least one coagulation factor, the method comprising the following steps: a. mixing the previously obtained sample from said patient with a reagent composition comprising tissue factor, phospholipids, t-PA, and one or more activator(s) of the intrinsic coagulation pathway chosen from: ellagic acid, silica, FIXa, FXIa, and optionally one or more coagulation factors lacking in the patient, in particular chosen from: FVIII, FIX, FXI; then a. incubation of the mixture obtained in a., then b. triggering coagulation by adding calcium ions to the mixture incubated in step b., to allow the formation of a fibrin clot in the mixture, then lysis of the clot formed, and c. measurement of the kinetics of fibrin clot degradation during lysis in step c., and d. determination of a baseline level value of the fibrin clot at a time tl, the time tl being chosen to be located between the Tmax and the TL of the fibrin clot lysis curve, and of a degraded level value of the fibrin clot at a later time t2, the time t2 being chosen between 300 and 900 seconds after tl.

2. The method according to claim 1, characterized in that: i. when in step d. the kinetics of degradation of the fibrin clot due to its lysis is carried out by a measurement method by turbidimetry, the measurement of the degradation kinetics is carried out at a wavelength between 350 and 800 nm for a duration between 1400 and 3600 seconds from the triggering of coagulation by the addition of calcium ions, or ii. when in step d. the kinetics of degradation of the fibrin clot due to its lysis is carried out by a measurement method by viscoelastometry, the measurement of the degradation kinetics is carried out for a duration between 1400 and 3600 seconds from the triggering of coagulation by the addition of calcium ions.

3. Method according to any one of claims 1 to 2, characterized in that in step b. the incubation of the mixture obtained in a. is carried out between 20 and 39°C, preferably at 37°C, for 2 to 10 minutes, in particular 5 minutes, in particular at 37°C for 5 minutes, then the addition of calcium ions to the incubated mixture is carried out in an amount allowing a final concentration of calcium ions between 5 and 25 mM, preferably 17 mM.

4. A method according to any one of claims 1 to 3, characterized in that the sample is an undiluted sample of whole blood or plasma, in particular platelet-rich plasma or platelet-poor plasma, plasma containing platelet microparticles, erythrocytes or any other cell, preferably platelet-poor plasma.

5. Method according to any one of claims 1 to 4, characterized in that: a. If the sample is a whole blood sample, t2 is chosen 650 seconds after t1, and b. If the sample is a plasma sample, t2 is chosen 600 seconds after t1.

6. Method according to any one of claims 1 to 5, characterized in that the sample has a volume between 5qL and 500 qL, preferably between 50 qL and 400 qL, preferably between 50 qL and 300 qL, preferably between 100 qL and 300 qL, preferably about 200 qL.

7. Method according to any one of claims 1 and 3 to 6, characterized in that in step d, the realization of a degradation kinetics of the fibrin clot due to its lysis is carried out by a measurement method in particular selected from: a viscoelastic method, a rheometric method, an acoustic method, an optical method, a wave trace analysis method, a fluorometric method, a magnetic resonance method, a turbidimetric method, in particular is carried out by turbidimetry or by a viscoelastic method.

8. Method according to any one of claims 1 to 7, wherein the kinetics of fibrin clot degradation in step d. is carried out by turbidimetry, by optical density (OD) measurement at a wavelength of 540 nm, and wherein the time t1 is chosen in the range of 700 to 900 seconds from the triggering of coagulation by the addition of calcium ions and the time t2 is chosen in the range of 1100 to 1400 seconds from the triggering of coagulation by the addition of calcium ions.

9. Method according to claim 8, wherein t1 is defined as 750 seconds from the triggering of coagulation by the addition of calcium ions and t2 is defined as 1400 seconds from the triggering of coagulation by the addition of calcium ions.

10. A method according to any one of claims 1 to 7, wherein the kinetics of fibrin clot degradation in step d. is carried out by a viscoelastic measurement method, in particular by measuring a curve amplitude in millimeters, and wherein the time t1 is chosen in the range of 900 to 1200 seconds from the triggering of coagulation by the addition of calcium ions and the time t2 is chosen in the range of 1500 to 1800 seconds from the triggering of coagulation by the addition of calcium ions.

11. Method according to claim 10, wherein: a. tl is defined at 900 seconds from the triggering of coagulation by the addition of calcium ions and t2 is defined at 1500 seconds from the triggering of coagulation by the addition of calcium ions, or b. tl is defined at 1200 seconds from the triggering of coagulation by the addition of calcium ions and t2 is defined at 1800 seconds from the triggering of coagulation by the addition of calcium ions.

12. Method according to any one of claims 1 to 11, comprising an additional step f. of classifying the tested sample into a group reflecting a lysis profile of a fibrin clot, said lysis profile being determined on the basis of the values ​​measured at t1 and t2 in step e., with regard to a predefined classification model based on classification parameters obtained with training data processed under the same experimental conditions as those of the analyzed sample.

13. Method according to claim 12, wherein the classification of the tested sample into a group reflecting a lysis profile of a fibrin clot is carried out on the basis of a classification model obtained by unsupervised or supervised learning.

14. Method according to claim 13, characterized in that the classification model is obtained by a learning method selected from: a hierarchical analysis, in particular a centered and normalized hierarchical analysis, a K-means method, a quadratic discriminant analysis, logistic regression or a "random forest" method.

15. A data processing system or device comprising means for implementing at least step e of claim 1, and optionally also at least one other step of any one of the method claims 1 to 14, and optionally comprising means for providing as input and / or output the variables generated during these steps, for rendering the ranking result taking into account the variables provided as input, and optionally also comprising a device for measuring the kinetics of degradation of a fibrin clot, or using a device for measuring the kinetics of degradation of a clot fibrin, in particular remote, and optionally also including a processor adapted to implement said steps.

16. A computer program comprising program code instructions for executing the steps of the method according to any one of claims 1 to 14 when said program is executed on a computer, in particular a computer program comprising code instructions that cause a data processing system or a device according to claim 15, in particular a device including a kinetic measuring apparatus or a data processing system or a device using such a measuring apparatus, in particular remote, to execute at least step e of the method according to any one of claims 1 to 14, and optionally also at least one other of the steps of the method according to claims 1 to 14, for example step d of the method according to any one of claims 1 to 14.

17. A non-transient, computer-readable recording medium on which is recorded a program for implementing the method according to any one of claims 1 to 14 when that program is executed by a processor.

18. A kit adapted for implementing a method according to any one of claims 1 to 14, comprising: a. one or more of the following reagents: tissue factor, phospholipids, t-PA, an intrinsic coagulation pathway activator selected from elagic acid, silica, FIXa, FXIa, or several of these, calcium ions, b. Optionally, a coagulation factor chosen from factor VIII, factor IX, factor XI, or several of these, c. Optionally, one or more suitable swabs, d. Optionally, instructions for performing one or more fibrin clot degradation kinetics, and e. Optionally, a computer system and / or device and / or program and / or computer-readable data storage medium according to any one of claims 15 to 17, f. Optionally, instructions for implementing the method according to one of claims 1 to 14, g. Optionally, instructions relating to the use of a signal from a data carrier, for the implementation of a method according to one of claims 1 to 14.

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