Device and method for determining a fibrinogen concentration in blood

Abstract

The invention relates to a method for determining a fibrinogen concentration in a blood sample, comprising whole blood, the sample having been mixed with a reagent configured to cause fibrin formation, the method comprising: - a) forming images (I(t)) of the sample at various times (t); - b) establishing a correlation indicator (C(t)) between images acquired at various respective times (I(t), (I(t + dt))); - c) determining a variation as a function of time in the correlation indicator; - d) detecting a coagulation time (tcoag) on the basis of the variation as a function of time; - e) depending on the coagulation time, determining the fibrinogen concentration in the blood; the method being characterised in that: the reagent comprises batroxobin, the batroxobin concentration, after mixing, being between 25 and 1000 enzyme units per mL.

Description

[0001] Device and method for determining the concentration of fibrinogen in blood

[0002] Description

[0003] TECHNICAL FIELD

[0004] The technical field of the invention is the determination of a concentration of fibrinogen in the blood, and more specifically in whole blood, possibly diluted.

[0005] EARLIER ART

[0006] Fibrinogen is a glycoprotein whose plasma concentration is usually between 2 and 4 g / L. Fibrinogen, a precursor of fibrin, is an essential component of hemostasis. Under the action of thrombin, it enables the formation of a solid hemostatic thrombus.

[0007] Hemostasis disorders can develop rapidly during hemorrhagic shock, particularly in severe trauma. They can promote the maintenance or recurrence of bleeding. Early diagnosis of a coagulopathy allows for prompt treatment and can reduce transfusion requirements and improve the prognosis. Determining fibrinogen concentration is an important element in making such a diagnosis. Indeed, a decrease in fibrinogen concentration is an important clinical indicator in the management of hemorrhage.

[0008] In surgery, it has been shown that a decrease in serum fibrinogen levels can be an independent risk factor for bleeding. A decrease in serum fibrinogen below 1 g / L is considered common in hemorrhagic shock and is an indicator of bleeding severity.

[0009] The administration of fibrinogen concentrates may be recommended in cases of fibrinogenemia below 1.5 g / L.

[0010] Currently, plasma fibrinogen assays are performed in the laboratory using plasma, which necessitates a certain analysis time, generally exceeding 30 minutes. The reference method is the Clauss method, based on measuring the plasma clotting time after the addition of thrombin.

[0011] The measurement of fibrinogen in plasma can be performed using a hemostatic enzyme, such as batroxobin, as described in US patent 20220120769 or KR102154079. Batroxobin is not affected by the presence of an anticoagulant, such as heparin, which can interfere with the determination of fibrinogen levels. The fibrinogen assay is based on the clotting time. It is a simple and automated chronometric test that describes the conversion of fibrinogen to fibrin.

[0012] US9341636 and US9442125 (EP2233923) describe methods for detecting, by optical means, a coagulation instant.

[0013] The objective of the invention described below is to determine a simple and rapid method for determining fibrinogen concentration without requiring centrifugation to obtain plasma. The method can be used with whole blood or diluted blood containing red blood cells. The method is rapid, typically taking a few tens of seconds, and can be performed with just a few drops of blood using a simple device. The method is particularly suitable for point-of-care or operating room use, without requiring expensive analytical equipment.

[0014] DESCRIPTION OF THE INVENTION

[0015] A first object of the invention is a method for determining the concentration of fibrinogen in a blood sample, comprising whole blood, the sample having been mixed with a reagent configured to induce fibrin formation, the method comprising:

[0016] - a) formation of images of the sample at different times;

[0017] - b) establishment of a correlation indicator between images respectively acquired at different times;

[0018] - c) determination of a temporal evolution of the correlation indicator;

[0019] - d) detection of a characteristic instant, called the coagulation instant, from the temporal evolution;

[0020] - e) depending on the time of coagulation, determination of the concentration of fibrinogen in the blood.

[0021] The reagent may contain batroxobin, the concentration of batroxobin being, after mixing, between 25 and 1000 enzyme units per mL.

[0022] Preferably, the reagent contains arginine. The arginine concentration may be between 25 and 250 mM after mixing. Alternatively, the reagent may contain a peptide sequence containing arginine, said sequence having a coagulation-inhibiting effect, the arginine concentration being between 0.1 and 0.3 mM after mixing.

[0023] The reagent may contain a non-ionic polymer with a molar mass of less than 40,000 g / mol, at a concentration of between 10 and 50 g / L after mixing. The non-ionic polymer may contain at least one of the following polymers: dextran, polyethylene glycol, polyvinylpyrrolidone, or polyoxyethylene.

[0024] Advantageously, the reagent may include a calcium or manganese salt, or more generally a divalent cation salt.

[0025] The concentration of calcium salt and / or manganese salt can be between 5 mM and 50 mM after mixing.

[0026] Preferably, the pH of the sample, mixed with the reagent, is between 8 and 10 or between 8.5 and 10 or between 9 and 10.

[0027] According to one possibility:

[0028] - step d) involves detecting a break in the slope of the temporal evolution at different times;

[0029] - during step e), the coagulation time is selected based on the times at which a break in slope is detected;

[0030] - during step e), the fibrinogen concentration is determined by a previously established calibration function applied at the time of coagulation.

[0031] Step e) may include:

[0032] - ei) selection of a time at which a break in slope is detected according to predetermined selection criteria;

[0033] - e-ii) application of a linear regression, called prior linear regression, of the temporal evolution, taking into account a time range prior to the instant selected during ei);

[0034] - e-iii) application of a linear regression, called posterior linear regression, of the temporal evolution, taking into account a time range subsequent to the instant selected during ei);

[0035] - e-iv) determination of a time corresponding to the intersection of the forward and backward linear regressions, the time thus determined corresponding to the coagulation time. A second object of the invention is a fluidic chamber, intended to receive a blood sample by capillary action from an opening, the fluidic chamber comprising a reagent including batroxobin and arginine. The fluidic chamber may extend to a thickness of less than 1 cm, 5 mm, or 1 mm. The reagent may also include a calcium or manganese salt.

[0036] The reagent can be configured so that, after mixing with the sample, the amount of batroxobin is between 25 and 1000 enzyme units per mL. The reagent can be configured so that, after mixing with the sample, the amount of arginine is between 25 and 250 mM.

[0037] The reagent may contain a peptide sequence containing arginine, said sequence having a coagulation-inhibiting effect. The reagent is configured so that, after mixing with the sample, the arginine concentration is between 0.1 and 0.3 mM.

[0038] The reagent may contain a nonionic polymer with a molar mass less than 40,000 g / mol. The nonionic polymer may contain at least one of the following: dextran, polyethylene glycol, polyvinylpyrrolidone, or polyoxyethylene. The reagent is configured so that, after mixing with the sample, the concentration of the nonionic polymer is between 10 and 50 g / L.

[0039] The reagent may contain a calcium or manganese salt, or more generally a divalent cation salt. The reagent may be configured so that, after mixing with the sample, the concentration of calcium and / or manganese salt can be between 5 mM and 50 mM.

[0040] Preferably, the pH of the sample, mixed with the reagent, is between 8 and 10 or between 8.5 and 10 or between 9 and 10.

[0041] The reagent can be present in the fluidic chamber in liquid or lyophilized form.

[0042] A third object of the invention is a kit, intended for implementing a process according to the first object of the invention, and comprising:

[0043] - a fluidic chamber according to the second object of the invention;

[0044] - a reader comprising a light source, extending in front of an image sensor, the reader being configured to receive the fluidic chamber between the light source and the image sensor;

[0045] - a processing unit, configured to implement steps b) to e) of a method according to the first object of the invention from images acquired by the image sensor when the fluidic chamber is disposed between the light source and the image sensor. The distance between the image sensor and the fluidic chamber may be less than 1 cm or 5 mm or 1 mm.

[0046] A fourth object of the invention is a method for determining the concentration of a chemical or biological species in a biological liquid sample containing particles, the sample having been mixed with a reagent configured to cause coagulation of the biological liquid or agglutination of the particles in the biological liquid, the method comprising:

[0047] - a) formation of images of the sample at different times;

[0048] - b) establishment of a correlation indicator between images respectively acquired at different times;

[0049] - c) determination of a temporal evolution of the correlation indicator;

[0050] - d) detection of a characteristic instant, from the temporal evolution;

[0051] - e) depending on the characteristic time, determination of the concentration of the chemical or biological species in the biological fluid.

[0052] According to one possibility:

[0053] - step d) involves detecting a break in the slope of the temporal evolution at different times;

[0054] - during step e), the characteristic instant is selected according to the instants at which a break in slope is detected;

[0055] - during step e), the concentration of the chemical or biological species is determined by a previously established calibration function applied at the characteristic time.

[0056] The biological fluid can be blood, in particular pure or diluted whole blood, the particles being red blood cells.

[0057] The characteristic moment can be a moment of coagulation or a moment corresponding to a certain level of agglutination of particles of the biological fluid.

[0058] Step e) may include substeps ei) to e-iv) described in relation to the first object of the invention.

[0059] The invention will be better understood by reading the explanation of the examples of embodiment presented, in the continuation of the description, in connection with the figures listed below.

[0060] FIGURES

[0061] Figure IA schematically illustrates a device enabling the implementation of the invention.

[0062] Figure IB schematically represents a fluidic chamber.

[0063] Figure 2 represents an example of an image of an acquired blood sample. Figures 3A to 3D show curves representing an evolution of a correlation indicator over time in different configurations, and for different concentrations of fibrinogen.

[0064] Figure 3A and Figure 3B represent curves obtained for different concentrations of fibrinogen, respectively without and in the presence of arginine in the reagent.

[0065] Figure 3C shows curves obtained, for different concentrations of fibrinogen, without and in the presence of calcium lactate gluconate in the reagent.

[0066] Figure 3D represents correlation curves, for different concentrations of fibrinogen, with a formulation considered advantageous.

[0067] Figure 4A shows different image processing steps for images acquired by the image sensor. Figure 4B shows details of a specific method for determining coagulation time from a correlation curve.

[0068] Figures 5A to 5E illustrate steps in determining a clotting time from correlation curves.

[0069] PRESENTATION OF SPECIFIC IMPLEMENTATION METHODS

[0070] Figure IA schematically illustrates a device 1 for implementing the invention. The device includes a fluidic chamber 15, intended to receive the blood sample 10. The fluidic chamber 15 may be as described in US11027277. The fluidic chamber is intended to be positioned facing a light source 11 and an image sensor 12.

[0071] The fluidic chamber is shown schematically in Figure IB. The fluidic chamber includes a fluidic channel 15 cextending between an opening 150 and an analysis chamber 15 a The opening 150 of the fluidic chamber is designed to collect the sample to be analyzed. The fluidic channel 15 c is configured to drain the blood sample, particularly by capillary action, to the analysis chamber 15 a The analysis chamber 15 a is intended to be positioned between the light source 11 and the image sensor 12. The thickness of the analysis chamber, between the light source and the image sensor, is preferably less than 1 mm. It is preferably less than 500 µm. It is preferably greater than 50 µm or 100 µm. The thickness of the fluidic chamber is, for example, 150 µm. The sample volume occupying the analysis chamber is typically between 1 and 10 pL, or between 1 and 5 pL, for example, 2 pL or 3 pL.

[0072] The light source 11 and the image sensor 12 can be integrated into a reader 2 as described in US patent 10634585. The fluidic chamber 15 is then inserted into the reader. After the fluidic chamber has been inserted into the reader, the distance between the fluidic chamber and the image sensor is preferably less than 1 cm, or even 5 mm, or even 1 mm. The distance between the light source and the image sensor is preferably such that the light source appears point-like relative to the fluidic chamber. Preferably, there are no magnification and / or image-forming optics between the fluidic chamber and the image sensor. Each image is acquired by the image sensor in a lensless imaging configuration. This does not preclude the presence of focusing microlenses at each pixel of the image sensor.The distance between the light source and the fluidic chamber is preferably greater than 1 cm or 2 cm.

[0073] The device includes a processing unit 20, connected to the image sensor, and configured to perform image processing operations as described below, in connection with Figures 4A and 4B. The processing unit may include a microprocessor. It may, for example, be a computer. The processing unit may be integrated into the reader or separate from it.

[0074] The blood sample (10) is preferably a whole blood sample, or a diluted one, containing red blood cells. This is because the sample benefits from the ability of red blood cells to form a blood clot, under the influence of an added protein. The sample consists of one or more drops of whole blood. Therefore, it is necessary that the sample contain a sufficient quantity of red blood cells, for example, a hematocrit level greater than 5%.

[0075] The blood sample can be capillary blood, introduced directly into the fluidic chamber. It can also be venous blood, possibly collected in a citrated tube before being introduced into the fluidic chamber.

[0076] The invention implements principles similar to those described in US9341636, based on image analysis of a whole blood sample, possibly diluted, containing red blood cells. Under the effect of the coagulation reaction, the red blood cells form a clot, altering the acquired images. Clot formation is detected by analyzing the correlation between images acquired at different times.

[0077] The image sensor is configured to acquire images at a specific acquisition frequency, for example, between 1 Hz and 100 Hz, or even higher. Setting the acquisition frequency affects the temporal accuracy at which a clotting time can be determined. The clotting time is correlated with the fibrinogen concentration in the sample. After the clotting time has been established, the fibrinogen concentration in the sample can be determined by applying a predetermined calibration function.

[0078] During clot formation, successively acquired images tend to become increasingly correlated. One way to monitor clot formation is to track the evolution of a correlation indicator, representing the correlation Corr(t) between two images with time shifts of / (t) and / (t + dt). The time shift dt can be equal to one or more time increments, for example, five time increments, each time increment being the inverse of the acquisition frequency.

[0079] The red blood cell concentration in the sample is preferably greater than or equal to 5% hematocrit. Ideally, the hematocrit level is between 20% and 40%, for example 35%.

[0080] Prior to image acquisition, a reagent containing the active ingredients described below is added to the sample. The addition of the reagent to the sample creates a mixture. The reagent may be present in the fluidic chamber in lyophilized or liquid form. Alternatively, the reagent may be added to the sample in liquid form, for example, before the sample is introduced into the fluidic chamber.

[0081] The reagent contains a hemostatic enzyme. Preferably, the hemostatic enzyme is batroxobin, whether native or recombinant. One advantage of batroxobin is that it does not activate other coagulation factors, such as thrombin. Batroxobin cleaves fibrinogen to form fibrin monomers, which is a step in the coagulation process.

[0082] The amount of batroxobine in the mixture is preferably greater than 25 U / mL. The unit U designates one enzyme unit of batroxobine. The amount of batroxobine is preferably greater than 100 U / mL, or even greater than 150 U / mL. The amount of batroxobine is preferably less than 1000 U / mL, or even less than 900 U / mL, or even less than 800 U / mL, or even less than 700 U / mL, or even less than 600 U / mL, or even less than 500 U / mL. The preferred concentration range of batroxobine is from 130 U / mL to 400 U / mL or from 130 U / mL to 350 U / mL. The inventors observed that above 130 U / mL, the method provides good sensitivity, generally at or below 0.5 g / L of fibrinogen. One of the objectives of the invention is to measure a low level of fibrinogen, for example, a fibrinogen concentration below 0.5 g / L, and preferably in the range of 0.3 g / L or 0.4 g / L.

[0083] Preferably, an amino acid is added to modulate batroxobin activity. This may be arginine, or a peptide sequence containing arginine, particularly L-arginine, which slows down batroxobin activity. The concentration of pure arginine in the sample may be between 25 mM and 250 mM, and preferably between 70 and 250 mM.

[0084] The concentration of arginine can be lower, particularly when arginine is incorporated into a peptide sequence that has an inhibitory effect on coagulation, for example, on factor XIII. Thus, arginine can be incorporated into a compound, for example, GPRP (Glycine Proline Arginine Proline), GPR (Glycine Proline Arginine), or AR (Alanine Arginine). The amount of arginine can then be lower, for example, 0.1 to 0.3 mM.

[0085] Arginine can inhibit batroxobin by binding to batroxobin's active sites. Arginine can act as a competitive inhibitor by binding to the active site of batroxobin, blocking access to fibrinogen.

[0086] It is necessary that the pH of the reagent be basic, preferably between 8 and 10 or between 8.5 and 10 or between 9 and 10. It is considered that the optimal pH of batroxobin is 8.

[0087] The inventors found that when the pH is less than or equal to 8, batroxobin acts too quickly.

[0088] Arginine also has a protective function when the added compounds are lyophilized.

[0089] Comparative tests, which have the advantage of the presence of arginine in the reagent, are described later in connection with figures 3A and 3B.

[0090] Preferably, a divalent cation salt is added, preferably calcium lactate gluconate or another calcium salt, for example, calcium chloride or calcium acetate. Alternatively, the added salt is manganese acetate, or another manganese salt, for example, manganese sulfate, manganese chloride, or manganese carbonate. The concentration of the divalent cation salt added to the mixture is preferably between 5 and 25 mM. The use of such a salt hardens and stabilizes the clot formed during coagulation, which is particularly suitable for the image processing described later. The use of such a salt thus improves the sensitivity of the measurement.

[0091] It is estimated that divalent cations have an effect on the activation of factor XIII. Comparative tests, showing the advantage of calcium lactate gluconate in the reagent, are described later in connection with figure 3C.

[0092] Preferably, a low molecular weight nonionic polymer can be added to the sample. Low molecular weight is defined as less than 40,000 g / mol, and preferably less than 30,000 g / mol or 20,000 g / mol. Such a polymer could, for example, be one described in the publication by Armstrong J. K., "The hydronynamics radii of macromolecules and their effect on red blood cell aggregation," Biophysical Journal, Vol. 87, December 2004. It could, for example, be a small polysaccharide, such as a glucose polymer. It could, for example, be dextran with a molecular weight less than 40,000 Da, for example, dextran 10K (average molecular weight 10,000 Da). Such a compound limits the formation of erythrocyte rolls due to the binding between adjacent red blood cells.Other non-ionic polymers can be used, either alternatively or in addition, for example polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polysorbate (Tween 20 or Tween 80), and polyoxyethylene (POE). The concentration of the low molecular weight non-ionic polymer can be between 10 g / L and 50 g / L, and preferably between 20 g / L and 50 g / L, for example 35 g / L.

[0093] As previously mentioned, compounds added to whole or diluted blood can be added while already present, notably in the form of a lyophilized reagent, in the fluidic chamber. In this case, as previously discussed, arginine can have a protective function. Before lyophilization, a stabilizing and protective agent, for example D-trehalose (a disaccharide composed of two glucose molecules), can be added in the form of D-(+) Trehalose dihydrate, with a concentration in the reagent between 10 g / L and 30 g / L.

[0094] For further stabilization purposes, a zwitterionic detergent of the CHAPS type (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate) can also be used. The concentration of CHAPS in the reagent can be 0.025 g / L.

[0095] For stabilization purposes, in the case of lyophilized reagents intended for mixing with the analyzed blood, BSA (Bovine Serum Albumin) may be used, with a concentration in the reagent, for example, between 10 g / L and 40 g / L. Sodium azide may also be used as an antimicrobial agent and preservative, with a concentration in the reagent, for example, of 0.3 g / L.

[0096] It should be noted that when the reagent is intended to be lyophilized, before being mixed with blood, stabilizing or preservative agents, such as trehalose, and / or CHAPS and / or sodium azide, may be incorporated before lyophilization.

[0097] Experimental trials.

[0098] Different samples were prepared, each containing varying concentrations of fibrinogen. Each sample was made from defibrinated plasma. Defibrination involves removing fibrinogen from native plasma by centrifugation and / or heat treatment. The plasma underwent heat treatment (56°C - 30 minutes), followed by centrifugation (5 minutes - 10,000 G), then another heat treatment (56°C - 30 minutes), and finally another centrifugation (5 minutes - 10,000 G). G represents the acceleration due to gravity.

[0099] Defibrinated plasma was supplemented with a fibrinogen solution prepared from Clottafact 1.5 g / 100 mL powder (LFB), which contains human fibrinogen. The powder was solubilized with water for injection. Purification by dialysis was performed in HBS buffer (HEPES saline) to remove the citrate present in the Clottafact powder. The resulting fibrinogen solution was used to supplement the defibrinated plasma. Different samples were prepared, each with a different fibrinogen concentration.

[0100] On the other hand, a washed red blood cell concentrate was obtained from a CQI HEMA sample (supplier Diagast), produced from human red blood cells of known blood groups and phenotypes. The hematocrit was between 25% and 30%. After homogenization, the HEMA samples were centrifuged (7 minutes - 300 G), which removes the white blood cells. The plasma was then removed and replaced with physiological saline. The red blood cell sample was then homogenized and centrifuged (3 minutes - 2200 G), followed by three successive washes with physiological saline. This yielded a red blood cell concentrate pellet.

[0101] The red blood cells thus obtained were added to the plasma, defibrinated, and then supplemented to obtain a hematocrit level of 45%. Fibrinogen concentrations in the samples were confirmed by a reference method (Clauss method) after plasma extraction by centrifugation (15 minutes - 2500 G) and subsequent dilution 1 / 20 lème

[0102] A reagent containing various active ingredients was then prepared to test a method for detecting fibrinogen levels based on successive image acquisition of a blood sample representative of whole blood. The tests were performed using a 150 µm thick fluidic chamber, as described in US11027277. A 2 pL volume of reagent was introduced into the fluidic chamber and then lyophilized using a Cryotec benchtop pilot lyophilizer. Approximately 3 pL of whole blood was then added.

[0103] The reagent solution contained:

[0104] Batroxobine: 135 U / mL;

[0105] D-(+)-Trehalose dihydrate: 10 g / L;

[0106] - BSA: 19g / L

[0107] Dextran (10 kDa): 35 g / L

[0108] Sodium azide: 0.3 g / L

[0109] CHAPS (molecular weight 614.88): 0.025 g /

[0110] The pH of the reagent was 9.5 ± 0.5.

[0111] In the tests shown in Figures 3B, 3C, and 3D, the reagent contained arginine. In some tests shown in Figure 3C, and in the tests shown in Figure 3D, the reagent contained calcium lactate gluconate.

[0112] Drops of blood, with different known concentrations of fibrinogen, were introduced into different fluidic chambers. Images of each fluidic chamber were acquired, as described in relation to Figure IA, at an acquisition frequency of 10 Hz. Figure 2 is an example of an image obtained.

[0113] Figures 3A to 3D represent, for different fibrinogen concentrations, the evolution of a correlation indicator Corr(t) of two images I(t), I(t + dt) (ordinate axis) as a function of time (abscissa axis - unit 0.1 s). In this example, dt = 700 ms.

[0114] The dimensions of the fluidic chamber 15 were 6.5 mm x 3.5 mm (in a plane parallel to the image sensor), with a thickness of 150 µm. The analyzed sample volume was approximately 3 pL. The fluidic chamber was introduced into a measurement device as described in US10634585. After introduction, the fluidic chamber was positioned between a 640 x 480 pixel monochrome CMOS image sensor and a 670 nm LED light source. The distance between the fluidic chamber and the image sensor was on the order of 1 mm. The distance between the fluidic chamber and the light source was 5 cm.

[0115] In each image, a region of interest I'(t), I'(t + dt) was extracted. The position of the region of interest on the image was previously determined through experimental trials. The size of the region of interest was 100 pixels by 100 pixels.

[0116] Each region of interest has been standardized, so that

[0117] / '(t) is a local mean matrix: each pixel is assigned an average value of the pixels in an 11x11 neighborhood, centered on that pixel. The neighborhood is 5 pixels in each direction, X and Y. Normalization is optional, but preferred; xy denotes the coordinates of each pixel.

[0118] We then determined, for each pair of images I(t), I(t + dt), a correlation indicator C(t) such that:

[0119] Figure 3A shows the evolution of the correlation indicator over time. The onset of coagulation corresponds to a break in the slope. In Figure 3A, the shades of gray represent fibrinogen concentrations: high concentrations (curves a), normal concentrations (curve b), medium concentrations (curves c), and low concentrations (curves d). Fibrinogen concentrations range from 4.2 g / L (high concentrations), 3.0 g / L (normal concentrations), 1.4 g / L (medium concentrations), to 0.6 g / L (low concentrations).

[0120] The occurrence of coagulation leads to an increase in the correlation indicator. Coagulation is observed regardless of the fibrinogen concentration. Figure 3B was obtained with samples identical to those used in Figure 3A. However, the reagent contained arginine (148 mM). Improved separability and repeatability of the temporal evolution of the correlation indicator as a function of fibrinogen concentration are observed.

[0121] Figure 3C was obtained with samples containing a fibrinogen concentration of 1.4 g / L. The reagent contained arginine (149 mH / L) and various concentrations of calcium lactate gluconate, ranging from 0 to 25 mM. Figure 3C shows the time evolution of the correlation indicator for different calcium lactate gluconate concentrations: 0 mM, 5 mM, 10 mM, and 25 mM. The addition of calcium lactate gluconate straightens the curve representing the correlation over time, increasing its slope. The addition of calcium lactate gluconate, or other divalent cation salts, appears to be relevant for increasing the sensitivity of the method, particularly for addressing low fibrinogen concentrations, typically below 1.4 g / L.

[0122] The 3D figure was obtained with samples containing fibrinogen at concentrations ranging from 2.0 g / L, 1.3 g / L, 0.6 g / L, and 0.3 g / L. The reagent contained arginine (166 mM) and calcium lactate gluconate (4.5 mM). The reagent composition appears particularly advantageous, as it allows for good separation of the curves corresponding to different concentrations. It also allows for the treatment of low fibrinogen concentrations.

[0123] Figure 4A summarizes the main steps of a process according to the invention:

[0124] Step 100: Introduction of a whole blood sample, possibly diluted, into the fluidic chamber. The fluidic chamber contains the reagents described previously, which induce the coagulation reaction.

[0125] Step 110: acquisition of images / (t) at different times t, and in particular from an initial time, from which the sample enters the fluidic chamber.

[0126] Step 120: Selecting a region of interest in each image.

[0127] Step 130: determination of a correlation indicator (Corr t) between the regions of interest selected during each step 120.

[0128] Step 140: Determination of a clotting time tcoag from the temporal evolution of the correlation indicator; Step 150: From the clotting time tcoag, defined relative to the initial time, determination of a fibrinogen concentration C of the sample, using a previously established calibration function f. C = f tcoag)

[0129] We now describe an advantageous method for determining coagulation time from a curve representing the temporal evolution of the correlation indicator (step 140). This corresponds to substeps 141 to 147 shown in Figure 4B. These steps are implemented by treatment unit 20.

[0130] During substep 141, we have a raw curve (curve a), as shown in Figure 5A, representing the time evolution of the correlation indicator Corr(t).

[0131] Optionally, an artifact removal step can be implemented when points deviate significantly, for example by 3 standard deviations, from the curve.

[0132] In substep 142, a smoothed derivative is calculated. This involves calculating a time derivative at each instant, taking into account the difference between the average of the last 37 values, both before and after. mean (C(t) C(t - 37)) - mean (C(t) C(t + 37))

[0133] At

[0134] Figure 5B represents such a smoothed derivative as a function of time. The instant at which the smoothed derivative is maximum is marked.

[0135] In substep 143, smoothing is performed, for example using a Butterworth filter. A smoothed curve is obtained as shown in Figure 5A (curve b).

[0136] From the raw or smoothed curve, an initial time can be defined, corresponding to the filling of the fluidic chamber 15 by the sample. The initial time may notably correspond to a significant drop in the correlation indicator. The initial time can also be established by analyzing the images acquired by the image sensor, due to the absorption, by the sample, of the light emitted by the light source.

[0137] Substep 144 involves implementing a method for detecting instants corresponding to changes in slope. To this end, a Bayesian changepoint detection algorithm, as described in the Adams and MacKays publication "Bayesian online changepoint detection," is implemented. Applying the slope break algorithm leads to the determination of instants at which a change in slope has been detected. The algorithm is applied to the raw curve (curve a in Figure 5A), which corresponds to the upper curve in Figure 5C. Figure 5C also shows the evolution of a time function (lower curve) resulting from the algorithm, where each break corresponds to a slope break detected on the upper curve. Each instant detected from the lower curve has been plotted on the upper curve (vertical lines).

[0138] The Bayesian change point detection algorithm is a sensitive algorithm that can define a large number of change points. A selection operation is performed to identify the most significant change points. This is addressed in substep 145. The selection is based on selection criteria. The selected change points correspond, for example, to the most significant variations in the time function generated by the algorithm (lower curve in Figure 5C). Thus, selection criteria are applied to the time function generated by the algorithm to retain only the most significant changes in slope. Other selection criteria can be implemented: for example, times corresponding to correlation coefficients above a predetermined correlation threshold, such as 0, are eliminated.45, or corresponding to an analysis duration greater than a predetermined duration threshold, for example 160s.

[0139] The times selected at the end of sub-step 145 are shown in Figure 5D.

[0140] Among the selected time intervals shown in Figure 5D, the smoothed curve (curve b in Figure 5A) is used to determine, at each time interval selected following step 145, an angle 0. The angle is defined by applying a linear regression of the smoothed curve on either side of each selected time interval. The time interval, called the characteristic time interval, for which the angle is the largest is retained. This corresponds to the time interval outlined in dashes in Figure 5D. Angle 0 is represented in Figure 5D. The angular selection corresponds to substep 146.

[0141] The algorithm can be refined by adjusting the characteristic time, which is the subject of substep 147 and illustrated in Figure 5E. Before and after the characteristic time defined following step 146 and shown in Figure 5E, the curve C(t) undergoes linear regressions on the portion of the curve smoothed before or after the characteristic time, respectively. For the portion after the characteristic time, the regression is, for example, established between the characteristic time and the time corresponding to the maximum of the smoothed derivative (see Figure 5B). The same duration is chosen for the portion before the characteristic time. The "refined" characteristic time corresponds to the intersection of the two regressions. Thus, the coagulation time corresponds either to the characteristic time resulting from step 146 or, preferably, to the characteristic time refined following step 147.

[0142] It was noted that the refined characteristic time is shifted, by a few time increments, relative to the time resulting from the selection described in connection with substep 146. Adjusting the coagulation time, by determining an intersection between linear regressions on either side of the time determined by the Bayesian algorithm, allows us to gain in precision.

[0143] The method is applicable to whole blood, possibly diluted, whether venous or capillary. It can be used at the point of collection (home, doctor's office, operating room) and allows for quantification of fibrinogen concentration in approximately one minute.

[0144] Substeps 141 to 147 can be implemented on a curve representing an image correlation between two instants separated by a time lag, independently of the particular application of detection of the particular application of quantification of fibrinogen concentration, but can be applied in a detection of a chemical or biological species in the blood, depending on which the blood clotting time is likely to vary.

[0145] More generally, substeps 141 to 147 can be implemented to detect image correlation induced by blood coagulation or particle agglutination in a biological fluid. When the biological fluid is blood, the particles can be red blood cells.

Claims

DEMANDS 1. A method for determining the concentration of fibrinogen in a blood sample comprising whole blood, the sample having been mixed with a reagent configured to induce fibrin formation, the method comprising: - a) formation of images ( / (t) ) of the sample at different times (t); - b) establishment of a correlation indicator (Corr t)) between images respectively acquired at different times ( / (t), ( / (t + dt))); - c) determination of a temporal evolution of the correlation indicator; - d) detection of a coagulation instant (tcoag) from the temporal evolution; - e) depending on the time of coagulation, determination of the concentration of fibrinogen in the blood; the process being characterized in that: the reagent comprises batroxobin, the concentration of batroxobin, after mixing, being between 25 and 1000 enzyme units per mL, and in that the reagent comprises arginine.

2. A process according to claim 1, wherein the concentration of arginine, after mixing, is between 25 and 250 mM.

3. A method according to any one of the preceding claims wherein the reagent comprises a peptide sequence containing arginine, said sequence having a coagulation inhibitor effect, the concentration of arginine being, after mixing, between 0.1 and 0.3 mM.

4. A process according to any one of the preceding claims, wherein the reagent comprises a non-ionic polymer, of molar mass less than 40000 g / mol, the concentration being, after mixing, between 10 and 50 g / L.

5. A process according to claim 4, wherein the non-ionic polymer comprises at least one of the following polymers: Dextran, polyethylene glycol, polyvinylpyrrolidone, polyoxyethylene.

6. A process according to any one of the preceding claims, wherein the reagent comprises a calcium or manganese salt.

7. A process according to claim C>, wherein the concentration of the calcium salt and / or manganese salt, after mixing, is between 5 mM and 50 mM.

8. A method according to any one of the preceding claims, wherein the pH of the sample, mixed with the reagent, is between 8 and 10 or between 8.5 and 10 or between 9 and 10.

9. A method according to any one of the preceding claims, wherein - step d) involves detecting a break in the slope of the temporal evolution at different times; - during step e), the coagulation time is selected based on the times at which a break in slope is detected; - during step e), the fibrinogen concentration is determined by a previously established calibration function applied at the time of coagulation.

10. A method according to claim 9, wherein step e) comprises: - ei) selection of a time at which a break in slope is detected according to predetermined selection criteria; - e-ii) application of a linear regression, called prior linear regression, of the temporal evolution, taking into account a time range prior to the instant selected during ei); - e-iii) application of a linear regression, called posterior linear regression, of the temporal evolution, taking into account a time range subsequent to the instant selected during ei); - e-iv) determination of a time corresponding to an intersection of the previous linear regression and the subsequent linear regression, the time thus determined corresponding to the time of coagulation.

11. Kit, intended for implementing a method according to any one of claims 1 to 9, and comprising, - a fluidic chamber (15) comprising a reagent including batroxobin and arginine; - a reader (2), comprising a light source, extending in front of an image sensor, the reader being configured to receive the fluidic chamber between the light source and the image sensor; - a processing unit (20), configured to carry out steps b) to e) of a method according to any one of claims 1 to 9 from images acquired by the image sensor when the fluidic chamber is disposed between the light source and the image sensor.

12. Kit, according to claim 11, wherein the distance between the image sensor and the fluidic chamber is less than 1 cm or 5 mm or 1 mm.

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