Device and method for determining the concentration of fibrinogen in blood

Abstract

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) forming images of the sample at different times; b) establishing a correlation indicator between images respectively acquired at different times; c) determining a time evolution of the correlation indicator; d) detecting a coagulation time from the time evolution; e) as a function of the coagulation time, determining the concentration of fibrinogen in the blood; the method being characterized in that: the reagent comprises batroxobin, the concentration of batroxobin, after mixing, being between 25 and 1000 enzyme units per mL.

Description

Title of the invention: Device and method for determining fibrinogen concentration in blood. Technical field

[0001] The technical field of the invention is the determination of the concentration of fibrinogen in the blood, and more specifically in whole blood, possibly diluted. PRIOR ART

[0002] Fibrinogen is a glycoprotein whose plasma concentration is usually between 2 and 4 g / L. Fibrinogen, a precursor of fibrin, is an essential element of hemostasis. Under the action of thrombin, it enables the formation of a solid hemostatic thrombus.

[0003] 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 rapid 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.

[0004] 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 levels below 1 g / L is considered common in hemorrhagic shock and is an indicator of bleeding severity.

[0005] The administration of fibrinogen concentrates may be recommended in cases of fibrinogenemia below 1.5 g / L.

[0006] Currently, plasma fibrinogen assays are performed in the laboratory, using plasma, which imposes a certain analysis time, generally exceeding 30 minutes. The reference method is the Clauss method, based on measuring the plasma coagulation time after the addition of thrombin.

[0007] The fibrinogen assay in plasma can be performed using a hemostatic enzyme, for example, batroxobin, as described in US patent 20220120769 or KR10-2154079. Batroxobin is not affected by the presence of an anticoagulant, such as heparin, which could interfere with the determination of the fibrinogen level. The fibrinogen assay is based on the clotting time. It is a simple and automated chronometric test describing the conversion of fibrinogen to fibrin.

[0008] 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 and a simple device. The method is particularly suitable for point-of-care or operating room use, without requiring expensive analytical equipment. Description of the invention

[0009] 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: - a) formation of images of the sample at different times; - b) establishment of a correlation indicator between images respectively acquired at different times; - c) determination of a temporal evolution of the correlation indicator; - d) detection of a characteristic instant, called the coagulation instant, from the temporal evolution; - e) depending on the time of coagulation, determination of the concentration of fibrinogen in the blood.

[0010] The reagent may contain batroxobin, the concentration of batroxobin being, after mixing, between 25 and 1000 enzyme units per mL.

[0011] Preferably, the reagent comprises arginine. The concentration of arginine may be between 25 and 250 mM after mixing.

[0012] According to one possibility, the reagent comprises a peptide sequence containing arginine, said sequence having a coagulation inhibitory effect, the concentration of arginine being between 0.1 and 0.3 mM after mixing.

[0013] The reagent may comprise 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 comprise at least one of the following polymers: dextran, polyethylene glycol, polyvinylpyrrolidone, polyoxyethylene.

[0014] Advantageously, the reagent may comprise a calcium or manganese salt, or more generally a divalent cation salt.

[0015] The concentration of calcium salt and / or manganese salt can be between 5 mM and 50 mM after mixing.

[0016] 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.

[0017] According to one possibility: - 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.

[0018] Step e) may include: - 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.

[0019] 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.

[0020] 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.

[0021] The reagent may comprise 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.

[0022] The reagent may comprise a nonionic polymer with a molar mass of less than 40,000 g / mol. The nonionic polymer may comprise at least one of the following polymers: dextran, polyethylene glycol, polyvinylpyrrolidone, polyoxyethylene. The The reagent is configured so that, after mixing with the sample, the concentration of non-ionic polymer is between 10 and 50 g / L.

[0023] The reagent may comprise 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 salt and / or manganese salt may be between 5 mM and 50 mM.

[0024] 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.

[0025] The reagent may be present in the fluidic chamber in liquid or lyophilized state.

[0026] A third object of the invention is a kit, intended for the implementation of a process according to the first object of the invention, and comprising: - a fluidic chamber according to the second object of the invention; - 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; - a processing unit, configured to implement steps b) to e) of a process 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.

[0027] The distance between the image sensor and the fluidic chamber can be less than 1 cm or 5 mm or 1 mm.

[0028] 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: - a) formation of images of the sample at different times; - b) establishment of a correlation indicator between images respectively acquired at different times; - c) determination of a temporal evolution of the correlation indicator; - d) detection of a characteristic instant, from the temporal evolution; - e) as a function of the characteristic time, determination of the concentration of the chemical or biological species in the biological fluid.

[0029] According to one possibility: - step d) involves detecting a break in the slope of the temporal evolution at different times; - during step e), the characteristic instant is selected according to the instants at which a break in slope is detected; - during step e), the concentration of the chemical or biological species is determined by a previously established calibration function applied at the characteristic time.

[0030] The biological fluid may be blood, in particular pure or diluted whole blood, the particles being red blood cells.

[0031] 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.

[0032] Step e) may include substeps ei) to e-iv) described in relation to the first object of the invention.

[0033] The invention will be better understood upon reading the description of the exemplary embodiments presented later in this description, in connection with the figures listed below. FIGURES

[0034] Fig. 1A schematically illustrates a device enabling implementation of the invention.

[0035] Fig. 1B schematically represents a fluidic chamber.

[0036] Figure 2 represents an example of an image of an acquired blood sample

[0037] Figures 3A to 3D show curves representing the evolution of an indicator correlation over time in different configurations, and for different concentrations of fibrinogen.

[0038] Fig. 3A and Fig. 3B represent curves obtained for different concentrations of fibrinogen, respectively without and in the presence of arginine in the reagent.

[0039] Fig. 3C shows curves obtained for different concentrations of fibrinogen, without and in the presence of calcium lactate gluconate in the reagent.

[0040] Fig. 3D represents correlation curves, for different concentrations of fibrinogen, with a formulation considered advantageous.

[0041] Fig. 4A shows different image processing steps acquired by the image sensor.

[0042] Fig. 4B shows details of a particular way of determining a clotting time from a correlation curve.

[0043] Figures 5A to 5E illustrate steps in determining a coagulation time based on correlation curves. PRESENTATION OF SPECIFIC IMPLEMENTATION METHODS

[0044] Figure 1A 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 US 11027277. The fluidic chamber is intended to be positioned facing a light source 11 and an image sensor 12.

[0045] The fluidic chamber is schematically shown in [Fig. 1B]. The fluidic chamber comprises a fluidic channel 15c extending between an opening 15o and an analysis chamber 15a. The opening 15o of the fluidic chamber is intended to collect the sample to be analyzed. The fluidic channel 15c is configured to drain the blood sample, particularly by capillary action, to the analysis chamber 15a. The analysis chamber 15a is intended to be positioned between the light source 11 and the image sensor 2. 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.

[0046] 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.

[0047] 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 as a point 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.

[0048] The device includes a processing unit 20, connected to the image sensor, and configured to perform image processing operations as described below, with reference to 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.

[0049] 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%.

[0050] The blood sample may be capillary blood, introduced directly into the fluidic chamber. It may also be venous blood, possibly collected in a citrated tube before being introduced into the fluidic chamber.

[0051] 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.

[0052] The image sensor is configured to acquire images at an acquisition frequency, for example, between 1 Hz and 100 Hz, or even higher. Setting the acquisition frequency affects the temporal accuracy with 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, it is possible to determine the fibrinogen concentration in the sample by applying a predetermined calibration function.

[0053] 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 a correlation Corr(t) between two temporally offset images (q-¢ / f). The temporal offset dt can be equal to one or more temporal increments, for example, five temporal increments, each temporal increment being the inverse of the acquisition frequency.

[0054] The concentration of red blood cells in the sample is preferably greater than or equal to 5% hematocrit. Ideally, the hematocrit level is between 20% and 40%, for example 35%.

[0055] Prior to image acquisition, a reagent containing active ingredients, described below, is added to the sample. The addition of the reagent to the sample forms 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.

[0056] The reagent comprises 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 constitutes a step in coagulation.

[0057] 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 found that above 130 U / mL, the method provides good sensitivity, generally at or below 0.5g / L of fibrinogen.

[0058] One of the objectives of the invention is to perform a measurement of a low level of fibrinogen, for example a fibrinogen concentration of less than 0.5 g / L, and preferably in the order of 0.3 g / L or 0.4 g / L.

[0059] Preferably, an amino acid is added to modulate the activity of batroxobin. This may be arginine, or a peptide sequence containing arginine, particularly L-arginine, which slows down the activity of batroxobin. The concentration of arginine, supplied in pure form, in the sample may be between 25 mM and 250 mM, and preferably between 70 and 250 mM.

[0060] The concentration of arginine may 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 may be introduced into a compound, for example GPRP (Glycine Proline Arginine Proline), GPR (Glycine Proline Arginine), or AR (Alanine Arginine). The amount of arginine may then be lower, for example, 0.1 to 0.3 mM.

[0061] Arginine can inhibit batroxobin by binding to active sites of batroxobin. Arginine can act as a competitive inhibitor by binding to the active site of batroxobin, blocking access to fibrinogen.

[0062] 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 optimum pH of batroxobin is 8.

[0063] The inventors have found that when the pH is less than or equal to 8, batroxobin acts too quickly

[0064] Arginine also has a protective function when the added compounds are lyophilized.

[0065] Comparative tests, which have the advantage of the presence of arginine in the reagent, are described below in connection with Figures 3A and 3B.

[0066] Preferably, a salt of a divalent cation is added, preferably calcium lactate gluconate or another calcium salt, for example calcium chloride or calcium acetate. Alternatively, the added salt is acetate of Manganese, or another manganese salt, such as 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. Using such a salt thus improves the sensitivity of the measurement.

[0067] 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 [Fig.3C].

[0068] Preferably, a low molecular weight nonionic polymer may 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 may, 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 may, for example, be a small polysaccharide, such as a glucose polymer. It may, 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 bonding 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.

[0069] As previously stated, the compounds added to whole or diluted blood can be added while already present, particularly in the form of a lyophilized reagent, in the fluidic chamber. In this case, as previously mentioned, 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.

[0070] Also for stabilization purposes, a zwitterionic detergent of the CHAPS type (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate) may be used. The concentration of CHAPS in the reagent may be 0.025 g / L.

[0071] Also for stabilization purposes, in the case of lyophilization of the reagents intended to be mixed with the analyzed blood, BSA (Bovine Serum Albumin) may be used, the concentration in the reagent being for example between 10 g / L and 40 g / L.

[0072] Sodium azide can also be used as an antimicrobial agent and preservative, the concentration in the reagent being, for example, 0.3 g / L.

[0073] 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.

[0074] Experimental tests.

[0075] Different samples were prepared, containing different concentrations of fibrinogen. Each sample was made from defibrinated plasma. Defibrination consists of 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.

[0076] Defibrinated plasma was supplemented with a fibrinogen solution prepared from Clottafact 1.5 g / 100 mL powder (LFB), containing human fibrinogen. The powder was solubilized with water for injection. Purification by dialysis was performed in HBS buffer (HEPES saline buffer) 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.

[0077] 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 groups and phenotypes. The hematocrit was between 25% and 30%. After homogenization, the HEMA samples were subjected to centrifugation (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 subjected to centrifugation (3 minutes - 2200 G), as well as three successive washes with physiological saline. This yielded a pellet of red blood cell concentrate.

[0078] The red blood cells thus obtained were added to the plasma, defibrinated and then supplemented, so as to obtain a hematocrit level of 45%.

[0079] The fibrinogen concentrations in the samples were confirmed by a reference method (Clauss method), after extraction of the plasma by centrifugation (15 minutes - 2500 G), then dilution 1 / 20th

[0080] A reagent, comprising various active ingredients, was then prepared to test a method for detecting fibrinogen levels based on the acquisition of successive images of a blood sample representative of whole blood. The tests were carried out using a fluidic chamber with a thickness of 150 µm, as described in US 11027277. A volume of 2 pL of reagent was introduced into the fluidic chamber and then lyophilized using a Cryotec benchtop pilot lyophilizer. A volume of approximately 3 pL of whole blood was then added.

[0081] The reagent solution comprised: - Batroxobine: 135 U / mL; - D-(+)-Trehalose dihydrate: 10 g / L; BSA: 19g / L - Dextran (10 kDa): 35 g / L - Sodium azide: 0.3 g / L - CHAPS (molecular weight 614.88): 0.025 g /

[0082] The pH of the reagent was 9.5 ± 0.5.

[0083] In the tests shown in Figures 3B, 3C and 3D, the reagent contained arginine. In some of the tests shown in [Fig. 3C], and in the tests shown in [Fig. 3D], the reagent contained calcium lactate gluconate.

[0084] 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 [Fig. 1A], at an acquisition frequency of 10 Hz. [Fig. 2] is an example of an image obtained.

[0085] Figures 3A to 3D represent, for different concentrations of fibrinogen, an evolution of a correlation indicator Corr(t) of two images l(t + dt) (ordinate axis) as a function of time (abscissa axis - unit 0.1 s). In this example, dt = 700 ms.

[0086] The dimensions of the fluidic chamber 15 were 6.5 mm * 3.5 mm (in a plane parallel to the image sensor), with a thickness of 150 pm. The volume of sample analyzed was approximately 3 pL.

[0087] The fluidic chamber was introduced into a measuring device as described in US 10634585. After introduction, the fluidic chamber was positioned between a 640 x 480 pixel monochrome CMOS image sensor and a light source of the LED type, with an emission wavelength of 670 nm. 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.

[0088] In each image, a region of interest was extracted / I '(t + dt)- The position of the region of interest on the image was previously determined during experimental tests. The size of the region of interest was 100 pixels by 100 pixels.

[0089] Each region of interest has been standardized, so that [°090] I* x} (t+dt) = r X y(t + dt) -l(t+dt)

[0091] f is a local average matrix: each pixel is assigned an average value of the pixels in an 11x11 neighborhood centered on that pixel. The neighborhood consists of 5 pixels in each X and Y direction. Normalization is optional, but preferred. xy denotes the coordinates of each pixel.

[0092] We then determined, for each pair of images l(t+ dt)', a correlation indicator C ( t ) such that:

[0093] i\,wi\y(t+dt) CorrW -

[0094] Figure 3A shows the evolution of the correlation indicator over time. The occurrence of coagulation corresponds to a break in the slope. In Figure 3A, the shades of gray represent fibrinogen concentrations. Fibrinogen concentrations range from 4.2 g / L (high model samples), 3.0 g / L (normal model samples), 1.4 g / L (medium model samples), to 0.6 g / L (low model samples).

[0095] The occurrence of coagulation leads to an increase in the correlation indicator. It is observed that coagulation is always observable, regardless of the fibrinogen concentration.

[0096] Figure 3B was obtained using 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.

[0097] Figure 3C was obtained with samples containing a fibrinogen concentration of 1.4 g / L. The reagent contained arginine (149 mM) and various concentrations of calcium lactate gluconate, ranging from 0 to 25 mM. Figure 3C shows different time evolutions of the correlation indicator for different concentrations of calcium lactate gluconate: 0 mM, 5 mM, 10 mM, and 25 mM. It can be seen that the addition of calcium lactate gluconate straightens the curve representing the evolution of the correlation over time, i.e., it increases its slope. The addition of calcium lactate gluconate, or other divalent cation salts, appears relevant for increasing the sensitivity of the method, in order to address low fibrinogen concentrations, typically below 1.4 g / L.

[0098] Figure 3D was obtained with samples whose fibrinogen concentration ranged 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 composition of the This reagent appears particularly advantageous, as it allows for good separation of the curves corresponding to different concentrations. It also allows for the treatment of low levels of fibrinogen concentrations.

[0099] Figure 4A summarizes the main steps of a process according to the invention:

[0100] Step 100: introduction of a whole blood sample, possibly diluted, into the fluidic chamber. The fluidic chamber contains the reagents described above, inducing the coagulation reaction.

[0101] Step 110: Acquisition of images j(f) at different times t, and in particular from an initial time, from which the sample enters the fluidic chamber.

[0102] Step 120: selection of a region of interest in each image.

[0103] Step 130: determination of a correlation indicator Corr(t) between the regions of interest selected during each step 120.

[0104] Step 140: determination of a coagulation time tcoag from the temporal evolution of the correlation indicator;

[0105] Step 150: From the coagulation 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)

[0106] An advantageous method for determining coagulation time is now described from a curve representing the temporal evolution of the correlation indicator (step 140). This corresponds to substeps 141 to 147 shown in [Fig. 4B]. These steps are implemented by the processing unit 20.

[0107] During substep 141, a raw curve (curve a), as shown in Figure 5A, is obtained, representing the time evolution of the correlation indicator Cori^t)-

[0108] Optionally, an artifact removal step can be implemented when points deviate significantly, for example by 3 standard deviations, from the curve.

[0109] During substep 142, a calculation of a so-called smoothed derivative is performed. This involves performing a time derivative, at each instant, taking into account the difference between the average of the last 37 values, respectively before and after.

[0110] niean (C(t):C(t-37))-mean (C(t}.C(t+37)) to t

[0111] Figure 5B represents such a smoothed derivative as a function of time. The instant at which the smoothed derivative is maximum is indicated.

[0112] During substep 143, smoothing is performed, for example by Butterworth filtering. A smoothed curve is obtained as shown in [Fig. 5A] (curve b).

[0113] From the raw or smoothed curve, an initial instant can be defined, corresponding to the filling of the fluidic chamber 15 by the sample. The initial instant may, in particular, correspond to a significant drop in the correlation indicator. The initial instant 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.

[0114] Substep 144 consists of 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 of [Fig. 5A]), which corresponds to the upper curve of [Fig. 5C]. In [Fig. 5C], the evolution of a time function (lower curve) resulting from the algorithm is also shown, 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).

[0115] 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 select the most significant change points. This is the subject of substep 145. The selection is carried out according to selection criteria. The selected change points correspond, for example, to the most significant variations in the time function resulting from the algorithm (lower curve of [Fig. 5C]). Thus, selection criteria are applied to the time function resulting from the algorithm to retain only the most significant changes in slope. Other selection criteria can be implemented: times corresponding to correlation indicators greater than a predetermined correlation threshold, for example 0, are eliminated.45, or corresponding to an analysis duration exceeding a predetermined duration threshold, for example 160s.

[0116] The times selected at the end of substep 145 are shown in [Fig. 5D].

[0117] Among the selected times shown in [Fig. 5D], the smoothed curve (curve b of [Fig. 5A]) is used to determine, at each time selected following step 145, an angle θ. The angle is defined by applying a linear regression of the smoothed curve on either side of each selected time. The time, referred to as time θ, is retained. characteristic, for which the angle is the largest. This corresponds to the instant circled by the dashed line in [Fig. 5D]. In [Fig. 5D], angle 0 is shown. Angular selection corresponds to substep 146.

[0118] 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 is subjected to 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 [Fig. 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.

[0119] Thus, the coagulation time corresponds either to the characteristic time resulting from step 146, or, and preferably, to the characteristic time refined following step 147.

[0120] It has been 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 for increased precision.

[0121] 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.

[0122] 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, according to which the blood clotting time is likely to vary.

[0123] 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 (j(f)) of the sample at different times (t); - b) establishment of a correlation indicator (CorrÇt)) between images respectively acquired at different times (j(f), + - c) determination of a temporal evolution of the correlation indicator; - d) detection of a coagulation time (tCOBff) from the temporal evolution; - e) as a function of the coagulation time, determination of the concentration of fibrinogen in the blood; the method being characterized in that: the reagent comprises batroxobin, the concentration of batroxobin, after mixing, being between 25 and 1000 enzyme units per mL.

2. A method according to claim 1, wherein the reagent comprises arginine.

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

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

5. A process according to any one of claims 2 to 5, 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.

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

7. A method according to any one of claims 2 to 6, wherein the reagent comprises a calcium or manganese salt.

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

9. 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.

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

11. A method according to claim 10, 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 a prior linear regression, of the time evolution, taking into account a time range prior to the time selected in ei); - e-iii) application of a linear regression, called a posterior linear regression, of the time evolution, taking into account a time range subsequent to the time selected in ei); - e-iv) determination of a time corresponding to an intersection of the prior linear regression and the posterior linear regression, the time thus determined corresponding to the time of coagulation.

12. A kit, intended for implementing a method according to any one of claims 1 to 11, and comprising,

13. - a fluidic chamber (15); - 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 implement steps b) to e) of a method according to any one of claims 1 to 11 from images acquired by the image sensor when the fluidic chamber is disposed between the light source and the image sensor. Kit, according to claim 12, wherein the distance between the image sensor and the fluidic chamber is less than 1 cm or 5 mm or 1 mm.

Images