Composition for hematological analyses.

Abstract

The invention relates to a composition for hematological analyses and its manufacturing process. The composition comprises an anticoagulant, in an amount of between 0.1% and 8% by weight, preferably between 2% and 6%; a salt in an amount of between 30% and 90% by weight, preferably between 50% and 80%; a pH buffer in an amount of between 0.1% and 50% by weight, preferably greater than or equal to 10%; and a surfactant in an amount of between 0.01% and 2% by weight, preferably between 0.05% and 1.5%, in solid form. The invention also relates to a solution obtained from the solid composition.

Description

Title of the invention: Composition for hematological analyses.

[0001] The invention relates to a composition for hematological analyses and its manufacturing process. The invention also relates to a solution prepared from the composition of the invention and intended for use in hematological analysis devices.

[0002] Hematology devices use specific reagents to analyze biological samples. The biological sample is preferably a blood sample, but it can also be, for example, cerebrospinal fluid, bone marrow fluid, or urine. The sample may contain particles of any kind (cells, proteins, biomarkers, etc.) that will need to be identified and counted. Prior to analysis, the biological sample can be diluted and treated with one or more reagents. The sample is then conveyed, for example, to an automated flow analyzer that uses electrical and optical measurements to count and differentiate the cells present in the sample to be analyzed.

[0003] The sample is then conveyed to an analyzer, or, in the case of specific blood analyses, to a hematology analyzer. The analyzer begins by dispensing sample volumes into trays that already contain, or receive shortly thereafter or simultaneously, a reagent. This reagent is used, for example, to detect one or more elements of interest present in the sample, or to characterize these elements (volume determination, hemoglobin assay, etc.). It may, in particular, be a diluent, a lysis reagent, a staining reagent, a coating reagent, or a cleaning reagent.

[0004] The reagent is thus an essential element in the functioning of biological analysis devices, and in particular hematology analyzers. It must be present at all times and, in particular, the diluent in considerable quantities.

[0005] One of the problems that arises is the need for frequent replenishment of this reagent. For example, when the reagent is diluent manufactured and / or sold at the working concentration (ready to use), a laboratory or hospital may need to plan for high-volume replenishment in a single day. This requires dedicated storage space, and sometimes even staff dedicated to replenishment. Furthermore, staff encounter handling difficulties due to the heavy loads to be handled and moved. Most laboratories, however, have limited space, and it is usually difficult to keep large quantities of diluent readily available. In addition, it is problematic to have to store used containers until they can be removed from the laboratory.

[0006] An attempt has been made to develop preparations of concentrated diluents. The document WO19222620 discloses a product of this type.

[0007] However, the preparation of these concentrated diluents was hampered by maximum concentrations reaching only about 25 times the working concentration. This is mainly due to problems with non-homogeneous mixtures.

[0008] Another problem with known concentrated diluents is the presence of harmful components, such as formaldehyde, imidazole, glutaraldehyde, or sodium azide used as a preservative. This presents a health risk to personnel as well as problems for the environment. Prior art products are therefore classified as serious health hazards and are typically marked with hazard pictograms. This leads to transport and / or storage problems, particularly due to the need for specific packaging.

[0009] The present invention improves the situation.

[0010] Thus, the invention relates to a composition for hematology analyses comprising: - at least one anticoagulant, in an amount between 0.1% and 8% by weight, preferably between 2% and 6%; - at least one salt in a quantity between 30% and 90% by weight, preferably between 50% and 80%; - at least one pH buffer in an amount between 0.1% and 50% by weight, preferably greater than or equal to 10%; and - at least one surfactant in an amount between 0.01% and 2% by weight, preferably between 0.05% and 1.5%,

[0011] which is in solid form.

[0012] The invention thus provides a reagent in solid form for the field of hematology (in particular in the form of a pellet, powder, or granules). In a preferred embodiment, the invention provides a solid diluent-type reagent that is a pioneering product in the field of hematology. It can be used directly by simple dissolution in a solvent, such as demineralized water, and thus avoids the problems of the prior art.

[0013] In one embodiment, the anticoagulant is a chelating agent, preferably EDTA, or one selected from K2-EDTA, K3-EDTA, Na2EDTA, T acid-citrate-dextrose (ACD), lithium heparin (LH), sodium heparin (NH3) and sodium citrate.

[0014] In a preferred embodiment, the weight of the anticoagulant (which is preferably a chelating agent) is between approximately 3% and approximately 4%; the weight of the salt is between approximately 50% and approximately 70%; the weight of the pH buffer is between approximately 30% and approximately 45%; and the weight of the surfactant is between approximately 0.05% and approximately 0.80%. In this embodiment, the amount of the salts can be adjusted according to the buffer. pH, and vice versa. The less salt there is, the more buffer will need to be added, and vice versa.

[0015] In another preferred embodiment, the salt is composed of a first salt, preferably NaCl, in an amount between 30% and 90% by weight, preferably between 50% and 80%, and a second salt, preferably KCl, in an amount between 0.1% and 90% by weight of said composition, preferably between 1% and 5%. The use of two salts has an effect on the pH. In particular, this allows for the use of less pH buffer, which optimizes costs.

[0016] In another preferred embodiment, the pH buffer is chosen to obtain a pH between 6.5 and 8.5, preferably between 7.5 and 8.5. The pH buffer can be chosen from the group consisting of K₂HPO₄·3H₂O, NaH₂PO₄·2H₂O, Na₂HPO₄·12H₂O, and KH₂PO₄, or a combination thereof. Preferably, the Tris buffer NH₂C(CH₂OH)₃, NH₂C(CH₂OH)₃HC₁ is used.

[0017] Thus, the pH buffer can be chosen from the group consisting of K2HPO4,3H2O, NaH2PO4,2H2O, Na2HPO4,12H2O, KH2PO4 and the Tris buffer NH2C(CH2OH)3, NH2 C(CH2OH)3HC1 or a combination thereof

[0018] In another preferred embodiment, the surfactant is selected from the group consisting of Tween 20, Tween 80, Tergitol 15-S-9, Tergitol 15-S-15, Brij L23, Brij 35, SB3-12, and SB3-14. Combinations of surfactants are considered, in particular, to improve the stability or to increase the penetration of an active ingredient or a staining agent into cells. The choice of surfactant depends on the end application. Each surfactant has a specific interaction with the cell membrane, for example, to stiffen the membrane or to make the cell more spherical.

[0019] In another preferred embodiment, the composition has the following formulation:

[0020] [Tables 1] Anticoagulant: Chelating agent EDTA 4.10% by weight; Salt NaCl 65.57% by weight; KCl 3.28% by weight; pH buffer K₂HPO₄·3H₂O 24.59% by weight; NaH₂PO₄·2H₂O 1.64% by weight; Surfactant TWEEN 20 0.82% by weight

[0021] In another preferred embodiment, the composition has the following formulation:

[0022] [Tables2] Anticoagulant: Chelating agent EDTA 2.91% by weight; Salt NaCl 52.30% by weight; KCl 1.15% by weight; pH buffer Na2HPO4·12H2O 14.53% by weight; KH2PO4 29.05% by weight; Surfactant TWEEN 80 0.06% by weight

[0023] Preferably, the composition is in a form chosen from, a tablet, a powder or a granule.

[0024] In one embodiment, the composition of the invention consists of: - an anticoagulant (preferably a chelating agent) in an amount between 0.1% and 8% by weight, preferably between 2% and 6%; - a salt in a quantity between 30% and 90% by weight, preferably between 50% and 80%; - a pH buffer in an amount between 0.1% and 50% by weight, preferably greater than or equal to 10%; - a surfactant in an amount between 0.01% and 2% by weight, preferably between 0.05% and 1.5%; - and optionally traces of solvent.

[0025] The invention also relates to a solution for hematology analyses obtained by dissolving the composition described above comprising: - an anticoagulant (which is preferably a chelating agent, preferably EDTA), in an amount between 0.01% and 0.2% by weight, preferably between 0.05% and 0.15%; - a first salt, preferably NaCl, in a quantity between 1.15% and 3.5% by weight, preferably between 0.8% and 0.9%; - a second salt, preferably KC1, in an amount between 0.01% and 0.9% by weight, preferably between 0.02% and 0.04%; - a pH buffer in an amount between 0.01% and 0.9% by weight, preferably 0.5%; - a surfactant in an amount between 0.001% and 0.02% by weight, preferably between 0.01% and 0.015%; and - a solvent, preferably reverse osmosis water, in a remaining quantity to obtain 100% by weight of said solution.

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[0037] The solvent may contain lysis agents, staining agents, enzymes, or other elements commonly used in hematology analyses. Furthermore, the present invention relates to a method for manufacturing a composition for hematological analyses, comprising the following steps: a. the provision of the following components: - an anticoagulant (preferably a chelating agent), in an amount between 0.1% and 8% by weight of said composition, the anticoagulant being in the form of a fine powder having a grain size between approximately 0.8 mm and approximately 2.2 mm; - a salt, in a quantity between 30% and 90% by weight of said composition, the salt being in the form of a fine powder, fine powder having a grain size between approximately 0.8 mm and approximately 2.2 mm; - a pH buffer, in an amount between 0.1% and 50% by weight of said composition, the pH buffer being in the form of a fine powder having a grain size between approximately 0.8 mm and approximately 2.2 mm; - a liquid surfactant in an amount between 0.01% and 2% by weight of said composition, mixed with a solvent in a surfactant:solvent ratio between 1:10 and 1:40; b. mixing the anticoagulant with the pH buffer to obtain a fine powder mixture; c. the addition of the liquid surfactant mixed with the solvent to said fine powder mixture so as to obtain a pasty premix; d. the addition of salt to the said pre-mixed paste followed by homogenization to obtain a paste; and e. the resting of said paste to obtain said composition in a solid form. In a preferred embodiment, salt is composed of: - of a first salt, preferably NaCl, in a quantity between 30% and 90% by weight, preferably between 50% and 80%, and - of a second salt, preferably KCl, in a quantity between 0.1% and 90% by weight of said composition, preferably between 1% and 5%. In another preferred embodiment, the solvent is chosen from osmosis water, distilled water and ethanol. In another preferred embodiment, the process further includes a step of homogenizing the size of the grains of said salt to obtain said grain size between 0.8 mm and 2.2 mm. In a preferred embodiment of the invention, the homogenization of step d. comprises the coating of the salt grains by at least a part of the surfactant. Other features, details and advantages will appear upon reading the description. detailed description below, and analysis of the attached drawings on which:

[0038] [Fig-1] shows a microscopic photograph of a grain of the composition of the invention in granular form;

[0039] [Fig.2] shows a microscopic photograph of several grains of the com position of the invention in granular form;

[0040] [Fig.3] shows histograms of red blood cells (RBCs) obtained using a a reagent from the prior art and a solution prepared from the composition of the invention; and

[0041] [Fig.4] shows comparative blood counts between an analysis with a reagent of the state of the art and with a solution prepared from the composition of the invention.

[0042] The figures, tables, and description below essentially contain elements of a certain nature. The figures and tables form an integral part of the description and may therefore not only serve to better understand the present invention but also contribute to its definition, if necessary.

[0043] Optimal sample flow management has become an important element in laboratory organization. Two criteria are particularly important to consider: sample processing time (Tum Around Time) and the workload that each action imposes on staff (Full Time Equivalent).

[0044] The reagent supply influences these two parameters, as well as the carbon footprint, storage costs, and purchasing procedures. The reagent is often contained in large containers called cubitainers, which are directly connected to the analyzers.

[0045] Cubitainers are heavy and bulky and must be changed regularly by staff when empty. Since the cubitainers of diluted reagent are composed of approximately 80% water, reagent preparation systems have been developed to reduce the frequency of changing these cubitainers by filling them with diluted reagent produced from concentrated reagent and water available on-site in the laboratory.

[0046] The reagent must have precise characteristics to avoid distorting the measurements of the instruments in which it is used. For this reason, it is crucial that the preparation devices reliably produce a reagent with the desired concentration. However, the water or diluent used and the concentrated reagent often have different temperatures, which can also vary greatly depending on the laboratory and working methods. These differences are a source of errors related to the expansion of solution volumes with temperature in dosing systems. Indeed, for the same quantity of the same liquid, its volume can differ depending on the temperature. To overcome this To address the problem and other sources of error in dosing, existing devices rely on a range of physical measurements to validate the dilution carried out in the preparation device, such as conductivity and pH.

[0047] In a very general way, the present invention relates to a solid composition containing all the ingredients and / or chemicals necessary to constitute a reagent for use in a hematology analyzer. The solid composition may, in particular, be in the form of a pellet, powder, or granules. Mixing this solid composition with demineralized water reconstitutes the reagent in liquid form. The reagent is thus ready for use in a hematology analyzer. In other words, the reagent is reconstituted by adding demineralized water to the solid composition of the invention. Mixing the composition with demineralized water may preferably be followed by filtration. The reagent can then be used directly in a hematology analyzer, typically as a diluent, lysis, sheathing, or rinsing reagent.

[0048] The invention thus provides a solid composition for hematological analysis, comprising only the substances necessary for the preparation and analysis of a biological sample such as a blood sample, plasma, platelet-rich plasma (PRP) preparation, cerebrospinal fluid, pleural fluid, fluid obtained during a bone marrow aspiration, or synovial fluid. The solid composition of the invention comprises a chelator, a surfactant (also called a detergent), and a pH buffer.

[0049] More specifically, the composition of the invention comprises: - an anticoagulant, which is preferably a chelating agent (preferably EDTA), in an amount between 0.1% and 8% by weight, preferably between 2% and 6%; - a salt in a quantity between 30% and 90% by weight, preferably between 50% and 80%; - a pH buffer in an amount between 0.1% and 50% by weight, preferably greater than or equal to 10%; and - a surfactant in an amount between 0.01% and 2% by weight, preferably between 0.05% and 1.5%.

[0050] The composition of the invention is soluble in an inert liquid, such as reverse osmosis water, distilled water, or ethanol. In particular, this dissolution is facilitated by the surfactant, also called a detergent, which prevents crystallization. Nonionic surfactants are primarily used. However, other families of surfactants are possible, such as nonionic, cationic, anionic, and zwitterionic surfactants. The surfactant used in the invention allows nearly 100% of the composition to dissolve. Indeed, when the composition in solid form of the invention is brought into contact with an inert liquid, it dissolves in the liquid and passes into the mass of this liquid to give a homogeneous whole.

[0051] Thus, the present invention also relates to a solution for hematology analyses obtained by dissolving the solid composition described above. The solution of the invention comprises: - an anticoagulant, which is preferably a chelating agent, preferably EDTA, in an amount between 0.01% and 0.2% by weight, preferably between 0.05% and 0.15%; - a first salt, preferably NaCl, in a quantity between 1.15% and 3.5% by weight, preferably between 0.8% and 0.9%; - a second salt, preferably KC1, in an amount between 0.01% and 0.9% by weight, preferably between 0.02% and 0.04%; - a pH buffer in an amount between 0.01% and 0.9% by weight, preferably 0.5%; - a surfactant in an amount between 0.001% and 0.02% by weight, preferably between 0.01% and 0.015%; and - a solvent, preferably reverse osmosis water, in a remaining quantity to obtain 100% by weight of said solution.

[0052] The Applicant tested various surfactants which showed satisfactory results. These surfactants are listed in Table 3 below.

[0053] [Tables3] Surfactant Solid Concentration (%) Concentration in Reconstituted Composition (g / L) - Solution / Reagent Tergitol 15-S-9 0.128-0.134 0.01506 Brij L23 0.170-0.177 0.02 Brij 35 0.170-0.255 0.026 SB3-12 0.255-0.266 0.03 Tween 20 0.255-0.266 0.03 Tween 80 0.255-0.266 0.03

[0054] Once the solid composition is dissolved in demineralized water, it can be used as a solution for hematological analyses directly in a hematological analysis device. Indeed, the composition possesses all the necessary physicochemical characteristics to avoid distorting hematological analyses and / or measurements. Thus, the solution of the invention (obtained from the solid composition of the invention) possesses the required functions, particularly in terms of osmolarity, pH, The solution exhibits surface activity and conductivity. Other functions, such as lysis or staining, are possible. It meets the requirements for hypertonicity and hypotonicity concentration limits. For example, the salt concentration in the composition is not greater than 1.2% (12 g / L, 200 mM) – hypertonicity 400 mOsm / L – and is not less than 0.6% (6 g / L, 100 mM) – low hypotonicity 100 mOsm / L. Furthermore, the composition allows for cell analysis, counting, and differentiation by resistivity and / or optical measurements.

[0055] The Applicant tested the salt concentration limits in the composition, defined by the resistivity target of 60.4 Qm ± 2 (identified acceptable tolerance)

[0056] [Tables4] Resistivity (Ωm) Conductivity (S / m) Osmolarity (mOsm) Electrolyte Concentration XI (mM) Electrolyte Concentration in Solid Diluent Target Pref. 60.4 16.56 332 180 89.08% Min Pref. 58.4 17.12 342 170 90.09% Max Pref. 62.4 16.03 322 190 88.04% Limit Min 50 20 400 200 45-95% Limit Max 100 10 100 100 45-95%

[0057] In practice, the target is broadly an operating range between: maximum 200 mM, 400 mOsm / L, (hypertonicity) and minimum 100 mM, 100 mOsm / L (low hypotonicity).

[0058] More generally, the salt of the present invention encompasses so-called molten salts, that is to say, dissociated salts consisting solely of ions. This includes, in particular, electrolytes. The term "salt" in the invention can thus be read as "salt" or even "at least one electrolyte".

[0059] A major advantage of the composition of the invention is that it can be produced without harmful compounds. Therefore, the problems associated with prior art reagents related to safety, the environment, transport and / or storage do not arise.

[0060] Another advantage of the composition of the invention is the possibility of a preservative-free embodiment. The absence of preservatives in the product This helps avoid chemical risks. It allows for the provision of a composition free from any mention of hazardous classification and / or hazard pictogram labeling.

[0061] Another advantage is related to the solid form of the composition. Indeed, the solid form of the invention offers increased storage capacity. As such, the composition offers a dual benefit: on the one hand, it is stable over time, and on the other hand, it allows for concentrations well over 60 times the working concentration. In a particular embodiment of the invention, the working concentration reaches nearly 90 times (approximately 87.7 times for the example described), and 126 times within the limits of low hypotonicity to preserve the integrity of the sample (0.6% NaCl, 100 mM, 100 mOsm / L). This results in easier storage in a small space. It also results in ease of use.

[0062] In general, the composition and / or solution of the invention can be used in particle analysis devices. These analytical devices are of the biological analyzer type, for example, for the preparation, counting, and / or identification of particles. A particle is defined as any product that can be analyzed and that requires, for this analysis, the use of at least one reagent. Examples of particles include blood cells, crystals, or other molecules.

[0063] The invention also relates to the method of manufacturing the solid composition.

[0064] There are various processes in the prior art for manufacturing a com solid-state positioning. In particular, techniques commonly used in biotechnology and medicine are well-known. These include melt extrusion, electrospinning, spray drying, freeze-drying, freeze-thaw, and co-precipitation. However, all these manufacturing techniques require suspending the various chemical compounds in solution and generally adding an additive and / or a binder.

[0065] The invention proposes another type of manufacturing process. Thus, the composition of the invention can be achieved as follows:

[0066] a. the provision of the following components: - an anticoagulant, which is preferably a chelating agent, in an amount between 0.1% and 8% by weight of said composition, the anticoagulant being in the form of a fine powder having a grain size between about 0.8 mm and about 2.2 mm; - a salt, in a quantity between 30% and 90% by weight of said composition, the salt being in the form of a fine powder, fine powder having a grain size between approximately 0.8 mm and approximately 2.2 mm; - a pH buffer, in an amount between 0.1% and 50% by weight of said composition, the pH buffer being in the form of a fine powder having a grain size between approximately 0.8 mm and approximately 2.2 mm; - a liquid surfactant in an amount between 0.01% and 2% by weight of said composition, mixed with a solvent in a surfactant:solvent ratio between 1:10 and 1:40;

[0067] b. mixing the anticoagulant with the pH buffer so as to obtain a fine powder mixture;

[0068] c. the addition of the liquid surfactant mixed with the solvent to said fine powder mixture so as to obtain a pasty premix;

[0069] d. the addition of salt to said pre-pasty mixture followed by homogenization to obtain a paste; and

[0070] e. resting said paste to obtain said composition in a solid form.

[0071] Contrary to the prior art, the process of the invention is free from the addition of any binder or other additive. Furthermore, the process of the invention avoids freeze-drying. It also avoids a drying step. And, the components used contain no preservatives or other toxic elements.

[0072] The surfactant can advantageously coat the powder grains, in particular the salt and / or electrolyte used.

[0073] The hygroscopic properties of salt contribute significantly to shaping the composition of the invention. In particular, during the manufacturing process, at step e. of the paste resting, the ionic charges of the salt attract the solvent molecules. In this way, the composition of the invention is formulated in a solid form.

[0074] It may also be provided for a step of grinding said salt until a powder is obtained whose grain size is between approximately 0.8 mm and 2.2 mm.

[0075] A step for shaping the dough may be provided, for example for shaping into pellets or granules.

[0076] Salt can advantageously be composed of: - of a first salt, preferably NaCl, in a quantity between 30% and 90% by weight, preferably between 50% and 80%, and - of a second salt, preferably KC1, in an amount between 0.1% and 90% by weight of said composition, preferably between 1% and 5%.

[0077] Indeed, the combined use of NaCl with KCl has shown particularly good performance. In fact, this ensures the integrity of the sample while respecting the osmotic limit.

[0078] The solvent is preferably chosen from osmosis water, distilled water, and an organic solvent such as ethanol, methanol, or ethylene glycol. The choice is ge- generally linked to the planned hematology tests.

[0079] Advantageously, the process includes a step of homogenizing the size of the grains of said salt. Optimally, a grain size of between 0.8 mm and 2.2 mm is targeted.

[0080] In this description, when grain size is referred to, it refers to a size measured by microscopy. However, grain shape is polymorphic. Thus, since the shape can vary greatly (including spherical, ellipsoidal, cubic, regular or irregular prisms, etc.), sizes are expressed here by referring to a measurement of a distance taken across the top of a chosen face of a seed. In practice, the length from one edge to another is measured. Reference is generally not made to the diameter or a diagonal. This is exemplified in Figures 2 and 3. It is estimated that each face measured on a grain is representative, to within approximately 10%, of the other faces of that grain.

[0081] EXAMPLES OF ACHIEVEMENTS

[0082] EXAMPLE 1: Manufacturing process

[0083] A composition according to the invention was prepared according to the following preparatory protocol.

[0084] I. Mixing of liquid components:

[0085] 1.1. Tergitol (concentration between 0 and 3%, preferably 1.5%) + Water distilled (or organic solvent (Ethanol, methanol, ethylene glycol) pure or in water (concentration between 1 and 2%).)

[0086] II. Grinding of the powdered components (separately):

[0087] IL 1. NaCl (concentration between 30% and 90%, preferably between 60% and 90%) and KCl (concentration between 0% and 90%, preferably between 2.5% and 25%): homogenization and reduction of seed size (from more than 2.3 mm to a size between 0.8 and 2.2 mm);

[0088] III. Mixture of fine powders (0.8-2.2 mm):

[0089] EDTA (concentration between 0% and 8%, preferably between 1% and 6%), pH buffer (for example TRIZMA base, Tris HCl): seed size already quite reduced (concentration between 0% and 20%, preferably between 5% and 10%);

[0090] IV. Mixing of liquid components with fine powders;

[0091] V. Addition of powders (coarse grains, > 2.3 mm) and homogenization of the mixture;

[0092] VI. Product shaping (granule or pellet)

[0093] This process makes it possible to obtain in particular a granule composed of solid grains.

[0094] EXAMPLE 2: Granules

[0095] The process described in this example was implemented with different choices among the components mentioned in the general description above. Various granules were thus obtained that meet the definition of the composition of the invention.

[0096] Figure 1 shows a microscopic photograph of a grain of the composition of the invention in granular form. The microscope is the Olympus BX-51 Microscope model with a Nikon xl0 / 0.25 air-filled objective. The camera is an Olympus DP-23. The figure shows a grain with a diameter between 0.8 mm and 2.2 mm, more precisely approximately 1.8 mm x 1.8 mm.

[0097] Figure 2 shows a microscopic photograph of several grains of the composition of the invention in granular form. The microscope is the same one used for Figure 1. Grains with dimensions ranging from 0.8 mm to 2.2 mm can be seen.

[0098] The solid composition in granular form shown in Figures 1 and 2 offers increased stability and shelf life. This form also reduces transport and storage costs. In particular, it reduces the volume of water required and the associated transport costs. It is easy to use: simply dilute it with an inert liquid. All of this has a positive impact on the environment.

[0099] EXAMPLE 3: Preferred diluent formulations

[0100] A first solid composition according to the invention was carried out in the proportions indicated in the table below.

[0101] [Tables5] Chelating agent EDTA 4.10% by weight, Salt NaCl 65.57% by weight, KCl 3.28% by weight, pH buffer K₂HPO₄·3H₂O 24.59% by weight, NaH₂PO₄·2H₂O 1.64% by weight, Surfactant TWEEN 20 0.82% by weight

[0102] A solution for hematological analyses according to the invention was prepared from this first composition. For this purpose, the solid composition is dissolved in demineralized water. The resulting solution is in the proportions indicated in the table below.

[0103] [Tableauxô] Chelating agent EDTA 0.05% by weight, Salt NaCl 0.8% by weight, KCl 0.04% by weight, pH buffer K₂HPO₄·3H₂O 0.3% by weight, NaH₂PO₄·2H₂O 0.02% by weight, Surfactant TWEEN 20 0.01% by weight, Solvent Water 98.78% by weight

[0104] A second solid composition according to the invention was made in the proportions indicated in the table below.

[0105] [Tables?] Chelating agent EDTA 2.91% by weight, Salt NaCl 52.30% by weight, KCl 1.15% by weight, pH buffer Na2HPO4·12H2O 14.53% by weight, KH2PO4 29.05% by weight, Surfactant TWEEN 80 0.06% by weight

[0106] A solution for hematological analyses according to the invention was prepared from this second composition. For this purpose, the solid composition is dissolved in demineralized water. The resulting solution is in the proportions indicated in the table below.

[0107] [Tables8] Chelating agent EDTA 0.05% by weight, Salt NaCl 0.9% by weight, KCl 0.02% by weight, pH buffer Na2HPO4·12H2O 0.25% by weight, KH2PO4 0.50% by weight, Surfactant TWEEN 20 0.001% by weight, Solvent Water 98.279% by weight

[0108] The performance of the above compositions or solutions has shown particularly satisfactory performance.

[0109] EXAMPLE 3: Formulation of a lysis reagent with dye

[0110] [Tables9] Example Lyse3 Concentration % by weight Anticoagulant EDTA 1.45 Electrolyte NaCl 29.75 KCl 26.18 pH Buffer Na2HPO4·12H2O 9.00 NaH2PO4·2H2O 0.48 Surfactant Tween 20 23.80 Crosslinking Agent Glutaraldehyde 2.28 Lytic Agent Saponin 0.75 DTABr 6.15 Dye Chlorazole Black 0.16

[0111] EXAMPLE 4: Exploration Test

[0112] An exploratory test on ten blood samples was carried out to test the performance of the solid composition of the invention (in its dissolved form) in comparison with a conventional liquid diluent for hematology analyzer called "ABX Diluent".

[0113] The ABX Diluent was compared with the solid composition formulated according to the procedure described above, then reconstituted in liquid form as a hematological solution by adding osmosis water. In summary, a prior art hematological reagent is compared here with the reconstituted hematological reagent of the invention.

[0114] An example of a red blood cell (RBC) histogram is reported for the two reagents. The histograms were produced using a Yumizen H500-OT device available from HORIBA MEDICAL.

[0115] Figure 3 shows the GR histograms of the prior art reagent and the solution prepared from the composition of the invention. The two compositions exhibit almost the same performance, but the solid composition of the invention offers all the advantages discussed above.

[0116] This is confirmed by blood counts performed with the Yumizen H500-OT device available from HORIBA MEDICAL.

[0117] Figure 4 shows comparative blood counts between the prior art reagent ABX diluent and a solution (or reagent) prepared from the composition of the invention, referred to in the figure as "Reconstituted Solid Diluent." Indeed, the hematological analysis composition of the invention, being in solid form, is capable of being dissolved in demineralized water to give a diluent-type hematological solution. The composition can therefore be described as a "solid diluent."

[0118] In particular, [Fig.4] shows the correlations on the main CBC parameters (from the English Complete Blood Count for Numération Formulae Sanguine (NFS) or "hemogram") for the ten samples tested.

[0119] The table below shows the coefficients of determination for each measured parameter of [Fig.4]

[0120] [TableauxlO] Parameter R2 Reconstituted solid diluent WBC 0.996 RBC 0.998 HGB 0.998 MCV 0.999 HTC 0.998 MCH 0.991 RDWcv 0.976 RDWsd 0.948 PLT 0.976 MPV 0.982 PTC 0.979 PDW 0.961

[0121] A very good correlation is observed between the ABX Diluent product and the reconstituted solid Diluent of the invention on all parameters.

[0122] Quality control of the composition of the invention can be performed using the reconstituted reagent (i.e., the solution of the invention). Conductivity is a particularly important parameter that can serve as a basis for quality control. Furthermore, conductivity measurement is a relatively simple method to implement and allows for a continuous measurement process. Generally, existing hematology devices are regulated systems designed to maintain a set conductivity in the produced reagent. The conductivity is measured within a reservoir and compared to a reference value. If necessary, the devices are configured to add reverse osmosis water and / or reagent until the target conductivity is achieved. Existing devices therefore use a control loop to adjust the volumes of water and / or reagent until the solution reaches the target conductivity.

[0123] Other control options are conceivable: detergent concentration, surface tension test and / or verification of certain hematological parameters such as MCV or hemoglobin using control blood.

Claims

Claims

1. Composition for hematology analyses, characterized in that it comprises: - an anticoagulant in an amount of between 0.1% and 8% by weight, preferably between 2% and 6%; - a salt in an amount of between 30% and 90% by weight, preferably between 50% and 80%; - a pH buffer in an amount of between 0.1% and 50% by weight, preferably greater than or equal to 10%; and - a surfactant in an amount of between 0.01% and 2% by weight, preferably between 0.05% and 1.5%, and in that it is in solid form.

2. The composition of claim 1, wherein - the amount by weight of the anticoagulant is between about 3% and about 4%; - the amount by weight of the salt is between about 50% and about 70%; - the amount by weight of the pH buffer is between about 30% and about 45%; and - the amount by weight of the surfactant is between about 0.05% and about 0.80%.

3. Composition according to one of the preceding claims, in which the salt is composed of: - a first salt, preferably NaCl, in an amount of between 30% and 90% by weight, preferably between 50% and 80%, and - a second salt, preferably KCl, in an amount of between 0.1% and 90% by weight of said composition, preferably between 1% and 5%.

4. Composition according to one of the preceding claims, in which the anticoagulant is chosen from the group consisting of EDTA, K2-EDTA, K3-EDTA, Na2EDTA, acid-citrate-dextrose (ACD),

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11. lithium heparin (LH), sodium heparin (NH), and sodium citrate. Composition according to one of the preceding claims, in which the anticoagulant is a chelating agent. Composition according to one of the preceding claims, wherein the pH buffer is selected from the group consisting of K2HPO4,3H2O, NaH2PO4,2H2O, Na2HPO4,12H2O, KH2PO4 and Tris buffer NH2C(CH2 OH)3, NH2C(CH2OH)3HC1 or a combination thereof. Composition according to one of the preceding claims, wherein the surfactant is selected from the group consisting of Tween 20, Tween 80, Tergitol 15-S-9, Brij L23, Brij 35, and SB3-12. Composition according to claim 1, having the following formulation: Anticoagulant: Chelating agent EDTA 4.10% by weight Salt NaCl 65.57% by weight KC1 3.28% by weight Buffer pH K2HPO4.3H2O 24.59% by weight NaH2PO4.2H2O 1.64% by weight Surfactant TWEEN 20 0.82% by weight Composition according to claim 1, having the following formulation: Anticoagulant: Chelating agent EDTA 2.91% by weight Salt NaCl 52.30% by weight KC1 1.15% by weight Buffer pH Na2HPO4,12H2O 14.53% by weight KH2PO4 29.05% by weight Surfactant TWEEN 80 0.06% by weight Composition according to one of the preceding claims in a form chosen from a tablet, a powder or a granule. Composition according to one of the preceding claims consisting of: an anticoagulant in an amount of between 0.1% and 8% by weight, preferably between 2% and 6%; a salt in an amount of between 30% and 90% by weight, preferably between 50% and 80%; a buffer pH in an amount between 0.1% and 50% by weight, preferably greater than or equal to 10%; a surfactant in an amount between 0.01% and 2% by weight, preferably between 0.05% and 1.5%; and optionally traces of solvent.

12. Composition according to one of the preceding claims, free from preservative.

13. Solution for hematology analyses obtained by dissolving the composition according to one of claims 1 to 12 comprising: - an anticoagulant, in an amount of between 0.01% and 0.2% by weight, preferably between 0.05% and 0.15%; - a first salt, preferably NaCl, in an amount of between 1.15% and 3.5% by weight, preferably between 0.8% and 0.9%; - a second salt, preferably KC1, in an amount of between 0.01% and 0.9% by weight, preferably between 0.02% and 0.04%; - a pH buffer in an amount of between 0.01% and 0.9% by weight, preferably 0.5%; - a surfactant in an amount of between 0.001% and 0.02% by weight, preferably between 0.01% and 0.015%; and - a solvent, preferably osmosis water, in a quantity remaining to obtain 100% by weight of said solution.

14. Method for manufacturing a composition for hematology analyses, characterized in that it comprises the following steps: a. providing the following components: - an anticoagulant, in an amount of between 0.1% and 8% by weight of said composition, the anticoagulant being in the form of a fine powder having a grain size of between approximately 0.8 mm and approximately 2.2 mm; - a salt, in an amount of between 30% and 90% by weight of said composition, the salt being in the form of a fine powder having a grain size of between approximately 0.8 mm and approximately 2.2 mm; - a pH buffer, in an amount of between 0.1% and 50% by weight of said composition, the pH buffer being in the form of a fine powder having a grain size of between approximately

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18. 0.8 mm and about 2.2 mm; - a liquid surfactant in an amount of between 0.01% and 2% by weight of said composition, mixed with a solvent in a surfactant:solvent proportion of between 1:10 and 1:40 b. mixing the anticoagulant with the pH buffer to obtain a fine powder mixture; c. adding the liquid surfactant mixed with the solvent to said fine powder mixture so as to obtain a pasty premix; d. adding salt to said pasty premix followed by homogenization so as to obtain a paste; and e. resting said paste to obtain said composition in a solid form. The method of claim 14, wherein the salt is composed of: - a first salt, preferably NaCl, in an amount of between 30% and 90% by weight, preferably between 50% and 80%, and - a second salt, preferably KC1, in an amount of between 0.1% and 90% by weight of said composition, preferably between 1% and 5%. Method according to one of claims 15 and 16, in which the solvent is chosen from osmosis water, distilled water and an organic solvent. Method according to one of claims 14 to 16, further comprising a step of homogenizing the grain size of said salt to obtain said grain size of between 0.8 mm and 2.2 mm. Method according to one of claims 14 to 17, in which the homogenization of step d. comprises the coating of the salt grains with at least a portion of said surfactant.

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