Hemocytometer Calibration Standard

Abstract

To provide a standard solution for blood cell counter calibration, which offers superior calibration accuracy and storage stability.SOLUTION: A standard solution for blood cell counter calibration is provided, the solution containing resin particles having an average circularity of 0.99 or less and a volume average particle diameter of 2 μm or more and 10 μm or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a standard solution for calibrating a hemocytometer. [Background technology]

[0002] Knowing the hemoglobin concentration in blood is important for predicting and understanding the severity of disease, and is a major item in blood tests. Hemoglobin concentration is measured using a hemocytometer that uses a particle size measurement method such as the Coulter method or the electrical sensing zone method. This hemocytometer must be highly calibrated to accurately measure hemoglobin concentration.

[0003] To calibrate this hemocytometer, a calibration standard solution in which resin particles are dispersed in an activator solution is used. However, the resin particles dispersed in these hemocytometer calibration standard solutions are spherical, which differs from the flat shape of human red blood cells. Therefore, the measurement of hemoglobin concentration using conventional hemocytometers does not provide sufficient calibration accuracy (see, for example, Patent Document 1). Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a standard solution for calibrating a hemocytometer that has excellent calibration accuracy and storage stability. [Means for solving the problem]

[0005] In order to solve the above-mentioned problems, one aspect of the present invention is a standard solution for calibrating a hemocytometer containing resin particles, wherein the resin particles have an average circularity of 0.99 or less, a volume average particle size of 2 μm or more and 10 μm or less, and a coefficient of variation of particle size of the resin particles is 5.3% or more and 15% or less. The resin particles are made of base particles to which fine particles are attached. . [Effects of the Invention]

[0006] According to one aspect of the present invention, a standard solution for calibrating a hemocytometer having excellent calibration accuracy and storage stability can be obtained. DETAILED DESCRIPTION OF THE INVENTION

[0007] An embodiment of the present invention will now be described in detail.

[0008] <Hematology Counter Calibration Standard Solution> The standard solution for calibrating a hematocytometer according to this embodiment contains resin particles. In this specification, a hematocytometer refers to a device for measuring the hematocrit value of human blood. The hematocrit value indicates the ratio of the total volume of red blood cells to the total volume of blood, and is a test item used for diagnosing anemia, etc. Examples of methods for measuring the hematocrit value include the centrifugation method and the Coulter method.

[0009] In the centrifugation method, approximately 1 ml of anticoagulant-added blood is placed in a capillary pipette, the tip of the pipette is inserted to the bottom of a Wintrobe tube, and the blood is gradually removed until the tube reaches the "0" mark, after which the tube is centrifuged to separate the blood, and the hematocrit value is calculated from the percentage of red blood cell volume. In this centrifugation method, the volume of red blood cells is read directly after centrifugation, allowing the hematocrit value to accurately reflect differences in the shape of red blood cells.

[0010] The Coulter method allows for automated measurement of hematocrit, and hemocytometers incorporating this Coulter technology are widely used. The Coulter technology is a counting technique that relies on measuring the impedance fluctuations in a measuring cell when one or more particles are guided through it during flow. The number of impedance changes indicates the number of particles in the fluid, while the measured impedance value is proportional to the volume of the particles in the fluid.

[0011] Specifically, in the Coulter method, a wall with a single through-hole is placed in an electrolyte solution in which particles are dispersed, electrodes are placed on both sides, and a constant current is passed through the pore, and the electrolyte is drawn in from the inside of the pore with a constant suction force, causing the particles to pass through the pore together with the electrolyte. At this time, the electrolyte in the pore decreases by an amount equivalent to the volume of the particles, and the electrical resistance of the pore increases in proportion to the amount of electrolyte removed.

[0012] Since the current flowing through the pore is constant regardless of the presence of particles, the amount of voltage change is proportional to the amount of change in the pore's electrical resistance. The volume of the particles is measured from this voltage change, and the particle's equivalent spherical diameter is calculated from this volume, and the particle size distribution is displayed. In addition, by counting the voltage change, the number of particles that pass through the pore is counted. In other words, the Coulter method can quantitatively measure the blood cell concentration and hematocrit value in blood.

[0013] However, in conventional standard solutions for calibrating hemocytometers, the dispersed resin particles (hereinafter referred to as dispersed particles) are spherical, and therefore hemocytometers using an electrical sensing zone method such as the Coulter method cannot accurately determine the particle volume of flat, non-spherical dispersed particles such as human red blood cells.

[0014] Therefore, in this embodiment, the average circularity of the resin particles contained in the standard solution for calibrating a hemocytometer is set to 0.99 or less, preferably 0.90 to 0.987, and more preferably 0.95 to 0.985. In this specification, the average circularity is a value obtained by optically detecting a particle and dividing it by the perimeter of an equivalent circle with the same projected area.

[0015] When the average circularity of the resin particles contained in the standard solution for calibrating a hemocytometer is 0.99 or less, the shape of the resin particles becomes close to the shape of hemoglobin, and the standard solution for calibrating a hemocytometer can have a particle shape that is excellent in measurement accuracy and storage stability.

[0016] If the average circularity exceeds 0.99, the resin particles will be nearly spherical, which may reduce the measurement accuracy of the standard solution for calibrating a hemocytometer. If the average circularity is less than 0.90, the dispersed particles in the standard solution for calibrating a hemocytometer will have a shape that is far from spherical, which may reduce the measurement accuracy. Therefore, an average circularity of 0.90 or more is preferred.

[0017] In the standard solution for calibrating a hemocytometer of this embodiment, the resin particles contained in the standard solution for calibrating a hemocytometer have a volume average particle size of 2 μm to 10 μm, preferably 1.5 μm to 9 μm, and more preferably 3 μm to 8 μm. Here, the volume average particle size refers to the particle size at the point where the cumulative volume is 50% when the cumulative volume particle size distribution curve is calculated, assuming the total volume of the particles to be 100%.

[0018] When the volume average particle diameter of the resin particles contained in the hemocytometer calibration standard solution is 2 μm or more and 10 μm or less, the storage stability of the hemocytometer calibration standard solution can be stabilized. Note that if the volume average particle diameter is less than 2 μm, the resin particles tend to aggregate in the hemocytometer calibration standard solution, which may reduce dispersibility. Also, if the volume average particle diameter exceeds 10 μm, the resin particles tend to settle in the hemocytometer calibration standard solution, which may reduce dispersibility.

[0019] In the standard solution for calibrating a hemocytometer of this embodiment, the coefficient of variation of the particle size of the resin particles is preferably 15% or less, more preferably 13% or less, and even more preferably 11% or less. In this specification, the coefficient of variation of the particle size is expressed as a percentage by dividing the standard deviation of the particles by the average particle size of the particles. Here, the average particle size is the particle size at which the cumulative distribution on a volume basis measured by a laser diffraction / scattering method is 50%.

[0020] When the coefficient of variation of the particle size of the resin particles contained in the standard solution for calibrating a hemocytometer is 15% or less, the shape of the resin particles becomes closer to the shape of hemoglobin, thereby improving the measurement accuracy of the standard solution for calibrating a hemocytometer.

[0021] If the coefficient of variation of the particle size exceeds 15%, the shape of the resin particles will be far removed from the shape of hemoglobin, which may result in poor measurement accuracy. There is no lower limit to the coefficient of variation of the particle size, but it is sufficient if the coefficient of variation is at least 1%.

[0022] In this embodiment, the aspect ratio of the resin particles contained in the standard solution for calibrating a hemocytometer is preferably 1.3 or more and 5.0 or less, more preferably 1.4 or more and 4.8 or less, and even more preferably 1.5 or more and 4.5 or less. In this specification, the aspect ratio refers to the ratio of the length of the longest side (major axis) to the length of the shortest side (minor axis), and the larger this value, the higher the degree of irregularity of the resin particles.

[0023] When the aspect ratio of the resin particles contained in the standard solution for calibrating a hemocytometer is greater than 1.3 and less than or equal to 5.0, the shape of the resin particles becomes closer to the shape of hemoglobin, thereby further improving the measurement accuracy of the standard solution for calibrating a hemocytometer.

[0024] If the aspect ratio is less than 1.3, the resin particles will be nearly spherical, which may reduce the measurement accuracy of the standard solution for calibrating hemocytometers.If the aspect ratio is more than 5.0, the shape of the resin particles will be far removed from the shape of hemoglobin, which may reduce the measurement accuracy.

[0025] In the standard solution for calibrating a hemocytometer according to the present embodiment, the resin particles have carboxyl groups on their surfaces. The resin particles having carboxyl groups on their surfaces are not particularly limited. For example, low-molecular-weight polyester is preferred as the resin particles having carboxyl groups on their surfaces.

[0026] In this embodiment, the resin particles contained in the standard solution for calibrating a hemocytometer have carboxyl groups on their surfaces, which improves the shelf life of the standard solution for calibrating a hemocytometer and also improves the dispersion stability of the resin particles in the standard solution for calibrating a hemocytometer.

[0027] In the standard solution for calibrating a hemocytometer according to this embodiment, the density of carboxyl groups on the surface of the resin particles is 0.001 particles / Å 2 In this specification, the density of carboxyl groups on the surface of resin particles is calculated from the content of carboxyl groups in the resin converted from the acid value of the resin based on the specifications of JIS K0070 (1992).

[0028] In the standard solution for calibrating a hemocytometer according to this embodiment, the density of carboxyl groups on the surface of the resin particles is 0.001 particles / Å 2 If the above conditions are met, the dispersibility of the resin particles in the standard solution for calibrating a hemocytometer is increased, and the calibration accuracy and storage stability of the standard solution for calibrating a hemocytometer can be improved.

[0029] The content of resin particles in the standard solution for calibrating a hemocytometer is preferably 1.0 to 10 mass%, more preferably 1.5 to 7.5 mass%, and even more preferably 2.5 to 5.0 mass%, relative to 100 mass% of the standard solution for calibrating a hemocytometer.

[0030] By setting the content of resin particles in the standard solution for hemocytometer calibration to 1.0% by mass or more, the cold resistance of the resin particles is increased, improving the storage stability of the standard solution for hemocytometer calibration. Furthermore, by setting the content of resin particles having carboxyl groups on their surfaces to 10% by mass or less, the dispersion stability of the resin particles in the standard solution for hemocytometer calibration is improved.

[0031] <Resin particles and their manufacturing method> Here, an example of the resin particles (dispersed particles) contained in the standard solution for calibrating a hemocytometer of this embodiment will be described. The resin particles can be produced by the following method. First, a polyol and a polycarboxylic acid are heated to 150 to 280°C in the presence of a known esterification catalyst such as tetrabutoxy titanate or dibutyltin oxide, and the resulting water is distilled off, if necessary, under reduced pressure, to obtain a polyester having hydroxyl groups.

[0032] Next, this is reacted with polyisocyanate at 40 to 140°C to obtain a modified polyester resin (A) having an isocyanate group. Furthermore, this (A) is reacted with amines (B) at 40 to 140°C to obtain a polyester modified with a urea bond. When reacting with polyisocyanate and when reacting (A) with (B), a solvent can be used as needed.

[0033] Examples of solvents that can be used include those that are inactive against polyisocyanates, such as aromatic solvents such as toluene and xylene; ketones such as acetone, methyl ethyl ketone and methyl isobutyl ketone; esters such as ethyl acetate; amides such as dimethylformamide and dimethylacetamide; and ethers such as tetrahydrofuran.

[0034] When unmodified polyester (A') is used in combination, this (A') is produced in the same manner as in the case of the polyester having hydroxyl groups, and is dissolved and mixed in the solution obtained after the reaction of the urea-modified polyester.

[0035] The resin used in this embodiment is preferably a low-molecular polyester resin. In addition, from the viewpoint of a standard solution for calibrating a hemocytometer, the density of carboxyl groups on the resin surface is preferably 0.001 / Å. 2 It is preferable that this is equal to or greater than this.

[0036] In this case, the resin constituting the resin particles preferably has an acid value of 40 mgKOH / g or more. When the acid value of the resin is 40 mgKOH / g or more, the dispersibility of the resin particles in the standard solution for calibrating a hemocytometer is increased, thereby improving the calibration accuracy and storage stability of the standard solution for calibrating a hemocytometer.

[0037] The acid value of the resin means the content of carboxyl groups in the polyester resin, and is determined by neutralization titration in accordance with JIS K0070 (1992).

[0038] The dispersed particles of the standard solution for calibrating a hemocytometer can be formed by reacting a dispersion of an isocyanate-modified polyester (A) with an amine (B) in an aqueous medium.

[0039] Examples of a method for stably forming a dispersion of urea-modified polyester or isocyanate-modified polyester (A) in an aqueous medium include a method of adding components of dispersed particle raw materials of a standard solution for calibrating a hemocytometer, which comprises urea-modified polyester or isocyanate-modified polyester (A), to an aqueous medium and dispersing the components by shear force.

[0040] The modified polyester (A) may further be mixed with other dispersed particle components (hereinafter also referred to as dispersed particle raw materials) of a standard solution for calibrating a hemocytometer.

[0041] Examples of the dispersion particle raw material include a colorant, a colorant masterbatch, a release agent, a charge control agent, and an unmodified polyester (A'). These dispersion particle raw materials may be mixed when forming a dispersion in an aqueous medium, but it is preferable to mix the dispersion particle raw material of a standard solution for calibrating a hemocytometer in advance, and then add the mixture to the aqueous medium and disperse it.

[0042] The dispersing device is not particularly limited and may be any commercially available agitator or disperser.

[0043] Examples of such dispersing devices include batch dispersers such as Homogenizer (manufactured by IKA Corporation) and TK Auto Homo Mixer (manufactured by Tokushu Kika Kogyo Co., Ltd.); continuous dispersers such as Ebara Milder (manufactured by Arihara Manufacturing Co., Ltd.) and TK Pipeline Homo Mixer (manufactured by Tokushu Kika Kogyo Co., Ltd.); and high-pressure dispersers such as Microfluidizer (manufactured by Mizuho Kogyo Co., Ltd.), Nanomizer (manufactured by Nanomizer Co., Ltd.) and APV Gaulin (manufactured by Gaulin Co., Ltd.).

[0044] Of these, from the viewpoint of applying a uniform shear force, APV Gaulin, Homogenizer, TK Auto Homo Mixer, Ebara Milder and TK Pipeline Homo Mixer are preferred, and TK Auto Homo Mixer and TK Pipeline Homo Mixer are more preferred.

[0045] The temperature during dispersion is preferably 0 to 150° C., more preferably 5 to 98° C., and particularly preferably 10 to 60° C. When the temperature exceeds 100° C., the temperature is under pressure.

[0046] The standard solution for calibrating a hemocytometer of this embodiment may contain other optional components such as a dispersant, a thickener, or a viscosity reducer, as long as the object of the present invention is not impaired.

[0047] A dispersant is preferably used to emulsify and disperse the oil layer containing the dispersed particle components of the standard solution for calibrating a hemocytometer in an aqueous medium. A known surfactant (S) can be used as the dispersant. When surfactant (S) is used, its amount is preferably 0.0001 to 50% by weight, more preferably 0.0005 to 0.4% by weight, and even more preferably 0.001 to 0.3% by weight, based on the total weight of (A), (B), and (A').

[0048] As the surfactant (S), an anionic surfactant (S-1), a cationic surfactant (S-2), or the like can be used.

[0049] As the anionic surfactant (S-1), carboxylic acid or its salt, sulfate ester salt, carboxymethyl salt, sulfonate, phosphate ester salt, or the like can be used.

[0050] The carboxylic acid or salt thereof may be a saturated or unsaturated fatty acid having 8 to 22 carbon atoms or a salt thereof. Examples of saturated or unsaturated fatty acids having 8 to 22 carbon atoms include capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and ricinoleic acid, as well as mixtures of higher fatty acids obtained by saponifying coconut oil, palm kernel oil, rice bran oil, beef tallow, etc.

[0051] Examples of the salts of carboxylic acids include sodium salts, potassium salts, amine salts, ammonium salts, quaternary ammonium salts, and alkanolamine salts (for example, monoethanolamine salts, diethanolamine salts, triethanolamine salts, etc.).

[0052] Examples of sulfates that can be used include higher alcohol sulfates (sulfate of an aliphatic alcohol having 8 to 18 carbon atoms), higher alkyl ether sulfates (sulfate of an adduct of an aliphatic alcohol having 8 to 18 carbon atoms with 1 to 10 moles of ethylene oxide or propylene oxide), and the like.

[0053] Examples of salts of sulfate esters include sodium salts, potassium salts, amine salts, ammonium salts, quaternary ammonium salts, and alkanolamine salts (for example, monoethanolamine salts, diethanolamine salts, triethanolamine salts, etc.).

[0054] Examples of higher alcohol sulfates include octyl alcohol sulfate, decyl alcohol sulfate, lauryl alcohol sulfate, and stearyl alcohol sulfate.

[0055] Examples of the salt of the carboxymethylated product that can be used include salts of carboxymethylated aliphatic alcohols having 8 to 16 carbon atoms, and salts of carboxymethylated products of adducts of 1 to 10 moles of ethylene oxide or propylene oxide with aliphatic alcohols having 8 to 16 carbon atoms.

[0056] Examples of salts of carboxymethylated aliphatic alcohols include octyl alcohol carboxymethylated sodium salt, decyl alcohol carboxymethylated sodium salt, lauryl alcohol carboxymethylated sodium salt, Dobanol 23 carboxymethylated sodium salt, and tridecanol carboxymethylated sodium salt.

[0057] Examples of the salts of carboxymethylated products of 1 to 10 moles of ethylene oxide adducts of aliphatic alcohols include sodium carboxymethylated octyl alcohol ethylene oxide 3 mole adduct, sodium carboxymethylated lauryl alcohol ethylene oxide 4 mole adduct, sodium carboxymethylated Dobanol 23 ethylene oxide 3 mole adduct, and sodium carboxymethylated tridecanol ethylene oxide 5 mole adduct.

[0058] Examples of sulfonates that can be used include alkylbenzenesulfonates, alkylnaphthalenesulfonates, sulfosuccinic acid diester salts, α-olefinsulfonates, Igepon T type and sulfonates of other aromatic ring-containing compounds.

[0059] Examples of alkylbenzenesulfonates include sodium dodecylbenzenesulfonate.

[0060] Examples of alkylnaphthalene sulfonates include sodium dodecylnaphthalene sulfonate.

[0061] Examples of sulfosuccinic acid diester salts include sulfosuccinic acid di-2-ethylhexyl ester sodium salt.

[0062] Examples of sulfonates of aromatic ring-containing compounds include mono- or disulfonates of alkylated diphenyl ethers, and styrenated phenol sulfonates.

[0063] As the phosphate ester salt, a higher alcohol phosphate ester salt, a higher alcohol ethylene oxide adduct phosphate ester salt, etc. can be used.

[0064] Examples of higher alcohol phosphate salts include lauryl alcohol phosphate monoester disodium salt and lauryl alcohol phosphate diester sodium salt.

[0065] Examples of the higher alcohol ethylene oxide adduct phosphate ester salt include oleyl alcohol ethylene oxide 5 mole adduct phosphate monoester disodium salt.

[0066] These anionic surfactants (S-1) may be used alone or in combination of two or more.

[0067] As the cationic surfactant (S-2), a quaternary ammonium salt surfactant, etc., can be used. The quaternary ammonium salt surfactant can be obtained by, for example, reacting a tertiary amine having 3 to 40 carbon atoms with a quaternizing agent (for example, an alkylating agent such as methyl chloride, methyl bromide, ethyl chloride, benzyl chloride, or dimethyl sulfate, or ethylene oxide).

[0068] Examples of quaternary ammonium salt surfactants include lauryl trimethyl ammonium chloride, didecyl dimethyl ammonium chloride, lauryl dimethyl benzyl ammonium chloride (benzalkonium chloride), polyoxyethylene trimethyl ammonium chloride, and stearamidoethyl diethyl methyl ammonium methosulfate.

[0069] These anionic surfactants (S-1) may be used alone or in combination of two or more.

[0070] Furthermore, fine particles other than resin particles are preferably used as the dispersant in order to stabilize the dispersed particle base particles of the standard solution for calibrating a hemocytometer formed in an aqueous medium. Such fine particles are preferably added so that the coverage of the surface of the dispersed particle base particles in the standard solution for calibrating a hemocytometer is in the range of 10 to 90%.

[0071] Examples of microparticles include polymethyl methacrylate microparticles of 1 μm and 3 μm, polystyrene microparticles of 0.5 μm and 2 μm, poly(styrene-acrylonitrile) microparticles of 1 μm, and organic microparticles under the trade names PB-200H (manufactured by Kao Corporation), SGP (manufactured by Soken Co., Ltd.), Technopolymer SB (manufactured by Sekisui Plastics Co., Ltd.), SGP-3G (manufactured by Soken Co., Ltd.), and Micropearl (manufactured by Sekisui Fine Chemical Co., Ltd.).

[0072] Furthermore, inorganic fine particles such as tricalcium phosphate, calcium carbonate, titanium oxide, colloidal silica, and hydroxyapatite can also be used as the fine particles.

[0073] A polymeric protective colloid may be used as a dispersant in combination with these fine particles to stabilize the dispersion.

[0074] The polymer protective colloid is not particularly limited, and examples thereof include acids such as acrylic acid, methacrylic acid, α-cyanoacrylic acid, α-cyanomethacrylic acid, itaconic acid, crotonic acid, fumaric acid, maleic acid, and maleic anhydride, or (meth)acrylic monomers containing a hydroxyl group; β-hydroxyethyl acrylate, β-hydroxyethyl methacrylate, β-hydroxypropyl acrylate, β-hydroxypropyl methacrylate, γ-hydroxypropyl acrylate, γ-hydroxypropyl methacrylate, 3-chloro-2-hydroxypropyl acrylate, 3-chloro-2-hydroxypropyl methacrylate, diethylene glycol monoacrylate, diethylene glycol monomethacrylate, glycerin monoacrylate, glycerin monomethacrylate, N-methylolacrylamide, N-methylolacrylamide, and the like, vinyl alcohol, or ethers with vinyl alcohol; vinyl methyl ether, vinyl ethyl ether, and vinyl propyl ether. or esters of vinyl alcohol and a compound containing a carboxyl group; vinyl acetate, vinyl propionate, vinyl butyrate, etc.; acrylamide, methacrylamide, diacetone acrylamide, or methylol compounds thereof, etc.; acid chlorides such as acrylic acid chloride and methacrylic acid chloride; nitrogen-containing compounds such as vinylpyridine, vinylpyrrolidone, vinylimidazole, ethyleneimine, etc., or homopolymers or copolymers of compounds having a heterocyclic ring thereof; polyoxyethylene-based compounds such as polyoxyethylene, polyoxypropylene, polyoxyethylene alkylamines, polyoxypropylene alkylamines, polyoxyethylene alkylamides, polyoxypropylene alkylamides, polyoxyethylene nonylphenyl ether, polyoxyethylene lauryl phenyl ether, polyoxyethylene stearyl phenyl ester, polyoxyethylene nonylphenyl ester, etc.; celluloses such as methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, etc.

[0075] The dispersed particles of the standard solution for hemocytometer calibration of this embodiment are produced by a production method including a step of applying shear force while removing the organic solvent from the emulsified dispersion containing dispersed particles of the standard solution for hemocytometer calibration obtained as described above to deform the dispersed particles of the standard solution for hemocytometer calibration. In the step of deform-forming the dispersed particles of the standard solution for hemocytometer calibration, the average circularity of the dispersed particles in the standard solution for hemocytometer calibration is adjusted to 0.99 or less, preferably 0.92 to 0.96.

[0076] The average circularity of dispersed particles in a standard solution for calibrating a hemocytometer is measured using a flow particle image analyzer.

[0077] Specifically, 100-150 mL of water from which impurities have been removed is placed in a specified container, 0.1-0.5 mL of a surfactant is added as a dispersant, and approximately 0.1-9.5 g of the measurement sample is then added. The suspension containing the dispersed sample is dispersed in an ultrasonic disperser for approximately 1-3 minutes, and the dispersion concentration is adjusted to 3,000-10,000 particles / μL, and the shape and distribution of the dispersed particles in the hemocytometer calibration standard solution are measured.

[0078] A thickener is preferably used to increase the viscosity of the emulsified dispersion and to deform the shape of the resin particles (dispersed particles) contained in the standard solution for calibrating a hemocytometer.

[0079] Examples of thickeners include water-soluble thickeners such as polyvinyl alcohol, hydroxyethyl cellulose, hydroxypropyl cellulose, polyacrylates (alkali metal salts, organic amine salts, quaternary ammonium salts, etc.) (weight average molecular weight 3,000 to 3,000,000), carboxymethyl cellulose, polyethylene glycol (weight average molecular weight 2,000 to 200,000), and guar gum.

[0080] These thickeners may be used alone or in combination of two or more.

[0081] In the standard solution for calibrating a hemocytometer of this embodiment, any device that is generally commercially available as a mixer or disperser can be used as the device for applying shear force to the emulsified dispersion thickened by such a thickener, without any particular limitation.

[0082] Specific preferred examples include a mixer having 1 to 10 stages of paddle-type stirring blades, a helical ribbon mixer, a Max Blend mixer, etc. Of these, the helical ribbon mixer and the Max Blend mixer are preferred from the viewpoint of applying a uniform shear force.

[0083] The shear force varies depending on the viscosity of the emulsion dispersion, the time for applying the shear force, and the temperature at which it is applied, and can be appropriately selected. From the viewpoint of ease of deformation of the resin particles and ease of particle size control, for example, in the shear force applying device exemplified above, the shear force is preferably applied at a rotation speed of 70 to 50,000 rpm, more preferably 100 to 20,000 rpm, and even more preferably 500 to 10,000 rpm.

[0084] The conditions for the deforming step are preferably as follows: In the deforming step, the concentration of the organic solvent in the emulsion dispersion is preferably 2 to 10% by weight, more preferably 2.5 to 9% by weight, and even more preferably 3 to 8% by weight.

[0085] When the concentration of the organic solvent is 2% by weight or more, the dispersed particles in the standard solution for calibrating a hemocytometer are easily deformed, shortening the process of deforming. On the other hand, when the concentration of the organic solvent is 10% by weight or less, the particles are less likely to return to their original shape after deforming, and when the organic solvent is finally removed to form dispersed particles in the standard solution for calibrating a hemocytometer, the desired shape is easily obtained.

[0086] The concentration of the organic solvent at the start of the deforming step is preferably 3 to 10% by weight. If the starting concentration is 3% by weight or more, deforming occurs easily, and if it is 10% by weight or less, the generation of fine powder is reduced. The concentration of the organic solvent is adjusted to the desired concentration by removing the solvent. The concentration of the organic solvent can be measured using gas chromatography under the following conditions.

[0087] [Concentration measurement of organic solvents] Equipment: Gas chromatograph (SIMAZU GC-14A, manufactured by Shimadzu Corporation) Column: PEG 20M 20% Chromasorb W MESH 60-80 Column temperature: 100℃ Injection temperature: 180℃ Nitrogen gas flow rate: 40 ml / min Sample solution: 5% dimethylformamide solution Solution injection volume: 1μL Detector: FID30

[0088] A calibration curve was prepared using n-hexane as a standard substance to determine the organic solvent concentration. The method for desolvation during the heteromorphization step is not particularly limited as long as it can remove the solvent, such as atmospheric desolvation by blowing air or nitrogen into the liquid phase or reduced pressure desolvation, but reduced pressure desolvation is preferred because it can be performed at a relatively low temperature.

[0089] When the pressure is reduced, the degree of reduction is preferably 1 to 90 kPa, more preferably 5 to 50 kPa. As the solvent is removed, the viscosity of the emulsified dispersion increases, but in order to achieve good deformation, it is preferable to add the above-mentioned water-soluble thickener to the emulsified dispersion to adjust the viscosity.

[0090] The viscosity of the mixture of the emulsified dispersion and the thickener is preferably in the range of 5,000 to 50,000 mPa·s, more preferably 5,500 to 40,000 mPa·s, and even more preferably 6,000 to 35,000 mPa·s.

[0091] By having the viscosity within this range, it is possible to shorten the time required to apply shear force to deform the shape of the dispersed particles in the standard solution for hemocytometer calibration, and it is also possible to obtain a dispersion that is less likely to return to a spherical shape after deformation and has a stable shape after deformation.The viscosity of the mixed solution is measured using a B-type viscometer after being kept at 25°C in a thermostatic bath for 30 minutes.

[0092] After the end of the modification step, the organic solvent may be further removed from the emulsion dispersion, if necessary. The method for removing the solvent is not particularly limited.

[0093] The solvent can be removed by blowing in a carrier gas such as air or nitrogen at normal pressure to remove the organic solvent. Another method involves removing the organic solvent under reduced pressure using a vacuum pump or the like. Still another method involves pouring the emulsified dispersion that has undergone the deformation process into a liquid in which the dispersed particle components of the standard solution for hemocytometer calibration are insoluble but the solvent is soluble, such as water, lower alcohols such as methanol or ethanol, or mixtures thereof, and extracting and removing the organic solvent.

[0094] After the deforming step, the viscosity of the emulsion dispersion may be reduced using a viscosity reducer, if necessary.

[0095] Examples of viscosity reducing agents include enzymes such as α-glycanase (amylase, dextrase, pullulase, etc.) and β-glycanase (cellulase, β-1,3-glucanase, chitinase), etc. These viscosity reducing agents may be used alone or in combination of two or more.

[0096] The amount of the viscosity reducer added varies depending on the type of thickener, but from the viewpoints of productivity (thickening time) and production costs, it is preferably 1% by weight or less, more preferably 0.000001 to 0.1% by weight, and even more preferably 0.00001 to 0.01% by weight, relative to the weight of the emulsion dispersion.

[0097] The viscosity of the reduced emulsified dispersion is preferably 200 mPa s or less, more preferably 100 mPa s or less, and even more preferably 40 mPa s or less. When the viscosity of the emulsified dispersion is in this range, it is easy to handle in the process of obtaining dispersed particles of the standard solution for calibrating a hemocytometer, and is easy to wash in the washing process, which is performed as needed and will be described later.

[0098] The resin particles contained in the standard solution for calibrating a hemocytometer of this embodiment preferably have a particle size coefficient of variation [(standard deviation of particle size / average particle size) × 100 (%)] of 10% or less, and are usually provided with dispersion stability so that they can be mixed into an aqueous medium such as water or an aqueous electrolyte solution to form a single dispersion. Examples of electrolytes used in this aqueous electrolyte solution include sodium chloride and potassium chloride.

[0099] The dispersion stability of fine particles in a liquid phase depends on the acidic polar groups (for example, carboxyl groups) present on the surface of the fine particles, and the more acidic polar groups present on the surface of the fine particles, the better the dispersion stability.

[0100] The polymer particles to which dispersion stability has been imparted are not particularly limited, and examples of such polymer particles that can be preferably used include copolymers of hydrophilic monomers and polymer particles having dissociable groups or hydrophilic groups on the surface.

[0101] Examples of hydrophilic monomers that constitute the copolymer of hydrophilic monomers include ionic monomers such as acrylic acid, methacrylic acid, acrylamido-N-propanesulfonic acid, and sodium styrenesulfonate, each having a dissociable group such as a carboxyl group or a sulfonic acid group, or a hydrophilic group such as a hydroxyl group or an amide group, hydroxyethyl (meth)acrylate, (meth)acrylamide, and N-vinylpyrrolidone.

[0102] Furthermore, as the polymer particles having dissociable groups or hydrophilic groups on the surface, for example, polymer particles having dissociable groups or hydrophilic groups on the surface introduced by an emulsifier used as needed during emulsion polymerization are preferred.

[0103] The average particle size of standard particles is preferably 7 to 8 μm for blood cells, and 5.5 × 10 depending on the type of blood cell counter. 5 The concentration is adjusted to 2.5 x 107 cells / ml. [Example]

[0104] The present invention will be explained in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples. In the following, "parts" and "%" are by mass unless otherwise specified. In addition, various tests and evaluations were performed according to the following methods.

[0105] <Average circularity> Measurements were performed using a flow particle image analyzer (FPIA-2100, manufactured by Sysmex Corporation). 100-150 mL of water, from which impurities and solids had been removed, was placed in a designated container, 0.1-0.5 mL of a surfactant was added as a dispersant, and approximately 0.1-9.5 g of the sample to be measured was then added. The suspension containing the dispersed sample was dispersed in an ultrasonic disperser for approximately 1-3 minutes, and the dispersion concentration was adjusted to 3,000-10,000 particles / μL. The shape and distribution of dispersed particles in the standard solution for hemocytometer calibration were then measured.

[0106] <Volume average particle size> The volume-average particle size was measured by dynamic light scattering using a zeta potential / particle size measurement system (ELSZ-1000, Otsuka Electronics Co., Ltd.). First, 0.2 g of resin particle dispersion (standard solution for hemocytometer calibration) was taken and then diluted 100 times with ion-exchanged water. A portion of the resulting solution was placed in a quartz cell and set in a sample holder. Measurement was then performed at a temperature of 25°C, with dust cuts (number of times: 5, upper: 5, lower: 100), and an accumulation count of 70, to obtain the volume-average particle size of the solid content in the standard solution for hemocytometer calibration.

[0107] <Coefficient of variation of particle size> The particle size distribution of the dispersed particles (resin particles) was measured using a laser diffraction / scattering particle size distribution analyzer (LA-950, manufactured by Horiba, Ltd.), and the coefficient of variation of the particle size was calculated from the average particle size obtained by measuring the particle size distribution and the standard deviation of the particles using the formula [(standard deviation of particle size / average particle size) × 100 (%)].

[0108] <Aspect ratio> 1,000 particles were randomly selected from the electron microscope photograph, and the ratio was calculated from the average value of their major axis to minor axis.

[0109] <Carboxyl group density> The acid value of the raw material of the resin particles (low molecular weight polyester resin) was measured in accordance with the provisions of JIS K0070 (1992), and the content of carboxyl groups in the low molecular weight polyester resin was calculated from the obtained acid value. The density of carboxyl groups on the surface of the resin particles was calculated from the content of carboxyl groups.

[0110] <Synthesis of low molecular weight polyester> [Low molecular weight polyester 1] A reactor equipped with a condenser, stirrer, and nitrogen inlet was charged with 562 parts of bisphenol A ethylene oxide 2-mol adduct, 75 parts of bisphenol A propylene oxide 2-mol adduct, 87 parts of bisphenol A propylene oxide 3-mol adduct, 143 parts of terephthalic acid, 126 parts of adipic acid, and 2 parts of dibutyltin oxide, and reacted at 230°C under atmospheric pressure for 8 hours. The reaction was then continued for another 5 hours at a reduced pressure of 10-15 mmHg. After this, 69 parts of trimellitic anhydride was added to the reactor and reacted at 180°C and atmospheric pressure for 2 hours to obtain "Low Molecular Weight Polyester 1." The acid value of "Low Molecular Weight Polyester 1" was 40 mgKOH / g.

[0111] [Low molecular weight polyester 2] A reaction vessel equipped with a condenser, stirrer, and nitrogen inlet was charged with 362 parts of bisphenol A ethylene oxide 2-mol adduct, 75 parts of bisphenol A propylene oxide 2-mol adduct, 83 parts of bisphenol A propylene oxide 2-mol adduct, 143 parts of terephthalic acid, 126 parts of adipic acid, and 2 parts of dibutyltin oxide, and reacted at 230°C under atmospheric pressure for 8 hours. The reaction was then continued for another 5 hours at a reduced pressure of 10-15 mmHg. After this, 69 parts of trimellitic anhydride was added to the reaction vessel and reacted at 180°C and atmospheric pressure for 2 hours to obtain "Low Molecular Weight Polyester 2." The acid value of "Low Molecular Weight Polyester 2" was 10 mgKOH / g.

[0112] [Low molecular weight polyester 3] A reaction vessel equipped with a condenser, stirrer, and nitrogen inlet tube was charged with 255 parts of bisphenol A ethylene oxide 2-mol adduct, 480 parts of bisphenol A propylene oxide 2-mol adduct, 223 parts of terephthalic acid, 49 parts of adipic acid, and 2 parts of dibutyltin oxide, and the mixture was reacted at 230°C under atmospheric pressure for 8 hours, and then further reacted for 5 hours at a reduced pressure of 10-15 mmHg. After that, 1 part of trimellitic anhydride was added to the reaction vessel, and the mixture was reacted at 180°C and atmospheric pressure for 2 hours to obtain "Low Molecular Weight Polyester 3." The acid value of "Low Molecular Weight Polyester 3" was 0.1 mgKOH / g.

[0113] <Preparation of oil-based dispersion> [Oil-based dispersion 1] 155 parts of "Low Molecular Weight Polyester 1" and 80 parts of ethyl acetate were placed in a beaker and stirred to dissolve. 1.2 parts of isophorone diamine was added to this dispersion, and the mixture was stirred at 2,000 rpm using a disper for 3 minutes to obtain "Oil-Based Dispersion 1."

[0114] [Oil-based dispersion 2] 155 parts of "Low Molecular Weight Polyester 2" and 80 parts of ethyl acetate were placed in a beaker and stirred to dissolve. 1.2 parts of isophorone diamine was added to this dispersion, and the mixture was stirred at 2,000 rpm using a disper for 3 minutes to obtain "Oil-Based Dispersion 2."

[0115] [Oil-based dispersion 3] 155 parts of "Low Molecular Weight Polyester 3" and 80 parts of ethyl acetate were placed in a beaker and stirred to dissolve. 1.2 parts of isophorone diamine was added to this dispersion, and the mixture was stirred at 2,000 rpm using a disper for 3 minutes to obtain "Oil-Based Dispersion 3."

[0116] [Oil-based dispersion 4] 155 parts of "Low Molecular Weight Polyester 1" and 80 parts of ethyl acetate were placed in a beaker and stirred to dissolve. 1.7 parts of isophorone diamine was added to this dispersion, and the mixture was stirred at 2,000 rpm using a disper for 3 minutes to obtain "Oil-Based Dispersion 4."

[0117] [Oil-based dispersion 5] 155 parts of "Low Molecular Weight Polyester 2" and 80 parts of ethyl acetate were placed in a beaker and stirred to dissolve. 1.7 parts of isophorone diamine was added to this dispersion, and the mixture was stirred at 2,000 rpm using a disper for 3 minutes to obtain "Oil-Based Dispersion 5."

[0118] [Oil-based dispersion 6] 155 parts of "Low Molecular Weight Polyester 2" and 80 parts of ethyl acetate were placed in a beaker and stirred to dissolve. 1.5 parts of isophorone diamine was added to this dispersion, and the mixture was stirred at 2,000 rpm using a disper for 3 minutes to obtain "Oil-Based Dispersion 6."

[0119] [Oil-based dispersion 7] 155 parts of "Low Molecular Weight Polyester 2" and 80 parts of ethyl acetate were placed in a beaker and stirred to dissolve. 1.7 parts of isophorone diamine was added to this dispersion, and the mixture was stirred at 2,000 rpm using a disper for 3 minutes to obtain "Oil-Based Dispersion 7."

[0120] [Oil-based dispersion 8] 155 parts of "Low Molecular Weight Polyester 2" and 80 parts of ethyl acetate were placed in a beaker and stirred to dissolve. 2.0 parts of isophorone diamine was added to this dispersion, and the mixture was stirred at 2,000 rpm using a disper for 3 minutes to obtain "Oil-Based Dispersion 8."

[0121] <Preparation of aqueous dispersion medium> [Aqueous dispersion medium 1] 945 parts of pure water, 40 parts of a 20% aqueous dispersion of a styrene-methacrylic acid-butyl acrylate copolymer, 160 parts of a 50% aqueous solution of sodium dodecyldiphenyletherdisulfonate (Eleminol MON-7, manufactured by Sanyo Chemical Industries, Ltd.), and 90 parts of ethyl acetate were placed in a container and mixed at 1,000 rpm for 3 minutes using Robomics to obtain "aqueous dispersion medium liquid 1."

[0122] <Preparation of standard solution for hemocytometer calibration> [Example 1] 37 parts of "Oil-based Dispersion Liquid 1" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm for 5 minutes using a TK Homomixer, yielding "Dispersion Slurry 1." Subsequently, the organic solvent was removed from "Dispersion Slurry 1" at 25°C until the residual solvent concentration per 100 parts of resin solids was 15%, yielding "Dispersion Slurry 2."

[0123] Next, "Dispersion Slurry 2" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 3." The organic solvent was removed from "Dispersion Slurry 3" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 1."

[0124] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 1" was 7.5 μm, the coefficient of variation of particle size was 2.7%, the circularity was 0.970, and the aspect ratio was 1.5. The carboxyl group density on the particle surface, as determined by acid-base titration, was 12 / Å. 2 The measurement results are shown in Table 1.

[0125] [Example 2] 37 parts of "Oil-based Dispersion Liquid 2" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm for 5 minutes using a TK Homomixer, yielding "Dispersion Slurry 2." Subsequently, the organic solvent was removed from "Dispersion Slurry 2" at 25°C until the residual solvent concentration per 100 parts of resin solids was 10%, yielding "Dispersion Slurry 3."

[0126] Next, "Dispersion Slurry 3" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 4." The organic solvent was removed from "Dispersion Slurry 4" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 2."

[0127] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 2" was 7.8 μm, the coefficient of variation of particle size was 5.3%, the circularity was 0.985, and the aspect ratio was 1.7. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.340 particles / Å. 2 The measurement results are shown in Table 1.

[0128] [Example 3] 37 parts of "Oil-based Dispersion Liquid 2" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm with a TK Homomixer for 5 minutes to obtain "Dispersion Slurry 5." Subsequently, the organic solvent was removed from "Dispersion Slurry 5" at 25°C until the residual solvent concentration per 100 parts of resin solids was 30%, to obtain "Dispersion Slurry 6."

[0129] Next, "Dispersion Slurry 6" and a thickener at 5% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 7." The organic solvent was removed from "Dispersion Slurry 7" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and then the mixture was washed to obtain "Hemocytometer Calibration Standard Solution 3."

[0130] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 3" was 7.2 μm, the coefficient of variation of particle size was 6.5%, the circularity was 0.950, and the aspect ratio was 4.3. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.160 particles / Å. 2 The measurement results are shown in Table 1.

[0131] [Example 4] 37 parts of "Oil-based Dispersion Liquid 2" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm with a TK Homomixer for 5 minutes to obtain "Dispersion Slurry 5." Subsequently, the organic solvent was removed from "Dispersion Slurry 5" at 25°C until the residual solvent concentration per 100 parts of resin solids was 15%, to obtain "Dispersion Slurry 6."

[0132] Next, "Dispersion Slurry 6" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 8." The organic solvent was removed from "Dispersion Slurry 8" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and then the mixture was washed to obtain "Hemocytometer Calibration Standard Solution 4."

[0133] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 4" was 7.3 μm, the coefficient of variation of particle size was 10.3%, the circularity was 0.973, and the aspect ratio was 4.3. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.260 particles / Å. 2 The measurement results are shown in Table 1.

[0134] [Example 5] 37 parts of "Oil-based Dispersion Liquid 3" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm with a TK Homomixer for 5 minutes to obtain "Dispersion Slurry 9." Subsequently, the organic solvent was removed from "Dispersion Slurry 9" at 25°C until the residual solvent concentration per 100 parts of resin solids was 15%, to obtain "Dispersion Slurry 10."

[0135] Next, "Dispersion Slurry 10" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 11." The organic solvent was removed from "Dispersion Slurry 11" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 5."

[0136] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 5" was 7.4 μm, the coefficient of variation of particle size was 6.8%, the circularity was 0.965, and the aspect ratio was 3.2. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.001 particles / Å. 2 The measurement results are shown in Table 1.

[0137] [Example 6] 37 parts of "Oil-based Dispersion Liquid 4" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm for 5 minutes using a TK Homomixer to obtain "Dispersion Slurry 12." Subsequently, the organic solvent was removed from "Dispersion Slurry 12" at 25°C until the residual solvent concentration per 100 parts of resin solids was 15%, to obtain "Dispersion Slurry 13."

[0138] Next, "Dispersion Slurry 13" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 14." The organic solvent was removed from "Dispersion Slurry 14" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 6."

[0139] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 6" was 3.4 μm, the coefficient of variation of particle size was 2.7%, the circularity was 0.970, and the aspect ratio was 1.5. The carboxyl group density on the particle surface, as determined by acid-base titration, was 12,000 particles / Å. 2 The measurement results are shown in Table 1.

[0140] [Example 7] 37 parts of "Oil-based Dispersion Liquid 4" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm for 5 minutes using a TK Homomixer to obtain "Dispersion Slurry 15." Subsequently, the organic solvent was removed from "Dispersion Slurry 15" at 25°C until the residual solvent concentration per 100 parts of resin solids was 10%, to obtain "Dispersion Slurry 16."

[0141] Next, "Dispersion Slurry 16" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 17." The organic solvent was removed from "Dispersion Slurry 17" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 7."

[0142] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 7" was 3.1 μm, the coefficient of variation of particle size was 5.3%, the circularity was 0.985, and the aspect ratio was 1.7. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.340 particles / Å. 2 The measurement results are shown in Table 1.

[0143] [Example 8] 37 parts of "Oil-based Dispersion Liquid 6" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm for 5 minutes using a TK Homomixer, yielding "Dispersion Slurry 18." Subsequently, the organic solvent was removed from "Dispersion Slurry 18" at 25°C until the residual solvent concentration per 100 parts of resin solids was 30%, yielding "Dispersion Slurry 19."

[0144] Next, "Dispersion Slurry 19" and a thickener at 5% relative to the resin solid content were placed in a stainless steel container and mixed at 200 rpm for 30 minutes using a helical ribbon, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 20." The organic solvent was removed from "Dispersion Slurry 20" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 8."

[0145] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 8" was 4.3 μm, the coefficient of variation of particle size was 6.5%, the circularity was 0.950, and the aspect ratio was 4.3. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.160 particles / Å. 2 The measurement results are shown in Table 1.

[0146] [Example 9] 37 parts of "Oil-based Dispersion Liquid 6" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm for 5 minutes using a TK Homomixer, yielding "Dispersion Slurry 18." Subsequently, the organic solvent was removed from "Dispersion Slurry 18" at 25°C until the residual solvent concentration per 100 parts of resin solids was 15%, yielding "Dispersion Slurry 19."

[0147] Next, "Dispersion Slurry 19" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 20." The organic solvent was removed from "Dispersion Slurry 20" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 9."

[0148] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 9" was 4.5 μm, the coefficient of variation of particle size was 10.3%, the circularity was 0.973, and the aspect ratio was 4.3. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.260 particles / Å. 2 The measurement results are shown in Table 1.

[0149] [Example 10] 37 parts of "Oil-based Dispersion Liquid 7" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm with a TK Homomixer for 5 minutes to obtain "Dispersion Slurry 21." Subsequently, the organic solvent was removed from "Dispersion Slurry 21" at 25°C until the residual solvent concentration per 100 parts of resin solids was 15%, to obtain "Dispersion Slurry 22."

[0150] Next, "Dispersion Slurry 22" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 23." The organic solvent was removed from "Dispersion Slurry 23" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 10."

[0151] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 10" was 3.8 μm, the coefficient of variation of particle size was 6.8%, the circularity was 0.965, and the aspect ratio was 3.2. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.001 particles / Å. 2 The measurement results are shown in Table 1.

[0152] [Comparative Example 1] 33 parts of "Oil-based Dispersion Liquid 8" and 67 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm with a TK Homomixer for 5 minutes to obtain "Dispersion Slurry 24." Subsequently, the organic solvent was removed from "Dispersion Slurry 24" at 25°C until the residual solvent concentration per 100 parts of resin solids was 10%, to obtain "Dispersion Slurry 25."

[0153] Next, "Dispersion Slurry 25" and a thickener at 3% relative to the resin solid content were placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water were added to the container to obtain "Dispersion Slurry 26." The organic solvent was removed from "Dispersion Slurry 26" at 25°C until the residual solvent concentration relative to 100 parts of resin solid content was 1% or less, and the mixture was then washed to obtain "Hemocytometer Calibration Standard Solution 11."

[0154] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 11" was 1.5 μm, the coefficient of variation of particle size was 5.2%, the circularity was 0.985, and the aspect ratio was 1.5. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.001 particles / Å. 2 The measurement results are shown in Table 1.

[0155] Comparative Example 2 37 parts of "Oil-based Dispersion Liquid 2" and 63 parts of "Aqueous Dispersion Medium Liquid 1" were placed in a beaker and mixed at 12,000 rpm for 5 minutes using a TK Homomixer, yielding "Dispersion Slurry 27." Subsequently, the organic solvent was removed from "Dispersion Slurry 27" at 25°C until the residual solvent concentration per 100 parts of resin solids was 5%, yielding "Dispersion Slurry 28."

[0156] Next, "Dispersion Slurry 28" was placed in a stainless steel container and mixed using a helical ribbon at 200 rpm for 30 minutes, after which 100 parts of pure water was added to the container to obtain "Dispersion Slurry 29." The organic solvent was removed from "Dispersion Slurry 29" at 25°C until the residual solvent concentration per 100 parts of resin solids was 1% or less, and then the mixture was washed to obtain "Hemocytometer Calibration Standard Solution 12."

[0157] The volume average particle size of the particles dispersed in "Hemocytometer Calibration Standard Solution 12" was 7.4 μm, the coefficient of variation of particle size was 5.6%, the circularity was 0.995, and the aspect ratio was 1.0. The carboxyl group density on the particle surface, as determined by acid-base titration, was 0.13 / Å. 2 The measurement results are shown in Table 1.

[0158] [Table 1]

[0159] The prepared standard solutions 1 to 12 for calibrating a hemocytometer (Examples 1 to 10 and Comparative Examples 1 and 2) were evaluated for the calibration accuracy of the device using a hemocytometer based on the electrical resistance detection method, and the storage stability of the standard solutions for calibrating a hemocytometer over time was evaluated.

[0160] [Storage of standard solution for hemocytometer calibration] The storage stability of the standard solution for hemocytometer calibration was evaluated using an Elma PC602 hemocytometer, based on the change in the particle count measurements over time for the dispersion on the day of preparation and after 180 days of storage at room temperature. The results of the evaluation based on the following criteria are shown in Table 2. A rating of 2 or higher was considered to indicate that the standard solution for hemocytometer calibration had good storage stability. 3: Less than 10% 2: 10% or more, less than 20% 1:20% or more

[0161] [Measurement accuracy of blood cell counter] The measurement accuracy of the standard solution for calibrating a hemocytometer using the electrical resistance detection method was evaluated based on the discrepancy between the hematocrit value determined from the number of particles dispersed in the standard solution measured using an Elma PC602 hemocytometer and the hematocrit value determined by the centrifugation method. The results of the evaluation based on the following criteria are shown in Table 2. A rating of 2 or higher was considered to indicate that the calibration accuracy of the standard solution for calibrating a hemocytometer was good. 3: Less than 10% 2: 10% or more, less than 20% 1:20% or more

[0162] [Table 2]

[0163] From Tables 1 and 2, the average circularity is 0.95 or more and 0.985 or less, the volume average particle size is 2 μm or more and 10 μm or less, the coefficient of variation of the particle size is 2.7% or more and 10.3% or less, the aspect ratio is 1.3 or more and 5.0 or less, the density of the carboxyl group on the surface is 0.001, The density of the carboxyl groups is 0.001 / Å 2 More than 12 pieces / Å 2 The standard solutions for calibrating hemocytometers containing the following resin particles were excellent in both calibration accuracy and storage stability (Examples 1 to 10).

[0164] In contrast, a standard solution for calibrating a hemocytometer containing resin particles with a volume average particle size of less than 2 μm had poor storage stability (Comparative Example 1), and a standard solution for calibrating a hemocytometer containing resin particles with an average circularity of more than 0.99 had poor calibration accuracy (Comparative Example 2).

[0165] These results demonstrate that a standard solution for calibrating a hemocytometer made of resin particles with an average circularity of 0.99 or less and a volume average particle size of 2 μm to 10 μm can provide excellent calibration accuracy and storage stability.

[0166] Although the embodiments of the present invention have been described above, the present invention is not limited to the specific embodiments, and various modifications and changes are possible within the scope of the invention described in the claims. [Prior art documents] [Patent documents]

[0167] [Patent Document 1] Japanese Patent Application Publication No. 01-301166

Claims

1. A standard solution for calibrating a hemocytometer containing resin particles, the resin particles have an average circularity of 0.99 or less, The resin particles have a volume average particle size of 2 μm or more and 10 μm or less, the coefficient of variation of the particle size of the resin particles is 5.3% or more and 15% or less; The resin particles are formed by adhering fine particles to base particles. Standard solution for calibrating hemocytometers.

2. the resin particles have an average circularity of 0.95 or more and 0.985 or less; The standard solution for calibrating a hemocytometer according to claim 1.

3. The resin particles have an aspect ratio of 1.3 or more and 5.0 or less. The standard solution for calibrating a hemocytometer according to claim 1 or 2.

4. The resin particles have carboxyl groups on their surfaces. The standard solution for calibrating a hemocytometer according to any one of claims 1 to 3.

5. The density of the carboxyl groups is 0.001 / Å ２ That's all. The standard solution for calibrating a hemocytometer according to claim 4.

6. A hemocytometer comprising the standard solution for calibrating a hemocytometer according to any one of claims 1 to 5.

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