Hemolysis reagent, reagent kit, and method for classifying leukocytes

Abstract

To provide a hemolytic reagent, a white blood cell classifying reagent kit, and a method for classifying white blood cells, which can more accurately classify monocytes and lymphocytes, as well as neutrophils and eosinophils.SOLUTION: Disclosed is a hemolytic reagent comprising a nonionic surfactant represented by formula (I), where R1 represents an alkyl group, an alkenyl group, or an alkynyl group having 8 or more and 25 or less carbon atoms, R2 represents an oxygen atom, (COO) or a group represented by formula (II) below. In the formula (I), n is 23 or larger and 25 or smaller, or 30, with n of 23 or larger and 25 or smaller, the concentration of the nonionic surfactant is 1700 ppm or higher and 2300 ppm or lower, and with n of 30, the concentration of the nonionic surfactant is 1900 ppm or higher and 2300 ppm or lower.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a hemolytic reagent, and also to a reagent kit for white blood cell classification and a method for white blood cell classification. [Background technology]

[0002] Normal white blood cells are classified into five types: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Information such as the classification and number of white blood cells is useful information for examining the health condition of a subject. As a reagent for classifying and counting white blood cells, for example, the reagent described in Patent Document 1 is known. Patent Document 1 discloses a hemolysis reagent containing an aromatic organic acid and having a predetermined pH, and describes that the use of this reagent makes it possible to accurately classify monocytes and lymphocytes. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Application Publication No. 2014 / 0120530 Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present invention is to provide a hemolysis reagent, a leukocyte classification reagent kit, and a leukocyte classification method that can more accurately classify not only monocytes and lymphocytes, but also neutrophils and eosinophils. [Means for solving the problem]

[0005] The present invention provides a hemolysis reagent comprising a nonionic surfactant represented by the following formula (I), in which R1 is an alkyl group, an alkenyl group, or an alkynyl group having 8 to 25 carbon atoms, and R2 is an oxygen atom, (COO) or represented by the following formula (II), in which n in the formula (I) is 23 to 25 or 30, and when n is 23 to 25, the concentration of the nonionic surfactant is 1700 ppm to 2300 ppm, and when n is 30, the concentration of the nonionic surfactant is 1900 ppm to 2300 ppm.

[0006] R1-R2-(CH2CH2O) n -H formula (I)

[0007] [ka]

[0008] The present invention provides a leukocyte classification reagent kit comprising the above-mentioned hemolysis reagent and a staining reagent for fluorescently staining nucleic acid.The present invention also provides a leukocyte classification method using the above-mentioned reagent kit. Effect of the Invention

[0009] According to the present invention, there are provided a hemolysis reagent, a reagent kit and a method for classifying leukocytes, which are capable of accurately classifying monocytes and lymphocytes, and also accurately classifying neutrophils and eosinophils. [Brief description of the drawings]

[0010] [Figure 1A] FIG. 2 is a schematic diagram showing an example of a hemolyzing reagent according to the present embodiment. [Figure 1B] FIG. 1 is a schematic diagram showing an example of a reagent kit according to an embodiment of the present invention. [Diagram 2] 1 is an example of a scattergram when a normal blood sample is measured using the white blood cell classification reagent kit of the present embodiment. [Figure 3A] 1 is a graph showing the distance between lymphocyte populations and monocyte populations in Example 4. [Figure 3B]1 is a graph showing the distance between the neutrophil population and the eosinophil population in Example 4. [Figure 4] 1 is a graph showing the results of a scattergram of sample 1 in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [1. Hemolysis Reagent] One embodiment of the present invention is a hemolysis reagent. The hemolysis reagent is a reagent for hemolyzing red blood cells in a specimen and damaging the cell membrane of white blood cells to such an extent that a fluorescent dye can penetrate the cell membrane. The hemolysis reagent of this embodiment is preferably used in an automatic blood cell counter capable of classifying white blood cells contained in a whole blood specimen into five groups of lymphocytes, monocytes, neutrophils, eosinophils, and basophils using a flow cytometry method. For example, the automatic blood cell counter mixes a whole blood specimen as a blood specimen with a hemolysis reagent and a staining reagent that fluorescently stains nucleic acid to prepare a measurement specimen in which red blood cells are hemolyzed and white blood cells are stained. The measurement specimen is supplied to a flow cell, and optical information is obtained from the white blood cells by irradiating the white blood cells with light. Each white blood cell is classified into the above-mentioned five groups based on the acquired optical information. Such an automatic blood cell counter is disclosed, for example, in JP 2008-209383 A. This hemolytic reagent makes it possible to accurately classify lymphocytes and monocytes, while more accurately classifying neutrophils and eosinophils, in a measurement sample prepared by mixing the hemolytic reagent with a whole blood specimen using an automated blood cell counter.

[0012] The automated blood cell counter may be equipped with a function for measuring body fluid samples other than whole blood samples (e.g., ascites, synovial fluid, pleural effusion, cerebrospinal fluid, bone marrow fluid, bronchoalveolar lavage fluid, peritoneal lavage fluid, etc.) in addition to the function for measuring whole blood samples. In this case, the hemolytic reagent can also be used to prepare a measurement sample for white blood cells contained in body fluid samples other than whole blood samples. The hemolytic reagent makes it possible to effectively separate white blood cells, particularly monocytes, from other groups contained in body fluid samples other than whole blood samples. An automated blood cell counter equipped with a function for measuring body fluid samples other than whole blood samples is disclosed in, for example, JP 2008-209383 A.

[0013] The hemolysis reagent of this embodiment contains a nonionic surfactant represented by the following formula (I): In formula (I), R1 is an alkyl group, an alkenyl group, or an alkynyl group having 8 to 25 carbon atoms, and R2 is an oxygen atom, (COO), or represented by the following formula (II).

[0014] R1-R2-(CH2CH2O) n -H formula (I)

[0015] [ka]

[0016] In the hemolysis reagent, the solvent is not particularly limited as long as it can dissolve the nonionic surfactant represented by formula (I). For example, water, organic solvents, and mixtures thereof can be mentioned. For example, organic solvents include alcohols having 1 to 6 carbon atoms, ethylene glycol, diethylene glycol, polyethylene glycol, dimethyl sulfoxide (DMSO), etc.

[0017] The hemolysis reagent of the present embodiment may contain a buffer substance for maintaining a constant pH. Examples of the buffer include inorganic acid salts, organic acid salts, Good's buffers, and combinations thereof. Examples of the inorganic acid salts include phosphates, borates, and combinations thereof. Examples of the organic acid salts include citrates, malates, and combinations thereof. Examples of the Good's buffer include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, TAPS, and combinations thereof.

[0018] In the hemolysis reagent of this embodiment, n in formula (I) is 23 or more and 25 or less. More preferably, n in formula (I) is 23 or 25, and even more preferably, n is 23. The concentration of the nonionic surfactant represented by formula (I) is 1700 ppm or more, preferably 1750 ppm or more. The concentration of the nonionic surfactant represented by formula (I) is 2300 ppm or less, preferably 2200 ppm or less. In the hemolysis reagent of a further embodiment, the concentration of the nonionic surfactant represented by formula (I) is about 1700 ppm or more, preferably about 1750 ppm or more. The concentration of the nonionic surfactant represented by formula (I) is about 2300 ppm or less, preferably about 2200 ppm or less.

[0019] In one example of the hemolysis reagent of this embodiment, n in formula (I) is 23. In addition, the concentration of the nonionic surfactant represented by formula (I) is 1700 ppm or more, preferably 1750 ppm or more. In addition, the concentration of the nonionic surfactant represented by formula (I) is 2300 ppm or less, preferably 2200 ppm or less. In one example of the hemolysis reagent of a further embodiment, the concentration of the nonionic surfactant represented by formula (I) is about 1700 ppm or more, preferably about 1750 ppm or more. In addition, the concentration of the nonionic surfactant represented by formula (I) is about 2300 ppm or less, preferably about 2200 ppm or less.

[0020] In another example of the hemolysis reagent of this embodiment, n in formula (I) is 25. In addition, the concentration of the nonionic surfactant represented by formula (I) is 1700 ppm or more, preferably 1750 ppm or more. In addition, the concentration of the nonionic surfactant represented by formula (I) is 2300 ppm or less, preferably 2200 ppm or less. In another example of the hemolysis reagent of a further embodiment, the concentration of the nonionic surfactant represented by formula (I) is about 1700 ppm or more, preferably about 1750 ppm or more. In addition, the concentration of the nonionic surfactant represented by formula (I) is about 2300 ppm or less, preferably about 2200 ppm or less.

[0021] In one embodiment of the hemolysis reagent of this embodiment, n in formula (I) is 30. The concentration of the nonionic surfactant represented by formula (I) is 1900 ppm or more, preferably 2000 ppm or more, more preferably 2100 ppm or more. The concentration of the nonionic surfactant represented by formula (I) is 2300 ppm or less, preferably 2200 ppm. In one embodiment of the hemolysis reagent of a further embodiment, the concentration of the nonionic surfactant represented by formula (I) is about 1900 ppm or more, preferably about 2000 ppm or more, more preferably about 2100 ppm or more. The concentration of the nonionic surfactant represented by formula (I) is about 2300 ppm or less, preferably about 2200 ppm or less.

[0022] In the hemolysis reagent of this embodiment, the nonionic surfactant represented by formula (I) may be, for example, polyoxyethylene alkyl ether, polyoxyethylene sterol, polyoxyethylene castor oil, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene alkylamine, polyoxyethylene polyoxypropylene alkyl ether, or a combination thereof. Among them, it is preferable that polyoxyethylene alkyl ether is included. As the polyoxyethylene alkyl ether, it is preferable that polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, polyoxyethylene (30) cetyl ether, or at least one selected from the group consisting of polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, and a combination thereof, and further preferable that the nonionic surfactant is polyoxyethylene (23) cetyl ether. In the hemolysis reagent, the nonionic surfactant may be one type or two or more types. In addition, the hemolysis reagent may further contain a nonionic surfactant other than the nonionic surfactant represented by formula (I).

[0023] The hemolysis reagent of this embodiment may further contain a cationic surfactant. Examples of the cationic surfactant include quaternary ammonium salt surfactants, pyridium salt surfactants, and combinations thereof. As the quaternary ammonium salt surfactant, for example, a surfactant represented by formula (III) having a total carbon number of 9 to 30 is preferably used. The cationic surfactant contained in the hemolysis reagent may be one type, or two or more types.

[0024] [ka]

[0025] In formula (III), R1 is an alkyl or alkenyl group having 6 to 18 carbon atoms. R2 and R3 are the same or different and are an alkyl or alkenyl group having 1 to 4 carbon atoms. R4 is an alkyl or alkenyl group having 1 to 4 carbon atoms or a benzyl group. X - is a halogen ion.

[0026] In formula (III), R1 is preferably an alkyl or alkenyl group having 6, 8, 10, 12, or 14 carbon atoms, and particularly preferably a linear alkyl group. More specific examples of R1 include an octyl group, a decyl group, and a dodecyl group. R2 and R3 are preferably a methyl group, an ethyl group, or a propyl group. R4 is preferably a methyl group, an ethyl group, or a propyl group.

[0027] An example of the pyridinium salt surfactant is a surfactant represented by the formula (IV).

[0028] [ka]

[0029] In formula (IV), R1 is an alkyl or alkenyl group having 6 to 18 carbon atoms, and X - is a halogen atom.

[0030] In formula (IV), R1 is preferably an alkyl or alkenyl group having 6, 8, 10, 12 or 14 carbon atoms, and particularly preferably a linear alkyl group. More specific examples of R1 include an octyl group, a decyl group and a dodecyl group.

[0031] In the hemolysis reagent, the cationic surfactant concentration can be appropriately selected depending on the type of surfactant. The cationic surfactant concentration is 10 ppm or more. It is preferably 400 ppm or more, more preferably 500 ppm or more, and even more preferably 600 ppm or more. The cationic surfactant concentration is 10,000 ppm or less. It is preferably 1,000 ppm or less, more preferably 800 ppm or less, and even more preferably 700 ppm or less.

[0032] The hemolysis reagent of the present embodiment may further contain an aromatic organic acid. In this specification, the aromatic organic acid means an acid having at least one aromatic ring in the molecule and its salt. Examples of the aromatic organic acid include aromatic carboxylic acids and aromatic sulfonic acids. Examples of the aromatic carboxylic acids include phthalic acid, benzoic acid, salicylic acid, hippuric acid, salts thereof, and combinations thereof. Examples of the aromatic sulfonic acids include p-aminobenzenesulfonic acid, benzenesulfonic acid, salts thereof, and combinations thereof. The aromatic organic acid contained in the hemolysis reagent may be one type or two or more types. In addition, the aromatic organic acid may exhibit a buffering effect. When an aromatic organic acid exhibiting a buffering effect is used, the addition of a buffer is optional and may be combined with the above-mentioned buffer.

[0033] When the hemolysis reagent further contains an aromatic organic acid, the concentration of the aromatic organic acid is not particularly limited, but is preferably 20 mM or more, more preferably 25 mM or more, from the viewpoint of the ability to distinguish monocytes and lymphocytes. The concentration of the aromatic organic acid contained in the hemolysis reagent is preferably 50 mM or less, more preferably 45 mM or less.

[0034] In the hemolysis reagent of this embodiment, the pH is preferably 5.5 or more, more preferably 5.7 or more, and even more preferably 5.9 or more. In addition, in the hemolysis reagent, the pH is preferably 7.2 or less, more preferably 6.9 or less, and even more preferably 6.6 or less. The pH can be adjusted using a known base (such as sodium hydroxide) or acid (such as hydrochloric acid).

[0035] In the hemolysis reagent of this embodiment, the osmotic pressure is not particularly limited, but is preferably 150 mOsm / kg or less, more preferably 130 mOsm / kg or less, and most preferably 110 mOsm / kg or less, from the viewpoint of the hemolysis efficiency of red blood cells. A suitable osmotic pressure regulator may be added to adjust the osmotic pressure. Examples of the osmotic pressure regulator include sugars, amino acids, organic solvents, sodium chloride, and combinations thereof.

[0036] An example of the hemolytic reagent of this embodiment is shown in Fig. 1A. In Fig. 1A, 10 denotes a container in which the hemolytic reagent is stored. The container 10 may further be stored in a packaging box or the like. When the container 10 is stored in a packaging box, it may be stored together with an attached document describing the composition, usage method, storage method, etc. of the hemolytic reagent, a cushioning material for reducing external impact, and the like.

[0037] [2. White blood cell classification reagent kit] One embodiment of the present invention is a leukocyte classification reagent kit including a hemolytic reagent and a staining reagent for fluorescently staining nucleic acid. The hemolytic reagent included in the reagent kit is the hemolytic reagent described in [1. Hemolytic reagent].

[0038] In the reagent kit, the fluorescent dye for staining nucleic acid is not particularly limited and can be appropriately selected depending on the wavelength of light irradiated from the light source, etc. For example, propidium iodide, ethidium bromide, ethidium-acridine heterodimer, ethidium diazide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, trimethylenebis[[3-[[4-[[(3-methylbenzothiazol-3-ium)-2-yl]methylene]-1,4-dihydroquinolin]-1-yl]propyl]dimethylaminium] tetraiodide (TOTO-1), 4-[(3-methylbenzothiazol-2(3H)-ylidene)methyl]-1-[3-(trimethylaminio)propyl]quinolinium diiodide (TO -PRO-1), N,N,N',N'-tetramethyl-N,N'-bis[3-[4-[3-[(3-methylbenzothiazol-3-ium)-2-yl]-2-propenylidene]-1,4-dihydroquinolin-1-yl]propyl]-1,3-propanediaminium tetraiodide (TOTO-3), or 2-[3-[[1-[3-(trimethylaminio)propyl]-1,4-dihydroquinolin]-4-ylidene]-1-propenyl]-3-methylbenzothiazol-3-ium diiodide (TO-PRO-3), fluorescent dyes represented by the following general formula (V), and combinations thereof.

[0039] [ka]

[0040] In formula (V), R1 and R4 are the same or different and are a hydrogen atom, an alkyl group, an alkyl chain having a hydroxyl group, an alkyl chain having an ether group, an alkyl chain having an ester group, or a benzyl group which may have a substituent. R2 and R3 are the same or different and are a hydrogen atom, a hydroxyl group, a halogen, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an alkylsulfonyl group, or a phenyl group. Z is a sulfur atom, an oxygen atom, or a carbon atom having a methyl group. n is 0, 1, 2, or 3. X - is an anion.

[0041] In formula (V), the alkyl group may be either linear or branched. When either R1 or R4 is an alkyl group having 6 to 18 carbon atoms, the other is preferably a hydrogen atom or an alkyl group having less than 6 carbon atoms. Among alkyl groups having 6 to 18 carbon atoms, alkyl groups having 6, 8 or 10 carbon atoms are preferred.

[0042] In formula (V), examples of the substituent of the benzyl group of R1 and R4 include an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, or an alkynyl group having 2 to 20 carbon atoms. Among these, a methyl group or an ethyl group is particularly preferred.

[0043] In formula (V), examples of the alkenyl group of R2 and R3 include alkenyl groups having 2 to 20 carbon atoms. Examples of the alkoxy group of R2 and R3 include alkoxy groups having 1 to 20 carbon atoms. Among these, a methoxy group or an ethoxy group is particularly preferred.

[0044] In formula (V), the anion X - As, F - , Cl - , Br - and I - Halogen ions such as CF3SO3 - , BF4 - etc.

[0045] In the reagent kit, one type of fluorescent dye may be used, or two or more types may be used. The concentration of the fluorescent dye can be appropriately set according to the type of fluorescent dye. Usually, the concentration of the fluorescent dye is 0.01 pg / μL or more, preferably 0.1 pg / μL or more, and the concentration of the fluorescent dye is 100 pg / μL or less, preferably 10 pg / μL or less. For example, when a fluorescent dye represented by formula (I) is used as a fluorescent dye in one reagent, the concentration of the fluorescent dye in the reagent kit is preferably 0.2 pg / μL or more, more preferably 0.3 pg / μL or more. In addition, when a fluorescent dye represented by formula (I) is used as a fluorescent dye in one reagent, the concentration of the fluorescent dye in the reagent kit is preferably 0.6 pg / μL or less, more preferably 0.5 pg / μL or less.

[0046] In the reagent kit of this embodiment, the staining reagent may be a commercially available staining reagent for measuring white blood cells, such as Fluorocell WDF (Sysmex Corporation) or Stromatolyser 4DS (Sysmex Corporation).

[0047] In the reagent kit of this embodiment, the solvent of the reagent contained in the reagent kit is not particularly limited as long as it can dissolve the nonionic surfactant represented by formula (I) and / or the fluorescent dye. The details of the solvent are the same as those described above in [1. Hemolysis Reagent]. The solvent for dissolving the fluorescent dye is preferably an organic solvent from the viewpoint of storage stability.

[0048] In the reagent kit of this embodiment, the solvent of each reagent contained in the reagent kit may contain a buffer substance to keep the pH constant. The details of the buffer substance are the same as those described in [1. Hemolysis Reagent] above.

[0049] An example of the reagent kit of this embodiment is shown in FIG. 1B. In FIG. 1B, 30 denotes the reagent kit. 11 denotes a first container in which the hemolysis reagent described in [1. Hemolysis Reagent] is accommodated. 20 denotes a second container in which a staining reagent for fluorescently staining nucleic acid is accommodated. The reagent kit 30 may further be accommodated in a packaging box or the like. When the reagent kit 30 is accommodated in a packaging box, it may be accommodated together with an attached document describing the composition, usage method, storage method, etc. of each reagent included in the reagent kit, a buffer material for reducing external impact, and the like. The reagent kit 30 may include other reagents, such as a buffer solution, a calibrator, and the like.

[0050] [3. Classification of white blood cells] One embodiment of the present invention is a method for classifying white blood cells, which includes the steps of: irradiating a measurement sample prepared by mixing a hemolysis reagent, a staining reagent for staining nucleic acid, and a specimen containing white blood cells with light to detect optical information; and classifying the white blood cells based on the optical information.

[0051] In the method of this embodiment, the specimen is not particularly limited as long as it is a sample that contains white blood cells or may contain blood cells. The sample that contains white blood cells or may contain blood cells is a sample collected from a mammal, preferably a human. Examples of the specimen include whole blood, ascites, synovial fluid, pleural fluid, cerebrospinal fluid, bone marrow fluid, bronchoalveolar lavage fluid, peritoneal lavage fluid, urine, and samples collected by apheresis.

[0052] In the method of this embodiment, the specimen may be diluted with an appropriate aqueous solvent or may contain known additives, as long as the detection step and / or classification step described below is not hindered. Examples of aqueous solvents include, but are not limited to, water, saline, and buffer solutions. The buffer solution preferably has a buffering effect at a pH near neutral (e.g., a pH of 6 to 8). Examples of additives include, but are not limited to, anticoagulants. Examples of anticoagulants include K3-EDTA, EDTA, EGTA, TPEN, BAPTA, sodium citrate, warfarin, heparin, danaparoid, fondaparinux, and combinations thereof. If the specimen contains impurities, the impurities may be removed by known means such as centrifugation or filtration.

[0053] In the method of this embodiment, the order of mixing the hemolytic reagent, the staining reagent, and the specimen is not particularly limited. For example, the hemolytic reagent and the staining reagent may be mixed first, and then the mixture may be mixed with the specimen. Alternatively, the hemolytic reagent and the specimen may be mixed first, and then the mixture may be mixed with the staining solution. A part of the hemolytic reagent may be mixed with the specimen to prepare a mixture, and then the prepared mixture may be mixed with the staining solution, and finally the remaining hemolytic reagent may be mixed.

[0054] In the method of this embodiment, the mixing ratio of the hemolysis reagent, the staining reagent, and the specimen can be appropriately selected depending on the hemolysis reagent, the staining reagent, and the specimen. The mixing ratio of the hemolysis reagent, the staining reagent, and the specimen is preferably 1000:1 or more:1 or more, for example, expressed as a volume ratio. More preferably, it is 1000:10 or more:10 or more, and even more preferably, it is 1000:15 or more:15 or more. In the method of this embodiment, the mixing ratio of the hemolysis reagent, the staining reagent, and the specimen is preferably 1000:50 or less:50 or less, for example, expressed as a volume ratio. More preferably, it is 1000:30 or less:30 or less, and even more preferably, it is 1000:25 or less:25 or less. The mixing ratio of the staining reagent and the specimen may be the same or different.

[0055] In the method of this embodiment, it is preferable to incubate the measurement sample before irradiating it with light. The incubation conditions are not particularly limited and can be appropriately selected depending on the specimen. For example, the incubation temperature is 15°C or higher, preferably 30°C or higher. The incubation temperature is 50°C or lower, preferably 45°C or lower. The incubation time is 5 seconds or more, preferably 10 seconds or more, more preferably 15 seconds or more. The incubation time is 120 seconds or less, preferably 80 seconds or less, more preferably 40 seconds or less.

[0056] In the method of this embodiment, the measurement sample is irradiated with light to detect optical information (detection step). The detection step is preferably performed by a flow cytometer. In measurement using a flow cytometer, a signal is generated from particles in the measurement sample by irradiating the measurement sample in a flow cell with light. Optical information can be obtained by detecting the generated signal. Note that the signal may be appropriately corrected, amplified, converted, or otherwise processed before or after detection, and the signal that has been subjected to such processing is also included in the optical information.

[0057] In the detection process, the light irradiated to the measurement sample is not particularly limited, and a light source with a wavelength suitable for exciting the fluorescent dye is selected. For example, a red semiconductor laser, a blue semiconductor laser, an argon laser, a He-Ne laser, a mercury arc lamp, etc. are used. In particular, a semiconductor laser is preferable from the viewpoint of cost because it is very inexpensive compared to a gas laser.

[0058] In the detection step, the optical information is not particularly limited as long as it is generally used in flow cytometry. For example, scattered light information, fluorescent information, etc. may be used. Scattered light includes forward scattered light (e.g., scattered light with a light receiving angle of 0 degrees to about 20 degrees) and side scattered light (e.g., scattered light with a light receiving angle of about 20 degrees to about 90 degrees). Scattered light information and fluorescent information include pulse height, pulse area, pulse width, transmittance, Stokes shift, ratio, change over time, and values ​​correlated therewith of scattered light and fluorescent light. Side scattered light is not particularly limited as long as it reflects internal information such as the complexity of the cell structure, granular characteristics, nuclear structure, and degree of lobulation.

[0059] In the method of this embodiment, the white blood cells in the measurement sample are classified based on the optical information (classification step). In the classification step, the classification of the white blood cells is preferably performed by creating a scattergram with side scattered light information and fluorescence information on two axes, and analyzing the obtained scattergram using appropriate analysis software. For example, when a scattergram is drawn with side scattered light intensity (SSC) on the X axis and fluorescence intensity (SFL) on the Y axis, the white blood cells are classified into five groups, namely lymphocytes, monocytes, neutrophils, eosinophils, and basophils, as shown in FIG. 2.

[0060] The classification step preferably includes a step of classifying a neutrophil population, an eosinophil population, a monocyte population, and a lymphocyte population. It is more preferable to classify a population having a higher side scattered light intensity than that of a neutrophil population as an eosinophil population, and to classify a population having a lower side scattered light intensity than that of a monocyte population as a lymphocyte population. The side scattered light intensity of a population can be determined, for example, by taking the arithmetic mean of the side scattered light intensity values ​​of each cell contained in the population, or by calculating the median of the side scattered light intensity values ​​of each cell contained in the population.

[0061] The method of the present embodiment allows neutrophils and eosinophils to be classified with high accuracy. In one example, the difference between the side scattered light intensity of the neutrophil group and the side scattered light intensity of the eosinophil group is larger than that of the conventional method, allowing clear classification. In another example, the variation in values ​​related to the optical information of the neutrophil group and / or the eosinophil group is smaller than that of the conventional method, allowing clear classification.

[0062] The method of this embodiment allows for accurate classification of monocytes and lymphocytes. In one example, the difference between the side scattered light intensity of a monocyte population and the side scattered light intensity of a lymphocyte population is greater than in conventional methods, allowing for clear classification. In another example, the variation in values ​​relating to optical information of a monocyte population and / or a lymphocyte population is smaller than in conventional methods, allowing for clear classification.

[0063] In the method of this embodiment, the variation in the side scattered light intensity can be evaluated using a statistical index, such as the coefficient of variation, standard deviation, or variance.

[0064] In the method of this embodiment, the hemolysis reagent contains a nonionic surfactant represented by the following formula (I): In formula (I), R1 is an alkyl group, an alkenyl group, or an alkynyl group having 8 to 25 carbon atoms, and R2 is an oxygen atom, (COO), or represented by the following formula (II).

[0065] R1-R2-(CH2CH2O) n -H formula (I)

[0066] [ka]

[0067] In the method of this embodiment, n in formula (I) is 23 or more and 25 or less. Preferably, n in formula (I) is 23 or 25, more preferably, n in formula (I) is 23. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 1650 ppm or more, preferably 1700 ppm or more. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 2250 ppm or less, preferably 2150 ppm or less. In a further embodiment of the hemolysis reagent, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is about 1650 ppm or more, preferably about 1700 ppm or more. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is about 2250 ppm or less, preferably about 2150 ppm or less.

[0068] In one example of the method of this embodiment, n in formula (I) is 23. In addition, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 1650 ppm or more, preferably 1700 ppm or more. In addition, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 2250 ppm or less, preferably 2150 ppm or less. In one example of the method of a further embodiment, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is about 1650 ppm or more, preferably about 1700 ppm or more. In addition, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is about 2250 ppm or less, preferably about 2150 ppm or less.

[0069] In another example of the method of the embodiment, n in formula (I) is 25. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 1650 ppm or more, preferably 1700 ppm or more. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 2250 ppm or less, preferably 2150 ppm or less. In another example of the method of the further embodiment, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is about 1650 ppm or more, preferably about 1700 ppm or more. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is about 2250 ppm or less, preferably about 2150 ppm or less.

[0070] In one embodiment of the method of the present embodiment, n in formula (I) is 30. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 1850 ppm or more, preferably 1950 ppm or more, more preferably 2050 ppm or more. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 2250 ppm or less, preferably 2150 ppm or less. In one embodiment of the method of the further embodiment, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is about 1850 ppm or more, preferably about 1950 ppm or more, more preferably about 2050 ppm or more. The concentration of the nonionic surfactant represented by formula (I) in the measurement sample is about 2250 ppm or less, preferably about 2150 ppm or less.

[0071] The method of the present embodiment can improve the accuracy of classification of leukocytes not only between monocytes and lymphocytes, but also between neutrophils and eosinophils, when classifying and counting leukocytes. In addition, the method of one embodiment can improve the accuracy of classification of the monocyte population.

[0072] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0073] Example 1 The relationship between the POE chain length of the nonionic surfactant and the classification accuracy of the neutrophil and eosinophil populations, and the classification accuracy of the lymphocyte and monocyte populations was examined. The classification accuracy of the neutrophil and eosinophil populations was evaluated by calculating the difference between the side scattered light intensity of the eosinophil population and the side scattered light intensity of the neutrophil population (Eo-Ne difference). The classification accuracy of the monocyte and lymphocyte populations was evaluated by calculating the difference between the side scattered light intensity of the lymphocyte population and the side scattered light intensity of the monocyte population (Mo-Ly difference).

[0074] As the hemolysis reagent, dodecyltrimethylammonium chloride (hereinafter referred to as LTAC) (Tokyo Kasei Chemical Industry Co., Ltd.), potassium hydrogen phthalate (Wako Pure Chemical Industries, Ltd.), EDTA-2K (Chubu Cherest Co., Ltd.) and polyoxyethylene (n) cetyl ether (hereinafter referred to as POE (n) cetyl ether, n represents the degree of polymerization of polyoxyethylene (POE)) were mixed to obtain the composition shown in Table 1 below. The pH of the hemolysis reagent was adjusted to pH 6.0 using NaOH. Fluorocell WDF (Sysmex Co., Ltd.) was used as the staining reagent. POE (n) cetyl ether is a nonionic surfactant.

[0075] [Table 1]

[0076] Fifteen blood samples taken from healthy subjects were used as specimens. XN-20 (Sysmex) was used as a flow cytometer. A measurement sample was prepared by mixing 1000 μL of hemolysis reagent, 17 μL of specimen, and 20 μL of staining solution. The measurement conditions were in accordance with the settings of the WDF channel of XN-20 (Sysmex). A scattergram was created based on the measured values ​​obtained by the flow cytometer. The created scattergram was analyzed using an appropriate analysis software to identify each white blood cell population. The side scattered light intensity and fluorescence intensity of each white blood cell population were calculated from the number of cells contained in the identified white blood cell population, the side scattered light intensity, and the fluorescence intensity. The Mo-Ly difference and the Eo-Ne difference were calculated from the side scattered light intensity of each white blood cell population.

[0077] As the nonionic surfactant, POE(20) cetyl ether (Nikko Chemicals), POE(25) cetyl ether (Aoki Oil & Fat), POE(30) cetyl ether (Nikko Chemicals) or POE(40) cetyl ether (Nikko Chemicals) were used to prepare hemolytic reagents with the compositions shown in Table 1. Using each of the prepared hemolytic reagents, 15 samples were measured with a flow cytometer. For each hemolytic reagent, the average value of the Eo-Ne difference of the 15 samples and the average value of the Mo-Ly difference of the 15 samples were calculated based on the measured values. The results of Example 1 are shown in Table 2.

[0078] [Table 2]

[0079] As shown in Table 2, as the degree of polymerization of the POE chain of the POE(n) cetyl ether in the hemolysis reagent increased, the Eo-Ne difference tended to decrease and the Mo-Ly difference tended to increase.

[0080] Example 2 The relationship between the concentration of nonionic surfactant in the hemolysis reagent, Eo-Ne difference, and Mo-Ly difference was examined.

[0081] A hemolytic reagent was prepared in the same manner as in Example 1, except that POE (23) cetyl ether was used as the nonionic surfactant, the concentration of POE (23) cetyl ether was adjusted to 879 ppm, 1318 ppm, 1758 ppm, 1977 ppm, 2197 ppm, 2417 ppm or 2636 ppm, and the pH was adjusted to 6.1. A hemolytic reagent was prepared in the same manner as in Example 1, except that POE (25) cetyl ether was used as the nonionic surfactant, the concentration of POE (25) cetyl ether was adjusted to 879 ppm, 1318 ppm, 1758 ppm, 2197 ppm or 2636 ppm, and the pH was adjusted to 6.1. A hemolytic reagent was prepared in the same manner as in Example 1, when POE (30) cetyl ether was used as the nonionic surfactant. Using each prepared hemolytic reagent, three samples were measured using a flow cytometer. Based on the measured values, the average Eo-Ne difference of the three samples and the average Mo-Ly difference of the three samples were calculated. Regarding the nonionic surfactant in the hemolysis reagent, the results when POE(23) cetyl ether was used are shown in Table 3, the results when POE(25) cetyl ether was used are shown in Table 4, and the results when POE(30) cetyl ether was used are shown in Table 5.

[0082] [Table 3]

[0083] [Table 4]

[0084] [Table 5]

[0085] When POE(23) cetyl ether or POE(25) cetyl ether was used, the Eo-Ne difference tended to increase as the concentration of the nonionic surfactant increased.In addition, when POE(23) cetyl ether or POE(25) cetyl ether was used at concentrations of 1758 ppm to 2197 ppm, it was found that the classification of neutrophils and eosinophils, and the classification of monocytes and lymphocytes could be clearly performed.

[0086] Example 3 The relationship between the concentration of POE(23) cetyl ether and the Eo-Ne difference and the Mo-Ly difference was examined.

[0087] A hemolytic reagent was prepared in the same manner as in Example 1, except that POE (23) cetyl ether was used as the nonionic surfactant, the concentration of POE (23) cetyl ether was adjusted to 1758 ppm, 1933 ppm, 2021 ppm, 2109 ppm, 2197 ppm, or 2285 ppm, and the pH was adjusted to 6.2. A hemolytic reagent was prepared in the same manner as in Example 1 when POE (30) cetyl ether was used as the nonionic surfactant. Using each prepared hemolytic reagent, four samples were measured using a flow cytometer. Based on the measured values, the average value of the Eo-Ne difference of the four samples and the average value of the Mo-Ly difference of the four samples were calculated. For the nonionic surfactant in the hemolytic reagent, the results when POE (23) cetyl ether was used are shown in Table 6, and the results when POE (30) cetyl ether was used are shown in Table 7.

[0088] [Table 6]

[0089] [Table 7]

[0090] When POE(23) cetyl ether was used, the Eo-Ne difference tended to increase as the concentration of the nonionic surfactant increased.In addition, it was found that when POE(23) cetyl ether was used at concentrations of 1758 ppm to 2285 ppm, it was possible to clearly distinguish between neutrophils and eosinophils, and between monocytes and lymphocytes.

[0091] Example 4 The Eo-Ne differences and Mo-Ly differences of Reagent A, Reagent B, and Reagent C were compared with those of Reagent D.

[0092] A hemolytic reagent was prepared in the same manner as in Example 1, except that POE (23) cetyl ether was used as a nonionic surfactant, the POE (23) cetyl ether concentration was adjusted to 2021 ppm, and the pH was adjusted to 6.2 (hereinafter referred to as reagent A). A hemolytic reagent was prepared in the same manner as in Example 1, except that POE (25) cetyl ether was used as a nonionic surfactant, the POE (25) cetyl ether concentration was adjusted to 1758 ppm, and the pH was adjusted to 6.1 (hereinafter referred to as reagent B). POE (30) cetyl ether was used as a nonionic surfactant for the hemolytic reagent. In addition, a hemolytic reagent was prepared by adjusting the POE (30) cetyl ether concentration to 2144 ppm, LTAC to 668 ppm, potassium hydrogen phthalate to 5106 ppm, ADA to 1236 ppm, and adjusting the pH to 6.5 (hereinafter referred to as reagent C). A hemolysis reagent was prepared in the same manner as in Example 1 using POE(30) cetyl ether (hereinafter referred to as Reagent D). Using Reagent A, Reagent B, Reagent C, and Reagent D, 132 samples were measured with a flow cytometer in the same manner as in Example 1. Based on the measured values, the average value of the Eo-Ne difference of the 132 samples and the average value of the Mo-Ly difference of the 132 samples were calculated.

[0093] The results of Example 4 are shown in Figures 3A and 3B. As shown in Figure 3A, when any of reagents A, B, and C was used, the Eo-Ne difference was larger than when reagent D was used. As shown in Figure 3B, when any of reagents A, B, C, and D was used, the Mo-Ly difference was equivalent. It was found that by using reagents A, B, and C, monocytes and lymphocytes could be classified with the same accuracy as when reagent D was used, and neutrophils and eosinophils could be classified with higher accuracy than when reagent D was used.

[0094] Example 5 The variability of the monocyte population was compared between Reagent A and Reagent D. The variability of the monocyte population was evaluated by calculating the coefficient of variation (CV) (hereafter referred to as the coefficient of variation of the monocyte population) from the median (MED) and standard deviation (SD) of the fluorescence intensity of leukocytes classified into the monocyte population.

[0095] Measurements were performed using a flow cytometer in the same manner as in Example 1, except that Reagent A or Reagent D prepared in Example 4 was used, and that a total of six samples were used as samples, including four samples of ascites (samples 1 to 4), one sample of pleural fluid (sample 5), and one sample of synovial fluid (sample 6). The coefficient of variation of the monocyte population was calculated based on the measured values. The results are shown in Table 8.

[0096] [Table 8]

[0097] In all of the specimens 1 to 6, the coefficient of variation of the monocyte population was smaller when reagent A was used than when reagent D was used. As an example, the scattergram of specimen 1 is shown in FIG. 4. The area surrounded by the frame in the scattergram in FIG. 4 is the area where the plot of monocytes is expected to appear. In FIG. 4, SSC on the X-axis represents the side scattered light intensity, and SFL on the Y-axis represents the fluorescence intensity. As shown in FIG. 4, it was also confirmed on the scattergram that the variation in the fluorescence intensity of the monocyte population was smaller when reagent A was used than when reagent D was used. [Explanation of symbols]

[0098] 10: Container 11: 1st container 20: Second container 30: Reagent kit

Claims

1. The nonionic surfactant is represented by the following formula (I): R 1 -R 2 -(CH 2 CH 2 O) n -H Formula (I) R 1 is an alkyl group, an alkenyl group, or an alkynyl group having 8 to 25 carbon atoms, R 2 is an oxygen atom, (COO) or is represented by the following formula (II): 【Chemistry 1】 The n is 23 or more and 25 or less, or 30; When the n is 23 or more and 25 or less, the concentration of the nonionic surfactant is 1700 ppm or more and 2300 ppm or less, A hemolysis reagent for classifying leukocytes, wherein the concentration of the nonionic surfactant is 1900 ppm or more and 2300 ppm or less when the n is 30.

2. 2. The hemolyzing reagent according to claim 1, wherein n in formula (I) is 23 to 25.

3. 3. The hemolysis reagent according to claim 2, wherein the concentration of the nonionic surfactant is 1750 ppm or more and 2200 ppm or less.

4. 4. The hemolysis reagent according to claim 1, wherein the nonionic surfactant is at least one selected from the group consisting of polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, polyoxyethylene (30) cetyl ether, and the like.

5. The hemolysis reagent according to any one of claims 1 to 4, further comprising a cationic surfactant.

6. 6. The hemolysis reagent according to claim 5, wherein the cationic surfactant comprises a quaternary ammonium salt type surfactant or a pyridinium salt type surfactant.

7. The hemolysis reagent according to any one of claims 1 to 6, further comprising an aromatic organic acid.

8. 8. The hemolyzing reagent according to claim 7, wherein the aromatic organic acid is at least one selected from the group consisting of aromatic carboxylic acids, aromatic sulfonic acids, and salts thereof.

9. The hemolysis reagent according to any one of claims 1 to 8, which is used for classifying leukocytes into at least a population of monocytes, lymphocytes, neutrophils, and eosinophils.

10. A reagent kit for classifying white blood cells, comprising the hemolytic reagent for classifying white blood cells according to any one of claims 1 to 9 and a staining reagent for fluorescently staining nucleic acid.

11. The method includes a step of irradiating light onto a measurement sample prepared by mixing a hemolysis reagent, a staining reagent for fluorescently staining nucleic acid, and a specimen containing white blood cells, and detecting optical information; and a step of classifying the white blood cells based on the optical information. A method for classifying white blood cells, comprising: The hemolysis reagent contains a nonionic surfactant represented by the following formula (I): R 1 -R 2 -(CH 2 CH 2 O) n -H Formula (I) R 1 is an alkyl group, an alkenyl group, or an alkynyl group having 8 to 25 carbon atoms, R 2 is an oxygen atom, (COO) or a group represented by the following formula (II), 【Chemistry 2】 The n is 23 or more and 25 or less, or 30; When the n is 23 or more and 25 or less, the concentration of the nonionic surfactant in the measurement sample is 1650 ppm or more and 2250 ppm or less, A method for classifying leukocytes, wherein when the n is 30, the concentration of the nonionic surfactant in the measurement sample is 1850 ppm or more and 2250 ppm or less.

12. The classification method according to claim 11 , wherein the optical information is scattered light intensity and fluorescent intensity.

13. The classification method according to claim 11 or 12, wherein the optical information is side scattered light intensity.

14. The classification method according to any one of claims 11 to 13, wherein the classifying step includes a step of classifying the white blood cells into at least a neutrophil group, an eosinophil group, a monocyte group, and a lymphocyte group.

15. the optical information is side scattered light intensity, The classifying step comprises: classifying a population having a larger side scattered light intensity than the side scattered light intensity of the neutrophil population as the eosinophil population; The classification method according to claim 14, further comprising a step of classifying a population having a lower side scattered light intensity than the side scattered light intensity of the monocyte population as the lymphocyte population.

16. The classification method according to any one of claims 11 to 15, wherein n in formula (I) is 23 or more and 25 or less.

17. The classification method according to claim 16, wherein the concentration of the nonionic surfactant is 1750 ppm or more and 2200 ppm or less.

18. The classification method according to any one of claims 11 to 16, wherein the specimen is a sample collected by whole blood, ascites, synovial fluid, pleural effusion, cerebrospinal fluid, bone marrow fluid, bronchoalveolar lavage fluid, peritoneal lavage fluid, urine, apheresis, or the like.

19. The classification method according to any one of claims 11 to 18, wherein in the step of detecting the optical information, the measurement sample is detected by a flow cytometer.

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