Blood cell analysis method and blood cell analysis reagent

Abstract

The purpose of the present invention is to provide a means for making it possible to sort and count reticulocytes and platelets. The blood cells in a test specimen are stained with a fluorescent dye, fluorescence information, first scattered light information, and second scattered light information that are generated by irradiating the stained blood cells with light having a wavelength of from 315 nm to 600 nm and light having a wavelength of from 610 nm to 750 nm are acquired, and the fluorescence information, the first scattered light information, and the second scattered light information are acquired on the basis of the first scattered light information and the second scattered light information, and / or on the basis of the first scattered light information and the second scattered light information, and / or on the basis of the first scattered light information and the second scattered light information. The problem is solved by specifying blood cells other than leukocytes on the basis of the first scattered light information and the fluorescence information, and classifying reticulocytes and platelets from groups other than leukocytes on the basis of the fluorescence information and the second scattered light information.

Description

[Technical field]

[0001] The present invention relates to a blood cell analysis method and a blood cell analysis reagent. [Background technology]

[0002] Blood contains various blood cell components such as red blood cells, white blood cells, and platelets. Among these, platelets are anucleated cells with a diameter of 2 to 3 μm, and there are 150,000 to 350,000 / μL in normal blood. However, it is known that the number of platelets decreases in the blood of patients with idiopathic thrombocytopenic purpura or acute leukemia. Generally speaking, when the number of platelets in the blood becomes less than 10,000 / μL, it is judged that blood transfusion is required. Therefore, it is important to correctly classify and count platelets in clinical sites.

[0003] Reticulocytes are also contained in the blood. Reticulocytes are young red blood cells that have just been released into the peripheral blood after the erythroblast cells in the bone marrow have lost their nuclei. Reticulocytes usually become mature red blood cells in about 1 day. Reticulocytes have a small amount of RNA or organelles such as ribosomes and mitochondria that are not contained in mature red blood cells. The classification and counting of reticulocytes are extremely important in understanding the hematopoietic ability of the subject's bone marrow. In a state where bone marrow hematopoiesis is suppressed, such as by aplastic anemia, chemotherapy for malignant tumors, etc., the number of reticulocytes decreases. Alternatively, in a state where bone marrow hematopoiesis is hyperactive, such as in hemolytic anemia, the number of reticulocytes increases.

[0004] EXAMPLE 8 of Patent Document 1 describes measuring reticulocytes and platelets by irradiating a sample prepared using a dye that stains platelets and leukocytes and a dye that stains reticulocytes with blue (488 nm) light.

[0005] [Prior art literature]

[0006] [Patent Literature]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2021 / 0041341

[0008] [Summary of the Invention]

[0009] [Problems to be solved by the invention]

[0010] Leukocytes have an influence on the classification and counting of reticulocytes and platelets in the subject, but the influence of leukocytes has not been particularly considered in the prior art. The present invention aims to provide a means for suppressing the influence of leukocytes and enabling the classification and counting of reticulocytes and platelets.

[0011]

Methods for solving the problem

[0012] The present inventors have discovered that reticulocytes and platelets can be detected based on fluorescence information, first scattered light information, and second scattered light information generated by dyeing blood cells in a subject with a fluorescent dye and irradiating the dyed blood cells with light of a wavelength of not less than 315 nm and not more than 600 nm and light of a wavelength of not less than 610 nm and not more than 750 nm, thereby completing the inventions described in the following [1] to

[31] .

[0013] [1] A method for analyzing blood cells, comprising:

[0014] a step of acquiring first scattered light information, second scattered light information, and fluorescence information generated by irradiating a measurement sample containing particles dyed with a fluorescent dye with light of a first wavelength and light of a second wavelength,

[0015] a step of identifying blood cells other than white blood cells from particles in the measurement sample based on the first scattered light information and the second scattered light information and / or based on the first scattered light information and the fluorescence information; and

[0016] a step of classifying reticulocytes and platelets from the blood cells other than the white blood cells based on the fluorescence information and the second scattered light information;

[0017] The first wavelength is greater than or equal to 315 nm and less than or equal to 600 nm, and the second wavelength is greater than or equal to 610 nm and less than or equal to 750 nm.

[0018] [2] The method described in [1] above, wherein

[0019] The first scattered light information is information related to side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength,

[0020] The second scattered light information is information related to forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength,

[0021] The fluorescence information is information related to fluorescence generated from the fluorescent dye of the particles by irradiating the measurement sample with the light of the first wavelength.

[0022] [3] The method described in [1] or [2] above, wherein in the classification step, the blood cells other than the white blood cells are classified into reticulocytes, platelets and mature red blood cells.

[0023] [4] The method according to any one of [1] to [3] above, wherein

[0024] The first scattered light information is the intensity of the first side scattered light of the particle irradiated with the light of the first wavelength, the second scattered light information is the intensity of the front scattered light of the particle irradiated with the light of the second wavelength, and the fluorescence information is the intensity of the fluorescence of the fluorescent dye of the particle irradiated with the light of the first wavelength.

[0025] In the identifying step, blood cells other than the white blood cells are identified based on the first side scattered light intensity and the forward scattered light intensity or the fluorescence intensity.

[0026] [5] The method described in [4] above, wherein in the determination step, a scattergram based on the first side scattered light intensity and the forward scattered light intensity or the fluorescence intensity is prepared, and blood cells other than the white blood cells are determined in the scattergram.

[0027] [6] The method described in [4] or [5] above, wherein

[0028] In the determining step, particles in the measurement sample are classified into a first particle group and a second particle group based on the first side scattered light intensity and the forward scattered light intensity.

[0029] The second particle group is a group that exhibits a first side scattered light intensity and / or a forward scattered light intensity higher than that of the first particle group.

[0030] The first particle group is determined to be blood cells other than leukocytes.

[0031] [7] The method described in [4] or [5] above, wherein

[0032] In the determining step, particles in the measurement sample are classified into a first particle group and a second particle group based on the first side scattered light intensity and the fluorescence intensity.

[0033] The second particle group is a group that exhibits a first side scattered light intensity and / or a fluorescence intensity higher than that of the first particle group.

[0034] The first particle group is determined to be blood cells other than leukocytes.

[0035] [8] The method according to any one of [1] to [7] above, wherein

[0036] The second scattered light information is the intensity of forward scattered light from the particle irradiated with the light of the second wavelength, and the fluorescence information is the intensity of fluorescence of the fluorescent dye from the particle irradiated with the light of the first wavelength.

[0037] In the classification step, reticulocytes and platelets are classified from the blood cells other than the white blood cells based on the fluorescence intensity and the forward scattered light intensity.

[0038] [9] The method described in [8] above, wherein in the classification step, a scattergram based on the fluorescence intensity and the forward scattered light intensity is prepared, and in the scattergram, reticulocytes and platelets are classified from the blood cells other than the white blood cells.

[0039]

[10] The method described in [8] or [9] above, wherein

[0040] In the classification step, the blood cells other than the white blood cells are classified into a third particle group, a fourth particle group, and a fifth particle group based on the fluorescence intensity and the forward scattered light intensity.

[0041] The third particle group is a group showing a higher fluorescence intensity than the fifth particle group.

[0042] The fourth particle group is a group showing a lower forward scattered light intensity than the third particle group.

[0043] The third particle population was determined to be a population of reticulocytes, and the fourth particle population was determined to be a population of platelets.

[0044]

[11] The method according to

[10] above, wherein the fifth particle population is determined to be a population of mature red blood cells.

[0045]

[12] The method described in any one of [1] to

[11] above, wherein the step of determining further includes a step of determining white blood cells and classifying the white blood cells into subgroups.

[0046]

[13] The method described in

[12] above, wherein white blood cells are determined and classified into subgroups based on the first scattered light information and the second scattered light information, and / or based on the first scattered light information and the fluorescence information.

[0047]

[14] The method described in

[13] above, wherein

[0048] The first scattered light information is the intensity of the first side scattered light of the particle irradiated with the light of the first wavelength, the second scattered light information is the intensity of the front scattered light of the particle irradiated with the light of the second wavelength, and the fluorescence information is the intensity of the fluorescence of the fluorescent dye of the particle irradiated with the light of the first wavelength.

[0049] Based on the first side scattered light intensity and the forward scattered light intensity or the fluorescence intensity, the leukocytes are identified and classified into subgroups.

[0050]

[15] The method described in

[14] above, wherein a scatter plot based on the first lateral scattered light intensity and the forward scattered light intensity, and / or a scatter plot based on the first lateral scattered light intensity and the fluorescence intensity is prepared, in which the white blood cells are identified and classified into subgroups.

[0051]

[16] The method described in

[12] above, wherein in the step of obtaining, third scattered light information is also obtained.

[0052] The third scattered light information is information related to side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength,

[0053] the leukocytes are identified based on the first scattered light information and the second scattered light information, and / or based on the first scattered light information and the fluorescence information,

[0054] The white blood cells are classified into subgroups based on the second scattered light information and the third scattered light information and / or based on the fluorescence information and the third scattered light information.

[0055]

[17] The method described in

[16] above, wherein

[0056] The second scattered light information is the intensity of the forward scattered light of the particle irradiated with the light of the second wavelength, the third scattered light information is the intensity of the second side scattered light of the particle irradiated with the light of the second wavelength, and the fluorescence information is the intensity of the fluorescence of the fluorescent dye of the particle irradiated with the light of the first wavelength.

[0057] The leukocytes are classified into subgroups based on the second side scattered light intensity and the forward scattered light intensity or the fluorescence intensity.

[0058]

[18] The method described in

[17] above, wherein a scatter plot based on the second lateral scattered light intensity and the forward scattered light intensity, and / or a scatter plot based on the second lateral scattered light intensity and the fluorescence intensity is prepared, and in the scatter plot, white blood cells are classified into subgroups.

[0059]

[19] The method according to any one of

[12] to

[18] above, wherein the subpopulation includes at least two of a lymphocyte population, a monocyte population, and a granulocyte population.

[0060]

[20] The method described in any one of [1] to

[19] above, wherein the determining step further includes obtaining information indicating the presence or absence of platelet aggregation based on the first scattered light information and the second scattered light information.

[0061]

[21] The method described in

[20] above, wherein

[0062] The first scattered light information is the intensity of the side scattered light from the particle irradiated with the light of the first wavelength, and the second scattered light information is the intensity of the front scattered light from the particle irradiated with the light of the second wavelength.

[0063] In the step of determining, blood cells other than white blood cells are determined based on the forward scattered light intensity and the side scattered light intensity.

[0064] Based on the forward scattered light intensity, the blood cells other than the white blood cells are classified into a group containing red blood cells and a group containing platelets.

[0065] When the group containing platelets contains particles showing a forward scattered light intensity equal to or greater than a threshold value, information indicating the presence of platelet aggregation is obtained.

[0066]

[22] The method described in

[21] above, wherein

[0067] In the determining step, a scattergram based on the forward scattered light intensity and the side scattered light intensity is prepared, and in the scattergram, particles in the measurement sample are determined as a group containing red blood cells, a group containing platelets, and white blood cells.

[0068] When the group containing platelets contains particles showing forward scattered light intensity equal to or higher than a threshold value, information indicating the presence of platelet aggregation is obtained.

[0069]

[23] The method described in any one of [1] to

[22] above, wherein in the classification step, the platelets are further classified into mature platelets and immature platelets.

[0070]

[24] The method described in any one of [1] to

[23] above, wherein the classification step further includes determining large platelets from the platelets.

[0071]

[25] The method described in any one of [1] to

[24] above, further comprising the step of calculating the platelet distribution width and / or the mean platelet volume.

[0072]

[26] The method described in any one of [1] to

[25] above, wherein the fluorescent dye is selected from acridine compounds, anthocyanin compounds and styryl compounds.

[0073]

[27] The method described in

[26] above, wherein the acridine compound is a compound represented by the following formula (I):

[0074]

Chemistry 1

[0075]

[0076] (In the formula, R1~R 10 are independently hydrogen atoms, -NH2, -(CH2) n -NR 11 R 12 、-NH-R 13 -NR 11 R 12 、-OR 11 , -COOH, halogen, phenyl optionally having a substituent, alkyl having 1 to 18 carbon atoms or acylamino, but at least one of R3, R6 and R9 is independently -NH2, -(CH2) n -NR 11 R 12 , or phenyl substituted by -NH2,

[0077] R 11 and R 12 the same or different, are hydrogen atoms or alkyl groups having 1 to 18 carbon atoms,

[0078] R 13 is an alkyl group having 1 to 6 carbon atoms,

[0079] n is an integer from 0 to 6,

[0080] X - is the counter ion).

[0081]

[28] The method described in

[26] or

[27] above, wherein the anthocyanin compound is a compound represented by the following formula (II):

[0082]

Chemistry 2

[0083]

[0084] (wherein, R1 and R2 are the same or different and are alkyl having 1 to 18 carbon atoms, alkenyl having 3 to 20 carbon atoms, alkyl having 1 to 18 carbon atoms having a hydroxyl group, alkyl having 1 to 18 carbon atoms having a carboxyl group, alkyl having 1 to 18 carbon atoms having a sulfonic group, halogen, -(CH2) m -NR6R7, -(CH2) n -O-R8 or benzyl optionally having a substituent,

[0085] R3 and R4 are the same or different and are hydrogen, halogen, alkyl having 1 to 18 carbon atoms, phenyl optionally having a substituent, alkoxy having 1 to 6 carbon atoms, or -(CH2) n -O-R8,

[0086] R5 is a hydrogen atom, a halogen, an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms and having a hydroxyl group, an alkyl group having 1 to 6 carbon atoms and having a carboxyl group, an alkyl group having 1 to 6 carbon atoms and having a sulfonic group, a phenyl group optionally having a substituent, a benzyl group optionally having a substituent, -(CH2) n -O-R6,

[0087] R6 and R7 are the same or different and are a hydrogen atom or an alkyl group having 1 to 18 carbon atoms,

[0088] R8 is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, or a phenyl group which may have a substituent,

[0089] m is an integer from 1 to 18, n is an integer from 1 to 6,

[0090] X and Y are the same or different and are sulfur atoms, oxygen atoms, selenium atoms or CR9R 10 ,

[0091] R9 and R 10 are the same or different and are alkyl groups having 1 to 3 carbon atoms,

[0092] Z - is the counter ion).

[0093]

[29] The method according to any one of

[26] to

[28] above, wherein the styrene-based compound is:

[0094] A compound represented by the following formula (III):

[0095]

Chemistry 3

[0096]

[0097] (wherein, R1 is an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 3 to 20 carbon atoms, an alkyl group having 1 to 18 carbon atoms having a hydroxyl group, an alkyl group having 1 to 18 carbon atoms having a carboxyl group, an alkyl group having 1 to 18 carbon atoms having a sulfonic group, a halogen, -(CH2) p -NR5R6, -(CH2) q -O-R7 or benzyl optionally having a substituent,

[0098] R2 and R3 are the same or different and are hydrogen, halogen, an alkyl group having 1 to 18 carbon atoms, a phenyl group which may have a substituent, or an alkoxy group having 1 to 6 carbon atoms,

[0099]

Chemistry 4

[0100] yes

[0101] R4 is a hydrogen atom, a halogen, an alkyl group having 1 to 18 carbon atoms, a phenyl group optionally having a substituent, an alkoxy group having 1 to 6 carbon atoms, or -(CH2) m -O-R7,

[0102] R5 and R6 are the same or different and are a hydrogen atom or an alkyl group having 1 to 18 carbon atoms,

[0103] R7 is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, or a phenyl group which may have a substituent,

[0104] X is a sulfur atom, an oxygen atom, a selenium atom or CR8R9,

[0105] R8 and R9 are the same or different and are alkyl groups having 1 to 3 carbon atoms,

[0106] m is an integer from 1 to 18,

[0107] Z - is a counterion), or

[0108] A compound represented by the following formula (IV):

[0109]

Chemistry 5

[0110]

[0111] (In the formula, R1 and Z - As mentioned above,

[0112] R2 and R3 are the same or different and are hydrogen, halogen, alkyl having 1 to 18 carbon atoms, phenyl optionally having a substituent, or alkoxy having 1 to 6 carbon atoms, or R2 and / or R3 together with the benzene ring to which N is bonded form the above-mentioned N-containing heterocyclic ring,

[0113] n is an integer from 0 to 2).

[0114]

[30] The method described in any one of [1] to

[29] above, wherein the first wavelength is greater than 315 nm and less than 490 nm.

[0115]

[31] A reagent for blood cell analysis used in the method described in any one of [1] to

[30] above, which contains a fluorescent dye selected from acridine compounds, anthocyanin compounds and styryl compounds.

[0116] [Effects of the Invention]

[0117] The present invention provides a blood cell analysis method that suppresses the influence of leukocytes and enables classification and counting of reticulocytes and platelets, and a blood cell analysis reagent used in the method.

[0118] [Brief description of the attached drawings]

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[0141]

【 Fig.17 (A) is a schematic diagram showing the positions of each subgroup of leukocytes in the third scatter plot (a) in which the second side scattered light (SSC-2) intensity is set on the horizontal axis and the FSC-2 intensity is set on the vertical axis. (B) is a schematic diagram showing the positions of each subgroup of leukocytes in the third scatter plot (b) in which the SSC-2 intensity is set on the horizontal axis and the SFL-1 intensity is set on the vertical axis.

[0142]

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[0163]

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[0164] (Analysis System)

[0165] First, refer to Figure 1 , an analysis system suitable for the blood cell analysis method of this embodiment is described. The analysis system 500 includes a measurement unit 400 as a measurement device and an analysis unit 300 as an analysis device. The analysis unit 300 is, for example, a personal computer that integrates software for analyzing a subject to be measured. The analysis unit 300 also performs operation control of the measurement unit 400. The analysis system 500 may also be a structure in which the analysis unit 300 is provided in the measurement unit 400.

[0166] The measuring unit 400 is a unit for measuring a subject, and includes a flow cytometer. In the measuring unit 400, the subject and the reagent are mixed to prepare a measuring sample. In the preparation of the measuring sample, a staining reagent and a diluting reagent are used as reagents. The staining reagent contains a fluorescent dye for staining particles. The diluting reagent contains a buffer and a solvent. The details of the subject, the staining reagent and the diluting reagent are described later. In the analysis of particles in the subject of this analysis system, particles in the measuring sample stained with a fluorescent dye are analyzed. "Particles in the measuring sample" refer to formed components contained in the measuring sample that can be measured one by one by the FCM detection unit 460 described later. As particles in the measuring sample, cells, red blood cell ghosts, platelet aggregates, lipid particles, fungi, bacteria, etc. contained in the subject can be cited. The term "cell" includes various blood cells and platelets. Red blood cell ghosts refer to the remains of hemolyzed red blood cells. Red blood cell ghosts may also be generated by hemolysis other than hemolysis reagents, such as physical impact on the blood collection tube, low pH and low osmotic pressure of the measurement sample.

[0167] The FCM detection unit 460 (see Figure 2 ) measurement. The measurement obtains the fluorescence-related optical signal emitted by the fluorescent dye of the dyed particles in the measurement sample and the scattered light-related optical signal emitted by the particles. "Fluorescent dye of particles" refers to fluorescent dyes bound to particles by dyeing. The embodiment of the combination of particles and fluorescent dyes is not particularly limited as long as the particles and fluorescent dyes are integrated and measured by flow cytometry. The obtained optical signal is A / D converted to obtain digital data. The analysis unit 300 analyzes the digital data obtained by the measurement unit 400 to identify and / or classify the particles in the measurement sample.

[0168] Reference Figure 2, the structure of the flow system in the measuring unit 400 is described. The measuring unit 400 includes a sample preparation unit 440, a sample suction unit 450, and an FCM detection unit 460. The sample preparation unit 440 includes a reagent container 410, a reagent container 411, a reaction chamber 420, and a waste liquid chamber 430. For example, the reagent container 410 contains a dilution reagent, and the reagent container 411 contains a staining reagent. The reagent container 410 and the reagent container 411 are connected to the reaction chamber 420 by a liquid delivery tube. The sample preparation unit 440 injects the dilution reagent and the staining reagent into the reaction chamber 420 through each liquid delivery tube. The sample suction unit 450 includes a suction tube 200. The suction tube 200 sucks the sample contained in the blood collection tube 100 and discharges it into the reaction chamber 420. The reaction chamber 420 is connected to the flow cell 413 of the FCM detection unit 460 by a liquid delivery tube.

[0169] The reaction chamber 420 is a container for preparing the measurement sample. The test sample is prepared by mixing the test body, the staining reagent containing the fluorescent dye, and the diluent reagent in the reaction chamber 420. The measurement sample in the reaction chamber 420 is supplied to the flow cell 413 of the FCM detection unit 460 through the liquid delivery tube for measurement. The FCM detection unit 460 obtains various optical signals emitted from each particle in the measurement sample. After the measurement by the FCM detection unit 460 is completed, the measurement sample remaining in the reaction chamber 420 is discarded to the waste liquid chamber 430. Before preparing the following measurement sample, the reaction chamber 420 is cleaned by a cleaning mechanism not shown in the figure.

[0170] Reference Figure 3 , the electrical connection of each part in the measuring unit 400 is described. The sample preparation part 440, the subject suction part 450 and the FCM detection part 460 are connected to the interface (IF) part 488. The FCM detection part 460 is connected to the analog processing part 481 and the A / D conversion part 482. The analog processing part 481 processes the analog signal output from the FCM detection part 460, and the A / D conversion part 482 converts the analog signal output from the analog processing part 481 into a digital signal. In addition, the measuring unit 400 is connected to the analysis unit 300 via the IF part 489. The IF part 484, the IF part 488 and the IF part 489 are connected to the bus 485.

[0171] Reference Figure 4, the FCM detection unit 460, the analog processing unit 481 and the A / D conversion unit 482 are described. The FCM detection unit 460 includes a first light source 411a, a second light source 411b, a flow cell 413, dichroic mirrors 418a, 418b, 418c, side scattered light receiving elements 412a, 412b, a forward scattered light receiving element 416 and side fluorescent light receiving elements 422a, 422b. The first light source 411a and the second light source 411b emit light of different wavelengths. For example, the first light source 411a emits light of a first wavelength, and the second light source 411b emits light of a second wavelength. The first wavelength is preferably a wavelength absorbed by hemoglobin. Such a wavelength is, for example, between 315 nm and 600 nm, preferably between 400 nm and 490 nm, more preferably between 400 nm and 450 nm, and even more preferably between 400 nm and 410 nm. When the light of the above-mentioned wavelength domain is irradiated to the mature red blood cells or reticulocytes containing hemoglobin, since the light is absorbed by the hemoglobin in the blood cells, the parameters such as the intensity (peak value), pulse width, pulse area, etc. of the scattered light generated from the mature red blood cells or reticulocytes have a tendency to become smaller than the white blood cells that do not contain hemoglobin. The second wavelength is a wavelength different from the first wavelength, preferably a wavelength that is not absorbed by hemoglobin. Such a wavelength is, for example, above 610nm and below 750nm, preferably above 620nm and below 700nm, and more preferably above 633nm and below 643nm. By using both the light of the wavelength domain absorbed by hemoglobin and the light of the wavelength domain not absorbed by hemoglobin, it is believed that mature red blood cells or reticulocytes and white blood cells can be clearly distinguished. As the first and second light sources, for example, a semiconductor laser source can be used.

[0172] The measurement sample prepared in the reaction chamber 420 is made to flow into the flow cell 413 of the FCM detection unit 460. Figure 4 In the example of , the measurement sample is made to flow in a direction perpendicular to the paper surface. When the measurement sample flows in the flow cell 413, the light irradiated from the first light source 411a is reflected by the dichroic mirror 418a and irradiates each particle in the measurement sample flowing in the flow cell 413. The light emitted from the second light source 411b passes through the dichroic mirror 418a and irradiates each particle in the measurement sample flowing in the flow cell 413.

[0173] exist Figure 4In the example of , the forward scattered light receiving element 416 is configured to receive the forward scattered light emitted from the particle based on the light irradiated from the second light source 411b. Alternatively, the light receiving element 416 may be configured to receive the forward scattered light emitted from the particle based on the light irradiated from the first light source 411a. Alternatively, another light receiving element may be further configured to receive the forward scattered light emitted from the particle based on the light irradiated from the first light source 411a. Forward scattered light is, for example, scattered light at a light receiving angle of 0° to about 20°. The forward scattered light receiving element 416 is, for example, a photodiode. The side scattered light (first side scattered light) corresponding to the light irradiated from the first light source 411a is reflected by the dichroic mirror 418b and received by the side scattered light receiving element 412a. The side scattered light (second side scattered light) corresponding to the light irradiated from the second light source 411b is reflected by the dichroic mirror 418c and received by the side scattered light receiving element 412b. The side scattered light is, for example, scattered light at a light receiving angle of about 20° to about 90°. The side scattered light receiving elements 412a and 412b are, for example, photodiodes.

[0174] In the blood cell analysis method of this embodiment, a fluorescent dye excited by the first wavelength is used. The side fluorescence (first side fluorescence) corresponding to the light generated by the fluorescent dye being excited is transmitted through the dichroic mirror 418b and received by the side fluorescence light receiving element 422a. In the blood cell analysis method of this embodiment, a fluorescent dye excited by the second wavelength is not used. Figure 4 In the example of FIG. 4 , for the purpose of explaining the optical system, a configuration is shown that can receive the side fluorescence (second side fluorescence) corresponding to the light generated when the fluorescent dye is excited. That is, the second side fluorescence is transmitted through the dichroic mirror 418c and received by the side fluorescence light receiving element 422b. The side fluorescence light receiving elements 422a and 422b are, for example, avalanche photodiodes.

[0175] Reference Figure 5 The relationship between various lights emitted when light is irradiated onto particles P passing through the flow cell 413 and the optical system of the FCM detection unit 460 will be described. Figure 5 In the embodiment, the light irradiated from the first light source 411a is the light L1 of the first wavelength, and the light irradiated from the second light source 411b is the light L2 of the second wavelength. When the light L1 and L2 are irradiated to the particles P passing through the flow cell 413, forward scattered light corresponding to the light of the first wavelength and the light of the second wavelength, respectively, is generated from the particles P in the forward direction relative to the direction of travel of the light. Figure 5 In the example, since the light receiving element 416 receives the forward scattered light corresponding to the light of the second wavelength, only the second forward scattered light (FSC-2) corresponding to the light of the second wavelength is displayed, and the first forward scattered light (FSC-1) corresponding to the light of the first wavelength is omitted. Figure 5, the first lateral scattered light (SSC-1) corresponding to the light of the first wavelength and the second lateral scattered light (SSC-2) corresponding to the light of the second wavelength are generated laterally relative to the direction of travel of the light. In addition, when the particle P is dyed with the fluorescent dye excited by the first wavelength, the first lateral fluorescence (SFL-1) excited by the light of the first wavelength is generated from the fluorescent dye laterally relative to the direction of travel of the light. In the blood cell analysis method of this embodiment, the fluorescent dye excited by the second wavelength is not used. Figure 5 In the example of FIG. 1 , for the purpose of explaining the optical system, the state in which both SFL-1 and the second lateral fluorescence (SFL-2) excited by the light of the second wavelength occur laterally with respect to the traveling direction of the light are shown.

[0176] As described above, FSC-2, SSC-1, SFL-1, SSC-2 and SFL-2 receive light receiving elements 416, 412a, 422a, 412b and 422b respectively. Each light receiving element outputs a waveform-shaped electrical signal (also referred to as an analog signal) containing a pulse corresponding to the light receiving intensity. The analog signal corresponding to FSC-2 is also referred to as the "second forward scattered light signal", the analog signal corresponding to SSC-1 is also referred to as the "first side scattered light signal", the analog signal corresponding to SFL-1 is also referred to as the "first fluorescent signal", the analog signal corresponding to SSC-2 is also referred to as the "second side scattered light signal", and the analog signal corresponding to SFL-2 is also referred to as the "second fluorescent signal". When receiving FSC-1, the analog signal corresponding to FSC-1 is also referred to as the "first forward scattered light signal". One of the pulses of each analog signal corresponds to one particle (e.g., one cell).

[0177] The analog signals corresponding to the various lights are input to the analog processing unit 481, and are processed by noise removal, flattening, etc. The A / D conversion unit 482 samples the analog signals output from the analog processing unit 481 at a specified sampling rate (for example, sampling at 10 nanosecond intervals for 1024 minutes, sampling at 80 nanosecond intervals for 128 minutes, or sampling at 160 nanosecond intervals for 64 minutes, etc.). The A / D conversion unit 482 digitizes the sampled analog signals to generate waveform data. The A / D conversion unit 482 samples and digitizes the five analog signals corresponding to each cell flowing through the flow cell 413 to generate waveform data of the second forward scattered light signal, the first side scattered light signal, the first fluorescent signal, the second side scattered light signal, and the second fluorescent signal. When receiving FSC-1, the waveform data of the first forward scattered light signal is also generated. Furthermore, the A / D conversion unit 482 calculates the characteristic parameters representing the characteristics of the morphology of each cell from the waveform data of each signal. Examples of such characteristic parameters include a peak value (height of a pulse peak), a pulse width, a pulse area, a transmittance, a Stokes shift, a ratio, a temporal change, and values ​​related thereto.

[0178] The optical information may be the characteristic parameters mentioned above. The optical information includes fluorescence information, first scattered light information, second scattered light information and third scattered light information. Fluorescence information is information related to fluorescence generated from the fluorescent dye of the stained particles by irradiating the measurement sample with light of the first wavelength. The fluorescence information is not particularly limited as long as it is information reflecting the amount of fluorescent dye that stains the nucleic acid in the nucleated cells. As fluorescence information, the peak value and pulse area of ​​the first fluorescence signal are preferred, and the peak value of the first fluorescence signal is more preferred. In this specification, the peak value of the first fluorescence signal is also referred to as "first fluorescence intensity". The first fluorescence intensity can be described as the intensity of the fluorescence of the fluorescent dye of the particles irradiated with light of the first wavelength.

[0179] The first scattered light information is information related to side scattered light generated from particles by irradiating the measurement sample with light of the first wavelength. The second scattered light information is information related to forward scattered light generated from particles by irradiating the measurement sample with light of the second wavelength. The third scattered light information is information related to side scattered light generated from particles by irradiating the measurement sample with light of the second wavelength. The side scattered light related information is not particularly limited as long as it is information reflecting internal information such as the complexity of cell structure, particle characteristics, nuclear structure, and lobation. The forward scattered light related information is not particularly limited as long as it is information reflecting the size of the particle. As the first scattered light information, the peak value and pulse area of ​​the first side scattered light signal are preferred, and the peak value of the first side scattered light signal is more preferred. As the second scattered light information, the peak value and pulse area of ​​the second forward scattered light signal are preferred, and the peak value of the second forward scattered light signal is more preferred. As the third scattered light information, the peak value and pulse area of ​​the second side scattered light signal are preferred, and the peak value of the second side scattered light signal is more preferred. The peak value of the first side scattered light signal is also referred to as the “first side scattered light intensity”, the peak value of the second front scattered light signal is also referred to as the “second front scattered light intensity”, and the peak value of the second side scattered light signal is also referred to as the “second side scattered light intensity”. The first side scattered light intensity can be described as the intensity of the first side scattered light of particles irradiated with light of the first wavelength. The second front scattered light intensity can be described as the intensity of the front scattered light of particles irradiated with light of the second wavelength. The second side scattered light intensity can be described as the intensity of the second side scattered light of particles irradiated with light of the second wavelength.

[0180] Reference Figure 6 The analysis unit 300 is electrically connected to the measurement unit 400 via the interface unit 304. The interface unit 304 is, for example, a USB interface. The analysis unit 300 includes a control unit 301, a bus 302, a storage unit 303, an interface unit 304, a display unit 305, and an operation unit 306. The analysis unit 300 is, for example, operated by a personal computer (see Figure 1The analyzing unit 300 of the analyzing system 500 is configured to execute a program stored in the storage unit 303 to control the measuring unit 400 of the analyzing system 500. The analyzing unit 300 analyzes data including optical information obtained by the measuring unit 400 and displays the analysis result on the display unit 305. The analyzing unit 300 can also classify cells based on the optical information.

[0181] The storage unit 303 stores, for example, a program for controlling the measurement unit 400 and a program for analyzing data acquired by the measurement unit 400. The display unit 305 displays, for example, analysis results of data acquired by the measurement unit 400. The operation unit 306 includes a pointing device including a keyboard, a mouse, or a touch panel.

[0182] Reference Figure 7 , an example of the operation of the analysis system 500 is described, but the present invention is not limited to this example. In step S11, the control unit 301 of the analysis unit 300 receives an instruction to start measurement from the user via the operation unit 306. When the control unit 301 receives the instruction to start measurement, it sends instruction data instructing the start of measurement to the measurement unit 400. The measurement unit 400 receives the instruction data and performs measurement processing in step S12.

[0183] In the blood cell analysis method of this embodiment, for example, the following first embodiment and second embodiment are included. In the "first embodiment", a measurement sample prepared from a subject is measured, and the first scattered light information and the second scattered light information of the particles in the measurement sample are obtained. Next, based on the first scattered light information and the second scattered light information, blood cells and leukocytes other than leukocytes are determined from the above-mentioned particles. Furthermore, based on the fluorescence information and the second scattered light information, reticulocytes and platelets are classified from blood cells other than leukocytes. In the "second embodiment", a measurement sample prepared from a subject is measured, and the first scattered light information and the fluorescence information of the particles in the measurement sample are obtained. Next, based on the first scattered light information and the fluorescence information, blood cells and leukocytes other than leukocytes are determined from the above-mentioned particles. Furthermore, reticulocytes and platelets are classified from blood cells other than leukocytes in the same manner as in the first embodiment.

[0184] (Measurement Process)

[0185] about Figure 7 For an example of the measurement process in the first embodiment and the second embodiment, refer to step S12. Figure 8Description, but not limited to this example. In step S21, the control unit 301 of the analysis unit 300 modulates the measurement sample from the subject to the measurement unit 400. Specifically, the measurement unit 400 mixes the subject, the staining reagent and the dilution reagent in the reaction chamber 420 to modulate the measurement sample. In step S22, the control unit 301 causes the measurement unit 400 to export the measurement sample to the FCM detection unit 460 and perform optical measurement by flow cytometry. Thus, the measurement unit 400 detects optical information, such as waveform data of SSC-1, SSC-2, FSC-2 and SFL-1, for each particle in the measurement sample. In step S23, the control unit 301 in the measurement unit 400 sends the optical information to the analysis unit 300 to end the measurement process. Furthermore, the analysis unit 300 executes Figure 7 The analysis process of step S13 is performed. Examples of the analysis process of each embodiment will be described later.

[0186] (Subject)

[0187] The subject is a blood subject. Examples of blood subjects include whole blood and dilutions of whole blood. Whole blood is, for example, peripheral blood collected from a subject. The blood subject may also contain an anticoagulant. Examples of such anticoagulants include ethylenediaminetetraacetic acid (EDTA), EDTA salts (e.g., EDTA·2K, EDTA·2Na, etc.), sodium citrate, heparin, warfarin, etc. Dilutions of whole blood are obtained, for example, by diluting whole blood with a suitable aqueous solvent, preferably a diluting reagent described later. Examples of aqueous solvents include water, physiological saline, and aqueous solutions of buffers.

[0188] The particles in the measurement sample are derived from the blood components of the subject. In this specification, "blood components" include formed elements and abnormal cells known to be contained in the blood. Formed elements known to be contained in the blood are, for example, rare blood cells usually contained in the peripheral blood of healthy people. As such blood cells, for example, white blood cells, mature red blood cells, reticulocytes and platelets can be cited. "Mature red blood cells" are terminally differentiated red blood cells without nuclei and nucleic acids. The term "mature red blood cells" in this specification is used to distinguish from reticulocytes. In the case where mature red blood cells and reticulocytes are not particularly distinguished, in this specification, as a term including mature red blood cells and reticulocytes, it is sometimes referred to as "red blood cells" or "RBC". In this specification, the term "platelets" includes immature platelets and large platelets. "Immature platelets" are also called reticulocytes, which are young platelets that have just been released into the peripheral blood. Mature platelets do not have nucleic acids, and immature platelets have a small amount of ribonucleic acid. "Large platelets" are platelets with a diameter of more than 4 μm. Platelets are usually 2 to 3 μm in diameter. "Abnormal cells" refer to formed elements that do not normally appear in blood or body fluids. Abnormal cells also contain non-cellular particles and microorganisms. Examples of non-cellular particles include platelet aggregates, red blood cell ghosts, and lipid particles. Examples of microorganisms include bacteria and fungi. Platelet aggregation occurs in blood collection tubes due to the mixing of tissue fluid during blood collection, insufficient mixing, or the action of EDTA.

[0189] (Dilution reagent)

[0190] The diluent reagent used in the preparation of the measurement sample is described. The diluent reagent contains a buffer and a solvent. Preferably, the diluent reagent contains a solution of a buffer. As the solvent, water, physiological saline, etc. can be cited, and water is particularly preferred. The buffer preferably has a buffering effect in the range of pH 6 or more and 11 or less. Such a buffer can be selected from, for example, carboxylates, phosphates, Good's buffer, taurine, triethanolamine, etc. The pH of the diluent reagent is, for example, 6 or more and 11 or less, preferably 7 or more and 10 or less, and more preferably 8 or more and 9.5 or less. When the pH of the diluent reagent is above 6, it becomes difficult to hemolyze red blood cells in the measurement sample. Thereby, the occurrence of red blood cell shadows can be suppressed. In addition, when the pH of the diluent reagent is below 11, the nonspecific staining of red blood cells and red blood cell shadows by fluorescent dyes can be reduced. The preferred diluent reagent is an aqueous solution of the above-mentioned buffer with a pH of 6 or more and 11 or less.

[0191] In addition to the above-mentioned buffer, the diluent may also contain ingredients such as an osmotic pressure compensator, a dyeing accelerator, a polyvalent anion, and a preservative. The osmotic pressure compensator is a substance that can maintain the osmotic pressure of the diluent in a suitable range. As the osmotic pressure compensator, for example, alkali metal salts of organic acids such as propionic acid, glucose, mannose, and other sugars, alkali metal halides such as sodium chloride, and alkaline earth metal halides such as magnesium chloride can be cited. One or more osmotic pressure compensators can be used. When using the osmotic pressure compensator, the osmotic pressure compensator is preferably added to the diluent in such a way that the osmotic pressure of the diluent becomes 150mOsm / kg·H2O or more and 600mOsm / kg·H2O or less, and more preferably 200mOsm / kg·H2O or more and mOsm / kg·H2O or less.

[0192] The dyeing accelerator is a substance that can promote the permeability of fluorescent dye to blood cells. As the dyeing accelerator, for example, a surfactant can be mentioned, preferably a cationic surfactant. As the cationic surfactant, a quaternary ammonium salt type surfactant is particularly preferred, for example, decyl trimethyl ammonium bromide (DTAB), lauryl trimethyl ammonium chloride (LTAC), octyl trimethyl ammonium bromide, cetyl trimethyl ammonium chloride, myristyl trimethyl ammonium bromide, etc. can be mentioned. These cationic surfactants can also be used in one or more than two kinds.

[0193] The concentration of the cationic surfactant in the diluent is not particularly limited as long as it can inhibit hemolysis of erythrocytes. It can be appropriately determined, for example, in the range of 50 ppm to 20,000 ppm. Specifically, when DTAB is used, the concentration of DTAB in the diluent is preferably 500 ppm to 3,000 ppm. When LTAC is used, the concentration of LTAC in the diluent is preferably 100 ppm to 500 ppm. By including the cationic surfactant in the diluent at such a concentration, hemolysis of erythrocytes can be inhibited and staining of blood cells by fluorescent dyes can be promoted.

[0194] In order to suppress the non-specific staining of red blood cells by fluorescent dyes, polyvalent anions are added to the diluent reagent. As polyvalent anions, for example, sulfate ions, phosphate ions, carbonate ions, polycarboxylate ions, etc. can be mentioned. As compounds that can supply these ions, for example, citric acid, sulfuric acid, phosphoric acid, EDTA and alkali metal salts thereof can be mentioned. One or more polyvalent anions can also be used.

[0195] Examples of the preservative contained in the diluent include sodium 2-pyridylthio-1-oxide and β-phenethyl alcohol.

[0196] In the blood cell analysis method of the present embodiment, a commercially available dilution reagent may be used. Examples of commercially available dilution reagents include CELLPACK (registered trademark) DFL (Sysmex Corporation).

[0197] (Staining reagents and fluorescent dyes)

[0198] The dyeing reagent used in the preparation of the assay sample is described. A fluorescent dye suitable for the dyeing reagent is a fluorescent dye that can be excited by light of the first wavelength and can bind to nucleic acid. Such fluorescent dyes are known per se and can be selected from acridine compounds, anthocyanin compounds, and styryl compounds. By binding the fluorescent dye to nucleic acid, cells that do not have nucleic acid such as mature red blood cells or platelets and cells that have nucleic acid such as reticulocytes or leukocytes can be distinguished.

[0199] Examples of fluorescent dyes of acridine compounds include fluorescent dyes represented by the above formula (I). The "alkyl" group may be a linear or branched chain. 12 As examples of the alkyl group having 1 to 18 carbon atoms, there can be mentioned, for example, methyl, ethyl, propyl, isopropyl, t-butyl, n-butyl, isopentyl, neopentyl, t-pentyl, isohexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, etc. The number of carbon atoms in the alkyl group is preferably 1 to 10, more preferably 1 to 6, and particularly preferably 1 to 3. Preferred examples of the alkyl group include methyl, ethyl, propyl, isopropyl, t-butyl, n-butyl, isopentyl, neopentyl, t-pentyl, isohexyl, etc. Regarding R in formula (I) 13 The number of carbon atoms in the alkyl group is preferably 1 to 3.

[0200] "Halogen" is fluorine, chlorine, bromine, or iodine. "Haloalkyl" is an alkyl group in which at least one hydrogen is replaced by a halogen. When the haloalkyl group contains two or more halogens, the halogens may be the same or may contain multiple halogens. "Acylamino" is a group in which one hydrogen of an amino group is replaced by an acyl group (R-CO-NH-). Examples of acylamino groups include formylamino, acetylamino, propionylamino, butyrylamino, benzylamino, toluylamino, naphthoylamino, nicotinylamino, isonicotinoylamino, furoylamino, and the like.

[0201] Examples of the substituent of the phenyl group include -NH2, halogen, -OH, -SH, -CN, -NO2, -COOH, and an alkyl group having 1 to 6 carbon atoms. Examples of the counterion X- in formula (I) include F-, Cl- - Br-I - 、1 / 2SO4 2- 、NO3 - etc. In X -It is Cl - 、1 / 2SO4 2- or NO3 - When the fluorescent dye is used, an acid such as HCl, H2SO4 or HNO3 that can provide ions to the fluorescent dye may be added. - , lactic acid can also be added to the fluorescent dye.

[0202] Preferred fluorescent dyes of acridine compounds represented by the above-mentioned formula (I) include, for example, 2,7,9-trimethylacridine-3,6-diamine, 3,6-diaminoacridine, 3,6-diamino-10-methylacridine, 3,6-diamino-2,7,10-trimethylacridine, 3,6-diamino-2,7-dimethylacridine, N3,N3,N6,N6-trimethylacridine-3,6-diamine, 9-(4-aminophenyl)acridine-3-amine, N2,N2,N8,N8-tetramethylacridine-2,8-diamine, and 6,9-diamino-2-ethoxyacridine.

[0203] As fluorescent dyes of anthocyanin compounds, for example, the fluorescent dyes represented by the above-mentioned formula (II) can be mentioned. The alkyl group in the above-mentioned formula (II) is the same as that described for formula (I). "Alkenyl" can also be any of a straight chain or a branched chain. Regarding R1 and R2 in the above-mentioned formula (II), as alkenyl groups having 3 to 20 carbon atoms, for example, allyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, isopentenyl, hexenyl, heptenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, eicosenyl, etc. can be mentioned. The number of carbon atoms of the alkenyl group is preferably 3 to 14, more preferably 3 to 10, and particularly preferably 3 to 6.

[0204] The substituents of the phenyl group in the above formula (II) are the same as those described for the formula (I). Examples of the substituents of the benzyl group in the above formula (II) include -CN, -COOH, -NH2, -NO2, -OH, -SH, alkyl groups having 1 to 18 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, alkylamino groups having 1 to 6 carbon atoms, acylamino groups having 1 to 6 carbon atoms, halogen groups, and halogenated alkyl groups having 1 to 6 carbon atoms. Examples of the counter ion Z- in the above formula (II) include F-, Cl-, Br- - ,I - CF3SO3 - 、BF4 - 、ClO4 - wait.

[0205] Preferred fluorescent dyes of the anthocyanin compounds represented by the above formula (II) include, for example, 3-methyl-2-[(3-methyl-2-benzoxazolylidene)methyl]benzoxazolylium, 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolylium, 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]-6-methyl-benzothiazolylium, 3-ethyl-2-[(3-ethyl-2-benzoxazolylidene)methyl]benzothiazolylium, 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolium, 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]benzothiazolium, 3-methyl-2-[(1,3,3-trimethyl-2-indolylidene)methyl]benzothiazolium and 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolylidene)methyl]-3H-indolylium.

[0206] As fluorescent dyes of styrene-based compounds, for example, fluorescent dyes represented by the above-mentioned formulas (III) and (IV) can be mentioned. The substituents of the alkyl and phenyl groups in the above-mentioned formulas (III) and (IV) are the same as those described for the formula (I). In addition, the substituents of the alkenyl and benzyl groups are the same as those described for the above-mentioned formula (II). In the above-mentioned formula (IV), the heterocyclic ring formed by R2 and / or R3 together with the benzene ring to which N is bonded may be a five-membered ring or a six-membered ring. In addition, the heterocyclic ring may optionally have a substituent. As the substituent, an alkyl group having 1 to 3 carbon atoms can be mentioned. The counterion Z in the above-mentioned formulas (III) and (IV) - For example, F - , Cl - Br - 、I-、CF3SO3 - 、BF4 - 、ClO4 - Etc. As the fluorescent dye of the styryl compound represented by the above formula (III), for example, 3-ethyl-2-[(1-ethyl-2(1H)-quinolylidene)methyl]benzothiazolium can be mentioned. In addition, as the fluorescent dye of the styryl compound represented by the above formula (IV), for example, 2-[4-(dimethylamino)phenyl]-3,6-dimethyl-1,3-benzothiazol-3-ium can be mentioned.

[0207] The staining reagent preferably contains a solution of a fluorescent dye. The solvent is not particularly limited as long as it can dissolve the above-mentioned fluorescent dye. For example, water, an organic solvent, and a mixture thereof can be cited. As an organic solvent, a solvent that can be mixed with water is preferred, and examples thereof include alcohols having 1 to 6 carbon atoms, ethylene glycol, diethylene glycol, polyethylene glycol, dimethyl sulfoxide (DMSO), etc. The concentration of the fluorescent dye in the staining reagent can be appropriately determined corresponding to the type of fluorescent dye. The concentration of the fluorescent dye can be, for example, more than 100 ppm, preferably more than 500 ppm, and more preferably more than 1000 ppm. The concentration of the fluorescent dye is, for example, below 100,000 ppm, preferably below 50,000 ppm, and more preferably below 10,000 ppm.

[0208] As described above, since the fluorescent dye itself binds to the nucleic acid of the cell, in the blood cell analysis method of this embodiment, it is not necessary to use an antibody labeled with a fluorescent dye to stain the cells. Therefore, in the blood cell analysis method of this embodiment, the fluorescent dye does not contain an antibody.

[0209] A staining reagent containing the above-mentioned fluorescent dye can also be provided as a blood cell analysis reagent used in the blood cell analysis method of this embodiment. The blood cell analysis reagent of this embodiment can be, for example, in the form of a solution of the above-mentioned fluorescent dye contained in a container. Fig. 9 , 11 denotes a container containing the blood cell analysis reagent of the present embodiment. When the container 11 is contained in a packing box, it may also contain accompanying documents recording the composition, usage method, storage method, etc. of the blood cell analysis reagent, a buffer material for reducing external impact, etc.

[0210] A further embodiment relates to the use of a fluorescent dye for the production of a reagent for blood cell analysis. The details of the fluorescent dye and the reagent for blood cell analysis are as described above.

[0211] (Preparation of measurement sample)

[0212] The fluorescent dye is mixed with the test object and the diluent in such a way that the concentration (final concentration) in the measurement sample becomes a concentration within a specified range. The preferred final concentration of the fluorescent dye in the measurement sample is 500 ppm or less, more preferably 100 ppm or less, and more preferably 50 ppm or less. The preferred final concentration of the fluorescent dye in the measurement sample is 0.01 ppm or more, more preferably 0.1 ppm or more, and more preferably 1 ppm or more.

[0213] The mixing ratio of the dilution reagent, the staining reagent and the test object can be appropriately determined corresponding to the concentration of the fluorescent dye in the staining reagent or the type of the test object. The mixing ratio of the dilution reagent, the staining reagent and the test object is preferably, for example, 1000: 0.1 or more: 1 or more, expressed in volume ratio. More preferably, it is 1000: 0.5 or more: 5 or more, and more preferably, it is 1000: 1 or more: 10 or more. In addition, the mixing ratio of the dilution reagent, the staining reagent and the test object is preferably, for example, 1000: 50 or less: 100 or less, expressed in volume ratio. More preferably, it is 1000: 30 or less: 50 or less, and more preferably, it is 1000: 20 or less: 25 or less. The mixing ratio of the staining reagent and the test object may be the same or different.

[0214] The blood cell analysis method of the present embodiment makes it possible to detect reticulocytes and platelets without using a hemolytic reagent. Therefore, in the blood cell analysis method of the present embodiment, the measurement sample does not contain a hemolytic reagent. That is, in the blood cell analysis method of the present embodiment, the preparation of the measurement sample does not include a process of hemolyzing red blood cells. Among them, the hemolytic reagent refers to a reagent used to dissolve the red blood cells in the subject by mixing with the subject. The hemolytic reagent itself is well known, for example, it contains a surfactant at a concentration that can dissolve red blood cells.

[0215] (Analysis Processing of First Embodiment)

[0216] about Figure 7 Step S13, refer to Figures 10A to 17 The example of the analysis process of the first embodiment is described, but is not limited to this example. In this specification, mature red blood cells are also referred to as "mRBC", reticulocytes are also referred to as "RET", platelets are also referred to as "PLT", and white blood cells are also referred to as "WBC".

[0217] In the analysis process of the first embodiment, first, blood cells other than WBC are identified from particles in the measurement sample. In the blood cell analysis method of this embodiment, since a hemolytic reagent is not used, mRBC and RET are contained in the measurement sample. In addition, WBC and PLT are also contained in the measurement sample. That is, blood cells other than WBC refer to a particle group containing mRBC, RET and PLT. In the first embodiment, blood cells other than WBC are identified based on the first scattered light information and the second scattered light information. In this example, the first side scattered light intensity (also called "SSC-1 intensity") is used as the first scattered light information, and the second forward scattered light intensity (also called "FSC-2 intensity") is used as the second scattered light information.

[0218] The identification of blood cells other than WBC can be performed, for example, by analysis using a scattergram. The scattergram for identifying blood cells other than WBC based on the first and second scattered light information is also referred to as "first scattergram (a)". Fig. 10A In step S101, the analysis unit 300 makes a first scatter plot (a). The first scatter plot (a) is made, for example, based on the SSC-1 intensity and the FSC-2 intensity. For example, the analysis unit 300 makes a scatter plot with the SSC-1 intensity and the FSC-2 intensity as two axes based on the acquired optical information. Furthermore, in the made first scatter plot (a), the positions of the points corresponding to each particle in the measured sample are determined. The horizontal axis and the vertical axis of the first scatter plot (a) can also be displayed in either a logarithmic or a linear manner. Preferably, the horizontal axis and the vertical axis of the first scatter plot (a) are represented logarithmically.

[0219] In step S102, the analysis unit 300 classifies the particles in the measured sample into a first particle group and a second particle group based on the SSC-1 intensity and / or the FSC-2 intensity. The second particle group is a group that shows a higher SSC-1 intensity and / or FSC-2 intensity than the first particle group. Among them, the comparison of the scattered light intensity between the particle groups in the scatter plot is preferably performed, for example, by a statistical representative value of the scattered light intensity of each particle group. The statistical representative value of the scattered light intensity of a particle group is a value obtained from the scattered light intensity of the particles constituting the particle group, and can include, for example, a median value, an average value, a most frequent value, etc. Preferably, it is a median value.

[0220] In the first scatter plot (a), the first particle group appears in the area with low SSC-1 intensity and / or FSC-2 intensity. In addition, the second particle group appears in the area with high SSC-1 intensity and / or FSC-2 intensity. Fig.12 In the example, the first particle group appears in the area on the side with low SSC-1 intensity, and the second particle group appears in the area on the side with high SSC-1 intensity. In this figure, the first particle group is a group composed of particles in the area surrounded by the dotted line. Specifically, the first particle group includes a group containing mRBC and RET (also called "RBC-containing group") and a group containing PLT. That is, the first particle group is blood cells other than WBC. Furthermore, when the subject contains platelet aggregates, platelet aggregates are also included in the group containing PLT. In addition, the second particle group is a group of WBC. Next, the classification of particles in the measured sample in the first scatter plot (a) into the first particle group and the second particle group is explained.

[0221] Among the blood cells other than WBC, mRBC and RET (expressed as "RBC") and WBC are distinguished based on SSC-1 intensity and / or FSC-2 intensity. Among them, hemoglobin contained in RBC is known to absorb blue-violet light. When the RBC is irradiated with light of the first wavelength corresponding to blue-violet light, the scattered light generated from these blood cells becomes weaker than the scattered light generated when the light of the second wavelength corresponding to red light is irradiated. Therefore, in the past, red light was preferably used in blood cell analysis. On the one hand, when the WBC is irradiated with light of the first wavelength and light of the second wavelength, there is almost no difference in the intensity of the scattered light generated. The inventors of the present invention intend to use the above phenomenon to distinguish WBC and RBC. Specifically, by irradiating light of the first wavelength, a difference occurs in the first scattered light information between WBC and RBC. For example, when the first scattered light information is SSC-1 intensity, the SSC-1 intensity of RBC becomes lower due to absorption by hemoglobin. However, it does not affect the SSC-1 intensity of WBC. Therefore, the SSC-1 intensity of RBC becomes lower than that of WBC. Thus, WBC and RBC can be distinguished. In addition, among the blood cells other than WBC, PLT and WBC are also distinguished by either FSC-2 intensity or SSC-1 intensity. It is known that PLT is the smallest in size among the blood cells. Among them, the intensity of the forward scattered light generated by irradiating light to the blood cells reflects the size of the blood cells. Therefore, the forward scattered light intensity of PLT is lower than that of WBC. In addition, due to the difference in internal structure, the side scattered light intensity of PLT is lower than that of WBC. Thus, PLT and WBC can be distinguished. From this, it can be seen that in the first scattergram (a), when RBC and PLT are regarded as a group (i.e., the first particle group), the SSC-1 intensity and FSC-2 intensity of this group become lower than those of the WBC group. Thus, in the first scattergram (a), the particles in the measured sample are classified into the first particle group containing blood cells other than WBC and the second particle group containing WBC.

[0222] Reference Fig. 10A In step S103, the analysis unit 300 determines the first particle group as blood cells other than WBC. As described above, since the first particle group includes mRBC, RET and PLT, the first particle group can be determined as blood cells other than WBC. In addition, since the second particle group includes WBC, the second particle can be determined as WBC. For example, by accumulating measurement data for a plurality of subjects, the region where the first particle group appears can be predetermined in the first scattergram (a), and the particles appearing in the region can be determined as blood cells other than WBC.

[0223] In this way, Fig. 10AIn the analysis process shown in , the blood cells other than WBC are identified by classifying the particles in the measurement sample into WBC and blood cells other than WBC. In step S103 , the data of WBC is excluded from the data of the particles in the measurement sample by identifying the blood cells other than WBC.

[0224] (Platelet Aggregation Test)

[0225] Reference Fig. 10B , the process proceeds to step S201. In this analysis process, information showing the presence or absence of platelet aggregation is obtained. In step S201, the analysis unit 300 classifies blood cells other than WBC into a group containing RBC and a group containing PLT based on the FSC-2 intensity. Since PLT is a blood cell smaller in size than mRBC and RET, the FSC-2 intensity of the group containing PLT is lower than that of the group containing RBC. Therefore, it can be classified into a group containing RBC and a group containing PLT based on the FSC-2 intensity. Refer to Fig.13A In the first scattergram (a), there are distributed RBC-containing groups, PLT-containing groups, and WBC-containing groups. Alternatively, by accumulating measurement data for a plurality of subjects, the region where the RBC-containing group appears and the region where the PLT-containing group appears can be predetermined in the first scattergram (a), and particles appearing in each region can be determined as the RBC-containing group and the PLT-containing group.

[0226] Reference Fig. 13B When platelet aggregation is contained in the test body, particles distributed in a manner extending from the group containing PLT to the group containing WBC are confirmed in the first scattergram (a). Fig. 13B In the figure, the particles that appear in the area surrounded by the dotted line are platelet aggregates. Platelet aggregates are aggregates of multiple platelets that are larger than normal platelets. Therefore, platelet aggregate particles show a higher forward scattered light intensity than platelets. Therefore, based on the FSC-2 intensity, it can be determined whether the PLT-containing group contains platelet aggregates. Fig. 10B In step S202, the analysis unit 300 determines whether the PLT-containing group contains particles that display an FSC-2 intensity above the threshold. Specifically, the FSC-2 intensity of all particles constituting the PLT-containing group is compared with a threshold value corresponding to the FSC-2 intensity. Particles that display an FSC-2 intensity above this threshold are suspected to be platelet aggregates. That is, the PLT-containing group is suspected to contain platelet aggregates. In step S202, when there are particles that display an FSC-2 intensity above the threshold, the process proceeds to step S203. In step S202, when there are no particles that display an FSC-2 intensity above the threshold, the process proceeds to step S203. Fig. 10C Step S301.

[0227] In a further embodiment, particles showing FSC-2 intensities above the threshold may also be counted. In step S202, when the number of particles showing FSC-2 intensities above the threshold is above a specified value, the process proceeds to step S203. In addition, when the number of particles showing FSC-2 intensities above the threshold is less than the specified value, the process proceeds to step S204. Fig. 10C The designated value is a value for determining whether a particle showing an FSC-2 intensity above a threshold value is platelet aggregation or not.

[0228] In step S203, the analysis unit 300 outputs information indicating the presence of platelet aggregation. For example, a symbol called "PLTc?" may be output to the display unit 305. Thereafter, the process proceeds to Fig. 10C Furthermore, not displaying the mark on the display unit 305 may be information indicating the absence of platelet aggregation.

[0229] (Reticulocyte and platelet detection)

[0230] After identifying blood cells other than WBC, the process proceeds to Fig. 10C Step S301. In this analysis process, based on the fluorescence information and the second scattered light information, blood cells other than WBC are classified into a group of RET and a group of PLT. Thus, RET and PLT are determined. Blood cells other than WBC may also be classified into a group of RET, a group of PLT, and a group of mRBC. Thus, RET, PLT, and mRBC are determined. Next, refer to Fig. 10C , the case where blood cells other than WBC are classified into a RET group, a PLT group, and an mRBC group will be described.

[0231] Classification of blood cells other than WBC can be performed, for example, by analysis using a scatter plot. A scatter plot for classifying RET groups and PLT groups from blood cells other than WBC based on fluorescence information and the second scattered light information is also referred to as a "second scatter plot." In this example, the first lateral fluorescence intensity (also referred to as "SFL-1 intensity") is used as fluorescence information, and the FSC-2 intensity is used as the second scattered light information. The second scatter plot is created based on the SFL-1 intensity and the FSC-2 intensity. Refer to Fig. 10CIn step S301, the analysis unit 300 creates a second scattergram. For example, the analysis unit 300 creates a scattergram with SFL-1 intensity and FSC-2 intensity as two axes based on the acquired optical information. Furthermore, in the created second scattergram, the positions of the points corresponding to the blood cells other than WBC are determined. The horizontal axis and the vertical axis of the second scattergram can also be displayed in either logarithmic or linear form. When the horizontal axis and the vertical axis of the second scattergram are expressed in logarithmic form, it is suitable for the analysis of PLT. When the horizontal axis and the vertical axis of the second scattergram are expressed in linear form, it is suitable for the analysis of RET and mRBC.

[0232] In step S302, the analysis unit 300 classifies blood cells other than WBC into the third particle group, the fourth particle group, and the fifth particle group based on the SFL-1 intensity and the FSC-2 intensity. The third particle group is a group showing a higher SFL-1 intensity than the fifth particle group. The fourth particle group is a group showing a lower FSC-2 intensity than the third particle group. The comparison of scattered light intensity and fluorescence intensity between particle groups in the scatter plot is as described above.

[0233] exist Fig.14A An example showing the second scatter plot (logarithmic display) and the second scatter plot (linear display). In each of the second scatter plots, the third particle group, the fourth particle group, and the fifth particle group appear. In the second scatter plot (logarithmic representation), the third and fifth particle groups appear in the area on the side with high FSC-2 intensity, and the fourth particle group appears in the area on the side with low FSC-2 intensity. In the second scatter plot (linear representation), the fourth particle group appears in the area with low FSC-2 intensity values, and the third particle group appears in the area on the side with higher FSC-2 intensity than the fourth particle group. In any scatter plot, the third particle group appears in the area on the side with higher SFL-1 intensity than the fifth particle group. As shown Fig.14A As shown, the third particle group is a RET group, the fourth particle group is a PLT group, and the fifth particle group is an mRBC group. Next, the classification of blood cells other than WBC into the RET group, the mRBC group, and the PLT group in the second scattergram will be described.

[0234] As described above, since PLT is the smallest in size among blood cells, the forward scattered light intensity of PLT is lower than that of RET. Therefore, in the second scatter plot, the group of PLT appears in the area on the side where the FSC-2 intensity is lower than that of the group of RET. Thus, RET and PLT are distinguished based on the FSC-2 intensity. As described above, RET has a small amount of ribonucleic acid, and mRBC does not have a nucleus and nucleic acid. Therefore, the group of RET appears in the area where the fluorescence intensity is higher than that of the group of mRBC. Thus, mRBC and RET are distinguished based on the SFL-1 intensity. In this way, in the second scatter plot, blood cells other than WBC are classified into the third particle group containing RET, the fourth particle group containing PLT, and the fifth particle group containing mRBC.

[0235] As described above, the third particle group contains RET, the fourth particle group contains PLT, and the fifth particle group contains mRBC. Fig. 10C In step S303, the analysis unit 300 determines the third particle group as RET, the fourth particle group as PLT, and the fifth particle group as mRBC. When RET matures, it becomes mRBC, so it is difficult to strictly determine the boundary between the RET group and the mRBC group. For example, a threshold of SFL-1 intensity that distinguishes the mRBC group from the RET group can be set based on the range of SFL-1 intensity that can usually be displayed by mRBC or the average number of particles of mRBC. Such a threshold can also be determined as the boundary between the mRBC group and the RET group on the second scatter plot. Furthermore, based on the threshold and FSC-2 intensity, for example Fig. 14B The regions where mRBC and RET appear respectively are set similarly to the regions indicated by dotted lines in . The particles in the region where mRBC appears can be determined as mRBC. In addition, the particles in the region where RET appears can be determined as RET. For the 4th particle group, when the 3rd and 5th particle groups are clearly separated from the 4th particle group on the 2nd scatter plot, the user can also determine the 4th particle group as a group of PLT in the 2nd scatter plot. Alternatively, by accumulating measurement data for a plurality of subjects, the regions where the 3rd, 4th and 5th particle groups appear can be predetermined in the 2nd scatter plot, and the particles appearing in each region can be determined as RET, PLT and mRBC respectively. The determined RET, PLT and mRBC can also be counted as needed. The counting of cells is performed by the analysis unit 300.

[0236] (Platelet analysis, reticulocyte analysis and classification of white blood cells)

[0237] Reference Fig. 10D, the process proceeds to step S401. In this analysis process, the PLT group and the RET group are further analyzed, and the WBC group is classified into subgroups. In step S401, the analysis unit 300, in the second scatter plot (logarithmic display), determines immature platelets and large platelets from the PLT group based on the SFL-1 intensity and the FSC-2 intensity. As described above, mature platelets do not have nucleic acids, and immature platelets have a small amount of ribonucleic acid. Thus, among the PLT groups on the second scatter plot, the PLT that shows a high SFL-1 intensity can be determined as immature platelets. For example, Fig.15A As shown in (A), PLTs appearing in the area with high SFL-1 (area surrounded by dotted lines) in the second scatter plot can also be determined as immature platelets. PLTs other than immature platelets can be determined as mature platelets. Thus, the groups of PLTs are classified into immature platelets and mature platelets. The determined immature platelets can also be counted. When the number of platelets is obtained by counting the groups of PLTs, the immature platelet ratio (IPF) can be calculated. IPF is the ratio of the number of platelets appearing in the area with high fluorescence intensity (the area surrounded by the square of solid lines) to the number of platelets. IPF is known to be an indicator of platelet production ability.

[0238] Since large platelets are larger than normal PLTs, PLTs showing high FSC-2 intensity among the PLT groups on the second scattergram can be identified as large platelets. Fig.15A As shown in (B), PLTs appearing in the area with high FSC-2 (area surrounded by dotted lines) in the second scatter plot can also be determined as large platelets. PLTs other than large platelets can be determined as PLTs of normal size. Thus, groups of PLTs are classified into large platelets and platelets of normal size. Determined large platelets can also be counted. When the number of platelets is obtained by counting the groups of PLTs, the large platelet ratio (P-LCR) can be calculated. P-LCR is the ratio of the number of platelets appearing in the area with high forward scattered light intensity (area surrounded by the dotted rectangle) to the number of platelets. P-LCR is known to be an indicator of platelet production capacity and congenital diseases.

[0239] Reference Fig. 10D , the process proceeds to step S402. In step S402, the analysis unit 300 obtains the platelet distribution width (PDW) and the mean platelet volume (MPV) based on the FSC-2 intensity and particle number of the PLT group. PDW is the distribution of the forward scattered light intensity of the PLT group and is an indicator of the deviation of the platelet size. MPV is the average value of the forward scattered light intensity of the PLT group and is an indicator of the size of the PLT. Fig.15AAs shown in (C), PDW and MPV can also be obtained by analyzing the PLT group on the second scatter plot. PDW is the length of the PLT group in the vertical direction. In addition, MPV is the average value calculated from the FSC-2 intensity of all particles constituting the PLT group. Alternatively, for the PLT group on the second scatter plot, as shown in Fig.15A As shown in (D), a histogram based on FSC-2 intensity and particle number is prepared. PDW is the length of the horizontal axis direction of the histogram.

[0240] Reference Fig. 10D , the process proceeds to step S403. In step S403, the analysis unit 300 classifies the RET group into particles appearing in the LFR region, particles appearing in the MFR region, and particles appearing in the HFR region based on the SFL-1 intensity. It is known that the more immature the RET is, the more nucleic acid (RNA) there is in the cell. When RET matures, the amount of RNA in the cell decreases. That is, the RNA amount of RET represents the maturity of RET. Among them, the SFL-1 intensity of the cell measured by FCM reflects the amount of nucleic acid in the cell. Thus, the maturity of RET can be analyzed based on the SFL-1 intensity. Refer to Fig. 15B In the second scatter plot (linear display), the region where RET appears can be divided into three regions: LFR, MFR, and HFR based on the SFL-1 intensity. RET appearing in each region can also be counted. Since the amount of RET nucleic acid appearing in the HFR region is the largest, it is the most immature RET.

[0241] The average value of the fluorescence intensity (SFL-1 intensity) and the average value of the forward scattered light intensity (FSC-2 intensity) can also be obtained for RET appearing in each region of LFR, MFR and HFR. Furthermore, the average value of the SFL-1 intensity and the FSC-2 intensity can also be obtained for the particles (i.e., mRBC) in the region where mRBC appears. The therapeutic effect on red blood cell diseases (such as pernicious anemia, iron deficiency anemia, etc.) can be monitored based on the average values ​​of the SFL-1 intensity and FSC-2 intensity of RET and mRBC appearing in each region according to the method described in Patent No. 4474135 (Patent No. 4474135 is incorporated into this specification by reference). Specifically, the average values ​​of the SFL-1 intensity and FSC-2 intensity of each region of LFR, MFR and HFR are plotted on a coordinate graph with SFL-1 intensity and FSC-2 intensity as the two axes, and an approximate straight line is obtained by the minimum second-order method. Furthermore, the value of the slope of this approximate straight line is obtained. By periodically obtaining the value of this slope from the date of initiation of treatment and monitoring changes in the value, the effect of treatment can be determined.

[0242] Reference Fig. 10D , the process proceeds to step S404. In this step, Fig. 10AThe WBC group determined in step S103 is classified into subgroups. In step S404, the analysis unit 300 classifies the WBC group into subgroups based on the SSC-1 intensity and the FSC-2 intensity. The subgroups of white blood cells are, for example, lymphocytes, monocytes, and granulocytes. Granulocytes are a general term for neutrophils, eosinophils, and basophils. In step S404, by enlarging the area where the WBC group appears in the first scattergram (a), as shown in FIG. Fig.16 As shown, it is confirmed that WBC is divided into each subgroup and distributed. In the figure, "Gran" represents granulocytes, "Mono" represents monocytes, and "Lym" represents lymphocytes. In this figure, WBC is classified into 3 subgroups, but it is not limited to this. WBC can be classified into at least two groups among the lymphocyte group, the monocyte group and the granulocyte group. For example, when WBC has an abnormality, there is a situation where a part of the subgroup of WBC is reduced to a degree that is difficult to classify. WBC can also be classified into two groups of lymphocytes or monocytes and granulocytes, for example.

[0243] The classification of WBC into subgroups can also be determined, for example, as follows. First, by accumulating measurement data for a plurality of subjects, the region where each subgroup of WBC appears is predetermined in the first scattergram (a). Then, the particles appearing in the region are determined as subgroups of WBC. If necessary, the cells contained in each subgroup of WBC can be counted. After the classification of WBC is performed, the analysis unit 300 ends the analysis process, and the process proceeds to Figure 7 Step S14.

[0244] (Modification of the classification process of white blood cells)

[0245] exist Fig. 10D In step S404, the WBC groups are classified based on the first scattered light information and the second scattered light information. Fig.11 and 17 , an example of analysis processing for classifying a group of WBCs into subgroups based on the second scattered light information and the third scattered light information is described. The scattergram for classifying a group of WBCs into subgroups based on the second scattered light information and the third scattered light information is also referred to as "third scattergram (a)". In this example, the second side scattered light intensity (also referred to as "SSC-2 intensity") is used as the third scattered light information. The third scattergram (a) is prepared, for example, based on the FSC-2 intensity and the SSC-2 intensity.

[0246] In step S501, the analysis unit 300 creates a third scatter plot (a). For example, based on the acquired optical information, the analysis unit 300 creates a scatter plot with SSC-2 intensity and FSC-2 intensity as two axes. Furthermore, in the created third scatter plot (a), the positions of the points corresponding to the particles in the WBC group are determined. The horizontal axis and vertical axis of the third scatter plot (a) can also be displayed either logarithmically or linearly. Preferably, the horizontal axis and vertical axis of the third scatter plot (a) are represented logarithmically. In step S502, the analysis unit 300 classifies the WBC group into lymphocytes, monocytes and granulocytes based on the SSC-2 intensity and the FSC-2 intensity. Among them, granulocytes refer to the general term for neutrophils, eosinophils and basophils. In the third scatter plot (a), as shown in FIG. Fig.17 As shown in (A), it is confirmed that WBC is divided into each subgroup and distributed. In this figure, WBC is classified into 3 subgroups, but it is not limited to this. WBC can be classified into at least two groups among the lymphocyte group, the monocyte group and the granulocyte group. WBC can also be classified into two groups of lymphocytes or monocytes and granulocytes, for example.

[0247] In a further embodiment, Fig.11 In the analysis process, the WBC groups can also be classified based on the fluorescence information and the third scattered light information. The scatter plot for classifying the WBC groups into subgroups based on the fluorescence information and the third scattered light information is also called "third scatter plot (b)". For example, the third scatter plot (b) is made based on the SFL-1 intensity and the SSC-2 intensity. Fig.11 In step S501, a third scatter plot (b) may be prepared instead of the third scatter plot (a). For example, the analysis unit 300 prepares a scatter plot with SSC-2 intensity on the horizontal axis and SFL-1 intensity on the vertical axis based on the optical information obtained. Furthermore, in the prepared third scatter plot (b), the position of the point corresponding to each particle in the WBC group is determined. The horizontal axis and the vertical axis of the third scatter plot (b) may also be displayed either logarithmically or linearly. Preferably, the horizontal axis and the vertical axis of the third scatter plot (b) are displayed logarithmically. In step S502, the analysis unit 300 classifies the WBC group into lymphocytes, monocytes and granulocytes based on the SSC-2 intensity and the SFL-1 intensity. In the third scatter plot (b), as Fig.17 As shown in (B), it is confirmed that WBC is divided into each subgroup and distributed. In this figure, WBC is classified into 3 subgroups, but it is not limited to this. The group of WBC can be classified into at least two groups among the group of lymphocytes, the group of monocytes and the group of granulocytes. WBC can also be classified into two groups of lymphocytes or monocytes and granulocytes, for example.

[0248] The classification of WBC into subgroups can also be determined, for example, as follows. First, by accumulating measurement data for a plurality of subjects, the regions where each subgroup of WBC appears are predetermined in the third scattergrams (a) and (b). Furthermore, the particles appearing in the regions are determined as subgroups of WBC.

[0249] If necessary, the cells contained in each subgroup of WBC can be counted. After the classification of WBC is performed, the analysis unit 300 ends the analysis process and the process proceeds to Figure 7 Step S14.

[0250] (Analysis Processing of Second Embodiment)

[0251] about Figure 7 Step S13, refer to Figures 18 to 20 The example of the analysis process for explaining the second embodiment is not limited to this example. In the second embodiment, blood cells other than WBC are identified from particles in the measurement sample based on the first scattered light information and the fluorescence information. In this example, the SSC-1 intensity is used as the first scattered light information, and the SFL-1 intensity is used as the fluorescence information. The identification of blood cells other than WBC can be performed, for example, by analysis using a scatter plot. The scatter plot for identifying blood cells other than WBC based on the first scattered light information and the fluorescence information is also referred to as the "first scatter plot (b)". Refer to Fig.18 In step S601, the analysis unit 300 creates a first scatter plot (b). The first scatter plot (b) is created, for example, based on the SSC-1 intensity and the SFL-1 intensity. For example, the analysis unit 300 creates a scatter plot with the SSC-1 intensity and the SFL-1 intensity as two axes based on the acquired optical information. Furthermore, in the created first scatter plot (b), the positions of the points corresponding to each particle in the measured sample are determined. The horizontal axis and the vertical axis of the first scatter plot (b) can also be displayed either logarithmically or linearly. Preferably, the horizontal axis and the vertical axis of the first scatter plot (b) are represented logarithmically.

[0252] In step S602, the analysis unit 300 classifies the particles in the measured sample into a first particle group and a second particle group based on the SSC-1 intensity and the SFL-1 intensity. The second particle group is a group that shows a higher SFL-1 intensity than the first particle group. Among them, the comparison of the fluorescence intensity between the particle groups in the scatter plot can be performed, for example, by a statistical representative value of the fluorescence intensity of each particle group. The statistical representative value of the fluorescence intensity of a particle group is a value obtained from the fluorescence intensity of the particles constituting the particle group, and can include, for example, a median value, an average value, a most frequent value, etc. Preferably, it is a median value. The statistical representative value of the fluorescence intensity of the particle group is calculated by the analysis unit 300.

[0253] In the first scattergram (b), the first particle group appears in the area where the SSC-1 intensity and / or SFL-1 intensity is low. In addition, the second particle group appears in the area where the SSC-1 intensity and / or SFL-1 intensity is high. Fig.19 In the example, the first particle group appears in the area on the side where the SSC-1 intensity and the SFL-1 intensity are low, and the second particle group appears in the area on the side where the SSC-1 intensity and the SFL-1 intensity are high. In this figure, the first particle group is a group composed of particles in the area surrounded by the dotted line. Specifically, the first particle group includes mRBC, RET and PLT. That is, the first particle group is blood cells other than WBC. In addition, the second particle group is a group of WBC. Among them, since WBC has a large amount of nucleic acid, it is strongly stained with the above-mentioned fluorescent dye. On the one hand, since mRBC and PLT do not contain nuclei and nucleic acids, they are almost not stained or weakly stained with the above-mentioned fluorescent dye. The amount of ribonucleic acid contained in RET is small. In addition, RET does not have a nucleus. Therefore, RET is weakly stained compared to WBC. In this way, there is a difference in fluorescence intensity between WBC and blood cells other than WBC. Therefore, in the first scatter plot (b), the particles in the measured sample are classified into the first particle group containing blood cells other than WBC and the second particle group containing WBC.

[0254] In step S603, the analysis unit 300 determines the first particle group as blood cells other than WBC. As described above, since the first particle group contains mRBC, RET and PLT, the first particle group can be determined as blood cells other than WBC. In addition, since the second particle group contains WBC, the second particle can be determined as WBC. For example, by accumulating measurement data for a plurality of subjects, the region where the first particle group appears can be predetermined in the first scattergram (b), and the particles appearing in the region can be determined as blood cells other than WBC.

[0255] In this way, Fig.18 In the analysis process shown in , the blood cells other than WBC are identified by classifying the particles in the measurement sample into WBC and blood cells other than WBC. In step S603 , the data of WBC is excluded from the data of the particles in the measurement sample by identifying the blood cells other than WBC.

[0256] After blood cells other than WBC are determined in step S603, the process proceeds to Fig. 10C Step S301. Further, as in the first embodiment, RET, PLT, and mRBC are determined from blood cells other than WBC. The details of this analysis process are the same as those described in the first embodiment. Fig. 10DIn the second embodiment, the analysis of PLT, the analysis of RET and the classification of WBC are performed in the same manner as in the first embodiment. The details of the analysis of PLT and RET are the same as those described in the first embodiment. Fig. 10D In step S404, based on the SSC-1 intensity and the SFL-1 intensity, Fig.18 The WBC group determined in step S603 is classified into subgroups. The subgroups of white blood cells are, for example, lymphocytes, monocytes, and granulocytes. In step S404, by enlarging the area where the WBC group appears in the first scattergram (b), as shown in FIG. Fig. 20 As shown in the figure, it is confirmed that WBC is divided into each subgroup and distributed. In this figure, WBC is classified into 3 subgroups, but it is not limited to this. WBC can be classified into at least two groups among the lymphocyte group, the monocyte group and the granulocyte group. For example, when WBC has an abnormality, a part of the subgroup of WBC is reduced to the point where it is difficult to classify. WBC can also be classified into two groups, for example, the lymphocyte or monocyte group and the granulocyte group.

[0257] The classification of WBC into subgroups can also be determined, for example, as follows. First, by accumulating measurement data for a plurality of subjects, the regions where each subgroup of WBC appears are predetermined in the first scattergram (b). Furthermore, the particles appearing in the region are determined as subgroups of WBC.

[0258] If necessary, the cells contained in each subgroup of WBC can be counted. After the classification of WBC is performed, the analysis unit 300 ends the analysis process and the process proceeds to Figure 7 Step S14.

[0259] Reference Figure 7 When the above analysis process is completed, the control unit 301 outputs the analysis result to the display unit 305 in step S14, and the process is completed. Figures 10A to 20 , the analysis process of the reticulocytes and platelets in the measurement sample will be described separately. However, the analysis process may be performed on a part of these, or all of the analysis processes may be performed.

[0260] The analysis result screen output to the display unit 305 may also include, for example, the number of reticulocytes, the number of platelets, IPF, P-LCR, PDW, MPV, a marker of platelet aggregation, the number of particles in each region of LFR, MFR and HFR, and the number of each subgroup of leukocytes (lymphocytes, monocytes and granulocytes). In addition to this information, the screen may also include a scatter plot made based on optical information. Furthermore, the screen may also include a marker based on the analysis result. For example, when particles suspected of platelet aggregation are determined, information showing the presence of platelet aggregation in the subject may be output. For example, a marker called "PLTc?" may also be output to the display unit 305.

[0261] Thus, the blood cell analysis method of this embodiment can provide, for example, information on the number of reticulocytes and platelets contained in a subject as analysis results of reticulocytes and platelets to medical practitioners such as physicians or testing technicians. In addition, analysis results of red blood cells and white blood cells can also be provided. Medical practitioners can determine whether to further examine the subject based on the obtained analysis results, for example. The preparation of the measurement sample and the analysis of the subject are all performed in vitro.

[0262] Next, the present invention will be described in detail by way of Examples, but the present invention is not limited to these Examples.

[0263] [Example]

[0264] In each of the following examples, the blood cell analysis method of this embodiment is performed using the following blood test sample, diluent reagent, staining reagent, and analysis device. The details are described in the following [Measurement Method].

[0265] [Blood test subject]

[0266] Whole blood (peripheral blood) was collected from multiple subjects using EDTA-2K blood collection tubes and stored at room temperature (25±2°C). When used, whole blood was separated from the blood collection tube and used as a blood sample. The details of the blood cell components and / or subjects contained in the blood sample are described in each example.

[0267] [Dilution reagent]

[0268] As a diluting reagent, CELLPACK (registered trademark) DFL (Sysmex Corporation) was used.

[0269] [Staining reagents]

[0270] A staining reagent was prepared by dissolving various fluorescent dyes in special grade ethylene glycol (1 L). The types of fluorescent dyes used are described in each example.

[0271] [Analysis device]

[0272] Prototypes of modified multi-item automatic blood cell analyzer XN-20 (Sysmex Co., Ltd.) and flow cytometer XF-1600 (Sysmex Co., Ltd.) were used as analysis devices. Each prototype was made as follows. XN-20 has a semiconductor laser source that emits red (639nm) light, a detector for red forward scattered light, a detector for red side scattered light, and a detector for fluorescence (light above 660nm) excited by red light. In this XN-20, a semiconductor laser source that emits bluish-violet (405nm) light, a detector for bluish-violet side scattered light, and a fluorescence detector that detects fluorescence (450 to 600nm) excited by bluish-violet light are added. In addition, this XN-20 is set to analyze the measurement data of the red and bluish-violet scattered light information and the fluorescence information generated from the fluorescent dye, and display the desired scatter plot. The XF-1600 is equipped with three semiconductor laser sources that emit blue (405nm), blue (488nm) and red (638nm) light, detectors for scattered light of each color, and detectors for fluorescence excited by each light. The semiconductor laser source that emits blue (488nm) light was removed from the XF-1600 and converted into an FCM system with two semiconductor laser sources. Each model has Figure 1 to Figure 3 The hardware shown in the FCM detection unit has Figure 4 composition.

[0273] [Measurement method]

[0274] In addition to using CELLPACK DFL and the above-mentioned staining reagent, the preparation and measurement of the measurement sample were carried out according to the manual attached to XN-20 and XF-1600. The measurement sample was prepared by mixing 1000 μL of CELLPACK DFL, 5.0 μL of whole blood and 20 μL of staining reagent. The measurement sample was incubated at 41°C for 30 seconds and then measured. The dilution ratio of the staining reagent in the measurement sample was 51.25 times. The final concentration of the fluorescent dye in the measurement sample is described in each example.

[0275] [Example 1: Blood cell analysis using acridine compounds]

[0276] A measurement sample is prepared using a staining reagent containing an acridine compound as a fluorescent dye, and reticulocytes and platelets in the measurement sample are classified by an analyzer.

[0277] (1) Measurement sample

[0278] For each of the above-mentioned blood test subjects, erythrocytes, reticulocytes, and platelets were pre-classified and counted by conventional methods, and the reticulocyte rate (ratio of the number of reticulocytes to 1000 erythrocytes: hereinafter also referred to as "RET%") was obtained. RET% is an indicator of anemia and hematopoietic abnormalities, and its normal range is 0.4 to 2.0%. The classification and counting of erythrocytes and reticulocytes by conventional methods were performed by measurement using a multi-item automatic blood cell analyzer XN-20 (Sysmex Corporation) and analysis using the RET channel. In addition, the classification and counting of platelets by conventional methods were performed by measurement using XN-20 and analysis using the PLT-F channel. In the classification and counting of platelets by XN-20, 6 times the amount of particles were measured compared to the classification and counting of erythrocytes and reticulocytes. Blood subjects with RET% of 0.21% and 5.56% (hereinafter, respectively referred to as "low RET% subject 1" and "high RET% subject 1") were selected from the above blood subjects, and the measurement samples were prepared as described above. As a staining reagent, an ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0279] (2) Analysis

[0280] Each measurement sample was measured by an analysis device (a modified XN-20 prototype) to obtain first scattered light information, second scattered light information, and fluorescence information.

[0281] The first scattered light information is the side scattered light intensity obtained by irradiating the particles in the measurement sample with a blue-violet laser (hereinafter also referred to as "side scattered light intensity (blue-violet)" or "V-SSC"). The second scattered light information is the forward scattered light intensity obtained by irradiating the particles in the measurement sample with a red laser (hereinafter also referred to as "forward scattered light intensity (red)" or "R-FSC"). The fluorescence information is the fluorescence intensity obtained by irradiating the particles in the measurement sample with a blue-violet laser (hereinafter also referred to as "fluorescence intensity (blue-violet)" or "V-SFL"). For each measurement sample, a scatter plot is prepared with the side scattered light intensity (blue-violet) on the horizontal axis and the forward scattered light intensity (red) on the vertical axis (hereinafter also referred to as "the first scatter plot (a)"). In addition, for each measurement sample, a scatter plot is prepared with the side scattered light intensity (blue-violet) on the horizontal axis and the fluorescence intensity (blue-violet) on the vertical axis (hereinafter also referred to as "the first scatter plot (b)"). The first scattergrams (a) and (b) of the high RET% subject 1 are shown in Fig.21A and 21B The first scatter plots (a) and (b) of the low RET% subject 1 are shown in Fig.22A and 22BIn the following figures, "WBC" represents white blood cells, "RBC" represents red blood cells, "PLT" represents platelets, "mRBC" represents mature red blood cells, and "RET" represents reticulocytes.

[0282] Reference Fig.21A In the first scatter plot (a), in the high RET% subject 1, white blood cells appear in the area where the V-SSC is high and the R-FSC is high. The group of red blood cells appears in the area where the V-SSC is lower than that of white blood cells and the R-FSC is high. The group of platelets appears in the area where the V-SSC and R-FSC are lower than those of white blood cells. Since platelets are smaller in size than white blood cells and red blood cells, the R-FSC of platelets becomes lower. Therefore, in the first scatter plot (a), the group of platelets appears in the area with low R-FSC. In addition, as mentioned above, since the hemoglobin of red blood cells absorbs the light of the first wavelength (blue-violet), the V-SSC of red blood cells is considered to be lower. Therefore, in the first scatter plot (a), the group of red blood cells is considered to appear in the area on the side where the V-SSC is lower than that of white blood cells. In this way, in the first scatter plot (a), white blood cells and blood cells other than white blood cells can be distinguished. In the first scatter plot (a), blood cells other than white blood cells are screened. The selected blood cells are displayed on a scattergram (hereinafter also referred to as a "second scattergram") with fluorescence intensity (blue-purple) on the horizontal axis and forward scattered light intensity (red) on the vertical axis. The horizontal and vertical axes of this second scattergram are logarithmic.

[0283] Reference Fig.21A In the second scatter plot, in the high RET% subject 1, the group of mature red blood cells appears in the area where V-SFL is low and R-FSC is high. The group of reticulocytes appears on the side where V-SFL is higher than the group of mature red blood cells. The group of platelets appears in the area where R-FSC is lower than the group of reticulocytes. In this way, by screening blood cells other than white blood cells, mature red blood cells, reticulocytes, and platelets can be classified without being affected by white blood cells. Fig.22A The first scattergram (a) and the second scattergram show that the results for the low RET% subject 1 are the same as those for the high RET% subject 1.

[0284] Reference Fig. 21B In the first scatter plot (b), in the high RET% subject 1, white blood cells appear in the area where V-SFL is high. In addition, the group of platelets and red blood cells appears in the area where V-SFL is lower than that of white blood cells. Mature red blood cells and platelets are anucleated cells, and the RNA of reticulocytes is small. In contrast, white blood cells have nuclei. Therefore, the V-SFL of the white blood cells is higher than that of the red blood cells group compared to the platelet group. Similar to the first scatter plot (a), white blood cells and blood cells other than white blood cells can be distinguished in the first scatter plot (b). Refer to Fig. 21BIn the second scatter plot, blood cells other than white blood cells are classified into mature red blood cells, reticulocytes, and platelets. In this way, by screening blood cells other than white blood cells, platelets, mature red blood cells, and reticulocytes can be classified without being affected by white blood cells. Fig. 22B The first scattergram (b) and the second scattergram showed the same results for the low RET% subject 1.

[0285] It is suggested that by classifying reticulocytes and platelets by excluding leukocytes as described above, the influence of leukocytes can be reduced when measuring reticulocytes and platelets. In addition, compared with the conventional method requiring two measurements, the present embodiment can classify reticulocytes and platelets in one measurement.

[0286] [Example 2: Blood cell analysis using anthocyanin compounds]

[0287] A measurement sample was prepared using a staining reagent containing a fluorescent dye different from that in Example 1, and reticulocytes and platelets in the measurement sample were classified by an analyzer.

[0288] (1) Measurement sample

[0289] Blood subjects with RET% of 0.61% and 4.33% (hereinafter, respectively referred to as "low RET% subject 2" and "high RET% subject 2") were selected from the above blood subjects, and the measurement samples were prepared as described above. As a staining reagent, an ethylene glycol solution of 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolium was used. The final concentration of the fluorescent dye in the measurement sample was 5.0 ppm.

[0290] (2) Analysis

[0291] As in Example 1, each measurement sample was measured by the analyzer (modified XN-20 prototype) to obtain the first scattered light information, the second scattered light information and the fluorescence information. Each information is the same as in Example 1. For each measurement sample, the first scattergrams (a) and (b) were prepared in the same manner as in Example 1. The first scattergrams (a) and (b) of the high RET% subject 2 are shown in FIG. Fig.23A and 23B The first scatter plots (a) and (b) of the low RET% subject 2 are shown in Fig.24A and 24B .

[0292] Reference Fig.23AIn the first scatter plot (a), in the high RET% subject 2, white blood cells appear in the area where V-SSC is high and R-FSC is high. In addition, the red blood cell group appears in the area where V-SSC is lower than that of white blood cells and R-FSC is high, and the platelet group appears in the area where V-SSC and R-FSC are lower than those of white blood cells. In this way, in the first scatter plot (a), white blood cells and blood cells other than white blood cells can be distinguished. In the first scatter plot (a), blood cells other than white blood cells are screened and their blood cells are displayed in the second scatter plot (logarithmic display). By screening blood cells other than white blood cells, in Fig.23A In the second scatter plot, platelets, mature red blood cells, and reticulocytes can be classified without being affected by white blood cells. Fig.24A The same result was obtained for low RET% subject 2. Fig. 23B In the first scatter plot (b), in the high RET% subject 2, white blood cells appear in the area where V-SFL is high. In addition, groups of platelets and red blood cells appear in the area where V-SFL is lower than that of white blood cells. Similar to the first scatter plot (a), white blood cells and blood cells other than white blood cells can be distinguished in the first scatter plot (b). In the first scatter plot (b), by screening blood cells other than white blood cells, platelets, mature red blood cells, and reticulocytes can be classified in the second scatter plot without being affected by white blood cells. Fig. 24B The same result was obtained for the low RET% subject 2. These results indicate that even when anthocyanin compounds are used as fluorescent dyes, platelets and reticulocytes can be classified in the same manner as in Example 1 using an acridine compound.

[0293] [Example 3: Blood cell analysis using various fluorescent dyes]

[0294] The measurement sample was prepared using a staining reagent containing a fluorescent dye different from that in Examples 1 and 2, and the reticulocytes and platelets in the measurement sample were classified by an analysis device. For comparison, analysis was also performed using staining reagents containing 2,7,9-trimethylacridine-3,6-diamine and 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolium.

[0295] (1) Measurement sample

[0296] For each of the above blood test subjects, platelet counts were obtained by measuring with XN-20 (Sysmex Co., Ltd.) and analyzing with the PLT-F channel. 3 / μL or less blood subjects. Among the selected blood subjects, two blood subjects (hereinafter referred to as "low PLT subject 1" and "low PLT subject 2" respectively) in which a group of impurities different from the group of platelets was confirmed in the scatter plot of the PLT-F channel were selected. The measurement samples were prepared from these blood subjects as described above. As fluorescent dyes of acridine compounds, 2,7,9-trimethylacridine-3,6-diamine, 3,6-diamino-2,7,10-trimethylacridine, 3,6-diamino-2,7-dimethylacridine, 3,6-diamino-2,7-dimethylacridine, 3,6-diaminoacridine and 3,6-diamino-10-methylacridine were used. As fluorescent dyes of anthocyanin compounds, 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzoxazolium, 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]benzothiazolium, 1,3,3-trimethyl-2-[(3-methyl-2-benzothiazolylidene)methyl]-3H-indolium and 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]-6-methyl-benzothiazolium were used. As staining reagents, ethylene glycol solutions of the respective fluorescent dyes were used. In the measurement sample, the final concentration of the fluorescent dye of the acridine compound was 1.0 ppm, and the final concentration of the fluorescent dye of the anthocyanin compound was 5.0 ppm.

[0297] (2) Analysis

[0298] Each measurement sample was measured by an analysis device (a modified XN-20 prototype) to obtain first scattered light information, second scattered light information, and fluorescence information. Each information is the same as in Example 1. A first scattergram (a) was prepared for the measurement samples prepared using 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzoxazolium, 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]benzothiazolium, 3,6-diaminoacridine, and 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolyllidene)methyl]-3H-indolium. The first scatter plot (b) is prepared for the measurement samples prepared using 2,7,9-trimethylacridine-3,6-diamine, 3,6-diamino-2,7,10-trimethylacridine, 3,6-diamino-2,7-dimethylacridine, 3,6-diamino-10-methylacridine and 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolylidene)methyl]-6-methyl-benzothiazolium. In the first scatter plots (a) and (b) of each measurement sample, blood cells other than white blood cells are screened and displayed on the second scatter plot. Two types of second scatter plots are prepared: linear representation and logarithmic representation. These scatter plots are shown in FIG. Fig.25A , 25B , 26A, 26B, 27A and 27B.

[0299] Fig.25A As an example of the first scattergram (a) and the second scattergram based thereon, the analysis results of the measurement sample prepared from the low PLT subject 1 using 3-ethyl-2-[(3-ethyl-2-benzothiazolylidene)methyl]benzothiazolium are shown. Fig.25A In the first scatter plot (a), the white blood cell group appears in the area where the V-SSC is high and the R-FSC is high. Fig.25A In the second scatter plot (linear display), the mature red blood cell group appears in the area with low V-SFL values, the platelet group appears in the area with low R-FSC values, and the reticulocyte group appears in the area with high R-FSC values. The debris group overlaps with the platelet group. Fig.25A In the second scatter plot (logarithmic display), the group of mature red blood cells appears in the area where R-FSC is high and V-SFL is low. The group of reticulocytes appears in the area where R-FSC is the same as that of the group of mature red blood cells and V-SFL is higher than that of the group of mature red blood cells. The group of platelets and the group of mixed substances appear together in the area where R-FSC is lower than that of mature red blood cells and reticulocytes, and the two groups can be distinguished. In this way, the second scatter plot display in logarithmic display is suitable for the classification and counting of platelets and the distinction between platelets and mixed substances.

[0300] Fig.25B As an example of the first scattergram (b) and the second scattergram based thereon, the analysis results of the measurement sample prepared from the low PLT subject 1 using 2,7,9-trimethylacridine-3,6-diamine are shown. Fig.25B In the first scatter plot (b), the white blood cell cluster appears in the area where V-SFL is high. Fig.25B In the second scatter plot (linear display), Fig.25A Similarly, in the second scatter plot (linear display), groups of mature red blood cells, reticulocytes, and platelets appear. Fig.25B In the second scatter plot (logarithmic display), each group of mature red blood cells, reticulocytes, platelets, and mixed substances appears. The group of platelets and the group of mixed substances are located at positions far from each other, and the two groups can be distinguished.

[0301] Fig.26A and 26B The first and second scattergrams of the measurement samples prepared from the low-PLT subject 1 using the above-mentioned 10 kinds of fluorescent dyes are shown. Fig.27A and 27B The first and second scatter plots of the measurement samples prepared from the low PLT subject 2 using the above-mentioned 10 fluorescent dyes are shown. Referring to these figures, when any fluorescent dye is used, in the first scatter plots (a) and (b), white blood cells and blood cells other than white blood cells can be distinguished. In addition, in the second scatter plot displayed linearly, each group of mature red blood cells, reticulocytes and platelets is confirmed. In the second scatter plot represented logarithmically, the group of platelets and the group of mixed substances are located at positions separated from each other, and the two groups can be distinguished. This shows that various fluorescent dyes of acridine compounds and anthocyanin compounds can be used to classify reticulocytes and platelets. In addition, it also shows that platelets and mixed substances can be distinguished.

[0302] [Example 4: Analysis of a platelet aggregation-containing measurement sample]

[0303] A measurement sample is prepared from a blood sample containing aggregated platelets, and the aggregated platelets in the measurement sample are detected by an analyzer.

[0304] (1) Measurement sample

[0305] Blood samples from healthy people and blood samples that were judged to be positive by visual observation of platelet aggregation under a microscope (hereinafter, each also referred to as "PLTc-negative sample" and "PLTc-positive sample") were selected. The measurement samples were prepared from these blood samples as described above. As a staining reagent, an ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0306] (2) Analysis

[0307] Each measurement sample is measured by an analysis device (a modified XN-20 prototype) to obtain fluorescence information, first scattered light information, second scattered light information, and third scattered light information. The fluorescence information, first scattered light information, and second scattered light information are the same as those in Example 1. The third scattered light information is the side scattered light intensity (hereinafter also referred to as "side scattered light intensity (red)" or "R-SSC") obtained by irradiating red laser light to the particles in the measurement sample. For each measurement sample, a first scatter plot (a) is prepared in the same manner as in Example 1. In addition, for comparison, a scatter plot is prepared with the side scattered light intensity (red) on the horizontal axis and the forward scattered light intensity (red) on the vertical axis. Each scatter plot is shown in FIG. Fig.28 In the figure, "PLTc?" indicates particles suspected to be platelet aggregates.

[0308] Reference Fig.28 In the scatter plot of the comparison of PLTc-negative subjects (A), the platelet group can be identified, and the white blood cells and red blood cells overlap. Therefore, the white blood cells and red blood cells cannot be classified. In addition, refer to Fig.28 In (B), in the scatter plot of the comparison of PLTc-positive subjects, a particle group was confirmed to be distributed in a manner extending from the platelet group to the overlapping group of white blood cells and red blood cells. However, it is impossible to distinguish whether the particle group is red blood cells or platelet aggregation. In this way, it is difficult to detect platelet aggregation based on the side scattered light intensity and forward scattered light intensity obtained only by red laser.

[0309] Reference Fig.28 (C) In the first scattergram (a) of the PLTc-negative subject, the white blood cell group, the red blood cell group, and the platelet group are clearly classified. Fig.28 (D), in the first scatter plot (a) of the PLTc-positive subject, the white blood cell group, the red blood cell group and the platelet group are also clearly classified. In addition, in the scatter plot of (D), in the area surrounded by the dotted line, a particle group that cannot be confirmed in the scatter plot of (C) appears. Since platelet aggregation is larger than the normal platelet size, the forward scattered light intensity becomes higher. Thus, the particle group in the area surrounded by the dotted line can be identified as platelet aggregation. In the scatter plot of (D), as a sign showing the appearance of particles suspected to be platelet aggregation, it is expressed as "PLTc?". In the first scatter plot (a), by comparing with the comparative scatter plot, the red blood cell group appears on the side with low V-SSC, and the appearance of platelet aggregation can be clearly confirmed. This indicates that platelet aggregation can be detected based on the side scattered light intensity obtained by the blue-violet laser and the forward scattered light intensity obtained by the red laser.

[0310] [Example 5: Classification and counting of white blood cells]

[0311] In Examples 1 to 3, blood cells other than leukocytes were screened in the first scattergram, and in Example 5, leukocytes were screened in the first scattergram. Furthermore, leukocytes were classified and counted.

[0312] (1) Measurement sample

[0313] The measurement sample was prepared from the blood sample of a healthy person as described above. As a staining reagent, an ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0314] (2) Analysis

[0315] Each measurement sample is measured by an analysis device (a modified XF-1600 prototype) to obtain fluorescence information, first scattered light information, second scattered light information, and third scattered light information. The fluorescence information, first scattered light information, and second scattered light information are the same as those in Example 1. The third scattered light information is the same as that in Example 4. In Example 5, the measurement time using the analysis device is extended to increase the number of particles analyzed. For each measurement sample, the first scatter plot (a) and (b) are prepared in the same manner as in Example 1. Each is shown in Fig.29 (A) and Fig.30 As shown in these figures, in the first scattergrams (a) and (b), leukocytes and blood cells other than leukocytes can be distinguished. In the first scattergrams (a) and (b), by counting the particles determined to be leukocytes, leukocytes can be counted.

[0316] Next, the white blood cells that appear in the first scatter plot (a) and (b) are screened. The white blood cells screened from the first scatter plot (a) are displayed in a scatter plot with the side scattered light intensity (red) on the horizontal axis and the forward scattered light intensity (red) on the vertical axis (hereinafter also referred to as the "third scatter plot (a)"). In addition, the white blood cells screened from the first scatter plot (b) are displayed in a scatter plot with the side scattered light intensity (red) on the horizontal axis and the fluorescence intensity (blue-purple) on the vertical axis (hereinafter also referred to as the "third scatter plot (b)"). Each is shown in Fig.29 (B) and Fig.30 (B). In the figure, "Gran" represents granulocytes, "Mono" represents monocytes, and "Lym" represents lymphocytes. Referring to each third scatter plot, it is shown that white blood cells can be classified into subgroups based on the side scattered light intensity and forward scattered light intensity obtained only by red laser. In addition, by counting the particles contained in each subgroup, granulocytes, monocytes, and lymphocytes can be counted.

[0317] The question was whether it was possible to classify leukocytes into subgroups in each first scattergram without screening leukocytes. The scattergrams showing the areas where leukocytes appear are magnified in the first scattergrams (a) and (b). Fig.29 (C) and Fig.30 (C). Referring to these figures, it is shown that by enlarging the area where the leukocytes appear in the first scattergram (a) and (b), the leukocytes can be classified into subgroups. In addition, by counting the particles contained in each subgroup, the granulocytes, monocytes and lymphocytes can be counted.

[0318] [Example 6: Classification and counting of white blood cells]

[0319] The correlation was investigated by comparing the white blood cell counts counted in the first scattergrams (a) and (b) with the white blood cell counts obtained by the conventional method. In the conventional method, the measurement sample was measured using a multi-item automatic blood cell analyzer XN-20 (Sysmex Corporation), and the white blood cells were counted by analysis using the WDF channel and the WNR channel.

[0320] (1) Measurement sample

[0321] The measurement sample is prepared as described above from 24 subjects including normal subjects and abnormal subjects. Normal subjects are blood subjects of healthy people. Abnormal subjects are blood subjects in which abnormalities are confirmed in blood cell components (except subjects with high values ​​of nucleated red blood cells). As a staining reagent, an ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine is used. The final concentration of the fluorescent dye in the measurement sample is 1.0 ppm. The measurement sample analyzed by the WDF channel of XN-20 is prepared by XN-20 using each of the above-mentioned 24 subjects, FLUOROCELL (registered trademark) WDF (Sysmex Co., Ltd.) as a staining reagent, and LYSERCELL (registered trademark) WDF (Sysmex Co., Ltd.) as a hemolysis reagent. The measurement samples analyzed by the WNR channel of XN-20 were prepared by XN-20 using each of the above-mentioned 24 test subjects, a staining reagent FLUOROCELL (registered trademark) WNR (Sysmex Corporation), and a hemolysis reagent LYSERCELL (registered trademark) WNR (Sysmex Corporation).

[0322] (2) Analysis

[0323] Each measurement sample is measured by an analysis device (a prototype of a modified XF-1600) to obtain the first scattered light information, the second scattered light information and the fluorescence information. Each information is the same as in Example 1. For each measurement sample, the first scatter plot (a) and (b) are prepared in the same manner as in Example 1. In the same manner as in Example 5, the white blood cells are counted in each scatter plot (a) and (b). In the conventional method, each measurement sample is measured by XN-20 and the white blood cells are counted by the WDF channel and the WNR channel. Among them, XN-20 has a semiconductor laser source that emits red (639nm) light, a detector for red forward scattered light, a detector for red side scattered light, and a detector for fluorescence (light above 660nm) excited by red light. In the measurement by XN-20, the fluorescence intensity (hereinafter also referred to as "fluorescence intensity (red)") obtained by irradiating the particles in the measurement sample with red laser, the side scattered light intensity (red) and the forward scattered light intensity (red) are obtained. In the WDF channel, blood cells are analyzed based on the side scattered light intensity (red) and the fluorescence intensity (red), and in the WNR channel, blood cells are analyzed based on the forward scattered light intensity (red) and the fluorescence intensity (red).

[0324] The correlation between the white blood cell counts obtained from the first scattergrams (a) and (b) and the white blood cell counts obtained from the WDF channel and the WNR channel was investigated. Fig.31 In addition, for the number of white blood cells, the correlation coefficients (r) between the first scatter plots (a) and (b) and the WDF channel and the WNR channel are shown in Table 1. Fig.31 As can be seen from Table 1, it is confirmed that the white blood cell counts obtained from the first scatter plots (a) and (b) are well correlated with the white blood cell counts obtained from the WDF channel and the WNR channel. In the blood cell analysis method of this embodiment, no hemolysis reagent is used, which means that the method can count white blood cells with the same degree of accuracy as the conventional method using a hemolysis reagent.

[0325]

Table 1

[0326] WDF Channel WNR Channel Scatter plot No. 1 (a) 0.94 0.94 Scatter plot No. 1 (b) 0.98 0.98

[0327] [Example 7: Analysis of platelets]

[0328] The cluster of platelets classified in the second scattergram is analyzed to obtain values ​​of various indexes related to platelets.

[0329] (1) Measurement sample

[0330] The measurement sample was prepared from the blood sample of a healthy person as described above. As a staining reagent, an ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0331] (2) Analysis

[0332] The sample was measured by an analyzer (a modified XN-20 prototype) to obtain fluorescence information, first scattered light information, and second scattered light information. Each information is the same as in Example 1. In the same manner as in Example 1, a first scattergram (a) was prepared to screen blood cells other than white blood cells. These blood cells are represented in a second scattergram (logarithmic display). An example of this second scattergram is shown in Fig.32 (A). In the second scatter plot, platelets are identified. In addition, the particles identified as platelets are counted to obtain the number of platelets. The group of platelets on the second scatter plot is screened to create a histogram based on forward scattered light intensity (red) and particle number. An example of this histogram is shown in Fig.32 (B).

[0333] Reference Fig.32 (A) Based on the distribution of platelets in the second scattergram, the immature platelet ratio (IPF) and the large platelet ratio (P-LCR) are obtained. Fig.32 (B), based on the histogram, the mean platelet volume (MPV) and the platelet distribution width (PDW) are obtained. An example of the values ​​of each index obtained is shown in Table 2. Thus, the values ​​of various indexes related to platelets can be obtained by the blood cell analysis method of this embodiment.

[0334]

Table 2

[0335] project Numeric unit MPV 9.3 f PDW 13.6 f P-LCR 17.9 ％ IPF 1.9 ％

[0336]

Explanation of symbols

[0337] 11: Reagents for blood cell analysis

[0338] 100: Blood collection tube

[0339] 200: Suction tube

[0340] 300: Analysis Unit

[0341] 400: Measurement unit

[0342] 410, 411: Reagent container

[0343] 420: Reaction Chamber

[0344] 430: Waste Room

[0345] 500: Analysis System

Claims

1. A blood cell analysis method comprising: a step of acquiring first scattered light information, second scattered light information, and fluorescence information generated by irradiating a measurement sample containing particles dyed with a fluorescent dye with light of a first wavelength and light of a second wavelength, a step of identifying blood cells other than white blood cells from particles in the measurement sample based on the first scattered light information and the second scattered light information and / or based on the first scattered light information and the fluorescence information; and a step of classifying reticulocytes and platelets from blood cells other than white blood cells based on the fluorescence information and the second scattered light information, The first wavelength is greater than 315 nm and less than 600 nm, The second wavelength is greater than 610 nm and less than 750 nm.

2. The method of claim 1, wherein The first scattered light information is information related to side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength, The second scattered light information is information related to forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength, The fluorescence information is information related to fluorescence generated from the fluorescent dye of the particles by irradiating the measurement sample with light of the first wavelength.

3. The method according to claim 1, wherein in the classification step, the blood cells other than white blood cells are classified into reticulocytes, platelets and mature erythrocytes.

4. The method of claim 1, wherein The first scattered light information is the intensity of the first side scattered light from the particle irradiated with the light of the first wavelength. The second scattered light information is the intensity of forward scattered light from the particle irradiated with the light of the second wavelength. The fluorescence information is the intensity of fluorescence from the fluorescent dye of the particle irradiated with the light of the first wavelength, In the identifying step, blood cells other than the white blood cells are identified based on the first side scattered light intensity and the forward scattered light intensity or the fluorescence intensity.

5. The method of claim 4, wherein In the determining step, a scattergram based on the first side scattered light intensity and the forward scattered light intensity or the fluorescence intensity is prepared, In the scattergram, blood cells other than the white blood cells are identified.

6. The method of claim 4 or 5, wherein In the determining step, particles in the measurement sample are classified into a first particle group and a second particle group based on the first side scattered light intensity and the forward scattered light intensity. The second particle group is a group that exhibits a first side scattered light intensity and / or a forward scattered light intensity higher than that of the first particle group. The first particle group is determined to be blood cells other than leukocytes.

7. The method of claim 4 or 5, wherein In the determining step, particles in the measurement sample are classified into a first particle group and a second particle group based on the first side scattered light intensity and the fluorescence intensity. The second particle group is a group that exhibits a first side scattered light intensity and / or a fluorescence intensity higher than that of the first particle group. The first particle group is determined to be blood cells other than leukocytes.

8. The method of claim 1, wherein The second scattered light information is the intensity of forward scattered light from the particle irradiated with the light of the second wavelength. The fluorescence information is the intensity of fluorescence from the fluorescent dye of the particle irradiated with the light of the first wavelength, In the classification step, reticulocytes and platelets are classified from the blood cells other than the white blood cells based on the fluorescence intensity and the forward scattered light intensity.

9. The method of claim 8, wherein In the classification step, a scattergram based on the fluorescence intensity and the forward scattered light intensity is prepared. In the scattergram, reticulocytes and platelets are classified from the blood cells other than the white blood cells.

10. The method of claim 8 or 9, wherein In the classification step, the blood cells other than the white blood cells are classified into a third particle group, a fourth particle group, and a fifth particle group based on the fluorescence intensity and the forward scattered light intensity. The third particle group is a group showing a higher fluorescence intensity than the fifth particle group, The fourth particle group is a group showing a lower forward scattered light intensity than the third particle group, The third particle group is determined to be a reticulocyte group, The fourth particle population is determined to be a platelet population. The method of claim 10 , wherein the fifth particle population is determined to be a population of mature red blood cells.

12. The method of claim 1, wherein the determining step further comprises a step of further determining white blood cells and classifying the white blood cells into subgroups.

13. The method of claim 12, wherein white blood cells are determined based on the first scattered light information and the second scattered light information, and / or based on the first scattered light information and the fluorescence information, and are classified into subgroups.

14. The method of claim 13, wherein The first scattered light information is the intensity of the first side scattered light from the particle irradiated with the light of the first wavelength. The second scattered light information is the intensity of forward scattered light from the particle irradiated with the light of the second wavelength. The fluorescence information is the intensity of fluorescence from the fluorescent dye of the particle irradiated with the light of the first wavelength, The leukocytes are identified based on the first side scattered light intensity and the forward scattered light intensity or the fluorescence intensity, and are classified into subgroups.

15. The method of claim 14, wherein: A scattergram based on the first side scattered light intensity and the forward scattered light intensity, and / or Scatter plot based on the first side scattered light intensity and the fluorescence intensity In the scatter plot, determining the white blood cells, and Classify the leukocytes into subgroups.

16. The method of claim 12, wherein In the step of obtaining, third scattered light information is further obtained. The third scattered light information is information related to side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength, determining the white blood cells based on the first scattered light information and the second scattered light information and / or based on the first scattered light information and the fluorescence information, The white blood cells are classified into subgroups based on the second scattered light information and the third scattered light information and / or based on the fluorescence information and the third scattered light information.

17. The method of claim 16, wherein The second scattered light information is the intensity of forward scattered light from the particle irradiated with the light of the second wavelength. The third scattered light information is the intensity of the second side scattered light from the particle irradiated with the light of the second wavelength. The fluorescence information is the intensity of fluorescence from the fluorescent dye of the particle irradiated with the light of the first wavelength, The leukocytes are classified into subgroups based on the second side scattered light intensity and the forward scattered light intensity or the fluorescence intensity.

18. The method of claim 17, wherein a scatter plot based on the second side scattered light intensity and the forward scattered light intensity and / or a scatter plot based on the second side scattered light intensity and the fluorescence intensity is prepared, and in the scatter plot, white blood cells are classified into subgroups.

19. The method of claim 12, wherein the subpopulation comprises at least two of a lymphocyte population, a monocyte population, and a granulocyte population.

20. The method according to claim 1, wherein the determining step further comprises acquiring information indicating the presence or absence of platelet aggregation based on the first scattered light information and the second scattered light information.

21. The method of claim 20, wherein The first scattered light information is the intensity of the side scattered light from the particle irradiated with the light of the first wavelength. The second scattered light information is the intensity of forward scattered light from the particle irradiated with the light of the second wavelength. In the step of specifying, blood cells other than white blood cells are specified based on the forward scattered light intensity and the side scattered light intensity. Based on the forward scattered light intensity, the blood cells other than the white blood cells are classified into a group containing red blood cells and a group containing platelets, When the platelet-containing group includes particles showing a forward scattered light intensity equal to or greater than a threshold value, information indicating the presence of platelet aggregation is acquired.

22. The method of claim 21, wherein In the determining step, a scattergram based on the forward scattered light intensity and the side scattered light intensity is prepared, In the scattergram, the particles in the measurement sample are identified as a group containing red blood cells, a group containing platelets, and white blood cells. When the platelet-containing group includes particles showing a forward scattered light intensity equal to or greater than a threshold value, information indicating the presence of platelet aggregation is acquired.

23. The method of claim 1, wherein in the classification step, the platelets are further classified into mature platelets and immature platelets.

24. The method of claim 1, wherein the classification process further includes determining large platelets from the platelets.

25. The method according to claim 1, further comprising the step of calculating platelet distribution width and / or mean platelet volume.

26. The method of claim 1, wherein the fluorescent dye is selected from the group consisting of acridine compounds, anthocyanin compounds, and styryl compounds.

27. The method of claim 26, wherein the acridine compound is a compound represented by the following formula (I): 【Chemistry 1】 In the formula, R1~R 10 are independently hydrogen atoms, -NH2, -(CH2) n -NR 11 R 12 、-NH-R 13 -NR 11 R 12 、-OR 11 , -COOH, halogen, phenyl optionally having a substituent, alkyl having 1 to 18 carbon atoms or acylamino, but at least one of R3, R6 and R9 is independently -NH2, -(CH2) n -NR 11 R 12 , or phenyl substituted by -NH2, R 11 and R 12 the same or different, are hydrogen atoms or alkyl groups having 1 to 18 carbon atoms, R 13 is an alkyl group having 1 to 6 carbon atoms, n is an integer from 0 to 6, X- is the counter ion.

28. The method according to claim 26, wherein the anthocyanin-based compound is a compound represented by the following formula (II): 【Chemistry 2】 In the formula, R1 and R2 are the same or different and are alkyl having 1 to 18 carbon atoms, alkenyl having 3 to 20 carbon atoms, alkyl having 1 to 18 carbon atoms having a hydroxyl group, alkyl having 1 to 18 carbon atoms having a carboxyl group, alkyl having 1 to 18 carbon atoms having a sulfonic group, halogen, -(CH2) m -NR6R7, -(CH2) n -O-R8 or benzyl optionally having a substituent, R3 and R4 are the same or different and are hydrogen, halogen, alkyl having 1 to 18 carbon atoms, phenyl optionally having a substituent, alkoxy having 1 to 6 carbon atoms, or -(CH2) n -O-R8, R5 is a hydrogen atom, a halogen, an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms and having a hydroxyl group, an alkyl group having 1 to 6 carbon atoms and having a carboxyl group, an alkyl group having 1 to 6 carbon atoms and having a sulfonic group, a phenyl group optionally having a substituent, a benzyl group optionally having a substituent, -(CH2) n -O-R6, R6 and R7 are the same or different and are a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, R8 is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, or a phenyl group which may have a substituent, m is an integer from 1 to 18, n is an integer from 1 to 6, X and Y are the same or different and are sulfur atoms, oxygen atoms, selenium atoms or CR9R 10 , R9 and R 10 are the same or different and are alkyl groups having 1 to 3 carbon atoms, Z - It is a counter ion.

29. The method of claim 26, wherein the styrene-based compound is: A compound represented by the following formula (III): 【Chemistry 3】 In the formula, R1 is an alkyl group having 1 to 18 carbon atoms, an alkenyl group having 3 to 20 carbon atoms, an alkyl group having 1 to 18 carbon atoms having a hydroxyl group, an alkyl group having 1 to 18 carbon atoms having a carboxyl group, an alkyl group having 1 to 18 carbon atoms having a sulfonic group, halogen, -(CH2) p -NR5R6, -(CH2) q -O-R7 or benzyl optionally having a substituent, R2 and R3 are the same or different and are hydrogen, halogen, an alkyl group having 1 to 18 carbon atoms, a phenyl group which may have a substituent, or an alkoxy group having 1 to 6 carbon atoms, 【Chemistry 4】 yes R4 is a hydrogen atom, a halogen, an alkyl group having 1 to 18 carbon atoms, a phenyl group optionally having a substituent, an alkoxy group having 1 to 6 carbon atoms, or -(CH2) m -O-R7, R5 and R6 are the same or different and are a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, R7 is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms, or a phenyl group which may have a substituent, X is a sulfur atom, an oxygen atom, a selenium atom or CR8R9, R8 and R9 are the same or different and are alkyl groups having 1 to 3 carbon atoms, m is an integer from 1 to 18, Z- is the counterion, or A compound represented by the following formula (IV): 【Chemistry 5】 In the formula, R1 and Z - As mentioned above, R2 and R3 are the same or different and are hydrogen, halogen, alkyl having 1 to 18 carbon atoms, phenyl optionally having a substituent, or alkoxy having 1 to 6 carbon atoms, or R2 and / or R3, together with the benzene ring to which N is bonded, form a heterocyclic ring containing the N, n is an integer from 0 to 2.

30. The method of claim 1, wherein the first wavelength is greater than 315 nm and less than 490 nm.

31. A reagent for blood cell analysis used in the method according to claim 1, comprising a fluorescent dye selected from the group consisting of acridine compounds, anthocyanin compounds and styryl compounds.

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