Blood cell analysis method and blood cell analysis reagent

Abstract

To provide means that enables classification and counting of reticulocytes and platelets.SOLUTION: The above challenge is cleared by following the steps of; staining blood cells in a sample with a fluorescent dye; acquiring fluorescence information, first scattered light information, and second scattered light information generated by irradiating the stained blood cells with light of a wavelength in a range of 315 nm to 600 nm, inclusive, and light of a wavelength in a range of 610 nm to 750 nm, inclusive; identifying blood cells other than white blood cells 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 fluorescence information; and classifying reticulocytes and platelets from populations other than white blood cells on the basis of the fluorescence information and the second scattered light information.SELECTED DRAWING: Figure 21B

Description

[Technical field]

[0001] The present invention relates to a method for analyzing blood cells, and to a reagent for analyzing blood cells. [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 normal blood contains 150,000 to 350,000 / μL. However, it is known that the number of platelets is reduced in the blood of patients with idiopathic thrombocytopenic purpura or acute leukemia. In general, when the number of platelets in blood falls below 10,000 / μL, it is determined that a blood transfusion is necessary. Therefore, accurate classification and counting of platelets is important in clinical practice.

[0003] Blood also contains reticulocytes. Reticulocytes are young red blood cells that are released into peripheral blood immediately after erythroblast cells in the bone marrow are enucleated. Reticulocytes usually become mature red blood cells in about one day. Reticulocytes contain small amounts of ribonucleic acid, ribosomes, mitochondria, and other organelles that are not contained in mature red blood cells. Classification and counting of reticulocytes is extremely important in understanding the hematopoietic ability of the bone marrow of a subject. For example, in a state where myelopoiesis is suppressed by aplastic anemia or chemotherapy for malignant tumors, the number of reticulocytes decreases. Alternatively, in a state where myelopoiesis is enhanced by hemolytic anemia, the number of reticulocytes increases.

[0004] EXAMPLE 8 of Patent Document 1 describes that a sample prepared using a dye for staining platelets and white blood cells and a dye for staining reticulocytes is irradiated with blue (488 nm) light to measure reticulocytes and platelets. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Patent Application Publication No. 2021 / 0041341 Summary of the Invention [Problem to be solved by the invention]

[0006] Depending on the specimen, white blood cells may affect the classification and counting of reticulocytes and platelets, but the influence of white blood cells has not been particularly taken into consideration in conventional techniques. The present invention aims to provide a means for suppressing the influence of white blood cells and enabling classification and counting of reticulocytes and platelets. [Means for solving the problem]

[0007] The inventors of the present invention discovered that reticulocytes and platelets can be detected based on the fluorescence information, first scattered light information, and second scattered light information generated by staining blood cells in a specimen with a fluorescent dye and irradiating the stained blood cells with light having a wavelength of 315 nm or more and 600 nm or less and light having a wavelength of 610 nm or more and 750 nm or less, and thus completed the inventions described below in [1] to

[31] .

[0008] [1] acquiring first scattered light information, second scattered light information, and fluorescent light information generated by irradiating a measurement sample containing particles stained with a fluorescent dye with light of a first wavelength and light of a second wavelength; identifying blood cells other than white blood cells from among 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 fluorescent light information; classifying reticulocytes and platelets from blood cells other than white blood cells based on the fluorescent light information and the second scattered light information; wherein the first wavelength is equal to or greater than 315 nm and equal to or less than 600 nm, and the second wavelength is equal to or greater than 610 nm and equal to or less than 750 nm.

[0009] [2] the first scattered light information is information about side scattered light generated from the particle by irradiating the measurement sample with light of the first wavelength, the second scattered light information is information about forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength, The method according to [1] above, wherein the fluorescence information is information regarding fluorescence generated from a fluorescent dye of the particle by irradiating the measurement sample with light of the first wavelength.

[0010] [3] The method according to [1] or [2] above, wherein in the classifying step, blood cells other than white blood cells are classified into reticulocytes, platelets and mature red blood cells.

[0011] [4] the first scattered light information is an intensity of first side scattered light from the particle irradiated with light of the first wavelength, the second scattered light information is an intensity of forward scattered light from the particle irradiated with light of the second wavelength, and the fluorescence information is an intensity of fluorescence from a fluorescent dye of the particle irradiated with light of the first wavelength, The method according to any one of the above [1] to [3], wherein 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 fluorescent light intensity.

[0012] [5] The method according to [4] above, wherein in the identifying step, a scattergram is created based on the first side scattered light intensity and the forward scattered light intensity or the fluorescent light intensity, and blood cells other than the white blood cells are identified in the scattergram.

[0013] [6] In the identifying step, the particles in the measurement sample are classified into a first particle population and a second particle population based on the first side scattered light intensity and the forward scattered light intensity; the second particle population is a population that exhibits a first side scattered light intensity and / or a forward scattered light intensity higher than that of the first particle population, The method according to [4] or [5] above, wherein the first particle population is identified as blood cells other than white blood cells.

[0014] [7] In the identifying step, the particles in the measurement sample are classified into a first particle population and a second particle population based on the first side scattered light intensity and the fluorescent light intensity; the second particle population is a population that exhibits a first side scattered light intensity and / or a fluorescence intensity higher than that of the first particle population, The method according to [4] or [5] above, wherein the first particle population is identified as blood cells other than white blood cells.

[0015] [8] the second scattered light information is an intensity of forward scattered light from the particle irradiated with light of the second wavelength, and the fluorescence information is an intensity of fluorescence from a fluorescent dye of the particle irradiated with light of the first wavelength; The method according to any one of the above [1] to [7], wherein in the classifying step, reticulocytes and platelets are classified from blood cells other than white blood cells based on the fluorescence intensity and the forward scattered light intensity.

[0016] [9] The method according to [8] above, wherein in the classification step, a scattergram is created based on the fluorescence intensity and the forward scattered light intensity, and reticulocytes and platelets are classified from blood cells other than white blood cells in the scattergram.

[0017]

[10] In the classifying 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 fluorescent light intensity and the forward scattered light intensity; the third particle population is a population exhibiting a higher fluorescence intensity than the fifth particle population, the fourth particle population is a population that exhibits a lower forward scattered light intensity than the third particle population, The method according to [8] or [9] above, wherein the third particle population is identified as a reticulocyte population, and the fourth particle population is identified as a platelet population.

[0018]

[11] The method according to

[10] above, wherein the fifth particle population is identified as a population of mature red blood cells.

[0019]

[12] In the identifying step, further identifying leukocytes; The method according to any one of the above [1] to

[11] , further comprising a step of classifying the leukocytes into subpopulations.

[0020]

[13] The method according to

[12] above, wherein white blood cells are identified and classified into subpopulations 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.

[0021]

[14] the first scattered light information is an intensity of first side scattered light from the particle irradiated with light of the first wavelength, the second scattered light information is an intensity of forward scattered light from the particle irradiated with light of the second wavelength, and the fluorescence information is an intensity of fluorescence from a fluorescent dye of the particle irradiated with light of the first wavelength, The method according to

[13] above, wherein the white blood cells are identified and classified into subpopulations based on the first side scattered light intensity and the forward scattered light intensity or the fluorescent light intensity.

[0022]

[15] The method according to

[14] above, further comprising creating a scattergram based on the first side scattered light intensity and the forward scattered light intensity, and / or creating a scattergram based on the first side scattered light intensity and the fluorescence intensity, identifying the white blood cells in the scattergram, and classifying the white blood cells into subpopulations.

[0023]

[16] In the acquiring step, third scattered light information is further acquired; the third scattered light information is information regarding side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength, identifying 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 fluorescent light information; The method according to claim 12, wherein the white blood cells are classified into subpopulations 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.

[0024]

[17] the second scattered light information is an intensity of forward scattered light from the particle irradiated with light of the second wavelength, the third scattered light information is an intensity of second side scattered light from the particle irradiated with light of the second wavelength, and the fluorescence information is an intensity of fluorescence from a fluorescent dye of the particle irradiated with light of the first wavelength, The method according to

[16] above, wherein the white blood cells are classified into subpopulations based on the second side scattered light intensity and the forward scattered light intensity or the fluorescent light intensity.

[0025]

[18] The method according to

[17] above, further comprising creating a scattergram based on the second side scattered light intensity and the forward scattered light intensity, and / or creating a scattergram based on the second side scattered light intensity and the fluorescence intensity, and classifying white blood cells into subpopulations in the scattergram.

[0026]

[19] The method according to any one of

[12] to

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

[0027]

[20] The method according to any one of [1] to

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

[0028]

[21] The first scattered light information is an intensity of side scattered light from the particle irradiated with light of the first wavelength, and the second scattered light information is an intensity of forward scattered light from the particle irradiated with light of the second wavelength, In the step of identifying, blood cells other than white blood cells are identified based on the forward scattered light intensity and the side scattered light intensity; classifying the blood cells other than the white blood cells into a group containing red blood cells and a group containing platelets based on the forward scattered light intensity; The method according to claim 20, wherein, when the platelet-containing population contains particles exhibiting a forward scattered light intensity equal to or greater than a threshold, information indicating the presence of platelet aggregation is obtained.

[0029]

[22] In the identifying step, a scattergram is created based on the forward scattered light intensity and the side scattered light intensity, and in the scattergram, particles in the measurement sample are identified as a group including red blood cells, a group including platelets, and white blood cells; The method according to claim 21, wherein when the platelet-containing population contains particles that exhibit a forward scattered light intensity equal to or greater than a threshold, information indicating the presence of platelet aggregation is obtained.

[0030]

[23] The method according to any one of the above-mentioned [1] to

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

[0031]

[24] The method according to any one of [1] to

[23] above, further comprising identifying large platelets from the platelets in the classifying step.

[0032]

[25] The method according to any one of [1] to

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

[0033]

[26] The method according to any one of the above [1] to

[25] , wherein the fluorescent dye is selected from the group consisting of an acridine compound, a cyanine compound, and a styryl compound.

[0034]

[27] The acridine compound has the following formula (I): [ka] (In the formula, R1~R 10 are independently a hydrogen atom, -NH2, -(CH2) n -NR 11 R 12 , -NH-R 13 -NR 11 R 12 , -OR 11, -COOH, halogen, a phenyl group which may have a substituent, an alkyl group having 1 to 18 carbon atoms, or an acylamino group, provided that at least one of R3, R6, and R9 is independently -NH2, -(CH2) n -NR 11 R 12 or a phenyl group substituted with -NH2; R 11 and R 12 are the same or different and each is a hydrogen atom or an alkyl group 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) The method according to

[26] above, wherein the compound is represented by the formula:

[0035]

[28] The cyanine compound has the following formula (II): [ka] (In the formula, R1 and R2 are the same or different and each represents 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 and a hydroxyl group, an alkyl group having 1 to 18 carbon atoms and a carboxyl group, an alkyl group having 1 to 18 carbon atoms and a sulfo group, a halogen, -(CH2) m -NR6R7, -(CH2) n -O-R8 or an optionally substituted benzyl group, R3 and R4 are the same or different and each represent a hydrogen atom, a halogen atom, an alkyl group having 1 to 18 carbon atoms, a phenyl group which may have a substituent, an alkoxy group 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 a hydroxyl group, an alkyl group having 1 to 6 carbon atoms and a carboxy group, an alkyl group having 1 to 6 carbon atoms and a sulfo group, a phenyl group which may have a substituent, a benzyl group which may have a substituent, -(CH2) n -O-R6, R6 and R7 are the same or different and each is 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, and n is an integer from 1 to 6, X and Y may be the same or different and each represents a sulfur atom, an oxygen atom, a selenium atom, or a CRR 10 and R9 and R 10 are the same or different and are alkyl groups having 1 to 3 carbon atoms, Z - is the counter ion) The method according to the above

[26] or

[27] , wherein the compound is represented by the formula:

[0036]

[29] The styryl compound is represented by the following formula (III): [ka] (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 and a hydroxyl group, an alkyl group having 1 to 18 carbon atoms and a carboxyl group, an alkyl group having 1 to 18 carbon atoms and a sulfo group, a halogen, -(CH2) p -NR5R6, -(CH2) q -O-R7 or an optionally substituted benzyl group, R2 and R3 are the same or different and each represents a hydrogen atom, a halogen atom, 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; [ka] and R4 is a hydrogen atom, a halogen atom, an alkyl group having 1 to 18 carbon atoms, a phenyl group which may have 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 each is 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 each is an alkyl group having 1 to 3 carbon atoms; m is an integer from 1 to 18; Z - is the counter ion) or a compound represented by the following formula (IV): [ka] (Wherein, R1 and Z - is as above, R2 and R3 are the same or different and each is a hydrogen atom, a 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, or R2 and / or R3 together with the benzene ring to which N is bonded form a heterocycle containing the N, n is an integer from 0 to 2. The method according to any one of the above

[26] to

[28] , wherein the compound is represented by the following formula:

[0037]

[30] The method according to any one of the above [1] to

[29] , wherein the first wavelength is 315 nm or more and 490 nm or less.

[0038]

[31] A reagent for blood cell analysis for use in the method according to any one of the above [1] to

[30] , comprising a fluorescent dye selected from the group consisting of an acridine compound, a cyanine compound and a styryl compound. Effect of the Invention

[0039] According to the present invention, there is provided a blood cell analysis method which suppresses the influence of white blood cells and enables classification and counting of reticulocytes and platelets, and a blood cell analysis reagent used in this method. [Brief description of the drawings]

[0040] [Figure 1]1 is a perspective view showing an analysis system suitable for the blood cell analysis method of the present embodiment. [Diagram 2] FIG. 2 is a diagram showing a fluid circuit including a specimen aspirating section, a sample preparing section, and a flow cytometer (FCM) detecting section in a measuring unit. [Diagram 3] FIG. 2 is a block diagram showing a configuration of a measurement unit. [Figure 4] 4 is a schematic diagram showing an example of the configuration of an optical system of an FCM detection unit. FIG. [Diagram 5] FIG. 2 is a schematic diagram showing that various types of light emitted when light is irradiated onto particles passing through a flow cell are received by the optical system of the FCM detection unit. [Figure 6] FIG. 2 is a block diagram showing a configuration of an analysis unit. [Figure 7] 4 is a flowchart showing the flow of operations of the analysis system. [Figure 8] 13 is a flowchart showing a procedure of a measurement process. [Figure 9] FIG. 2 is a diagram showing an example of a blood cell analysis reagent according to the present embodiment. [Figure 10A] 4 is a flowchart showing a procedure of an analysis process according to the first embodiment. [Figure 10B] 4 is a flowchart showing a procedure of an analysis process according to the first embodiment. [Figure 10C] 4 is a flowchart showing a procedure of an analysis process according to the first embodiment. [Figure 10D] 4 is a flowchart showing a procedure of an analysis process according to the first embodiment. [Figure 11] 13 is a flowchart showing a modified example of the white blood cell classification process. [Figure 12] FIG. 1 is a schematic diagram illustrating positions where a first particle population and a second particle population appear in a first scattergram (a) in which the horizontal axis represents the first side scattered light (SSC-1) intensity and the vertical axis represents the second forward scattered light (FSC-2) intensity. [Figure 13A] FIG. 2 is a schematic diagram illustrating the positions at which a population containing RBCs, a population containing PLTs, and a second particle population appear in a first scattergram (a). [Figure 13B]FIG. 2 is a schematic diagram illustrating the positions where platelet aggregation appears in the first scattergram (a). [Figure 14A] (A) is a schematic diagram illustrating positions where the third, fourth, and fifth particle populations appear in a second scattergram (logarithmic display) with the horizontal axis representing the logarithm of the first side fluorescence (SFL-1) intensity (SFL-1(log)) and the vertical axis representing the logarithm of the second forward scattered light intensity (FSC-2(log)). (B) is a schematic diagram illustrating positions where the third, fourth, and fifth particle populations appear in a second scattergram (linear display) with the horizontal axis representing the SFL-1 intensity and the vertical axis representing the FSC-2 intensity. [Figure 14B] (A) is a schematic diagram illustrating the regions in which mRBC and RET appear in the second scattergram (logarithmic display). (B) is a schematic diagram illustrating the regions in which mRBC and RET appear in the second scattergram (linear display). [Figure 15A] (A) is a schematic diagram illustrating the area where immature platelets appear in the second scattergram (logarithmic display). (B) is a schematic diagram illustrating the area where large platelets appear in the second scattergram (logarithmic display). (C) is a schematic diagram illustrating the sorting of platelets in the second scattergram (logarithmic display). (D) is an example of a histogram created based on the FSC-2 intensity and particle number of sorted platelets. In the figure, "MPV" is the platelet mean volume, and "PDW" is the platelet distribution width. [Figure 15B] FIG. 13 is a schematic diagram illustrating the region where mRBC appears, the LFR (Low Fluorescence Rate) region, the MFR (Middle Fluorescence Rate) region, and the HFR (High Fluorescence Rate) region in the second scattergram (linear display). [Figure 16] FIG. 2 is a schematic diagram illustrating the positions where each subpopulation of leukocytes appears in the first scattergram (a). [Figure 17](A) is a schematic diagram illustrating the positions where each subpopulation of leukocytes appears in a third scattergram (a) with the horizontal axis representing second side scattered light (SSC-2) intensity and the vertical axis representing FSC-2 intensity, and (B) is a schematic diagram illustrating the positions where each subpopulation of leukocytes appears in a third scattergram (b) with the horizontal axis representing SSC-2 intensity and the vertical axis representing SFL-1 intensity. [Figure 18] 13 is a flowchart showing a procedure of an analysis process according to a second embodiment. [Figure 19] FIG. 13 is a schematic diagram illustrating the positions where a first particle population and a second particle population appear in a first scattergram (b) in which the horizontal axis represents SSC-1 intensity and the vertical axis represents SFL-1 intensity. [Figure 20] FIG. 2 is a schematic diagram illustrating the positions where each subpopulation of leukocytes appears in the first scattergram (b). [Figure 21A] 1 shows a first scattergram (a) and a second scattergram for high RET% sample 1 of Example 1. [Figure 21B] 1 shows a first scattergram (b) and a second scattergram for high RET% sample 1 of Example 1. [Figure 22A] 1 shows a first scattergram (a) and a second scattergram for low RET% sample 1 of Example 1. [Figure 22B] 1 shows a first scattergram (b) and a second scattergram for low RET% sample 1 of Example 1. [Figure 23A] 1 shows a first scattergram (a) and a second scattergram for high RET% sample 2 of Example 2. [Figure 23B] 1 shows the first scattergram (b) and the second scattergram for high RET% sample 2 of Example 2. [Figure 24A] 1 shows a first scattergram (a) and a second scattergram for low RET% sample 2 of Example 2. [Figure 24B] 1 shows the first scattergram (b) and the second scattergram for low RET% sample 2 of Example 2. [Figure 25A] 1 shows a first scattergram (a) and a second scattergram (b) for low PLT sample 1 stained with 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium in Example 3. [Figure 25B] 1 shows the first scattergram (b) and the second scattergram for low PLT sample 1 stained with 2,7,9-trimethylacridine-3,6-diamine in Example 3. [Figure 26A] 1 shows a first scattergram (a) and a second scattergram for low PLT sample 1 stained with various fluorescent dyes in Example 3. [Figure 26B] 13 shows a first scattergram (b) and a second scattergram for low PLT sample 1 stained with various fluorescent dyes in Example 3. [Figure 27A] 1 shows a first scattergram (a) and a second scattergram for low PLT sample 2 stained with various fluorescent dyes in Example 3. [Figure 27B] 13 shows a first scattergram (b) and a second scattergram for low PLT sample 2 stained with various fluorescent dyes in Example 3. [Figure 28] FIG. 11 shows a comparative scattergram and a first scattergram (a) for PLTc-negative and PLTc-positive samples from Example 4. [Figure 29] 1 shows a first scattergram (a) and a third scattergram (a) for a blood sample from a healthy subject in Example 5. [Diagram 30] 13 shows the first scattergram (b) and the third scattergram (b) for a blood sample from a healthy subject in Example 5. [Diagram 31] 13 is a graph showing the correlation between the count values ​​of white blood cells in normal and abnormal samples in Example 6 using the first scattergrams (a) and (b) and the count values ​​using the conventional method (WDF channel and WNR channel). [Diagram 32] (A) is a second scattergram (logarithmic display) for the blood sample of a healthy subject in Example 7. (B) is a histogram for the selected platelets. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] (Analysis System) First, an analysis system suitable for the blood cell analysis method of this embodiment will be described with reference to Fig. 1. The analysis system 500 includes a measurement unit 400, which is a measurement device, and an analysis unit 300, which is an analysis device. The analysis unit 300 is, for example, a personal computer in which software for analyzing a sample to be measured is installed. The analysis unit 300 also controls the operation of the measurement unit 400. The analysis system 500 may be configured such that the analysis unit 300 is provided within the measurement unit 400.

[0042] The measurement unit 400 is a unit for measuring a specimen, and includes a flow cytometer. In the measurement unit 400, a specimen is mixed with a reagent to prepare a measurement specimen. A staining reagent and a dilution reagent are used as reagents to prepare the measurement specimen. The staining reagent includes a fluorescent dye for staining particles. The dilution reagent includes a buffer and a solvent. Details of the specimen, the staining reagent, and the dilution reagent will be described later. In the analysis of particles in a specimen by this analysis system, particles in the measurement specimen stained with a fluorescent dye are analyzed. The "particles in the measurement specimen" refer to formed elements contained in the measurement specimen that can be individually measured by the FCM detection unit 460 described later. Examples of particles in the measurement specimen include cells, red blood cell ghosts, platelet aggregates, lipid particles, fungi, bacteria, and the like contained in the specimen. The term "cells" includes various blood cells and platelets. Red blood cell ghosts refer to remnants of hemolyzed red blood cells. Erythrocyte ghosts can also be caused by hemolysis due to factors other than the hemolyzing reagent, such as physical impact on the blood collection tube, and low pH and low osmotic pressure of the measurement sample.

[0043] The measurement sample is measured by the FCM detection section 460 (see FIG. 2) of the measurement unit 400, which will be described later. Through the measurement, an optical signal related to the fluorescence emitted from the fluorescent dye of the dyed particles in the measurement sample and an optical signal related to the scattered light emitted from the particles are obtained. The "fluorochrome of the particle" refers to the fluorescent dye bound to the particle by dyeing. The manner of binding between the particle and the fluorescent dye is not particularly limited as long as the particle and the fluorescent dye are measured as a unit by the flow cytometer. 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.

[0044] The configuration of the fluid system in the measurement unit 400 will be described with reference to FIG. 2. The measurement unit 400 includes a sample preparation section 440, a specimen aspirating section 450, and an FCM detection section 460. The sample preparation section 440 includes a reagent container 410, a reagent container 411, a reaction chamber 420, and a waste 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 liquid supply tubes. The sample preparation section 440 injects the dilution reagent and the staining reagent into the reaction chamber 420 through the respective liquid supply tubes. The specimen aspirating section 450 includes an aspirating tube 200. The aspirating tube 200 aspirates the specimen contained in the blood collection tube 100 and discharges it into the reaction chamber 420. The reaction chamber 420 is connected to a flow cell 413 of the FCM detection section 460 by the liquid supply tube.

[0045] The reaction chamber 420 is a container for preparing a measurement sample. In the reaction chamber 420, a specimen, a staining reagent containing a fluorescent dye, and a dilution reagent are mixed to prepare a measurement sample. The measurement sample in the reaction chamber 420 is supplied to a flow cell 413 of the FCM detection unit 460 via a liquid supply tube and measured. The FCM detection unit 460 acquires various optical signals emitted from individual particles 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 in the waste liquid chamber 430. The reaction chamber 420 is washed by a washing mechanism (not shown) before the next measurement sample is prepared.

[0046] 3, the electrical connections of the various parts in the measuring unit 400 will be described. The sample preparing section 440, the specimen aspirating section 450 and the FCM detecting section 460 are connected to an interface (IF) section 488. The FCM detecting section 460 is connected to an analog processing section 481 and an A / D converting section 482. The analog processing section 481 processes an analog signal output from the FCM detecting section 460, and the A / D converting section 482 converts an analog signal output from the analog processing section 481 into a digital signal. The measuring unit 400 is also connected to the analyzing unit 300 via an IF section 489. The IF section 484, the IF section 488 and the IF section 489 are connected to a bus 485.

[0047] The FCM detection unit 460, the analog processing unit 481, and the A / D conversion unit 482 will be described with reference to FIG. 4. The FCM detection unit 460 includes a first light source 411a, a second light source 411b, a flow cell 413, dichroic mirrors 418a, 418b, and 418c, side scattered light receiving elements 412a and 412b, a forward scattered light receiving element 416, and side fluorescent light receiving elements 422a and 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, 315 nm or more and 600 nm or less, preferably 400 nm or more and 490 nm or less, more preferably 400 nm or more and 450 nm or less, and even more preferably 400 nm or more and 410 nm or less. When light in the above wavelength range is irradiated to mature red blood cells or reticulocytes containing hemoglobin, the light is absorbed by the hemoglobin in the blood cells, so that parameters such as the intensity (peak value), pulse width, and pulse area of ​​the scattered light generated from the mature red blood cells or reticulocytes tend to be smaller than those of white blood cells not containing hemoglobin. The second wavelength is a wavelength different from the first wavelength, and is preferably a wavelength that is not absorbed by hemoglobin. Such a wavelength is, for example, 610 nm or more and 750 nm or less, preferably 620 nm or more and 700 nm or less, and more preferably 633 nm or more and 643 nm or less. It is believed that by using both light in a wavelength range that is absorbed by hemoglobin and light in a wavelength range that is not absorbed by hemoglobin, it is possible to clearly distinguish between mature red blood cells and reticulocytes and white blood cells. As the first and second light sources, for example, semiconductor laser light sources can be used.

[0048] The measurement sample prepared in the reaction chamber 420 is made to flow through the flow cell 413 of the FCM detection unit 460. In the example of FIG. 4, the measurement sample is made to flow in a direction perpendicular to the paper surface. While the measurement sample is flowing through the flow cell 413, light irradiated from the first light source 411a is reflected by the dichroic mirror 418a and is irradiated onto individual particles in the measurement sample flowing through the flow cell 413. Light emitted from the second light source 411b is transmitted through the dichroic mirror 418a and is irradiated onto individual particles in the measurement sample flowing through the flow cell 413.

[0049] In the example of FIG. 4, the forward scattered light receiving element 416 is arranged 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 arranged 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 arranged to receive the forward scattered light emitted from the particle based on the light irradiated from the first light source 411a. The forward scattered light is, for example, scattered light having a light receiving angle of 0 degrees to about 20 degrees. 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. Side scattered light (second side scattered light) corresponding to the light emitted 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 having a light receiving angle of about 20 degrees to about 90 degrees. The side scattered light receiving elements 412a and 412b are, for example, photodiodes.

[0050] The blood cell analysis method of this embodiment uses a fluorescent dye excited by a first wavelength. Side fluorescence (first side fluorescence) corresponding to light generated by excitation of the fluorescent dye is transmitted through the dichroic mirror 418b and received by the side fluorescence receiving element 422a. The blood cell analysis method of this embodiment does not use a fluorescent dye excited by a second wavelength, but in the example of FIG. 4, for the purpose of explaining the optical system, a configuration capable of receiving side fluorescence (second side fluorescence) corresponding to light generated by excitation of the fluorescent dye is shown. That is, the second side fluorescence is transmitted through the dichroic mirror 418c and received by the side fluorescence receiving element 422b. The side fluorescence receiving elements 422a and 422b are, for example, avalanche photodiodes.

[0051] With reference to FIG. 5, the relationship between various types of light emitted when a particle P passing through the flow cell 413 is irradiated with light and the optical system of the FCM detection unit 460 will be described. In FIG. 5, the light irradiated from the first light source 411a is light L1 of a first wavelength, and the light irradiated from the second light source 411b is light L2 of a second wavelength. When the light L1 and L2 are irradiated to the particle 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 is generated from the particle P forward with respect to the traveling direction of the light. In the example of FIG. 5, the light receiving element 416 receives forward scattered light corresponding to the light of the second wavelength, so only the second forward scattered light (FSC-2) corresponding to the light of the second wavelength is shown, and the first forward scattered light (FSC-1) corresponding to the light of the first wavelength is omitted. 5, a first side scattered light (SSC-1) corresponding to the light of the first wavelength and a second side scattered light (SSC-2) corresponding to the light of the second wavelength are generated laterally with respect to the traveling direction of the light. When the particle P is stained with a fluorescent dye excited by the first wavelength, a first side fluorescence (SFL-1) excited by the light of the first wavelength is generated from the fluorescent dye laterally with respect to the traveling direction of the light. In the blood cell analysis method of this embodiment, a fluorescent dye excited by the second wavelength is not used, but in the example of FIG. 5, for the purpose of explaining the optical system, a state in which both SFL-1 and a second side fluorescence (SFL-2) excited by the light of the second wavelength are generated laterally with respect to the traveling direction of the light are shown.

[0052] As described above, FSC-2, SSC-1, SFL-1, SSC-2, and SFL-2 are received by the light receiving elements 416, 412a, 422a, 412b, and 422b, respectively. Each light receiving element outputs a waveform-shaped electric signal (also called an analog signal) including a pulse according to the intensity of the received light. The analog signal corresponding to FSC-2 is also called the "second forward scattered light signal", the analog signal corresponding to SSC-1 is also called the "first side scattered light signal", the analog signal corresponding to SFL-1 is also called the "first fluorescent signal", the analog signal corresponding to SSC-2 is also called the "second side scattered light signal", and the analog signal corresponding to SFL-2 is also called the "second fluorescent signal". When FSC-1 is received, the analog signal corresponding to FSC-1 is also called the "first forward scattered light signal". One pulse of each analog signal corresponds to one particle (e.g., one cell).

[0053] The analog signals corresponding to the various types of light are input to the analog processing unit 481, where noise removal, smoothing, and other processing are performed. The A / D conversion unit 482 samples the analog signals output from the analog processing unit 481 at a predetermined sampling rate (for example, sampling of 1024 points at 10 nanosecond intervals, sampling of 128 points at 80 nanosecond intervals, or sampling of 64 points at 160 nanosecond intervals). The A / D conversion unit 482 digitizes the sampled analog signals to generate waveform data. The A / D conversion unit 482 samples and digitizes five types of analog signals corresponding to individual cells flowing through the flow cell 413 to generate waveform data of a second forward scattered light signal, a first side scattered light signal, a first fluorescent light signal, a second side scattered light signal, and a second fluorescent light signal. When FSC-1 is received, waveform data of the first forward scattered light signal is also generated. Furthermore, the A / D conversion unit 482 calculates characteristic parameters representing the morphological characteristics of individual cells from the waveform data of each signal. Such characteristic parameters include, for example, peak value (height of the pulse peak), pulse width, pulse area, transmittance, Stokes shift, ratio, change over time, and values ​​correlated therewith.

[0054] The optical information may be the above-mentioned characteristic parameters. The optical information includes fluorescence information, first scattered light information, second scattered light information, and third scattered light information. The fluorescence information is information on the 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 that reflects the amount of the fluorescent dye that stains the nucleic acid in the nucleated cells. As the fluorescence information, the peak value and pulse area of ​​the first fluorescent signal are preferable, and the peak value of the first fluorescent signal is more preferable. In this specification, the peak value of the first fluorescent signal is also referred to as the "first fluorescent intensity". The first fluorescent intensity can be said to be the intensity of the fluorescence from the fluorescent dye of the particles irradiated with light of the first wavelength.

[0055] The first scattered light information is information on the 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 on the forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength. The third scattered light information is information on the side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength. The information on the side scattered light is not particularly limited as long as it reflects internal information such as the complexity of the cell structure, granule characteristics, nuclear structure, and degree of lobulation. The information on the forward scattered light is not particularly limited as long as it reflects the size of the particles. As the first scattered light information, the peak value and pulse area of ​​the first side scattered light signal are preferable, and the peak value of the first side scattered light signal is more preferable. As the second scattered light information, the peak value and pulse area of ​​the second forward scattered light signal are preferable, and the peak value of the second forward scattered light signal is more preferable. As the third scattered light information, the peak value and pulse area of ​​the second side scattered light signal are preferable, and the peak value of the second side scattered light signal is more preferable. 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 forward scattered light signal is also referred to as the "second forward 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 said to be the intensity of the first side scattered light from a particle irradiated with light of the first wavelength. The second forward scattered light intensity can be said to be the intensity of the forward scattered light from a particle irradiated with light of the second wavelength. The second side scattered light intensity can be said to be the intensity of the second side scattered light from a particle irradiated with light of the second wavelength.

[0056] Referring to FIG. 6, the analysis unit 300 is electrically connected to the measurement unit 400 via an 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, configured by a personal computer (see the analysis unit 300 in FIG. 1), and controls the measurement unit 400 of the analysis system 500 by executing a program stored in the storage unit 303. The analysis unit 300 analyzes data including optical information acquired by the measurement unit 400, and displays the analysis results on the display unit 305. The analysis unit 300 may classify cells based on the optical information.

[0057] The storage unit 303 stores, for example, a program for controlling the measurement unit 400, a program for analyzing data acquired by the measurement unit 400, etc. The display unit 305 displays, for example, the analysis results of the data acquired by the measurement unit 400. The operation unit 306 includes a pointing device including a keyboard, a mouse, or a touch panel.

[0058] An example of the operation of the analysis system 500 will be described with reference to Fig. 7, but 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 a user via the operation unit 306. Upon receiving the instruction to start measurement, the control unit 301 transmits instruction data instructing the measurement unit 400 to start measurement. The measurement unit 400 receives the instruction data and executes a measurement process in step S12.

[0059] The blood cell analysis method of this embodiment includes, for example, the following first and second embodiments. In the "first embodiment", a measurement sample prepared from a specimen is measured, and first scattered light information and second scattered light information of particles in the measurement sample are obtained. Next, blood cells other than white blood cells and white blood cells are identified from the above particles based on the first scattered light information and the second scattered light information. Then, reticulocytes and platelets are classified from blood cells other than white blood cells based on the fluorescent information and the second scattered light information. In the "second embodiment", a measurement sample prepared from a specimen is measured, and first scattered light information and fluorescent information of particles in the measurement sample are obtained. Next, blood cells other than white blood cells and white blood cells are identified from the above particles based on the first scattered light information and the fluorescent information. Then, reticulocytes and platelets are classified from blood cells other than white blood cells in the same manner as in the first embodiment.

[0060] (Measurement processing) With respect to step S12 in FIG. 7, an example of the measurement process of the first and second embodiments will be described with reference to FIG. 8, but is not limited to this example. In step S21, the control unit 301 of the analysis unit 300 causes the measurement unit 400 to prepare a measurement sample from the specimen. Specifically, the measurement unit 400 mixes the specimen, the staining reagent, and the dilution reagent in the reaction chamber 420 to prepare the measurement sample. In step S22, the control unit 301 causes the measurement unit 400 to guide the measurement sample to the FCM detection unit 460 and perform optical measurement by the flow cytometry method. As a result, 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 causes the measurement unit 400 to transmit the optical information to the analysis unit 300, and ends the measurement process. Then, the analysis unit 300 performs the analysis process of step S13 in FIG. 7. An example of the analysis process in each embodiment will be described later.

[0061] (Specimen) The specimen is a blood specimen. Examples of blood specimens include whole blood and diluted whole blood. The whole blood is, for example, peripheral blood collected from a subject. The blood specimen may 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. The diluted whole blood is obtained, for example, by diluting the whole blood with an appropriate aqueous solvent, preferably a dilution reagent described below. Examples of the aqueous solvent include water, physiological saline, and an aqueous solution of a buffer.

[0062] The particles in the measurement sample are derived from the blood components of the specimen. In this specification, the term "blood components" includes formed elements known to be contained in blood and abnormal cells. Formed elements known to be contained in blood are, for example, blood cells that are usually contained in the peripheral blood of healthy people. Examples of such blood cells include white blood cells, mature red blood cells, reticulocytes, and platelets. "Mature red blood cells" are terminally differentiated red blood cells that do not have a nucleus and nucleic acid. In this specification, the term "mature red blood cells" is used to distinguish them from reticulocytes. When there is no particular distinction between mature red blood cells and reticulocytes, in this specification, the term "red blood cells" or "RBCs" may be used to include mature red blood cells and reticulocytes. In this specification, the term "platelets" includes immature platelets and large platelets. "Immature platelets" are also called reticulated platelets, and are young platelets immediately after being released into peripheral blood. Mature platelets do not contain nucleic acid, while immature platelets contain a small amount of ribonucleic acid. "Large platelets" are platelets with a diameter of 4 μm or more. 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 include 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 a blood collection tube due to contamination of tissue fluid during blood collection, insufficient mixing, or the action of EDTA.

[0063] (Diluted Reagent) The dilution reagent used in the preparation of the measurement sample will be described. The dilution reagent contains a buffer and a solvent. Preferably, the dilution reagent contains a solution of a buffer. Examples of the solvent include water and physiological saline, and water is particularly preferred. The buffer preferably has a buffering effect in the range of pH 6 to 11. Such a buffer can be selected from, for example, carboxylates, phosphates, Good's buffer, taurine, triethanolamine, and the like. The pH of the dilution reagent is, for example, 6 to 11, preferably 7 to 10, and more preferably 8 to 9.5. When the pH of the dilution reagent is 6 or more, red blood cells are less likely to be hemolyzed in the measurement sample. This can suppress the generation of red blood cell ghosts. In addition, when the pH of the dilution reagent is 11 or less, nonspecific staining of red blood cells and red blood cell ghosts by a fluorescent dye can be reduced. A preferred dilution reagent is an aqueous solution of the above buffer, having a pH of 6 to 11.

[0064] In addition to the above-mentioned buffer, the diluted reagent may further contain components such as an osmotic pressure compensator, a dyeing promoter, a polyvalent anion, and a preservative. The osmotic pressure compensator is a substance that can maintain the osmotic pressure of the diluted reagent in an appropriate range. Examples of the osmotic pressure compensator include an alkali metal salt of an organic acid such as propionic acid, a sugar such as glucose or mannose, an alkali metal halide such as sodium chloride, and an alkaline earth metal halide such as magnesium chloride. One or more types of osmotic pressure compensators can be used. When an osmotic pressure compensator is used, the osmotic pressure compensator is added to the diluted reagent so that the osmotic pressure of the diluted reagent is preferably 150 mOsm / kg·H2O to 600 mOsm / kg·H2O, more preferably 200 mOsm / kg·H2O to 600 mOsm / kg·H2O.

[0065] The staining accelerator is a substance that can enhance the permeability of the fluorescent dye into blood cells. Examples of the staining accelerator include surfactants, preferably cationic surfactants. As the cationic surfactant, quaternary ammonium salt surfactants are particularly preferred, such as decyltrimethylammonium bromide (DTAB), lauryltrimethylammonium chloride (LTAC), octyltrimethylammonium bromide, cetyltrimethylammonium chloride, and myristyltrimethylammonium bromide. These cationic surfactants may be used alone or in combination of two or more.

[0066] The concentration of the cationic surfactant in the dilution reagent is not particularly limited as long as it can suppress hemolysis of red blood cells. For example, it can be appropriately determined from the range of 50 ppm to 20000 ppm. Specifically, when DTAB is used, the concentration of DTAB in the dilution reagent is preferably 500 ppm to 3000 ppm. When LTAC is used, the concentration of LTAC in the dilution reagent is preferably 100 ppm to 500 ppm. By including the cationic surfactant in the dilution reagent at such a concentration, hemolysis of red blood cells can be suppressed and staining of blood cells with a fluorescent dye can be promoted.

[0067] Polyvalent anions are added to the dilution reagent to suppress non-specific staining of red blood cells by fluorescent dyes. Examples of polyvalent anions include sulfate ions, phosphate ions, carbonate ions, polycarboxylate ions, etc. Examples of compounds that can supply these ions include citric acid, sulfuric acid, phosphoric acid, EDTA, and alkali metal salts thereof. One or more types of polyvalent anions may be used.

[0068] Preservatives contained in the dilution reagent include, for example, sodium 2-pyridylthio-1-oxide, β-phenethyl alcohol, and the like.

[0069] In the blood cell analysis method of this embodiment, a commercially available dilution reagent may be used, for example, Cellpack (registered trademark) DFL (Sysmex Corporation).

[0070] (Staining reagents and fluorescent dyes) The staining reagent used in the preparation of the measurement sample will be described. A fluorescent dye suitable for the staining reagent is a fluorescent dye that can be excited by light of a first wavelength and can bind to nucleic acid. Such fluorescent dyes are known and can be selected from, for example, acridine compounds, cyanine compounds, and styryl compounds. The binding of the fluorescent dye to nucleic acid makes it possible to distinguish between cells that do not contain nucleic acid, such as mature red blood cells and platelets, and cells that contain nucleic acid, such as reticulocytes and white blood cells.

[0071] The fluorescent dye of the acridine compound is, for example, the fluorescent dye represented by the above formula (I). The "alkyl group" may be either linear or branched. R1 to R 12 With regard to the above, examples of the alkyl group having 1 to 18 carbon atoms include methyl, ethyl, propyl, isopropyl, t-butyl, n-butyl, isopentyl, neopentyl, t-pentyl, isohexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, and octadecyl. 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. Preferable examples of the alkyl group include methyl, ethyl, propyl, isopropyl, t-butyl, n-butyl, isopentyl, neopentyl, t-pentyl, and isohexyl. R in formula (I) 13 With respect to the above, the alkyl group preferably has 1 to 3 carbon atoms.

[0072] "Halogen" refers to fluorine, chlorine, bromine, or iodine. "Haloalkyl group" refers to an alkyl group in which at least one hydrogen atom is replaced by a halogen atom. When a haloalkyl group contains two or more halogen atoms, the halogen atoms may be the same or may be of different types. "Acylamino group" refers to a group (R-CO-NH-) in which one hydrogen atom of an amino group is replaced by an acyl group. Examples of acylamino groups include formylamino, acetylamino, propionylamino, butyrylamino, benzoylamino, toluoylamino, naphthoylamino, nicotinoylamino, isonicotinoylamino, and furoylamino.

[0073] 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. - For example, F - , Cl - , Br - , I - , 1 / 2SO4 2- , NO3 - X - Cl - , 1 / 2SO4 2- Or NO3 - In the case where the counter ion X is a fluorescer, an acid capable of providing these ions, such as HCl, H2SO4, or HNO3, may be added to the fluorescent dye. - As an alternative, lactate may be added to the fluorescent dye.

[0074] Preferred examples of the fluorescent dye of the acridine compound represented by formula (I) above include 2,7,9-trimethylacridine-3,6-diamine, 3,6-diaminoacridine, 3,6-diamino-10-methylacridinium, 3,6-diamino-2,7,10-trimethylacridinium, 3,6-diamino-2,7-dimethylacridinium, 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.

[0075] The fluorescent dye of the cyanine compound may be, for example, a fluorescent dye represented by the above formula (II). The alkyl group in the above formula (II) is the same as that described for formula (I). The "alkenyl group" may be either linear or branched. As for R1 and R2 in the above formula (II), examples of the alkenyl group having 3 to 20 carbon atoms include allyl, propenyl, isopropenyl, butenyl, isobutenyl, pentenyl, isopentenyl, hexenyl, heptenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, octadecenyl, and icosenyl. The number of carbon atoms in the alkenyl group is preferably 3 to 14, more preferably 3 to 10, and particularly preferably 3 to 6.

[0076] The substituents of the phenyl group in the above formula (II) are the same as those described for formula (I). The substituents of the benzyl group in the above formula (II) include, for example, -CN, -COOH, -NH2, -NO2, -OH, -SH, an alkyl group having 1 to 18 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, an alkylamino group having 1 to 6 carbon atoms, an acylamino group having 1 to 6 carbon atoms, halogen, and a haloalkyl group having 1 to 6 carbon atoms. The counter ion Z in the above formula (II) - For example, F - , Cl - , Br - , I - , CF3SO3 - , BF4 - , ClO4- etc.

[0077] Examples of the fluorescent dye of the cyanine compound represented by the above formula (II) include 3-methyl-2-[(3-methyl-2-benzoxazolinylidene)methyl]benzoxazolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]-6-methyl-benzothiazolium, and 3-ethyl-2-[(3-ethyl-2-benzoxazolinylidene)methyl] Benzoxazolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzoxazolium, 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]benzothiazolium, 3-methyl-2-[(1,3,3-trimethyl-2-indolinylidene)methyl]benzothiazolium and 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolinylidene)methyl]-3H-indolinium are preferred.

[0078] Examples of fluorescent dyes of styryl compounds include fluorescent dyes represented by the above formulae (III) and (IV). The substituents of the alkyl group and phenyl group in the above formulae (III) and (IV) are the same as those described for formula (I). The substituents of the alkenyl group and benzyl group are the same as those described for formula (II). In the above formula (IV), the heterocycle 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. The heterocycle may have a substituent. Examples of the substituent include an alkyl group having 1 to 3 carbon atoms. The counter ion Z in the above formulae (III) and (IV) may be a five-membered ring or a six-membered ring. - For example, F - , Cl - , Br - , I - , CF3SO3 - , BF4 - , ClO4 -Examples of the fluorescent dye of the styryl compound represented by the above formula (III) include 3-ethyl-2-[(1-ethyl-2(1H)-quinolilidene)methyl]benzothiazolium. Examples of the fluorescent dye of the styryl compound represented by the above formula (IV) include 2-[4-(dimethylamino)phenyl]-3,6-dimethyl-1,3-benzothiazol-3-ium.

[0079] 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. Examples include water, organic solvents, and mixtures thereof. The organic solvent is preferably a solvent that can be mixed with water, and examples of the organic solvent include alcohols having 1 to 6 carbon atoms, ethylene glycol, diethylene glycol, polyethylene glycol, dimethyl sulfoxide (DMSO), and the like. The concentration of the fluorescent dye in the staining reagent can be appropriately determined depending on the type of the fluorescent dye. The concentration of the fluorescent dye can be, for example, 100 ppm or more, preferably 500 ppm or more, and more preferably 1000 ppm or more. The concentration of the fluorescent dye is, for example, 100,000 ppm or less, preferably 50,000 ppm or less, and more preferably 10,000 ppm or less.

[0080] As described above, the fluorescent dye itself binds to the nucleic acid of the cells, so in the blood cell analysis method of this embodiment, it is not necessary to stain the cells with an antibody labeled with a fluorescent dye. Therefore, in the blood cell analysis method of this embodiment, the fluorescent dye does not contain an antibody.

[0081] A staining reagent containing the above-mentioned fluorescent dye may 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 may be in the form of, for example, a solution of the above-mentioned fluorescent dye housed in a container. With reference to FIG. 9, 11 indicates a container in which the blood cell analysis reagent of this embodiment is housed. When the container 11 is housed in a packaging box, a package insert describing the composition, usage method, storage method, etc. of the blood cell analysis reagent, a cushioning material for reducing external impact, etc. may also be housed.

[0082] A further embodiment relates to the use of the fluorescent dye for the manufacture of a reagent for blood cell analysis. Details of the fluorescent dye and the reagent for blood cell analysis are given above.

[0083] (Preparation of measurement samples) The fluorescent dye is mixed with the specimen and the dilution reagent so that the concentration (final concentration) in the measurement sample is within a predetermined 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 even 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 even more preferably 1 ppm or more.

[0084] The mixing ratio of the dilution reagent, the staining reagent, and the specimen can be appropriately determined depending on the concentration of the fluorescent dye in the staining reagent and the type of specimen. The mixing ratio of the dilution reagent, the staining reagent, and the specimen is preferably, for example, 1000:0.1 or more:1 or more, expressed as a volume ratio. More preferably, it is 1000:0.5 or more:5 or more, and even more preferably, it is 1000:1 or more:10 or more. Furthermore, the mixing ratio of the dilution reagent, the staining reagent, and the specimen is preferably, for example, 1000:50 or less:100 or less, expressed as a volume ratio. More preferably, it is 1000:30 or less:50 or less, and even more preferably, it is 1000:20 or less:25 or less. The mixing ratio of the staining reagent and the specimen may be the same or different.

[0085] The blood cell analysis method of this embodiment enables detection of reticulocytes and platelets without using a hemolytic reagent. Therefore, in the blood cell analysis method of this embodiment, the measurement sample does not contain a hemolytic reagent. That is, in the blood cell analysis method of this embodiment, the measurement sample preparation does not include a step of hemolyzing red blood cells. Here, the hemolytic reagent is a reagent that dissolves red blood cells in a specimen by mixing with the specimen. The hemolytic reagent itself is known, and contains, for example, a surfactant at a concentration capable of dissolving red blood cells.

[0086] (Analysis process of the first embodiment) Regarding step S13 in Fig. 7, an example of the analysis process of the first embodiment will be described with reference to Figs. 10A to 17, but is not limited to this example. In this specification, mature red blood cells are also called "mRBC", reticulocytes are also called "RET", platelets are also called "PLT", and white blood cells are also called "WBC".

[0087] In the analysis process of the first embodiment, first, blood cells other than WBC are identified from particles in a measurement sample. In the blood cell analysis method of this embodiment, since a hemolytic reagent is not used, the measurement sample includes mRBC and RET. The measurement sample also includes WBC and PLT. In other words, blood cells other than WBC are a particle group including 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, a first side scattered light intensity (also referred to as "SSC-1 intensity") is used as the first scattered light information, and a second forward scattered light intensity (also referred to as "FSC-2 intensity") is used as the second scattered light information.

[0088] Identification of blood cells other than WBC can be performed, for example, by analysis using a scattergram. A scattergram for identifying blood cells other than WBC based on the first and second scattered light information is also called a "first scattergram (a)". Referring to FIG. 10A, in step S101, the analysis unit 300 creates a first scattergram (a). The first scattergram (a) is created based on, for example, SSC-1 intensity and FSC-2 intensity. For example, the analysis unit 300 creates a scattergram with SSC-1 intensity and FSC-2 intensity as two axes based on the acquired optical information. Then, in the created first scattergram (a), the position of a point corresponding to each particle in the measurement sample is determined. The horizontal and vertical axes of the first scattergram (a) may be displayed either logarithmically or linearly. Preferably, the horizontal and vertical axes of the first scattergram (a) are displayed logarithmically.

[0089] In step S102, the analysis unit 300 classifies the particles in the measurement sample into a first particle population and a second particle population based on the SSC-1 intensity and / or FSC-2 intensity. The second particle population is a population that exhibits higher SSC-1 intensity and / or FSC-2 intensity than the first particle population. Here, the comparison of the scattered light intensity between particle populations in the scattergram is preferably performed, for example, by a statistical representative value of the scattered light intensity of each particle population. The statistical representative value of the scattered light intensity of one particle population is a value obtained from the scattered light intensity of particles that constitute the particle population, and examples of the statistical representative value include the median, the average value, and the mode. The median is preferable.

[0090] In the first scattergram (a), the first particle group appears in an area with low SSC-1 intensity and / or low FSC-2 intensity. Also, the second particle group appears in an area with high SSC-1 intensity and / or high FSC-2 intensity. In the example of FIG. 12, the first particle group appears in an area with low SSC-1 intensity, and the second particle group appears in an area with high SSC-1 intensity. In this figure, the first particle group is a group consisting of particles in an area surrounded by a dashed line. Specifically, the first particle group includes a group including mRBC and RET (also called a "group including RBC") and a group including PLT. That is, the first particle group is blood cells other than WBC. In addition, if the specimen contains platelet aggregates, the group including PLT also includes platelet aggregates. Also, the second particle group is a group of WBC. The reason why the particles in the measurement sample are classified into a first particle population and a second particle population in the first scattergram (a) will be explained below.

[0091] Among blood cells other than WBC, mRBC and RET (hereinafter referred to as "RBC") are discriminated from WBC based on SSC-1 intensity and / or FSC-2 intensity. Here, hemoglobin contained in RBC is known to absorb blue-violet light. When RBC is irradiated with light of a first wavelength corresponding to blue-violet light, the scattered light generated from these blood cells is weaker than the scattered light generated when light of a second wavelength corresponding to red light is irradiated. For this reason, red light has been preferably used for blood cell analysis in the past. On the other hand, when 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 present inventors have conceived of discriminating between WBC and RBC by utilizing the above phenomenon. Specifically, a difference is generated in the first scattered light information between WBC and RBC by irradiating with light of the first wavelength. For example, when the first scattered light information is SSC-1 intensity, the SSC-1 intensity of RBC is lower due to absorption by hemoglobin. However, the SSC-1 intensity of WBC is not affected. Therefore, the SSC-1 intensity of RBC is lower than that of WBC. This allows discrimination between WBC and RBC. Furthermore, among blood cells other than WBC, PLT and WBC can be discriminated by both FSC-2 intensity and SSC-1 intensity. PLT is known to be the smallest blood cell. Here, the intensity of forward scattered light generated by irradiating light on blood cells reflects the size of the blood cells. Therefore, PLT has a lower forward scattered light intensity than WBC. Furthermore, due to the difference in internal structure, PLT has a lower side scattered light intensity than WBC. Therefore, PLT and WBC can be discriminated. For these reasons, when RBC and PLT are regarded as one group (i.e., a first particle group) in the first scattergram (a), the SSC-1 intensity and FSC-2 intensity of this group are lower than those of the WBC group. Therefore, in the first scattergram (a), the particles in the measurement sample are classified into a first particle population including blood cells other than WBCs, and a second particle population including WBCs.

[0092] 10A, in step S103, the analysis unit 300 identifies 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 identified as blood cells other than WBC. Also, since the second particle group includes WBC, the second particles can be identified as WBC. For example, by accumulating measurement data for a plurality of samples, a region in which the first particle group appears in the first scattergram (a) may be determined in advance, and particles appearing in that region may be identified as blood cells other than WBC.

[0093] 10A, particles in a measurement sample are classified into WBCs and blood cells other than WBCs, thereby identifying blood cells other than WBCs. In step S103, since blood cells other than WBCs are identified, the data of WBCs is excluded from the data of particles in the measurement sample.

[0094] (Detection of platelet aggregation) Referring to FIG. 10B, the process proceeds to step S201. In this analysis process, information indicating the presence or absence of platelet aggregation is obtained. In step S201, the analysis unit 300 classifies blood cells other than WBCs into a group including RBCs and a group including PLTs based on the FSC-2 intensity. Since PLTs are blood cells smaller in size than mRBCs and RETs, the group including PLTs has a lower FSC-2 intensity than the group including RBCs. Therefore, the group including RBCs and the group including PLTs can be classified based on the FSC-2 intensity. Referring to FIG. 13A, the group including RBCs, the group including PLTs, and the group of WBCs are distributed on the first scattergram (a). By accumulating measurement data for multiple samples, the region in which the group including RBCs appears and the region in which the group including PLTs appears in the first scattergram (a) may be determined in advance, and particles that appear in each region may be identified as the group including RBCs and the group including PLTs.

[0095] With reference to FIG. 13B, when platelet aggregates are contained in the specimen, particles are observed on the first scattergram (a) that are distributed so as to extend from the group containing PLTs to the group containing WBCs. In FIG. 13B, particles that appear in the area surrounded by the dashed line are platelet aggregates. Platelet aggregates are an aggregate of multiple platelets, and are larger in size than normal platelets. Therefore, particles of platelet aggregates show a higher forward scattered light intensity than platelets. Therefore, it is possible to determine whether or not platelet aggregates are contained in the group containing PLTs based on the FSC-2 intensity. With reference to FIG. 10B, in step S202, the analysis unit 300 determines whether or not the group containing PLTs includes particles that show an FSC-2 intensity equal to or greater than a threshold value. Specifically, the FSC-2 intensity of all particles that constitute the group containing PLTs is compared with a threshold value corresponding to the FSC-2 intensity. Particles that show an FSC-2 intensity equal to or greater than this threshold value are suspected to be platelet aggregates. In other words, the group containing PLTs is suspected to include platelet aggregates. In step S202, if there are particles exhibiting an FSC-2 intensity equal to or greater than the threshold, the process proceeds to step S203. In step S202, if there are no particles exhibiting an FSC-2 intensity equal to or greater than the threshold, the process proceeds to step S301 in FIG. 10C.

[0096] In a further embodiment, particles exhibiting FSC-2 intensity equal to or greater than a threshold may be counted. In step S202, if the number of particles exhibiting FSC-2 intensity equal to or greater than the threshold is equal to or greater than a predetermined value, the process proceeds to step S203. If the number of particles exhibiting FSC-2 intensity equal to or greater than the threshold is less than a predetermined value, the process proceeds to step S301 of FIG. 10C. Here, the predetermined value is a value for determining whether the particles exhibiting FSC-2 intensity equal to or greater than the threshold are platelet aggregates.

[0097] In step S203, the analysis unit 300 outputs information indicating the presence of platelet aggregation. For example, a flag "PLTc?" may be output to the display unit 305. The process then proceeds to step S301 in FIG. 10C. Note that not displaying the flag on the display unit 305 may be information indicating the absence of platelet aggregation.

[0098] (detection of reticulocytes and platelets) After identifying blood cells other than WBC, the process proceeds to step S301 in Fig. 10C. In this analysis process, a RET population and a PLT population are classified from blood cells other than WBC based on the fluorescence information and the second scattered light information. This identifies RET and PLT. Blood cells other than WBC may be classified into a RET population, a PLT population, and a mRBC population. This identifies RET, PLT, and mRBC. With reference to Fig. 10C, the case of classifying blood cells other than WBC into a RET population, a PLT population, and a mRBC population will be described below.

[0099] Classification of blood cells other than WBC can be performed, for example, by analysis using a scattergram. A scattergram for classifying a RET group and a PLT group from blood cells other than WBC based on the fluorescence information and the second scattered light information is also called a "second scattergram". In this example, the first side fluorescence intensity (also called "SFL-1 intensity") is used as the fluorescence information, and the FSC-2 intensity is used as the second scattered light information. The second scattergram is created based on the SFL-1 intensity and the FSC-2 intensity. Referring to FIG. 10C, in step S301, the analysis unit 300 creates a second scattergram. For example, the analysis unit 300 creates a scattergram with the SFL-1 intensity and the FSC-2 intensity as the two axes based on the acquired optical information. Then, in the created second scattergram, the positions of points corresponding to blood cells other than WBC are determined. The horizontal and vertical axes of the second scattergram may be displayed in either logarithmic or linear. When the horizontal and vertical axes of the second scattergram are displayed in logarithmic scale, it is suitable for PLT analysis. When the horizontal and vertical axes of the second scattergram are displayed in linear scale, it is suitable for RET and mRBC analysis.

[0100] In step S302, the analysis unit 300 classifies blood cells other than WBC into a third particle group, a fourth particle group, and a fifth particle group based on the SFL-1 intensity and the FSC-2 intensity. The third particle group is a group that shows a higher SFL-1 intensity than the fifth particle group. The fourth particle group is a group that shows a lower FSC-2 intensity than the third particle group. The comparison of scattered light intensity and fluorescent intensity between particle groups in the scattergram is as described above.

[0101] FIG. 14A shows examples of the second scattergram (logarithmic display) and the second scattergram (linear display). In each of the second scattergrams, a third particle group, a fourth particle group, and a fifth particle group appear. In the second scattergram (logarithmic display), the third and fifth particle group appear in an area with high FSC-2 intensity, and the fourth particle group appears in an area with low FSC-2 intensity. In the second scattergram (linear display), the fourth particle group appears in an area with low FSC-2 intensity, and the third particle group appears in an area with higher FSC-2 intensity than the fourth particle group. In both scattergrams, the third particle group appears in an area with higher SFL-1 intensity than the fifth particle group. As shown in Fig. 14A, the third particle population is a RET population, the fourth particle population is a PLT population, and the fifth particle population is an mRBC population. The fact that blood cells other than WBCs are classified into a RET population, a mRBC population, and a PLT population in the second scattergram will be described below.

[0102] As described above, PLTs are the smallest blood cells, and therefore PLTs have a lower forward scattered light intensity than RETs. Therefore, in the second scattergram, the PLT population appears in an area with a lower FSC-2 intensity than the RET population. As a result, RET and PLTs are discriminated based on the FSC-2 intensity. As described above, RETs have a small amount of ribonucleic acid, but mRBCs have no nuclei or nucleic acids. Therefore, the RET population appears in an area with a higher fluorescence intensity than the mRBC population. As a result, mRBCs and RETs are discriminated based on the SFL-1 intensity. Thus, in the second scattergram, blood cells other than WBCs are classified into a third particle population including RETs, a fourth particle population including PLTs, and a fifth particle population including mRBCs.

[0103] As described above, the third particle group includes RET, the fourth particle group includes PLT, and the fifth particle group includes mRBC. Therefore, referring to FIG. 10C, in step S303, the analysis unit 300 identifies the third particle group as RET, the fourth particle group as PLT, and the fifth particle group as mRBC. Since RET becomes mRBC when it matures, it is difficult to strictly determine the boundary between the RET group and the mRBC group. For example, a threshold value of SFL-1 intensity that separates the mRBC group from the RET group may be set based on the range of SFL-1 intensity that mRBC can normally exhibit and the average particle number of mRBC. Such a threshold value may be set as the boundary between the mRBC group and the RET group on the second scattergram. Then, based on the threshold value and the FSC-2 intensity, the area in which each of mRBC and RET appears may be set, for example, as the area indicated by the dashed line in FIG. 14B. Particles in the region where the set mRBC appears can be identified as mRBC. Also, particles in the region where the set RET appears can be identified as RET. Regarding the fourth particle group, if the third and fifth particle groups and the fourth particle group are clearly separated on the second scattergram, the user may identify the fourth particle group in the second scattergram as a group of PLT. Alternatively, by accumulating measurement data on a plurality of specimens, the regions in which the third, fourth and fifth particle groups appear in the second scattergram may be determined in advance, and the particles appearing in each region may be identified as RET, PLT and mRBC, respectively. If necessary, the identified RET, PLT and mRBC may be counted. The cell count is performed by the analysis unit 300.

[0104] (Platelet analysis, reticulocyte analysis and white blood cell classification) Referring to FIG. 10D, the process proceeds to step S401. In this analysis process, the PLT population and the RET population are further analyzed to classify the WBC population into subpopulations. In step S401, the analysis unit 300 identifies immature platelets and large platelets from the PLT population based on the SFL-1 intensity and the FSC-2 intensity in the second scattergram (logarithmic display). As described above, mature platelets do not have nucleic acid, but immature platelets have a small amount of ribonucleic acid. Therefore, among the PLT population on the second scattergram, PLTs that show high SFL-1 intensity can be identified as immature platelets. For example, as shown in FIG. 15A (A), PLTs that appear in an area where SFL-1 is high (area surrounded by a dashed line) in the second scattergram may be identified as immature platelets. PLTs other than immature platelets can be identified as mature platelets. As a result, the PLT population is classified into immature platelets and mature platelets. The identified immature platelets may be counted. If the platelet count is obtained by counting the PLT population, the immature platelet fraction (IPF) can be calculated. The IPF is the ratio of the number of platelets that appeared in the area of ​​high fluorescence intensity (area surrounded by a solid line rectangle) to the total platelet count. The IPF is known as an index of platelet production capacity.

[0105] Since large platelets are larger in size than normal PLTs, PLTs showing high FSC-2 intensity among the group of PLTs on the second scattergram can be identified as large platelets. For example, as shown in FIG. 15A (B), PLTs appearing in the region of high FSC-2 (region surrounded by dashed line) in the second scattergram may be identified as large platelets. PLTs other than large platelets can be identified as PLTs of normal size. This allows the group of PLTs to be classified into large platelets and platelets of normal size. The identified large platelets may be counted. When the group of PLTs is counted to obtain the platelet count, the large platelet ratio (P-LCR) can be calculated. The P-LCR is the ratio of the number of platelets appearing in the region of high forward scattered light intensity (region surrounded by dashed square) to the platelet count. The P-LCR is known as an index of platelet production ability and congenital diseases.

[0106] Referring to FIG. 10D, the process proceeds to step S402. In step S402, the analysis unit 300 acquires a platelet distribution width (PDW) and a platelet mean volume (MPV) based on the FSC-2 intensity and particle number of the PLT population. The PDW is the distribution of the forward scattered light intensity of the PLT population, and is an index representing the variation in the size of the platelets. The MPV is the average value of the forward scattered light intensity of the PLT population, and is an index representing the size of the PLT. As shown in FIG. 15A (C), the PDW and MPV may be acquired by analyzing the PLT population on the second scattergram. The PDW is the length in the vertical axis direction of the PLT population. Moreover, the MPV is the average value calculated from the FSC-2 intensity of all particles constituting the PLT population. Alternatively, a histogram based on the FSC-2 intensity and the particle number may be created for the PLT population on the second scattergram, as shown in FIG. 15A (D). The PDW is the length in the horizontal axis direction of the histogram.

[0107] Referring to FIG. 10D, the process proceeds to step S403. In step S403, the analysis unit 300 classifies the RET population 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 younger the RET, the greater the amount of nucleic acid (RNA) in the cell. As the RET matures, the amount of RNA in the cell decreases. That is, the RNA amount of the RET indicates the maturity of the RET. Here, the SFL-1 intensity of the cell measured by FCM reflects the amount of nucleic acid in the cell. Therefore, the maturity of the RET can be analyzed based on the SFL-1 intensity. Referring to FIG. 15B, in the second scattergram (linear display), the area where the RET appears can be divided into three areas, LFR, MFR, and HFR, based on the SFL-1 intensity. The RET appearing in each area may be counted. The RET appearing in the HFR area has the greatest amount of nucleic acid, and is therefore the most immature RET.

[0108] For RET appearing in each of the LFR, MFR and HFR regions, the average value of the fluorescence intensity (SFL-1 intensity) and the average value of the forward scattered light intensity (FSC-2 intensity) may be obtained. Furthermore, for particles (i.e., mRBC) in the region where mRBC appears, the average values ​​of the SFL-1 intensity and the FSC-2 intensity may be obtained. According to the method described in Japanese Patent No. 4474135, the therapeutic effect on red blood cell diseases (e.g., pernicious anemia, iron deficiency anemia, etc.) can be monitored based on the average values ​​of the SFL-1 intensity and the FSC-2 intensity of RET and mRBC appearing in each region (Japanese Patent No. 4474135 is incorporated herein by reference). Specifically, the average values ​​of the SFL-1 intensity and the FSC-2 intensity in each of the LFR, MFR and HFR regions are plotted on a graph with the SFL-1 intensity and the FSC-2 intensity on the two axes, and an approximate straight line is obtained by the least squares method. Then, the value of the slope of this approximate straight line is obtained. The value of this slope is obtained periodically from the day treatment is started, and the effect of treatment can be determined by monitoring the change in this value.

[0109] Referring to FIG. 10D, the process proceeds to step S404. In this step, the WBC population identified in step S103 of FIG. 10A is classified into subpopulations. In step S404, the analysis unit 300 classifies the WBC population into subpopulations based on SSC-1 intensity and FSC-2 intensity. The subpopulations of white blood cells are, for example, lymphocytes, monocytes, and granulocytes. Granulocytes are a general term for neutrophils, eosinophils, and basophils. In step S404, the area in which the WBC population appears in the first scattergram (a) is enlarged, so that the WBCs are distributed in each subpopulation as shown in FIG. 16. In the figure, "Gran" represents granulocytes, "Mono" represents monocytes, and "Lym" represents lymphocytes. In this figure, the WBCs are classified into three subpopulations, but this is not limiting. WBCs can be classified into at least two populations of lymphocytes, monocytes, and granulocytes. For example, when there is an abnormality in WBCs, some of the WBC subpopulations may be reduced to such an extent that classification is difficult. WBCs may be classified into two populations, for example, lymphocytes or monocytes, and granulocytes.

[0110] The classification of WBC into subpopulations may be determined, for example, as follows. First, the measurement data for a plurality of samples is accumulated to determine in advance the area in the first scattergram (a) in which each WBC subpopulation appears. Then, the particles appearing in the area are identified as the WBC subpopulation. If necessary, the cells contained in each WBC subpopulation may be counted. After performing the WBC classification, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG. 7.

[0111] (Modification of white blood cell classification process) In step S404 in Fig. 10D, the WBC population was classified based on the first scattered light information and the second scattered light information. An example of an analysis process for classifying the WBC population into subpopulations based on the second scattered light information and the third scattered light information will be described with reference to Figs. 11 and 17. The scattergram for classifying the WBC population into subpopulations based on the second scattered light information and the third scattered light information is also called the "third scattergram (a)". In this example, the second side scattered light intensity (also called "SSC-2 intensity") is used as the third scattered light information. The third scattergram (a) is created based on, for example, the FSC-2 intensity and the SSC-2 intensity.

[0112] In step S501, the analysis unit 300 creates a third scattergram (a). For example, the analysis unit 300 creates a scattergram with SSC-2 intensity and FSC-2 intensity as two axes based on the acquired optical information. Then, in the created third scattergram (a), the position of a point corresponding to each particle in the WBC population is determined. The horizontal and vertical axes of the third scattergram (a) may be displayed in either logarithmic or linear. Preferably, the horizontal and vertical axes of the third scattergram (a) are displayed in logarithmic. In step S502, the analysis unit 300 classifies the WBC population into lymphocytes, monocytes, and granulocytes based on the SSC-2 intensity and the FSC-2 intensity. Here, granulocytes are a general term for neutrophils, eosinophils, and basophils. In the third scattergram (a), it is observed that the WBCs are distributed in subpopulations as shown in FIG. 17 (A). In this figure, WBCs are classified into three subpopulations, but are not limited thereto. WBCs may be classified into at least two of a lymphocyte population, a monocyte population, and a granulocyte population. WBCs may be classified into, for example, two populations: a lymphocyte or monocyte population, and a granulocyte population.

[0113] In a further embodiment, in the analysis process of FIG. 11, the WBC population may be classified based on the fluorescence information and the third scattered light information. A scattergram for classifying the WBC population into subpopulations based on the fluorescence information and the third scattered light information is also called a "third scattergram (b)". For example, the third scattergram (b) is created based on the SFL-1 intensity and the SSC-2 intensity. With reference to FIG. 11, in step S501, the third scattergram (b) may be created instead of the third scattergram (a). For example, the analysis unit 300 creates a scattergram with the SSC-2 intensity on the horizontal axis and the SFL-1 intensity on the vertical axis based on the acquired optical information. Then, in the created third scattergram (b), the position of a point corresponding to each particle in the WBC population is determined. The horizontal and vertical axes of the third scattergram (b) may be displayed in either a logarithmic or linear manner. Preferably, the horizontal and vertical axes of the third scattergram (b) are displayed in logarithmic scale. In step S502, the analysis unit 300 classifies the WBC population into lymphocytes, monocytes, and granulocytes based on the SSC-2 intensity and the SFL-1 intensity. In the third scattergram (b), the WBCs are distributed in subpopulations as shown in FIG. 17(B). In this figure, the WBCs are classified into three subpopulations, but this is not limited thereto. The WBC population may be classified into at least two of a lymphocyte population, a monocyte population, and a granulocyte population. The WBCs may be classified into two groups, for example, a lymphocyte or monocyte population, and a granulocyte population.

[0114] The classification of WBC into subpopulations may be performed, for example, as follows: First, by accumulating measurement data for a plurality of samples, the regions in which each WBC subpopulation appears in the third scattergrams (a) and (b) are determined in advance, and then the particles appearing in the regions are identified as the WBC subpopulations.

[0115] If necessary, the cells contained in each WBC subpopulation may be counted. After performing WBC classification, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG.

[0116] (Analysis process of the second embodiment) With respect to step S13 in FIG. 7, an example of the analysis process of the second embodiment will be described with reference to FIGS. 18 to 20, but is not limited to this example. In the second embodiment, blood cells other than WBC are identified from particles in a measurement sample based on the first scattered light information and the fluorescence information. In this example, SSC-1 intensity is used as the first scattered light information, and SFL-1 intensity is used as the fluorescence information. Identification of blood cells other than WBC can be performed, for example, by analysis using a scattergram. A scattergram for identifying blood cells other than WBC based on the first scattered light information and the fluorescence information is also called a "first scattergram (b)". With reference to FIG. 18, in step S601, the analysis unit 300 creates a first scattergram (b). The first scattergram (b) is created based on, for example, the SSC-1 intensity and the SFL-1 intensity. For example, the analysis unit 300 creates a scattergram with the SSC-1 intensity and the SFL-1 intensity as two axes based on the acquired optical information. Then, in the created first scattergram (b), the position of a point corresponding to each particle in the measurement sample is determined. The horizontal and vertical axes of the first scattergram (b) may be displayed either logarithmically or linearly. Preferably, the horizontal and vertical axes of the first scattergram (b) are displayed logarithmically.

[0117] In step S602, the analysis unit 300 classifies particles in the measurement 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. Here, the comparison of the fluorescence intensity between particle groups in the scattergram 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 one particle group is a value obtained from the fluorescence intensity of particles that constitute the particle group, and examples of the statistical representative value include the median, the average value, and the mode. The median is preferable. The statistical representative value of the fluorescence intensity of the particle group is calculated by the analysis unit 300.

[0118] In the first scattergram (b), the first particle group appears in an area where the SSC-1 intensity and / or SFL-1 intensity are low. Also, the second particle group appears in an area where the SSC-1 intensity and / or SFL-1 intensity are high. In the example of FIG. 19, the first particle group appears in an area where the SSC-1 intensity and SFL-1 intensity are low, and the second particle group appears in an area where the SSC-1 intensity and SFL-1 intensity are high. In this figure, the first particle group is a group consisting of particles in the area surrounded by a dashed line. Specifically, the first particle group includes mRBC, RET, and PLT. That is, the first particle group is blood cells other than WBC. Also, the second particle group is a group of WBC. Here, WBC has a large amount of nucleic acid, so it is strongly stained by the above-mentioned fluorescent dye. On the other hand, mRBC and PLT do not contain nuclei and nucleic acids, so they are hardly stained or are stained weakly by the above fluorescent dyes. RET contains ribonucleic acid, but in small amounts. Also, RET does not have a nucleus. Therefore, RET is stained weakly compared to WBC. Thus, there is a difference in fluorescence intensity between WBC and blood cells other than WBC. Therefore, in the first scattergram (b), the particles in the measurement sample are classified into a first particle group containing blood cells other than WBC and a second particle group containing WBC.

[0119] In step S603, the analysis unit 300 identifies 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 identified as blood cells other than WBC. Also, since the second particle group includes WBC, the second particles can be identified as WBC. For example, by accumulating measurement data for a plurality of samples, a region in which the first particle group appears in the first scattergram (b) may be determined in advance, and particles appearing in that region may be identified as blood cells other than WBC.

[0120] 18, particles in a measurement sample are classified into WBCs and blood cells other than WBCs, thereby identifying blood cells other than WBCs. In step S603, since blood cells other than WBCs are identified, the data of WBCs is excluded from the data of particles in the measurement sample.

[0121] After identifying blood cells other than WBC in step S603, the process proceeds to step S301 in FIG. 10C. Then, RET, PLT, and mRBC are identified from blood cells other than WBC in the same manner as in the first embodiment. Details of this analysis process are the same as those described for the first embodiment. With reference to FIG. 10D, in the second embodiment, PLT analysis, RET analysis, and WBC classification are performed in the same manner as in the first embodiment. Details of the PLT and RET analysis processes are the same as those described for the first embodiment. In the classification of WBC, in step S404 in FIG. 10D, the WBC group identified in step S603 in FIG. 18 is classified into subgroups based on SSC-1 intensity and SFL-1 intensity. The subgroups of white blood cells are, for example, lymphocytes, monocytes, and granulocytes. In step S404, the area in which the WBC group appears in the first scattergram (b) is enlarged, so that the WBC is distributed in each subgroup as shown in FIG. 20. In this figure, WBCs are classified into three subpopulations, but are not limited thereto. WBCs can be classified into at least two of a lymphocyte population, a monocyte population, and a granulocyte population. For example, when there is an abnormality in WBCs, some of the WBC subpopulations may decrease to such an extent that classification is difficult. WBCs may be classified into two groups, for example, a lymphocyte or monocyte population, and a granulocyte population.

[0122] The classification of WBC into subpopulations may be performed, for example, as follows: First, by accumulating measurement data for multiple samples, the regions in which each WBC subpopulation appears in the first scattergram (b) are determined in advance, and then particles appearing in those regions are identified as WBC subpopulations.

[0123] If necessary, the cells contained in each WBC subpopulation may be counted. After performing WBC classification, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG.

[0124] 7, when the above-mentioned analysis process is completed, the control unit 301 outputs the analysis result to the display unit 305 in step S14, and ends the process. As described above, the processes for analyzing reticulocytes and platelets in the measurement sample have been individually explained using Figs. 10A to 20. However, it is sufficient that only a part of the analysis process is performed, or all of the analysis process may be performed.

[0125] The analysis result screen output to the display unit 305 may include, for example, the reticulocyte count, the platelet count, the IPF, the P-LCR, the PDW, the MPV, a platelet aggregation flag, the particle count in each region of the LFR, the MFR and the HFR, and the number of each subpopulation of white blood cells (lymphocytes, monocytes and granulocytes). In addition to this information, the screen may also include a scattergram created based on the optical information. Furthermore, the screen may include a flag based on the analysis result. For example, when a particle suspected of being a platelet aggregate is identified, information indicating the presence of platelet aggregation in the sample may be output. For example, a flag "PLTc?" may be output to the display unit 305.

[0126] In this way, the blood cell analysis method of the present embodiment can provide medical professionals such as doctors and laboratory technicians with information on the number of reticulocytes and platelets contained in a sample as the analysis results of reticulocytes and platelets. It can also provide analysis results of red blood cells and white blood cells. From the obtained analysis results, medical professionals can decide, for example, whether to perform further tests on the sample. The above-mentioned preparation of the measurement sample and analysis of the sample are all performed in vitro.

[0127] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0128] In each of the following examples, the blood cell analysis method of this embodiment was carried out using the following blood samples, dilution reagents, staining reagents and analyzer. Details are as described in the "Measurement Method" below.

[0129] [Blood sample] 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 taken from the blood collection tubes and used as a blood sample. Details of the blood cell components and / or subjects contained in the blood samples are described in each example.

[0130] [Diluted Reagent] As a dilution reagent, Cellpack (registered trademark) DFL (Sysmex Corporation) was used.

[0131] [Staining reagent] Various fluorescent dyes were dissolved in 1 L of special grade ethylene glycol to prepare staining reagents. The types of fluorescent dyes used were described in each example.

[0132] [Analyzer] A prototype of a multi-item automated blood cell analyzer XN-20 (Sysmex Corporation) and a flow cytometer XF-1600 (Sysmex Corporation) were used as the analyzers. Each prototype was made as follows. The XN-20 was equipped with a semiconductor laser light source that emits red (639 nm) light, a detector for red forward scattered light, a detector for red side scattered light, and a detector for fluorescence excited by red light (light of 660 nm or more). A semiconductor laser light source that emits blue-violet (405 nm) light, a detector for blue-violet side scattered light, and a fluorescence detector that detects fluorescence excited by blue-violet light (450 to 600 nm) were added to this XN-20. In addition, this XN-20 analyzed the measurement data detected for red and blue-violet scattered light information and fluorescence information generated by the fluorescent dye, and displayed the desired scattergram. The XF-1600 was equipped with three semiconductor laser light sources that emitted blue-violet (405 nm), blue (488 nm), and red (638 nm) light, respectively, a detector for scattered light of each color, and a detector for fluorescent light excited by each light. The semiconductor laser light source that emitted blue (488 nm) light was removed from this XF-1600, and it was remodeled into an FCM system equipped with two semiconductor laser light sources. Each prototype had the hardware shown in Figures 1 to 3, and the FCM detection unit had the configuration shown in Figure 4.

[0133] [Measurement method] Except for using Cellpack DFL and the above staining reagent, the preparation and measurement of the measurement sample were performed 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 measured after incubating at 41° C. for 30 seconds. 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 was described in each example.

[0134] Example 1: Blood cell analysis using acridine compounds A measurement sample was prepared using a staining reagent containing an acridine compound as a fluorescent dye, and reticulocytes and platelets in the measurement sample were classified using an analyzer.

[0135] (1) Measurement sample For each of the above blood samples, red blood cells, reticulocytes, and platelets were classified and counted in advance by the conventional method, and the reticulocyte rate (the ratio of reticulocytes to 1000 red blood cells: hereinafter also referred to as "RET%") was obtained. RET% is an indicator of anemia and hematopoietic abnormality, and its normal range is 0.4 to 2.0%. Classification and counting of red blood cells and reticulocytes by the conventional method was performed by measurement with a multi-item automated blood cell analyzer XN-20 (Sysmex Corporation) and analysis in the RET channel. Classification and counting of platelets by the conventional method was performed by measurement with the XN-20 and analysis in the PLT-F channel. In classification and counting of platelets by the XN-20, six times the amount of particles was measured compared to classification and counting of red blood cells and reticulocytes. From the above blood samples, blood samples with RET% of 0.21% and 5.56% (hereinafter also referred to as "low RET% sample 1" and "high RET% sample 1", respectively) were selected and measurement samples were prepared as described above. An ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used as the staining reagent. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0136] (2) Analysis Each measurement sample was measured using an analyzer (a prototype modified from the XN-20) to obtain first scattered light information, second scattered light information, and fluorescence information.

[0137] The first scattered light information was the side scattered light intensity (hereinafter also referred to as "side scattered light intensity (blue-violet)" or "V-SSC") obtained by irradiating particles in the measurement sample with a blue-violet laser. The second scattered light information was the forward scattered light intensity (hereinafter also referred to as "forward scattered light intensity (red)" or "R-FSC") obtained by irradiating particles in the measurement sample with a red laser. The fluorescence information was the fluorescence intensity (hereinafter also referred to as "fluorescence intensity (blue-violet)" or "V-SFL") obtained by irradiating particles in the measurement sample with a blue-violet laser. For each measurement sample, a scattergram (hereinafter also referred to as "first scattergram (a)") was created with the side scattered light intensity (blue-violet) on the horizontal axis and the forward scattered light intensity (red) on the vertical axis. In addition, for each measurement sample, a scattergram (hereinafter also referred to as "first scattergram (b)") was created with the side scattered light intensity (blue-violet) on the horizontal axis and the fluorescence intensity (blue-violet) on the vertical axis. The first scattergrams (a) and (b) of the high RET% sample 1 are shown in Figures 21A and 21B, respectively. The first scattergrams (a) and (b) of the low RET% sample 1 are shown in Figures 22A and 22B, respectively. Hereinafter, in the figures, "WBC" stands for white blood cells, "RBC" stands for red blood cells, "PLT" stands for platelets, "mRBC" stands for mature red blood cells, and "RET" stands for reticulocytes.

[0138] Referring to the first scattergram (a) of FIG. 21A, in the high RET% sample 1, the white blood cells appeared in an area where the V-SSC was high and the R-FSC was high. The red blood cell group appeared in an area where the V-SSC was lower than that of the white blood cells and the R-FSC was high. The platelet group appeared in an area where the V-SSC and R-FSC were lower than that of the white blood cells. Since the platelets are smaller in size than the white blood cells and the red blood cells, the R-FSC of the platelets was low. Therefore, in the first scattergram (a), the platelet group appeared in an area where the R-FSC was low. Also, as described above, the hemoglobin of the red blood cells absorbs the first wavelength (blue-purple) light, so it is considered that the V-SSC of the red blood cells was low. Therefore, in the first scattergram (a), it is considered that the red blood cell group appeared in an area where the V-SSC is lower than that of the white blood cells. In this way, in the first scattergram (a), it was possible to distinguish between white blood cells and blood cells other than white blood cells. In the first scattergram (a), blood cells other than white blood cells were selected. The selected blood cells were displayed on a scattergram (hereinafter also referred to as a "second scattergram") with the fluorescence intensity (blue-purple) on the horizontal axis and the forward scattered light intensity (red) on the vertical axis. The horizontal and vertical axes of this second scattergram were displayed logarithmically.

[0139] Referring to the second scattergram in FIG. 21A, in the high RET% sample 1, the mature red blood cell group appeared in the region with low V-SFL and high R-FSC. The reticulocyte group appeared on the higher V-SFL side than the mature red blood cell group. The platelet group appeared in the region with lower R-FSC than the reticulocyte group. In this way, by selecting blood cells other than white blood cells, mature red blood cells, reticulocytes, and platelets could be classified without being influenced by white blood cells. Referring to the first scattergram (a) and the second scattergram in FIG. 22A, the low RET% sample 1 also showed the same results as the high RET% sample 1.

[0140] Referring to the first scattergram (b) in FIG. 21B, in the high RET% sample 1, white blood cells appeared in an area with a high V-SFL. Furthermore, the platelet and red blood cell groups appeared in an area with a lower V-SFL than the white blood cells. Mature red blood cells and platelets are non-nucleated cells, and reticulocytes have a small amount of RNA, whereas white blood cells have a nucleus. Therefore, the V-SFL of the white blood cells was higher than the platelet and red blood cell groups. As with the first scattergram (a), the first scattergram (b) also made it possible to distinguish white blood cells from blood cells other than white blood cells. Referring to the second scattergram in FIG. 21B, blood cells other than white blood cells were classified into mature red blood cells, reticulocytes, and platelets. In this way, by selecting blood cells other than white blood cells, platelets, mature red blood cells, and reticulocytes could be classified without being affected by white blood cells. Referring to the first scattergram (b) and the second scattergram in FIG. 22B, the same results were obtained for the low RET% sample 1.

[0141] As described above, it was suggested that the influence of white blood cells can be reduced when measuring reticulocytes and platelets by classifying reticulocytes and platelets while excluding white blood cells. Furthermore, in the present embodiment, reticulocytes and platelets can be classified in one measurement, whereas the conventional method requires two measurements.

[0142] Example 2: Blood cell analysis using cyanine compounds A measurement sample was prepared using a staining reagent containing a fluorescent dye different from that in Example 1, and the reticulocytes and platelets in the measurement sample were classified using an analyzer.

[0143] (1) Measurement sample From the above blood samples, blood samples with RET% of 0.61% and 4.33% (hereinafter also referred to as "low RET% sample 2" and "high RET% sample 2", respectively) were selected, and measurement samples were prepared as described above. An ethylene glycol solution of 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium was used as the staining reagent. The final concentration of the fluorescent dye in the measurement sample was 5.0 ppm.

[0144] (2) Analysis As in Example 1, each measurement sample was measured using an analytical device (a prototype modified from XN-20) to obtain first scattered light information, second scattered light information, and fluorescence information. Each piece of information was the same as in Example 1. As in Example 1, first scattergrams (a) and (b) were created for each measurement sample. The first scattergrams (a) and (b) for high RET% sample 2 are shown in Figures 23A and 23B, respectively. The first scattergrams (a) and (b) for low RET% sample 2 are shown in Figures 24A and 24B, respectively.

[0145] Referring to the first scattergram (a) in FIG. 23A, in the high RET% sample 2, the white blood cells appeared in the region where the V-SSC was high and the R-FSC was high. Also, the red blood cell group appeared in the region where the V-SSC was lower than that of the white blood cells and the R-FSC was high, and the platelet group appeared in the region where the V-SSC and R-FSC were lower than those of the white blood cells. Thus, in the first scattergram (a), it was possible to distinguish the white blood cells from the blood cells other than the white blood cells. In the first scattergram (a), the blood cells other than the white blood cells were selected, and these blood cells were displayed in the second scattergram (logarithmic display). By selecting the blood cells other than the white blood cells, the platelets, mature red blood cells, and reticulocytes could be classified in the second scattergram in FIG. 23A without being influenced by the white blood cells. Referring to FIG. 24A, the same results were obtained for the low RET% sample 2. Referring to the first scattergram (b) in FIG. 23B, in the high RET% sample 2, white blood cells appeared in the region where the V-SFL was high. Moreover, the platelet and red blood cell groups appeared in the region where the V-SFL was lower than that of the white blood cells. As in the first scattergram (a), the first scattergram (b) also allowed discrimination between white blood cells and blood cells other than white blood cells. By selecting blood cells other than white blood cells in the first scattergram (b), the platelets, mature red blood cells, and reticulocytes could be classified in the second scattergram without being influenced by white blood cells. Referring to FIG. 24B, the same results were obtained for the low RET% sample 2. These results showed that even when a cyanine compound was used as a fluorescent dye, platelets and reticulocytes could be classified in the same way as in Example 1, in which an acridine compound was used.

[0146] Example 3: Blood cell analysis using various fluorescent dyes Measurement samples were prepared using staining reagents containing fluorescent dyes different from those in Examples 1 and 2, and the reticulocytes and platelets in the measurement samples were classified using an analytical device. For comparison, analyses were also performed using staining reagents containing 2,7,9-trimethylacridine-3,6-diamine and 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium, respectively.

[0147] (1) Measurement sample For each of the above blood samples, platelet counts were obtained in advance by measurement using XN-20 (Sysmex Corporation) and analysis using the PLT-F channel. 3Blood samples with a platelet count of 0.01 mg / μL or less were selected. Among the selected blood samples, two blood samples were selected in which a population of impurities different from the platelet population was observed in the scattergram of the PLT-F channel (hereinafter, these are also referred to as "low PLT sample 1" and "low PLT sample 2" respectively). Measurement samples were prepared from these blood samples as described above. As fluorescent dyes of acridine compounds, 2,7,9-trimethylacridine-3,6-diamine, 3,6-diamino-2,7,10-trimethylacridinium, 3,6-diamino-2,7-dimethylacridinium, 3,6-diaminoacridine and 3,6-diamino-10-methylacridinium were used. As the fluorescent dyes of the cyanine compounds, 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzoxazolium, 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]benzothiazolium, 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolinylidene)methyl]-3H-indolium and 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]-6-methyl-benzothiazolium were used. As the staining reagent, an ethylene glycol solution of each fluorescent dye was 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 cyanine compound was 5.0 ppm.

[0148] (2) Analysis Each measurement sample was measured by an analyzer (a prototype modified from XN-20) to obtain the first scattered light information, the second scattered light information, and the fluorescence information. Each information was the same as that in Example 1. For the measurement samples prepared using 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzoxazolium, 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]benzothiazolium, 3,6-diaminoacridine, and 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolinylidene)methyl]-3H-indolium, the first scattergram (a) was created. For the measurement samples prepared using 2,7,9-trimethylacridine-3,6-diamine, 3,6-diamino-2,7,10-trimethylacridinium, 3,6-diamino-2,7-dimethylacridinium, 3,6-diamino-10-methylacridinium, and 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]-6-methyl-benzothiazolium, the first scattergram (b) was created. In the first scattergrams (a) and (b) of each measurement sample, blood cells other than white blood cells were selected and displayed in the second scattergram. Two types of second scattergrams, linear and logarithmic, were created. These scattergrams are shown in Figures 25A, 25B, 26A, 26B, 27A, and 27B.

[0149] FIG. 25A shows an example of a first scattergram (a) and a second scattergram based thereon, which shows the analysis results of a measurement sample prepared from a low PLT specimen 1 using 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium. Referring to the first scattergram (a) in FIG. 25A, a group of white blood cells appeared in an area with high V-SSC and high R-FSC. Referring to the second scattergram (linear display) in FIG. 25A, a group of mature red blood cells appeared in an area with low V-SFL, a group of platelets appeared in an area with low R-FSC, and a group of reticulocytes appeared in an area with high R-FSC. A group of impurities (debris) overlapped with a group of platelets. Referring to the second scattergram (logarithmic display) in FIG. 25A, the mature red blood cell group appeared in an area where the R-FSC was high and the V-SFL was low. The reticulocyte group appeared in an area where the R-FSC was similar to that of the mature red blood cell group and the V-SFL was higher than that of the mature red blood cell group. Although both the platelet group and the impurity group appeared in an area where the R-FSC was lower than that of the mature red blood cells and the reticulocyte group, the two groups were distinguishable. Thus, it was shown that the second scattergram in logarithmic display is suitable for classification and counting of platelets and for distinguishing between platelets and impurities.

[0150] FIG. 25B shows an example of the first scattergram (b) and a second scattergram based thereon, which shows the analysis result of a measurement sample prepared from a low PLT specimen 1 using 2,7,9-trimethylacridine-3,6-diamine. Referring to the first scattergram (b) in FIG. 25B, a group of white blood cells appeared in the region of high V-SFL. In the second scattergram (linear display) in FIG. 25B, the groups of mature red blood cells, reticulocytes, and platelets appeared, as in the second scattergram (linear display) in FIG. 25A. In the second scattergram (logarithmic display) in FIG. 25B, the groups of mature red blood cells, reticulocytes, platelets, and impurities appeared. The group of platelets and the group of impurities appeared at positions distant from each other, and both groups were distinguishable.

[0151] 26A and 26B show the first and second scattergrams of the measurement sample prepared from the low PLT sample 1 using the above 10 kinds of fluorescent dyes. FIG. 27A and 27B show the first and second scattergrams of the measurement sample prepared from the low PLT sample 2 using the above 10 kinds of fluorescent dyes. Referring to these figures, in the first scattergrams (a) and (b), it was possible to distinguish white blood cells from blood cells other than white blood cells, regardless of which fluorescent dye was used. In addition, in the second scattergram displayed in linear form, each group of mature red blood cells, reticulocytes, and platelets was observed. In the second scattergram displayed in logarithmic form, the group of platelets and the group of impurities appeared at positions separated from each other, and both groups were distinguishable. In this way, it was shown that reticulocytes and platelets can be classified using various fluorescent dyes of acridine compounds and cyanine compounds. It was also shown that platelets can be distinguished from impurities.

[0152] Example 4: Analysis of a measurement sample containing platelet aggregation A measurement sample was prepared from a blood specimen containing platelet aggregation, and platelet aggregation in the measurement sample was detected by an analyzer.

[0153] (1) Measurement sample Blood samples from healthy subjects and blood samples that were judged to be positive for platelet aggregation by visual observation under a microscope were selected (hereinafter, also referred to as "PLTc negative samples" and "PLTc positive samples", respectively). Measurement samples were prepared from these blood samples as described above. An ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used as the staining reagent. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0154] (2) Analysis Each measurement sample was measured by an analyzer (a prototype modified from XN-20) 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 were the same as in Example 1. The third scattered light information was the side scattered light intensity (hereinafter also referred to as "side scattered light intensity (red)" or "R-SSC") obtained by irradiating particles in the measurement sample with a red laser. For each measurement sample, a first scattergram (a) was created in the same manner as in Example 1. For comparison, a scattergram was created in which the horizontal axis represents the side scattered light intensity (red) and the vertical axis represents the forward scattered light intensity (red). Each scattergram is shown in (A) to (D) of FIG. 28. In the figure, "PLTc?" represents particles suspected to be platelet aggregates.

[0155] Referring to FIG. 28(A), in the scattergram of the comparison of the PLTc-negative sample, a platelet group could be identified, but the white blood cells and red blood cells appeared overlapping. Therefore, the white blood cells and red blood cells could not be classified. Also, referring to FIG. 28(B), in the scattergram of the comparison of the PLTc-positive sample, a particle group was observed that extended from the platelet group to the overlapping group of white blood cells and red blood cells. However, it was not possible to determine whether the particle group was red blood cells or platelet aggregates. Thus, it was difficult to detect platelet aggregates based on the side scattered light intensity and forward scattered light intensity obtained only by the red laser.

[0156] Referring to FIG. 28(C), in the first scattergram (a) of the PLTc-negative specimen, the white blood cell group, the red blood cell group, and the platelet group were clearly classified. Referring to FIG. 28(D), in the first scattergram (a) of the PLTc-positive specimen, the white blood cell group, the red blood cell group, and the platelet group were also clearly classified. Furthermore, in the scattergram (D), a particle group that was not observed in the scattergram (C) appeared in the area surrounded by the dashed line. Platelet aggregates are larger in size than normal platelets, and therefore have a high forward scattered light intensity. Therefore, the particle group in the area surrounded by the dashed line could be identified as platelet aggregates. In the scattergram (D), "PLTc?" was displayed as a flag indicating that particles suspected to be platelet aggregates appeared. In the first scattergram (a), the appearance of platelet aggregation was clearly confirmed by the appearance of a group of red blood cells on the lower V-SSC side compared to the comparative scattergram. Thus, it was demonstrated 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.

[0157] Example 5: Leukocyte classification and counting In Examples 1 to 3, blood cells other than white blood cells were selected in the first scattergram, but white blood cells were selected in the first scattergram in Example 5. Furthermore, the white blood cells were classified and counted.

[0158] (1) Measurement sample Measurement samples were prepared from healthy blood samples as described above. An ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used as the staining reagent. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0159] (2) Analysis Each measurement sample was measured by an analyzer (a prototype modified from XF-1600) 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 were the same as in Example 1. The third scattered light information was the same as in Example 4. In Example 5, the measurement time by the analyzer was extended to increase the number of particles analyzed. For each measurement sample, first scattergrams (a) and (b) were created in the same manner as in Example 1. These are shown in FIG. 29(A) and FIG. 30(A), respectively. As shown in these figures, in the first scattergrams (a) and (b), it was possible to distinguish between white blood cells and blood cells other than white blood cells. By counting the particles identified as white blood cells in the first scattergrams (a) and (b), it was possible to count the white blood cells.

[0160] Next, the leukocytes appearing in the first scattergram (a) and (b) were selected. The leukocytes selected from the first scattergram (a) were displayed on a scattergram (hereinafter also referred to as "third scattergram (a)") with the horizontal axis representing the side scattered light intensity (red) and the vertical axis representing the forward scattered light intensity (red). The leukocytes selected from the first scattergram (b) were displayed on a scattergram (hereinafter also referred to as "third scattergram (b)") with the horizontal axis representing the side scattered light intensity (red) and the vertical axis representing the fluorescence intensity (blue-purple). These are shown in FIG. 29(B) and FIG. 30(B), respectively. In the figures, "Gran" represents granulocytes, "Mono" represents monocytes, and "Lym" represents lymphocytes. It was shown that the leukocytes could be classified into subpopulations based on the side scattered light intensity and forward scattered light intensity obtained by only the red laser, and the granulocytes, monocytes, and lymphocytes could be counted by counting the particles contained in each subpopulation.

[0161] We investigated whether white blood cells could be classified into subpopulations in each first scattergram without selecting the white blood cells. Scattergrams in which the areas where white blood cells appeared in the first scattergrams (a) and (b) were enlarged are shown in FIG. 29(C) and FIG. 30(C). With reference to these figures, it was shown that white blood cells could be classified into subpopulations by enlarging the areas where white blood cells appeared in the first scattergrams (a) and (b). In addition, granulocytes, monocytes, and lymphocytes could be counted by counting the particles contained in each subpopulation.

[0162] Example 6: Leukocyte classification and counting The white blood cell counts counted in the first scattergrams (a) and (b) were compared with the white blood cell counts obtained by the conventional method to examine the correlation. In the conventional method, the measurement sample was measured using a multi-parameter automated blood cell analyzer XN-20 (Sysmex Corporation), and the white blood cells were counted by analysis in the WDF channel and WNR channel.

[0163] (1) Measurement sample Measurement samples were prepared from 24 samples, including normal and abnormal samples, as described above. The normal samples were blood samples from healthy individuals. The abnormal samples were blood samples with abnormalities in blood cell components (excluding samples with high levels of nucleated red blood cells). An ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used as the staining reagent. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm. Measurement samples analyzed by the WDF channel of the XN-20 were prepared by the XN-20 using each of the 24 samples described above, the staining reagent Fluorocell (registered trademark) WDF (Sysmex Corporation), and the hemolysis reagent Lysercell (registered trademark) WDF (Sysmex Corporation). The measurement samples to be analyzed using the WNR channel of the XN-20 were prepared using the XN-20 with each of the 24 specimens listed above, the staining reagent Fluorocell (registered trademark) WNR (Sysmex Corporation), and the hemolysis reagent Lysacell (registered trademark) WNR (Sysmex Corporation).

[0164] (2) Analysis Each measurement sample was measured by an analyzer (a prototype modified from XF-1600) to obtain first scattered light information, second scattered light information, and fluorescence information. Each piece of information was the same as in Example 1. For each measurement sample, first scattergrams (a) and (b) were created in the same manner as in Example 1. White blood cells were counted in each scattergram (a) and (b) in the same manner as in Example 5. In the conventional method, each measurement sample was measured by XN-20, and white blood cells were counted by the WDF channel and the WNR channel. Here, XN-20 was equipped with a semiconductor laser light source that emits red (639 nm) light, a detector for red forward scattered light, a detector for red side scattered light, and a detector for fluorescence (light of 660 nm or more) excited by red light. In the XN-20 measurements, the particles in the measurement sample were irradiated with a red laser to obtain the fluorescence intensity (hereinafter referred to as "fluorescence intensity (red)"), side scattered light intensity (red), and forward scattered light intensity (red). In the WDF channel, blood cells were analyzed based on the side scattered light intensity (red) and fluorescence intensity (red), and in the WNR channel, blood cells were analyzed based on the forward scattered light intensity (red) and fluorescence intensity (red).

[0165] 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 examined. The results are shown in FIG. 31. In addition, the correlation coefficient (r) between the first scattergrams (a) and (b) and the WDF channel and the WNR channel for the white blood cell count is shown in Table 1. As can be seen from FIG. 31 and Table 1, the white blood cell counts obtained from the first scattergrams (a) and (b) were found to have a good correlation with the white blood cell counts obtained from the WDF channel and the WNR channel. Although the blood cell analysis method of this embodiment does not use a hemolytic reagent, it was shown that this method can count white blood cells with the same accuracy as the conventional method using a hemolytic reagent.

[0166] [Table 1]

[0167] Example 7: Platelet Analysis The platelet population classified in the second scattergram was analyzed to obtain values ​​of various platelet-related indices.

[0168] (1) Measurement sample Measurement samples were prepared from healthy blood samples as described above. An ethylene glycol solution of 2,7,9-trimethylacridine-3,6-diamine was used as the staining reagent. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0169] (2) Analysis The measurement sample was measured using an analyzer (a prototype modified from XN-20) to obtain fluorescence information, first scattered light information, and second scattered light information. Each piece of information was the same as in Example 1. As in Example 1, a first scattergram (a) was created, and blood cells other than white blood cells were selected. These blood cells were displayed on a second scattergram (logarithmic display). An example of this second scattergram is shown in FIG. 32(A). Platelets were identified in the second scattergram. In addition, the particles identified as platelets were counted to obtain the platelet count. A group of platelets on the second scattergram was selected, and a histogram based on the forward scattered light intensity (red) and the number of particles was created. An example of this histogram is shown in FIG. 32(B).

[0170] With reference to (A) of Figure 32, the immature platelet fraction (IPF) and large platelet fraction (P-LCR) were obtained based on the platelet distribution in the second scattergram. With reference to (B) of Figure 32, the mean platelet volume (MPV) and platelet distribution width (PDW) were obtained based on the histogram. An example of the obtained values ​​of each index is shown in Table 2. In this way, it was demonstrated that the blood cell analysis method of this embodiment can obtain values ​​of various indexes related to platelets.

[0171] [Table 2] [Explanation of symbols]

[0172] 11 Blood cell analysis reagents 100 blood collection tube 200 suction tube 300 analytical units 400 measurement units 410, 411 Reagent container 420 Reaction Chamber 430 Waste chamber 500 Analysis System

Claims

1. acquiring first scattered light information, second scattered light information, and fluorescent light information generated by irradiating a measurement sample containing particles stained with a fluorescent dye with light of a first wavelength and light of a second wavelength; identifying blood cells other than white blood cells from among 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 fluorescent light information; classifying reticulocytes and platelets from blood cells other than white blood cells based on the fluorescent light information and the second scattered light information; wherein the first wavelength is 315 nm or more and 600 nm or less, and the second wavelength is 610 nm or more and 750 nm or less.

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

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

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

5. The method according to claim 4 , wherein in the identifying step, a scattergram is created based on the first side scattered light intensity and the forward scattered light intensity or the fluorescent light intensity, and blood cells other than the white blood cells are identified in the scattergram.

6. In the identifying step, particles in the measurement sample are classified into a first particle population and a second particle population based on the first side scattered light intensity and the forward scattered light intensity; the second particle population exhibits a first side scattered light intensity and / or a forward scattered light intensity higher than the first particle population, The method of claim 4 or 5, wherein the first particle population is identified as blood cells other than white blood cells.

7. In the step of identifying, the particles in the measurement sample are classified into a first particle population and a second particle population based on the first side scattered light intensity and the fluorescent light intensity; the second particle population is a population that exhibits a first side scattered light intensity and / or a fluorescence intensity higher than that of the first particle population, The method of claim 4 or 5, wherein the first particle population is identified as blood cells other than white blood cells.

8. the second scattered light information being an intensity of forward scattered light from the particle irradiated with light of the second wavelength, and the fluorescence information being an intensity of fluorescence from a fluorescent dye of the particle irradiated with light of the first wavelength, 2. The method according to claim 1, wherein in the classifying step, reticulocytes and platelets are classified from blood cells other than white blood cells based on the fluorescence intensity and the forward scattered light intensity.

9. 9. The method according to claim 8, wherein in the classifying step, a scattergram is created based on the fluorescence intensity and the forward scattered light intensity, and reticulocytes and platelets are classified from blood cells other than white blood cells in the scattergram.

10. In the classifying 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 fluorescent light intensity and the forward scattered light intensity; the third particle population is a population exhibiting a higher fluorescence intensity than the fifth particle population, the fourth particle population is a population that exhibits a lower forward scattered light intensity than the third particle population, 10. The method of claim 8 or 9, wherein the third particle population is identified as a population of reticulocytes and the fourth particle population is identified as a population of platelets.

11. The method of claim 10, wherein the fifth particle population is identified as a population of mature red blood cells.

12. In the identifying step, the leukocytes are further identified, The method of claim 1, further comprising the step of classifying the white blood cells into subpopulations.

13. 13. The method of claim 12, further comprising identifying and classifying white blood cells into subpopulations 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.

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

15. The method according to claim 14, further comprising creating a scattergram based on the first side scattered light intensity and the forward scattered light intensity, and / or creating a scattergram based on the first side scattered light intensity and the fluorescence intensity, identifying the white blood cells in the scattergram, and classifying the white blood cells into subpopulations.

16. In the acquiring step, third scattered light information is further acquired, the third scattered light information is information regarding side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength, identifying 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 fluorescent light information; 13. The method of claim 12, further comprising classifying the white blood cells into subpopulations 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 second scattered light information is an intensity of forward scattered light from the particle irradiated with light of the second wavelength, the third scattered light information is an intensity of second side scattered light from the particle irradiated with light of the second wavelength, and the fluorescence information is an intensity of fluorescence from a fluorescent dye of the particle irradiated with light of the first wavelength, 17. The method of claim 16, further comprising classifying the white blood cells into subpopulations based on the second side scattered light intensity and the forward scattered light intensity or the fluorescent light intensity.

18. The method according to claim 17, further comprising creating a scattergram based on the second side scattered light intensity and the forward scattered light intensity, and / or creating a scattergram based on the second side scattered light intensity and the fluorescence intensity, and classifying white blood cells into subpopulations in the scattergram.

19. 13. The method of claim 12, wherein the subpopulations include at least two of a lymphocyte population, a monocyte population, and a granulocyte population.

20. The method according to claim 1 , further comprising, in the step of identifying, obtaining information indicative of the presence or absence of platelet aggregation based on the first scattered light information and the second scattered light information.

21. the first scattered light information is an intensity of side scattered light from the particle irradiated with light of the first wavelength, and the second scattered light information is an intensity of forward scattered light from the particle irradiated with light of the second wavelength, In the step of identifying, blood cells other than white blood cells are identified based on the forward scattered light intensity and the side scattered light intensity; classifying the blood cells other than the white blood cells into a group containing red blood cells and a group containing platelets based on the forward scattered light intensity; 21. The method of claim 20, wherein information indicative of the presence of platelet aggregation is obtained when the platelet-containing population includes particles that exhibit a forward scattered light intensity above a threshold value.

22. In the identifying step, a scattergram is created based on the forward scattered light intensity and the side scattered light intensity, and particles in the measurement sample are identified in the scattergram as a group including red blood cells, a group including platelets, and white blood cells; 22. The method of claim 21, wherein information indicative of the presence of platelet aggregation is obtained when the platelet-containing population contains particles that exhibit a forward scattered light intensity above a threshold value.

23. 2. The method of claim 1, wherein in said step of sorting, said platelets are further sorted into mature platelets and immature platelets.

24. 2. The method of claim 1, wherein said classifying step further comprises identifying large platelets from said platelets.

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

26. 2. The method of claim 1, wherein the fluorescent dye is selected from the group consisting of an acridine compound, a cyanine compound, and a styryl compound.

27. The acridine compound has the following formula (I): 【Chemistry 1】 (In the formula, R 1 ~R 10 are independently a hydrogen atom, -NH 2 , -(CH 2 ) n -NR 11 R 12 , -NH-R 13 -NR 11 R 12 , -OR 11 , -COOH, halogen, a phenyl group which may have a substituent, an alkyl group having 1 to 18 carbon atoms, or an acylamino group, where R 3 , R 6 and R 9 At least one of is independently -NH 2 , -(CH 2 ) n -NR 11 R 12 , or -NH 2 is a phenyl group substituted with R 11 and R 12 are the same or different and each is a hydrogen atom or an alkyl group 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) The method according to claim 26, wherein the compound is represented by the formula:

28. The cyanine compound is represented by the following formula (II): 【Chemistry 2】 (In the formula, R 1 and R 2 are the same or different and each represents 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 and a hydroxyl group, an alkyl group having 1 to 18 carbon atoms and a carboxyl group, an alkyl group having 1 to 18 carbon atoms and a sulfo group, a halogen, -(CH 2 ) m -NR 6 R 7 , -(CH 2 ) n -OR 8 or an optionally substituted benzyl group, R 3 and R 4 are the same or different and each represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 18 carbon atoms, a phenyl group which may have a substituent, an alkoxy group having 1 to 6 carbon atoms, or -(CH 2 ) n -OR 8 and R 5 represents a hydrogen atom, a halogen, an alkyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms and a hydroxyl group, an alkyl group having 1 to 6 carbon atoms and a carboxyl group, an alkyl group having 1 to 6 carbon atoms and a sulfo group, a phenyl group which may have a substituent, a benzyl group which may have a substituent, -(CH 2 ) n -OR 6 and R 6 and R 7 are the same or different and each is a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, R 8 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, and n is an integer from 1 to 6; X and Y are the same or different and each represents a sulfur atom, an oxygen atom, a selenium atom, or a CR 9 R 10 and R 9 and R 10 are the same or different and each is an alkyl group having 1 to 3 carbon atoms, Z - is the counter ion) The method according to claim 26, wherein the compound is represented by the formula:

29. The styryl compound is represented by the following formula (III): 【Chemistry 3】 (In the formula, R 1 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 and a hydroxyl group, an alkyl group having 1 to 18 carbon atoms and a carboxyl group, an alkyl group having 1 to 18 carbon atoms and a sulfo group, a halogen, -(CH 2 ) p -NR 5 R 6 , -(CH 2 ) q -OR 7 or an optionally substituted benzyl group, R 2 and R 3 are the same or different and each represents a hydrogen atom, a halogen atom, 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】 and R 4 is a hydrogen atom, a halogen atom, an alkyl group having 1 to 18 carbon atoms, a phenyl group which may have a substituent, an alkoxy group having 1 to 6 carbon atoms, or -(CH 2 ) m -OR 7 and R 5 and R 6 are the same or different and each is a hydrogen atom or an alkyl group having 1 to 18 carbon atoms, R 7 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 CR 8 R 9 and R 8 and R 9 are the same or different and each is an alkyl group having 1 to 3 carbon atoms, m is an integer from 1 to 18; Z - is the counter ion) or a compound represented by the following formula (IV): 【Chemistry 5】 (In the formula, R 1 and Z - is as above, R 2 and R 3 are the same or different and are a hydrogen atom, a halogen atom, 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, or R 2 and / or R 3 forms a heterocycle containing N together with the benzene ring to which N is bonded, n is an integer from 0 to 2. The method according to claim 26, wherein the compound is represented by the formula:

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

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

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