Blood cell analysis method, blood cell analysis reagent, blood cell analysis reagent kit, and method of fluorescently staining blood cells

Abstract

To provide means of performing blood cell analysis using a reagent that can be commonly used for classification of white blood cells and classification of reticulocytes and platelets, and can be used for blood cell analysis using multiple types of light.SOLUTION: The above challenge is cleared by staining blood cells in a sample using a cyanine-based fluorescent dye that can be excited by light of a wavelength in a range of 315 nm to 600 nm, inclusive, and can bind to nucleic acids.SELECTED DRAWING: Figure 18

Description

[Technical field]

[0001] The present invention relates to a method for analyzing blood cells, a reagent for analyzing blood cells, a reagent kit for analyzing blood cells, and a method for fluorescently staining blood cells. [Background technology]

[0002] Blood contains various blood cell components such as red blood cells, white blood cells, and platelets. Analysis of these blood cells can provide information useful for clinical diagnosis of various diseases. A method using an automated blood cell analyzer is known for analyzing blood cells. In analysis using an automated blood cell analyzer, it is common to measure samples prepared using dedicated reagents corresponding to the types of blood cells. However, from the viewpoint of simplifying the test and the equipment, analysis using reagents that can be commonly used for multiple types of blood cells is desired. For example, Patent Document 1 describes that white blood cells are classified and counted using one type of fluorescent dye and a hemolysis reagent, and that reticulocytes and platelets are classified and counted using the same fluorescent dye and a dilution reagent. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2022 / 100717 Summary of the Invention [Problem to be solved by the invention]

[0004] In recent years, a method for analyzing blood cells by irradiating them with multiple lights of different wavelengths using an automated blood cell analyzer equipped with multiple light sources has become known. Therefore, a blood cell analysis method using a reagent that can be used for such blood cell analysis using multiple lights is desired. The present invention aims to provide a blood cell analysis method using a reagent that can be used in common for classifying white blood cells and reticulocytes and platelets, and that can be used for blood cell analysis using multiple lights. [Means for solving the problem]

[0005] The present inventors have discovered that a cyanine fluorescent dye that can be excited by light with a wavelength of 315 nm or more and 600 nm or less and that can bind to nucleic acids can be used in common for classifying white blood cells and for classifying reticulocytes and platelets, and can also be used in blood cell analysis using multiple types of light, and have completed the inventions described in the following [1] to

[19] .

[0006] [1] A step of irradiating a measurement sample containing particles stained with a fluorescent dye with light to acquire fluorescent information and scattered light information generated by the particles; classifying blood cells from particles in the measurement sample based on the fluorescence information and the scattered light information; wherein the fluorescent dye has the following formula (I):

[0007] [ka]

[0008] (In the formula, R1 and R2 are the same or different and each represents a halogen, 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, -(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) m -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) A blood cell analysis method represented by the formula:

[0009] [2] The method according to [1] above, wherein in the formula (I), when one of X and Y is an oxygen atom, the remaining one is a sulfur atom, a selenium atom, or C(CH3)2.

[0010] [3] The method according to [1] or [2] above, wherein the fluorescent dye is at least one selected from the group consisting of 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]-6-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-methyl-2-[(1,3,3-trimethyl-2-indolinylidene)methyl]benzothiazolium and 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolinylidene)methyl]-3H-indolium.

[0011] [4] The measurement sample is prepared by mixing the fluorescent dye, a hemolysis reagent, and whole blood; The method according to any one of the above-mentioned [1] to [3], wherein in the classifying step, leukocytes in the measurement sample are classified into subpopulations.

[0012] [5] The light has a first wavelength of 315 nm or more and 600 nm or less, the fluorescence information is information about fluorescence generated from a fluorescent dye of the particle by irradiating the measurement sample with light of the first wavelength, The method according to [4] above, wherein the scattered light information is information regarding side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength.

[0013] [6] The light has a first wavelength of 315 nm or more and 600 nm or less and a second wavelength of 610 nm or more and 750 nm or less, the fluorescence information is information about fluorescence generated from a fluorescent dye of the particle by irradiating the measurement sample with light of the first wavelength, The method according to [4] above, wherein the scattered light information is information regarding side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength, and information regarding side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.

[0014] [7] The fluorescence information is a fluorescence intensity, and the scattered light information is a side scattered light intensity, The method according to [5] or [6] above, wherein in the classifying step, the leukocytes in the measurement sample are classified into subpopulations based on the fluorescence intensity and the side scattered light intensity.

[0015] [8] The method according to [7] above, further comprising creating a scattergram based on the fluorescence intensity and the side scattered light intensity, and classifying the leukocytes into subpopulations in the scattergram.

[0016] [9] The method according to [8] above, wherein the subpopulations include a population of lymphocytes, a population of monocytes and a population of granulocytes.

[0017]

[10] The method according to [8] above, wherein the subpopulations include a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population and a basophil population.

[0018]

[11] The measurement sample is prepared by mixing the fluorescent dye, a dilution reagent, and whole blood; The method according to any one of the above-mentioned [1] to [3], wherein in the classifying step, reticulocytes and platelets are classified from particles in the measurement sample.

[0019]

[12] The method according to

[11] above, wherein the measurement sample does not contain a hemolytic reagent.

[0020]

[13] The light has a first wavelength of 315 nm or more and 600 nm or less and a second wavelength of 610 nm or more and 750 nm or less, the fluorescence information is information about fluorescence generated from a fluorescent dye of the particle by irradiating the measurement sample with light of the first wavelength, The method according to

[11] or

[12] above, wherein the scattered light information is information about forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.

[0021]

[14] The fluorescence information is fluorescence intensity, and the scattered light information is forward scattered light intensity, The method according to claim 13, wherein in the classifying step, reticulocytes, platelets and mature red blood cells are classified from the particles in the measurement sample based on the fluorescence intensity and the forward scattered light intensity.

[0022]

[15] The method according to

[14] above, further comprising creating a scattergram based on the fluorescence intensity and the forward scattered light intensity, and classifying particles in the measurement sample in the scattergram into a group of reticulocytes, a group of platelets, and a group of mature red blood cells.

[0023]

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

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

[0024]

[17] A reagent for blood cell analysis which contains the fluorescent dye represented by the formula (I) and is used in the method according to any one of the above [1] to

[16] .

[0025]

[18] A reagent kit for blood cell analysis, comprising a staining reagent containing the fluorescent dye represented by the formula (I) and a hemolysis reagent, for use in the method according to any one of the above [1] to

[16] .

[0026]

[19] The following formula (I):

[0027] [ka]

[0028] (In the formula, R1 and R2 are the same or different and each represents a halogen, 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, -(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) m -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) A method for fluorescently staining blood cells, comprising contacting blood cells with a fluorescent dye represented by the formula: Effect of the Invention

[0029] According to the present invention, there is provided a blood cell analysis method using a fluorescent dye that can be commonly used for classifying white blood cells and for classifying platelets and reticulocytes, and that can also be used for blood cell analysis using a plurality of lights. There are also provided a blood cell analysis reagent and a blood cell analysis reagent kit for use in this method. There is also provided a method for fluorescently staining blood cells using the above-mentioned fluorescent dye. [Brief description of the drawings]

[0030] [Figure 1] 1 is a perspective view showing an analysis system suitable for the sample 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] 4 is a flowchart showing the procedure of a measurement process according to the first embodiment. [Figure 9]10 is a flowchart showing the procedure of a measurement process according to a second embodiment.In the drawing, "WBC" stands for white blood cells. [Figure 10] This is the absorption spectrum of the fluorescent dye in ethanol. [Figure 11A] FIG. 2 is a diagram showing an example of a blood cell analysis reagent according to the present embodiment. [Figure 11B] FIG. 1 is a diagram showing an example of a blood cell analysis reagent kit according to an embodiment of the present invention. [Figure 12] 4 is a flowchart showing a procedure of an analysis process according to the first embodiment. [Figure 13] (A) and (C) are schematic diagrams illustrating the positions where each subpopulation of leukocytes appears in a first scattergram (a) with the horizontal axis representing the first side scattered light (SSC-1) intensity and the vertical axis representing the first fluorescent light (SFL-1) intensity. (B) and (D) are schematic diagrams illustrating the positions where each subpopulation of leukocytes appears in a first scattergram (b) with the horizontal axis representing the second side scattered light (SSC-2) intensity and the vertical axis representing the SFL-1 intensity. Granulocytes are a general term for neutrophils, eosinophils, and basophils. [Figure 14] 13 is a flowchart showing a procedure of an analysis process according to a second embodiment. [Figure 15A] (A) is a schematic diagram illustrating the positions where the RET, mRBC, and PLT populations appear in a second scattergram (logarithmic display) with the horizontal axis representing the logarithm of the first fluorescent 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 the positions where the RET, mRBC, and PLT 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 15B] (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 16]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 17] (A) is a schematic diagram illustrating an example of a region where immature platelets appear in the second scattergram (logarithmic display), and (B) is a schematic diagram illustrating an example of a region where large platelets appear in the second scattergram (logarithmic display). [Figure 18] 1 shows a scattergram of Comparative Example 1 and first scattergrams (a) and (b) of Example 1. [Figure 19] 1A and 1B are first scattergrams of Example 1. [Figure 20] Scattergrams of Comparative Examples 2 and 3 and a second scattergram of Example 2. [Figure 21] 2 is a second scattergram of Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] (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.

[0032] The measurement unit 400 is a unit for measuring a specimen, and includes a flow cytometer. In the measurement unit 400, a measurement specimen is prepared by mixing a specimen with a reagent. When classifying white blood cells in a specimen, a staining reagent and a hemolysis reagent are used as reagents in the preparation of the measurement specimen. When classifying reticulocytes and platelets in a specimen, a staining reagent and a dilution reagent are used as reagents in the preparation of the measurement specimen. The staining reagent includes a fluorescent dye for staining particles. The hemolysis reagent includes a surfactant and a solvent. The dilution reagent includes a buffer and a solvent. Details of the specimen, the staining reagent, the hemolysis 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. Erythrocyte ghosts can also be caused by hemolysis caused by factors other than the hemolysis reagent, such as physical impact on the blood collection tube, low pH and low osmotic pressure of the measurement sample.

[0033] 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.

[0034] 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 reagent containers 410, 411, and 414, a reaction chamber 420, and a waste liquid chamber 430. For example, the reagent container 410 contains a dilution reagent, the reagent container 411 contains a hemolysis reagent, and the reagent container 414 contains a staining reagent. The reagent containers 410, 411, and 414 are each connected to the reaction chamber 420 by a liquid supply tube. The sample preparation section 440 injects the dilution reagent and the staining reagent into the reaction chamber 420 through each liquid supply tube. 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 the FCM detection unit 460 via a liquid transfer tube.

[0035] 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 hemolysis reagent or a dilution reagent are mixed to prepare a measurement sample. The measurement sample in the reaction chamber 420 is supplied to 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.

[0036] 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.

[0037] 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. 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. For example, a semiconductor laser light source can be used as the first and second light sources.

[0038] 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.

[0039] 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.

[0040] The fluorescent dye represented by the above formula (I) is excited by a first wavelength. Side fluorescence (first side fluorescence) corresponding to the light generated by the excitation of the fluorescent dye is transmitted through the dichroic mirror 418b and received by the side fluorescent light receiving element 422a. In the blood cell analysis method of this embodiment, a fluorescent dye excited by a second wavelength is not used, 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 the light generated by the 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 fluorescent light receiving element 422b. The side fluorescent light receiving elements 422a and 422b are, for example, avalanche photodiodes.

[0041] 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.

[0042] 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).

[0043] 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.

[0044] The optical information may be the above-mentioned characteristic parameters. The optical information includes fluorescence information and 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.

[0045] As the scattered light information, information on a plurality of types of scattered light, for example, first scattered light information, second scattered light information, and third scattered light information may be acquired. For example, the first scattered light information may be information on side scattered light generated from particles by irradiating the measurement sample with light of a first wavelength. Also, the second scattered light information may be information on side scattered light generated from particles by irradiating the measurement sample with light of a second wavelength. Also, the third scattered light information may be information on forward scattered light generated from particles by irradiating the measurement sample with light of the second wavelength. The information on 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 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 side scattered light signal are preferable, and the peak value of the second side scattered light signal is more preferable. As the third 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. In this specification, the peak value of the first side scattered light signal is also called the "first side scattered light intensity", the peak value of the second side scattered light signal is also called the "second side scattered light intensity", and the peak value of the second forward scattered light signal is also called the "second forward 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 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. 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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 under hemolytic conditions is measured, and white blood cells in the measurement sample are classified. In the "second embodiment," the measurement process and analysis process differ depending on the measurement target specified by the user. When white blood cells are specified, the same measurement process and analysis process as in the first embodiment are performed. When reticulocytes and platelets are specified, a measurement sample prepared under non-hemolytic conditions is measured, and reticulocytes and platelets are classified from the particles in the measurement sample.

[0050] (Measurement process of the first embodiment) Regarding step S12 in FIG. 7, an example of the measurement process of the first embodiment 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 under hemolysis conditions. Specifically, the measurement unit 400 mixes the specimen, the staining reagent, and the hemolysis reagent in the reaction chamber 420 to prepare a 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.

[0051] (Measurement process of the second embodiment) Regarding step S12 in FIG. 7, an example of the measurement process of the second embodiment will be described with reference to FIG. 9, but is not limited to this example. In step S31, the control unit 301 of the analysis unit 300 accepts the designation of the measurement target from the user via the operation unit 306. The measurement target is a type of blood cell to be analyzed, for example, white blood cells, reticulocytes, and platelets. The input of the designation of the measurement target can be received based on a selection screen of the measurement target displayed on the display unit 305, for example. Specifically, the control unit 301 displays a selection screen for designating the measurement target on the display unit 305. The selection screen allows the user to designate white blood cells, or reticulocytes and platelets, as the measurement target. After the designation of the measurement target, the measurement is performed by the user deciding to start the measurement displayed on the screen. In step S31, if the control unit 301 accepts the designation of white blood cells as the measurement target, in step S32, the control unit 301 causes the measurement unit 400 to prepare a measurement sample from the specimen under hemolysis conditions. The measurement sample under hemolytic conditions is prepared as described above. When the control unit 301 receives a designation of reticulocytes and platelets as the measurement targets in step S31, the control unit 301 causes the measurement unit 400 to prepare a measurement sample from the specimen under non-hemolytic conditions in step S33. Specifically, the measurement unit 400 mixes the specimen, the staining reagent, and the dilution reagent in the reaction chamber 420 to prepare a measurement sample. Under non-hemolytic conditions, no hemolytic reagent is used. Steps S34 and S35 are the same as steps S22 and S23, respectively. After the measurement process is completed, the analysis unit 300 executes the analysis process of step S13 in FIG. 7. Examples of the analysis process of each embodiment will be described later.

[0052] (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.

[0053] 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 normally 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 "RBC" may be used to include mature red blood cells and reticulocytes. "Reticulocytes" are young red blood cells immediately after erythroblast cells in the bone marrow are denucleated and released into the peripheral blood. 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 present in mature erythrocytes. In this specification, the term "platelets" includes immature platelets and large platelets. "Immature platelets", also called reticulated platelets, are young platelets immediately after being released into peripheral blood. Mature platelets do not contain nucleic acid, but 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.

[0054] "Abnormal cells" refer to formed elements that do not normally appear in blood. Examples of abnormal cells include immature granulocytes. Immature granulocytes are precursor cells of granulocytes, and include promyelocytes, myelocytes, and metamyelocytes. Immature granulocytes are usually present in bone marrow, but may appear in blood due to myeloid leukemia, bone metastasis of cancer, severe infections, and other causes. When immature granulocytes may be contained in a blood sample, the term "white blood cells" also includes immature granulocytes. 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 aggregates occur in blood collection tubes due to contamination of tissue fluid during blood collection, insufficient mixing, or the action of EDTA.

[0055] (Hemolysis Reagent) The hemolytic reagent used in the preparation of the measurement sample will be described. The hemolytic reagent is a reagent for dissolving red blood cells in a specimen by mixing with the specimen. The hemolytic reagent contains a surfactant and is used in combination with a staining reagent containing a fluorescent dye. The surfactant can hemolyze the red blood cells in the specimen and can damage the cell membranes of cells other than the red blood cells to an extent that the fluorescent dye can penetrate the cell membranes. Examples of surfactants include nonionic surfactants, cationic surfactants, and combinations thereof. The hemolytic reagent preferably contains a nonionic surfactant.

[0056] An example of the nonionic surfactant is one represented by the following formula (II): R1-R2-(CH2CH2O) n -H (II) (In the formula, R1 is an alkyl group, an alkenyl group, or an alkynyl group having 8 to 25 carbon atoms; R2 is an oxygen atom, -(COO)-, or a group represented by the following formula (III):

[0057] [ka] and n is an integer between 10 and 50.

[0058] The concentration of the nonionic surfactant represented by formula (II) in the hemolysis reagent is, for example, 0.5 g / L or more and 5 g / L or less. Specific examples of the nonionic surfactant represented by formula (II) include polyoxyethylene alkyl ether, polyoxyethylene sterol, polyoxyethylene castor oil, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene alkylamine, polyoxyethylene polyoxypropylene alkyl ether, and combinations thereof. Among them, polyoxyethylene alkyl ether is preferable. The nonionic surfactant contained in the hemolysis reagent may be one type or two or more types. In addition, the hemolysis reagent may further contain a nonionic surfactant other than the nonionic surfactant represented by formula (II).

[0059] The hemolysis reagent may further contain a cationic surfactant. Examples of the cationic surfactant include quaternary ammonium salt surfactants, pyridium salt surfactants, and combinations thereof. Examples of the quaternary ammonium salt surfactant include surfactants represented by the following formula (IV) having a total carbon number of 9 to 30. The cationic surfactant contained in the hemolysis reagent may be one type, or two or more types.

[0060] [ka]

[0061] In formula (IV), R1 is an alkyl or alkenyl group having 6 to 18 carbon atoms; R2 and R3 are the same or different and are an alkyl or alkenyl group having 1 to 4 carbon atoms; R4 is an alkyl or alkenyl group having 1 to 4 carbon atoms or a benzyl group; and X - is a halogen ion. A "halogen" is fluorine, chlorine, bromine, or iodine.

[0062] In formula (IV), R1 is preferably an alkyl group or alkenyl group having 6, 8, 10, 12, or 14 carbon atoms, and is particularly preferably a linear alkyl group. More specific examples of R1 include an octyl group, a decyl group, and a dodecyl group. R2 and R3 may be the same or different, and are preferably a methyl group, an ethyl group, or a propyl group. R4 is preferably a methyl group, an ethyl group, or a propyl group.

[0063] An example of the pyridinium salt surfactant is a surfactant represented by the formula (V).

[0064] [ka]

[0065] In formula (V), R1 is an alkyl or alkenyl group having 6 to 18 carbon atoms; - is a halogen ion.

[0066] In formula (V), R1 is preferably an alkyl or alkenyl group having 6, 8, 10, 12 or 14 carbon atoms, and is particularly preferably a linear alkyl group. More specific examples of R1 include an octyl group, a decyl group and a dodecyl group.

[0067] The concentration of the cationic surfactant in the hemolysis reagent can be appropriately selected depending on the type of surfactant, and is, for example, 10 ppm or more and 10,000 ppm or less.

[0068] The hemolysis reagent may contain a buffer to keep the pH constant. Examples of the buffer include inorganic acid salts, organic acid salts, Good's buffers, and combinations thereof. Examples of the inorganic acid salts include phosphate, borate, and combinations thereof. Examples of the organic acid salts include citrate, malate, and combinations thereof. Examples of the Good's buffer include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricine, Tris, Bicine, TAPS, and combinations thereof.

[0069] The hemolysis reagent may further contain an aromatic organic acid. The concentration of the aromatic organic acid in the hemolysis reagent is not particularly limited. In this specification, the aromatic organic acid means an acid having at least one aromatic ring in the molecule and its salt. Examples of the aromatic organic acid include aromatic carboxylic acids and aromatic sulfonic acids. Examples of the aromatic carboxylic acids include phthalic acid, benzoic acid, salicylic acid, hippuric acid, salts thereof, and combinations thereof. Examples of the aromatic sulfonic acids include p-aminobenzenesulfonic acid, benzenesulfonic acid, salts thereof, and combinations thereof. The aromatic organic acid contained in the hemolysis reagent may be one type or two or more types. In addition, the aromatic organic acid may exhibit a buffering effect. When an aromatic organic acid exhibiting a buffering effect is used, the addition of a buffering agent is optional, and it may be combined with the above-mentioned buffering agent.

[0070] The hemolysis reagent is preferably a liquid reagent. The solvent is not particularly limited as long as it can dissolve each component such as the surfactant. Examples of the solvent include water, organic solvents, and mixtures thereof. Examples of the organic solvent include solvents that are miscible with water, such as alcohols having 1 to 6 carbon atoms, ethylene glycol, diethylene glycol, polyethylene glycol, and DMSO.

[0071] The pH of the hemolysis reagent is not particularly limited. The pH of the hemolysis reagent is, for example, 4.5 or more, and preferably 5 or more. The pH of the hemolysis reagent is, for example, 11 or less, and preferably 10 or less. A known base (such as sodium hydroxide) or acid (such as hydrochloric acid) can be used to adjust the pH.

[0072] In the hemolysis reagent, the osmotic pressure is not particularly limited, but from the viewpoint of the efficiency of hemolysis of red blood cells, it is preferably 150 mOsm / kg·H2O or less. To adjust the osmotic pressure, an appropriate osmotic pressure regulator may be added. Examples of the osmotic pressure regulator include sugars, amino acids, organic solvents, sodium chloride, and combinations thereof.

[0073] The hemolytic reagent may be a commercially available hemolytic reagent for measuring blood cells, such as Lyzacell WDF (Sysmex Corporation) or Lyzacell WDFII (Sysmex Corporation).

[0074] (Diluted Reagent) The dilution reagent used in the preparation of the measurement sample will be described. 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 buffers, 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.

[0075] 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 alkali metal salts of organic acids such as propionic acid, sugars such as glucose and mannose, alkali metal halides such as sodium chloride, and alkaline earth metal halides 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 preferably added to the diluted reagent so that the osmotic pressure of the diluted reagent is 150 mOsm / kg·H2O or more and 600 mOsm / kg·H2O or less.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] Preservatives contained in the dilution reagent include, for example, sodium 2-pyridylthio-1-oxide, β-phenethyl alcohol, and the like.

[0080] In the blood cell analysis method of this embodiment, a commercially available dilution reagent may be used, such as Cellpack (registered trademark) DFL (Sysmex Corporation).

[0081] (Staining reagents and fluorescent dyes) The staining reagent used in the preparation of the measurement sample will be described. The fluorescent dye suitable for the staining reagent is the fluorescent dye represented by the above formula (I). This fluorescent dye is a cyanine fluorescent dye that can be excited by light of the first wavelength and can bind to nucleic acid. The fluorescent dye binds to nucleic acid, making 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. In addition, since the maximum absorption wavelength of the fluorescent dye represented by the above formula (I) is within the range of the first wavelength, interference between the fluorescence from the fluorescent dye and scattered light corresponding to the light of the second wavelength is suppressed.

[0082] The "alkyl group" may be either linear or branched. Regarding R1 to R4 and R6 to R8 in formula (I), 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. Regarding R5 in formula (I), the number of carbon atoms in the alkyl group is preferably 1 to 3.

[0083] The "alkenyl group" may be either linear or branched. Regarding R1 and R2 in the above formula (I), 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.

[0084] Examples of the substituent of the phenyl group include -NH2, halogen, -OH, -SH, -CN, -NO2, -COOH, and an alkyl group having 1 to 6 carbon atoms. Examples of the substituent of the benzyl group include -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, a halogen, and a haloalkyl group having 1 to 6 carbon atoms. A "haloalkyl group" is an alkyl group in which at least one hydrogen atom is substituted with a halogen. When a haloalkyl group contains two or more halogens, the halogens may be the same or may contain multiple types.

[0085] In the above formula (I), when one of X and Y is an oxygen atom, the remaining one is preferably a sulfur atom, a selenium atom, or C(CH3)2. - For example, F - , Cl - , Br - , I - , CF3SO3 - , BF4 - , ClO4 - etc.

[0086] Examples of the fluorescent dye represented by the above formula (I) 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. zolium, 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. The absorption spectra of these fluorescent dyes in ethanol are shown in Figure 10. The maximum absorption wavelengths of these fluorescent dyes in ethanol are also shown in Table 1.

[0087] [Table 1]

[0088] Preferred fluorescent dyes are 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]-6-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-methyl-2-[(1,3,3-trimethyl-2-indolinylidene)methyl]benzothiazolium and 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolinylidene)methyl]-3H-indolium.

[0089] 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 can be, for example, 100,000 ppm or less, preferably 50,000 ppm or less, and more preferably 10,000 ppm or less.

[0090] 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.

[0091] 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. 11A, 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.

[0092] The staining reagent containing the above-mentioned fluorescent dye and the above-mentioned hemolytic reagent may be provided as a reagent kit for blood cell analysis used in the blood cell analysis method of this embodiment. The reagent kit for blood cell analysis of this embodiment may be in a form in which, for example, the above-mentioned fluorescent dye solution and the above-mentioned hemolytic reagent are each contained in a container. With reference to FIG. 11B, 21 indicates the reagent kit for blood cell analysis of this embodiment, 22 indicates a first container containing a fluorescent dye solution, and 23 indicates a second container containing a hemolytic reagent. The reagent kit for blood cell analysis of this embodiment may be contained in a packaging box. When the reagent kit 21 is contained in a packaging box, the above-mentioned package insert, the above-mentioned buffer material, etc. may also be contained. The reagent kit for blood cell analysis of this embodiment may further include the above-mentioned dilution reagent, calibrator, etc.

[0093] A further embodiment relates to the use of the fluorescent dye for the manufacture of a reagent for blood cell analysis. The details of the fluorescent dye and the reagent for blood cell analysis are as described above. A further embodiment relates to the use of the fluorescent dye and the hemolytic reagent for the manufacture of a reagent kit for blood cell analysis. The details of the fluorescent dye, the hemolytic reagent, and the reagent kit for blood cell analysis are as described above.

[0094] (Preparation of measurement samples under hemolytic conditions) The fluorescent dye represented by the above formula (I) is mixed with the specimen and the hemolysis 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 1000 ppm or less, preferably 100 ppm or less, more preferably 10 ppm or less. The preferred final concentration of the fluorescent dye in the measurement sample is 0.001 ppm or more, more preferably 0.01 ppm or more, and even more preferably 0.1 ppm or more.

[0095] The mixing ratio of the hemolysis 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 hemolysis reagent, the staining reagent, and the specimen is preferably 1000:1 or more:1 or more, for example, expressed as a volume ratio. More preferably, it is 1000:10 or more:10 or more, and even more preferably, it is 1000:15 or more:15 or more. Furthermore, the mixing ratio of the hemolysis reagent, the staining reagent, and the specimen is preferably 1000:50 or less:50 or less, for example, expressed as a volume ratio. More preferably, it is 1000:30 or less:30 or less, and even more preferably, it is 1000:25 or less:25 or less. The mixing ratio of the staining reagent and the specimen may be the same or different.

[0096] Red blood cells are the most abundant blood cells in blood samples. Since the hemolysis reagent dissolves red blood cells, the influence of red blood cells can be excluded in the analysis of blood cells that are not lysed by the hemolysis reagent (e.g. white blood cells). In addition, the action of the surfactant contained in the hemolysis reagent promotes the staining of white blood cells with the fluorescent dye. Thus, preparation of the measurement sample under hemolysis conditions is suitable for the analysis of white blood cells.

[0097] (Preparation of measurement samples under non-hemolytic conditions) The fluorescent dye represented by the above formula (I) is mixed with the specimen and the dilution reagent so that the final concentration is within a predetermined range. The preferred final concentration of the fluorescent dye in the measurement sample is 500 ppm or less, preferably 100 ppm or less, more preferably 50 ppm or less. The preferred final concentration of the fluorescent dye in the measurement sample is 0.01 ppm or more, preferably 0.1 ppm or more, more preferably 1 ppm or more.

[0098] 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.

[0099] Since reticulocytes are dissolved by a hemolytic reagent in the same manner as mature red blood cells, preparation of a measurement sample under non-hemolytic conditions is suitable for analysis of reticulocytes. Conventionally, reticulocytes and platelets could be classified and counted by a single measurement, but the number of platelets obtained was not accurate depending on the specimen. Therefore, in order to count platelets more accurately, a separate measurement was performed under conditions suitable for measuring platelets. On the other hand, in the blood cell analysis method of the present embodiment, reticulocytes and platelets can be classified and counted more accurately by a single measurement of a measurement sample prepared under non-hemolytic conditions. Thus, preparation of a measurement sample under non-hemolytic conditions is suitable for analysis of reticulocytes and platelets. In classification of reticulocytes and platelets by the blood cell analysis method of the present embodiment, the measurement sample does not contain a hemolytic reagent. That is, the preparation of the measurement sample does not include a process of hemolyzing red blood cells.

[0100] A further embodiment relates to a method for fluorescently staining blood cells, comprising contacting blood cells with a fluorescent dye represented by the above formula (I). In this method, blood cells can be stained with the fluorescent dye by mixing the fluorescent dye with a specimen containing blood cells. In this method, the above staining reagent may be used as the fluorescent dye. In addition, when mixing the fluorescent dye with the specimen, the above hemolytic reagent or dilution reagent may be further mixed. The concentrations and mixing ratios of the fluorescent dye and each reagent in the mixing are the same as those described for the preparation of the measurement sample above. The details of the fluorescent dye, specimen, hemolytic reagent, dilution reagent, and measurement sample are as described above.

[0101] (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 Fig. 12 and Fig. 13, but is not limited to this example. White blood cells are also called "WBC", mature red blood cells are also called "mRBC", and reticulocytes are also called "RET".

[0102] With reference to FIG. 12, in the analysis process of the first embodiment, WBCs in a measurement sample are classified into subpopulations. In the first embodiment, the measurement sample is prepared using a hemolysis reagent, and therefore mRBCs and RETs in the measurement sample are hemolyzed to become red blood cell ghosts. The classification of WBCs into subpopulations is performed by identifying each subpopulation of WBCs from particles in the measurement sample based on the fluorescence information and the first scattered light information, and / or based on the fluorescence information and the second scattered light information. In this example, the first side fluorescence intensity (also referred to as "SFL-1 intensity") is used as the fluorescence information, the first side scattered light intensity (also referred to as "SSC-1 intensity") is used as the first scattered light information, and the second side scattered light intensity (also referred to as "SSC-2 intensity") is used as the second scattered light information.

[0103] Classification of WBCs into subpopulations can be performed, for example, by analysis using a scattergram. A scattergram for classifying WBCs into subpopulations based on the fluorescence information and the first scattered light information is also called a "first scattergram (a)". A scattergram for classifying WBCs into subpopulations based on the fluorescence information and the second scattered light information is also called a "first scattergram (b)". In the analysis process of the first embodiment, either the first scattergram (a) or (b) is created. Alternatively, both the first scattergrams (a) and (b) may be created.

[0104] Referring to FIG. 12, in step S101, the analysis unit 300 creates a first scattergram (a) based on the acquired optical information. The first scattergram (a) is a scattergram with SSC-1 intensity and SFL-1 intensity on two axes. In step S101, the analysis unit 300 may create a first scattergram (b) based on the acquired optical information. The first scattergram (b) is a scattergram with SSC-2 intensity and SFL-1 intensity on two axes. The horizontal and vertical axes of the first scattergrams (a) and (b) may be displayed either logarithmically or linearly. Preferably, the horizontal axes of the first scattergrams (a) and (b) are displayed logarithmically, and the vertical axes are displayed linearly. In step S101, the positions of points corresponding to each particle in the measurement sample are determined in the created first scattergram (a). When the first scattergram (b) is created, the positions of points in the first scattergram (b) corresponding to each particle in the measurement sample are determined.

[0105] In step S102, the analysis unit 300 identifies each subpopulation of white blood cells from the particles in the measurement sample based on the determined SFL-1 intensity and the SSC-1 intensity or SSC-2 intensity of each point. At least a lymphocyte population, a monocyte population, and a granulocyte population are identified as the subpopulations. As described above, granulocytes include neutrophils, eosinophils, and basophils. If it is possible to classify granulocytes, at least one of a neutrophil population, an eosinophil population, and a basophil population is further identified. Preferably, a lymphocyte population, a monocyte population, a neutrophil population, and an eosinophil population are identified from the particles in the measurement sample. More preferably, a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population are identified from the particles in the measurement sample. If the measurement sample contains immature granulocytes, in addition to each subpopulation of white blood cells, an immature granulocyte population may be identified. The algorithm for identifying each subpopulation of white blood cells is publicly known. For example, white blood cells can be classified into subpopulations by a program stored in the memory unit 303 of the analysis unit 300. In addition, since the positions where each subpopulation of white blood cells appears on a scattergram are publicly known, each subpopulation of white blood cells may be identified based on the scattergram. FIG. 13 shows an example in which white blood cells are classified into three or five subpopulations in the first scattergrams (a) and (b). In addition, in each scattergram, the area surrounded by a dashed line indicates the area where immature granulocytes appear. In the figure, "Lymp" is a group of lymphocytes, "Mono" is a group of monocytes, "Gran" is a group of granulocytes, "Neut" is a group of neutrophils, "Eo" is a group of eosinophils, and "Baso" is a group of basophils. In addition, "IG" represents immature granulocytes.

[0106] Referring to (A) and (B) of FIG. 13, three subpopulations of lymphocytes, monocytes, and granulocytes are distributed on the first scattergrams (a) and (b) as subpopulations of white blood cells. In addition, debris such as red blood cell ghosts appear in the region with low SFL-1 intensity. As can be seen from these figures, in both the first scattergrams (a) and (b), white blood cells can be classified into three subpopulations of lymphocytes, monocytes, and granulocytes. When immature granulocytes are contained in the measurement sample, the immature granulocytes appear in a region with higher SFL-1 intensity than the granulocyte population and higher SSC-1 intensity or SSC-2 intensity than the monocyte population, as shown by the dashed lines.

[0107] When granulocytes can be classified into subpopulations, white blood cells can be classified into, for example, five subpopulations. With reference to (C) and (D) of FIG. 13, in both the first scattergrams (a) and (b), five subpopulations of white blood cells, namely, lymphocytes, monocytes, neutrophils, eosinophils, and basophils, are distributed on the first scattergrams (a) and (b). When immature granulocytes are contained in the measurement sample, the immature granulocytes appear in an area having a higher fluorescence intensity than the neutrophil group and a higher SSC-1 intensity or SSC-2 intensity than the monocyte group, as shown by the dashed line. In the example of FIG. 13, white blood cells are classified into three or five subpopulations, but are not limited thereto. White blood cells may be classified into four subpopulations, namely, lymphocytes, monocytes, neutrophils, and eosinophils. If necessary, the cells contained in each subpopulation of white blood cells may be counted. The cell count is performed by the analysis unit 300. After performing the white blood cell classification, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG.

[0108] (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. 14 to 17, but is not limited to this example. In this specification, platelets are also called "PLT". With reference to FIG. 14, when the control unit 301 of the analysis unit 300 accepts the designation of RET and PLT as the measurement target in step S201, the process proceeds to step S202. In this case, since the measurement sample is prepared without using a hemolysis reagent, the measurement sample includes mRBC and RET. The measurement sample also includes WBC and PLT. In this analysis process, a RET group and a PLT group are classified from the particles in the measurement sample. As a result, RET and PLT are identified. Alternatively, a RET group, a PLT group, and a mRBC group may be classified from the particles in the measurement sample. As a result, RET, PLT, and RBC are identified. In this example, the classification of particles in a measurement sample into RET, PLT, and RBC populations based on the fluorescence information and the third scattered light information will be described below. As the fluorescence information, SFL-1 intensity is preferable. As the third scattered light information, second forward scattered light intensity (also called "FSC-2 intensity") is preferable.

[0109] The classification of particles in the measurement sample can be performed, for example, by analysis using a scattergram. A scattergram for classifying the RET group and the PLT group from the particles in the measurement sample based on the fluorescence information and the third scattered light information is also called a "second scattergram". In this example, the SFL-1 intensity is used as the fluorescence information, and the FSC-2 intensity is used as the third scattered light information. The second scattergram is created, for example, based on the SFL-1 intensity and the FSC-2 intensity. With reference to FIG. 14, in step S202, 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 position of the point corresponding to the particle in the measurement sample is determined. The horizontal and vertical axes of the second scattergram may be displayed either logarithmically or linearly. 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 RBC analysis.

[0110] In step S203, the analysis unit 300 classifies blood cells other than WBC into a RET group, a PLT group, and an mRBC group based on the SFL-1 intensity and FSC-2 intensity. The RET group is a group that shows a higher SFL-1 intensity than the mRBC group. The PLT group is a group that shows a lower FSC-2 intensity than the RET group. Here, the comparison of the fluorescence intensity and scattered light intensity between particle groups in the scattergram may be performed, for example, by a statistical representative value of the fluorescence intensity and scattered light intensity of each particle group. The statistical representative value of the fluorescence intensity and scattered light intensity of one particle group is a value obtained from the fluorescence intensity and scattered light intensity of the particles that constitute the particle group. Examples of the statistical representative value include the median, the average value, and the mode. The median is preferable.

[0111] FIG. 15A shows examples of the second scattergram (logarithmic display) and the second scattergram (linear display). In each of the second scattergrams, a RET group, a PLT group, and a mRBC group appear. In the second scattergram (logarithmic display), the RET and mRBC groups appear in an area with high FSC-2 intensity, and the PLT group appears in an area with low FSC-2 intensity. In the second scattergram (linear display), the PLT group appears in an area with low FSC-2 intensity, and the RET group appears in an area with higher FSC-2 intensity than the PLT group. In both scattergrams, the RET group appears in an area with higher SFL-1 intensity than the mRBC group. The following will explain how the particles in the measurement sample are classified into a RET group, a mRBC group, and a PLT group in the second scattergram.

[0112] 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, the particles in the measurement sample are classified into a RET population, a PLT population, and an mRBC population.

[0113] Referring to FIG. 14, in step S203, the analysis unit 300 identifies a RET population, a PLT population, and an mRBC population from particles in the measurement sample. Since RET becomes mRBC when it matures, it is difficult to strictly determine the boundary between the RET population and the mRBC population. For example, a threshold value of SFL-1 intensity for separating the mRBC population from the RET population may be set based on the range of SFL-1 intensity that mRBC can normally exhibit, the average particle number of mRBC, etc. Such a threshold value may be set as the boundary between the mRBC population and the RET population on the second scattergram. Then, based on the threshold value and the FSC-2 intensity, regions in which mRBC and RET appear may be set, for example, as the region indicated by the dashed line in FIG. 15B. Particles in the set region in which mRBC appears can be identified as mRBC. Also, particles in the set region in which RET appears can be identified as RET. For the PLT population, for example, when a population that appears in the region on the high FSC-2 intensity side and a population that appears in the region on the low FSC-2 intensity side are clearly separated on the second scattergram (logarithmic display), the user may specify the population that appears in the region on the low FSC-2 intensity side as PLT. Alternatively, by accumulating measurement data on multiple samples, the regions in which the RET, PLT, and mRBC populations appear in the second scattergram may be determined in advance, and the particles that appear in each region may be specified as RET, PLT, and mRBC, respectively. If necessary, the specified RET, PLT, and RBC may be counted. The cell count is performed by the analysis unit 300.

[0114] With reference to FIG. 14, the process proceeds to step S204. In step S204, 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. With reference to FIG. 16, in the second scattergram (linear display), the region where the RET appears can be divided into three regions, LFR, MFR, and HFR, based on the SFL-1 intensity. The RET appearing in each region may be counted. The RET appearing in the HFR region has the greatest amount of nucleic acid, and is therefore the most immature RET.

[0115] 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.

[0116] Referring to FIG. 14, the process proceeds to step S205. In this step, the PLT population is further analyzed. In step S205, 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 showing high SFL-1 intensity can be identified as immature platelets. For example, as shown in FIG. 17(A), PLTs appearing 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.

[0117] 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. 17B, 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 normal-sized PLTs. This allows the group of PLTs to be classified into large platelets and normal-sized platelets. 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. After performing the analysis of the PLTs, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG. 7.

[0118] 14, in step S201, when the analysis unit 300 control unit 301 accepts the designation of WBC as the measurement target, the process proceeds to step S101 in FIG 12. In this case, the analysis process (classification of WBC) of the first embodiment is executed. After executing the classification of WBC, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG 7.

[0119] With reference to Fig. 7, when the above analysis process is completed, in step S14, control unit 301 outputs the analysis result to display unit 305, and ends the process. As described above, the process for analyzing white blood cells in a measurement sample has been described using Figs. 12 and 13. Also, the process for analyzing reticulocytes and platelets in a measurement sample has been described using Figs. 14 to 17. However, it is sufficient that only a part of the analysis process is performed, or all of the analysis process may be performed.

[0120] The analysis result screen output to the display unit 305 may include, for example, the number of each subpopulation of white blood cells, the reticulocyte count, the platelet count, the particle count in each region of the LFR, MFR and HFR, the IPF, the P-LCR, etc. 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.

[0121] In this way, the blood cell analysis method of the present embodiment can provide medical personnel such as doctors and laboratory technicians with information on the number of each subpopulation of white blood cells contained in a sample as a result of white blood cell analysis. In addition, information on the number of reticulocytes and platelets contained in a sample as a result of reticulocyte and platelet analysis can be provided. Furthermore, analysis results on red blood cells can also be provided. From the obtained analysis results, medical personnel can decide, for example, whether or not 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.

[0122] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0123] Example 1: Classification of leukocyte subpopulations By using the blood cell analysis method of the present embodiment, which uses various fluorescent dyes excited by light of a first wavelength (315 nm or more and 600 nm or less), white blood cells in a blood sample were classified into subpopulations. In addition, the results of classification of white blood cells by a conventional method were compared.

[0124] (1) Blood samples Whole blood (peripheral blood) was collected from multiple subjects using EDTA-2K blood collection tubes and stored at room temperature (25±2°C). Whole blood was collected from the blood collection tubes and used as blood samples. In Example 1, blood samples that were determined to contain 2% or more immature granulocytes and 2% or more reticulocytes by analysis using a multi-item automated blood cell analyzer XR-20 (Sysmex Corporation) were used.

[0125] (2) Reagents As a hemolysis reagent, Lyzacel (registered trademark) WDFII (Sysmex Corporation) was used. The staining reagent was prepared by dissolving various fluorescent dyes in special grade ethylene glycol (1 L). The fluorescent dyes used were 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium, 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolinylidene)methyl]-3H-indolium, 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzoxazolium, 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]-6-methyl-benzothiazolium, and 3-ethyl-6-methyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]benzothiazolium. For comparison, Fluorocell (registered trademark) WDF (Sysmex Corporation) was used. Fluorocell WDF was a staining reagent containing a fluorescent dye excited by light of the second wavelength (610 nm or more and 750 nm or less). The measurement sample prepared using Fluorocell WDF and Lysercell WDFII is hereinafter also referred to as "Comparative Example 1."

[0126] (3) Analyzer A prototype modified from a flow cytometer XF-1600 (Sysmex Corporation) was used as the analysis device. The prototype was manufactured as follows. The XF-1600 was equipped with three semiconductor laser light sources emitting blue-violet (405 nm), blue (488 nm) and red (639 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 emitting blue (488 nm) light was removed from this XF-1600, and it was modified to an FCM system equipped with two semiconductor laser light sources. The prototype was equipped with the hardware shown in Figures 1 to 3, and the FCM detection unit had the configuration shown in Figure 4. For the measurement of Comparative Example 1, a multi-item automatic blood cell analyzer XR-20 (Sysmex Corporation) was used. The XR-20 was equipped with a semiconductor laser light source emitting 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 longer) excited by red light.

[0127] (4) Measurement method Except for using the above staining reagent, the preparation and measurement of the measurement sample were performed according to the manual attached to the XF-1600. The measurement sample was prepared by mixing 1000 μL of LyzaCell (registered trademark) WDFII, 17 μL of whole blood, and 0.5 to 2 μL of staining reagent. The final concentration of the fluorescent dye in the measurement sample was 0.1 to 0.5 ppm. The measurement sample was measured after incubation at 41° C. for 25 seconds. Data analysis was performed using software installed in the analyzer. Comparative Example 1 was measured using XR-20 (Sysmex Corporation) and analyzed using the XR-WDF channel.

[0128] (5) Analysis Each measurement sample was measured by an analysis device to obtain fluorescence information, first scattered light information, and second scattered light information. The fluorescence information was the fluorescence intensity obtained by irradiating particles in the measurement sample with a blue-violet laser (hereinafter also referred to as "fluorescence intensity (blue-violet)" or "V-SFL"). For Comparative Example 1, the fluorescence information was the fluorescence intensity obtained by irradiating particles in the measurement sample with a red laser (hereinafter also referred to as "fluorescence intensity (red)" or "R-SFL"). The first scattered light information was the side scattered light intensity obtained by irradiating particles in the measurement sample with a blue-violet laser (hereinafter also referred to as "side scattered light intensity (blue-violet)" or "V-SSC"). The second scattered light information was the side scattered light intensity obtained by irradiating particles in the measurement sample with a red laser (hereinafter also referred to as "side scattered light intensity (red)" or "R-SSC").

[0129] For each measurement sample, a scattergram (hereinafter also referred to as "first scattergram (a)") was created with the side scattered light intensity (blue-purple) on the horizontal axis and the fluorescence intensity (blue-purple) on the vertical axis. Also, for each measurement sample, a scattergram (hereinafter also referred to as "first scattergram (b)") was created with the side scattered light intensity (red) on the horizontal axis and the fluorescence intensity (blue-purple) on the vertical axis. For Comparative Example 1, a scattergram was created with the side scattered light intensity (red) on the horizontal axis and the fluorescence intensity (red) on the vertical axis. Each scattergram is shown in Figures 18 and 19. Here, the analysis result of Comparative Example 1 is the classification result of white blood cells by the conventional method. In Figure 18, "Lymp" represents lymphocytes, "Mono" represents monocytes, "Neut" represents neutrophils, "Eo" represents eosinophils, "Baso" represents basophils, and "IG" represents immature granulocytes.

[0130] Referring to FIG. 18, in the scattergram of Comparative Example 1, the white blood cells were classified into five subpopulations, namely, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Furthermore, a population of immature granulocytes was observed in an area with a higher fluorescence intensity than the population of neutrophils. Referring to FIGS. 18 and 19, in the analysis using any of the above-mentioned fluorescent dyes, the white blood cells were classified into five subpopulations in the first scattergrams (a) and (b). Furthermore, in any of the scattergrams, a population of immature granulocytes was observed in an area with a higher fluorescence intensity than the population of neutrophils and a higher side scattered light intensity than the population of monocytes. Thus, it was shown that the blood cell analysis method of this embodiment can classify white blood cells in the same way as the conventional method. Furthermore, it was shown that the white blood cells and each subpopulation can be counted by counting the particles contained in each classified population.

[0131] Example 2: Classification of reticulocytes and platelets The blood cell analysis method of the present embodiment using various fluorescent dyes excited by light of the first wavelength was used to classify reticulocytes and platelets in a blood sample, and the classification results of reticulocytes and platelets were compared with those of a conventional method.

[0132] (1) Blood samples and reagents In Example 2, the same blood sample as in Example 1 was used. Cellpack (registered trademark) DFL (Sysmex Corporation) was used as the dilution reagent. The staining reagent was prepared in the same manner as in Example 1. The fluorescent dye used was the same as in Example 1. For comparison, Fluorocell (registered trademark) RET (Sysmex Corporation) and Fluorocell (registered trademark) PLT (Sysmex Corporation) were used. Fluorocell RET and Fluorocell PLT were staining reagents containing a fluorescent dye excited by light of a second wavelength (610 nm or more and 750 nm or less). The measurement sample prepared using Fluorocell RET and Cellpack DFL is also referred to as "Comparative Example 2" below. The measurement sample prepared using Fluorocell PLT and Cellpack DFL is also referred to as "Comparative Example 3" below.

[0133] (2) Analytical equipment and measurement methods The analytical device used was a prototype modified from XF-1600 (Sysmex Corporation) as in Example 1. XR-20 (Sysmex Corporation) was used for the measurements in Comparative Examples 2 and 3. Except for the use of Cellpack DFL and the above-mentioned staining reagent, the preparation and measurement of the measurement sample were performed according to the manual attached to the XF-1600. The measurement sample was prepared by mixing 1000 μL of Cellpack DFL, 17 μL of whole blood, and 20 μL of staining reagent. The final concentration of the fluorescent dye in the measurement sample was 51.25 ppm. The measurement sample was measured after incubation at 41° C. for 30 seconds.

[0134] (3) Analysis Each measurement sample was measured by an analyzer to obtain fluorescence information and third scattered light information. The fluorescence information was fluorescence intensity (blue-purple). For Comparative Examples 2 and 3, fluorescence intensity (red) was obtained. The third scattered light information was 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. For each measurement sample, a scattergram (hereinafter also referred to as "second scattergram") was created with the fluorescence intensity (blue-purple) on the horizontal axis and the forward scattered light intensity (red) on the vertical axis. Two types of second scattergrams were created, with the horizontal axis and the vertical axis displayed as linear and logarithmic. Each scattergram is shown in Figures 20 and 21. Here, the analysis result of Comparative Example 2 is the classification result of reticulocytes by the conventional method, and the analysis result of Comparative Example 3 is the classification result of platelets by the conventional method.

[0135] With reference to FIG. 20, in the scattergram of Comparative Example 2, the mature red blood cell group appeared in the region where the R-SFL was low and the R-FSC was high. The reticulocyte group appeared on the side where the R-SFL was higher than that of the mature red blood cell group. The platelet group appeared in the region where the R-FSC was low. Thus, in the scattergram of Comparative Example 2, the mature red blood cells, reticulocytes, and platelets were clearly classified. With reference to FIGS. 20 and 21, in the analysis using any of the above fluorescent dyes, the mature red blood cells, reticulocytes, and platelets were clearly classified in the second scattergram displayed in a linear manner. Thus, it was shown that the blood cell analysis method of this embodiment can classify mature red blood cells and reticulocytes in the same way as the conventional method.

[0136] With reference to FIG. 20, in the scattergram of Comparative Example 3, the platelet population appeared in the region with low R-FSC and R-SFL. Thus, in the scattergram of Comparative Example 3, platelets were clearly classified from particles in the measurement sample. With reference to FIGS. 20 and 21, in the analysis using any of the above-mentioned fluorescent dyes, the mature red blood cell and reticulocyte populations appeared in the region with high R-FSC in the logarithmic second scattergram. Furthermore, the platelet population appeared in the region with lower R-FSC than the mature red blood cell and reticulocyte populations. The mature red blood cell and reticulocyte populations were clearly distinguishable from the platelet population. Thus, it was shown that the blood cell analysis method of this embodiment is capable of classifying platelets in the same manner as the conventional method. [Explanation of symbols]

[0137] 11 Blood cell analysis reagents 21 Blood cell analysis reagent kit 22 First container containing a staining reagent 23 Second container containing hemolysis reagent 100 blood collection tube 200 Suction tube 300 analytical units 400 measurement units 410, 411, 414 Reagent containers 420 Reaction Chamber 430 Waste chamber 500 Analysis System

Claims

1. A step of irradiating a measurement sample containing particles stained with a fluorescent dye with light to acquire fluorescent information and scattered light information generated from the particles; classifying blood cells from particles in the measurement sample based on the fluorescence information and the scattered light information; wherein the fluorescent dye has the following formula (I): 【Chemistry 1】 (In the formula, R 1 and R 2 are the same or different and each represents a halogen, 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, -(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 ) m -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) A blood cell analysis method represented by the formula:

2. In the formula (I), when one of X and Y is an oxygen atom, the other is a sulfur atom, a selenium atom, or a C(CH 3 ) 2 The method according to claim 1, wherein

3. 2. The method according to claim 1, wherein the fluorescent dye is at least one selected from the group consisting of 3-ethyl-2-[(3-ethyl-2-benzothiazolinylidene)methyl]benzothiazolium, 3-ethyl-2-[(3-ethyl-6-methyl-2-benzothiazolinylidene)methyl]-6-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-methyl-2-[(1,3,3-trimethyl-2-indolinylidene)methyl]benzothiazolium, and 1,3,3-trimethyl-2-[(3-methyl-2-benzoxazolinylidene)methyl]-3H-indolium.

4. the measurement sample is prepared by mixing the fluorescent dye, a hemolysis reagent, and whole blood; The method according to claim 1 , wherein in the classifying step, the leukocytes in the measurement sample are classified into subpopulations.

5. the light has a first wavelength of 315 nm or more and 600 nm or less, the fluorescence information is information about fluorescence generated from a fluorescent dye of the particle by irradiating the measurement sample with light of the first wavelength, The method according to claim 4 , wherein the scattered light information is information regarding side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength.

6. the light has a first wavelength of 315 nm or more and 600 nm or less and a second wavelength of 610 nm or more and 750 nm or less, the fluorescence information is information about fluorescence generated from a fluorescent dye of the particle by irradiating the measurement sample with light of the first wavelength, The method according to claim 4, wherein the scattered light information is information regarding side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength, and information regarding side scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.

7. the fluorescence information is a fluorescence intensity, the scattered light information is a side scattered light intensity, The method according to claim 5 or 6, wherein in the classifying step, the leukocytes in the measurement sample are classified into subpopulations based on the fluorescence intensity and the side scattered light intensity.

8. The method according to claim 7, further comprising creating a scattergram based on the fluorescence intensity and the side scattered light intensity, and classifying the white blood cells into subpopulations in the scattergram.

9. The method of claim 8, wherein the subpopulations include a population of lymphocytes, a population of monocytes and a population of granulocytes.

10. 9. The method of claim 8, wherein the subpopulations include a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population.

11. the measurement sample is prepared by mixing the fluorescent dye, a dilution reagent, and whole blood; The method according to claim 1 , wherein in the classifying step, reticulocytes and platelets are classified from particles in the measurement sample.

12. The method according to claim 11 , wherein the measurement sample does not contain a hemolysis reagent.

13. the light has a first wavelength of 315 nm or more and 600 nm or less and a second wavelength of 610 nm or more and 750 nm or less, the fluorescence information is information about fluorescence generated from a fluorescent dye of the particle by irradiating the measurement sample with light of the first wavelength, The method according to claim 11 , wherein the scattered light information is information about forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.

14. the fluorescence information is a fluorescence intensity, the scattered light information is a forward scattered light intensity, The method according to claim 13, wherein in the classifying step, reticulocytes, platelets and mature red blood cells are classified from the particles in the measurement sample based on the fluorescence intensity and the forward scattered light intensity.

15. The method according to claim 14, further comprising creating a scattergram based on the fluorescence intensity and the forward scattered light intensity, and classifying particles in the measurement sample in the scattergram into a reticulocyte population, a platelet population, and a mature red blood cell population.

16. 14. The method of claim 13, wherein the first wavelength is greater than or equal to 315 nm and less than or equal to 490 nm.

17. A reagent for blood cell analysis used in the method according to claim 1, comprising the fluorescent dye represented by formula (I).

18. A reagent kit for blood cell analysis for use in the method according to claim 1, comprising a staining reagent containing the fluorescent dye represented by formula (I) and a hemolysis reagent.

19. The following formula (I): 【Chemistry 2】 (In the formula, R 1 and R 2 are the same or different and each represents a halogen, 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, -(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 ) m -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) A method for fluorescently staining blood cells, comprising contacting blood cells with a fluorescent dye represented by the formula:

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