Platelet aggregation detection method

Abstract

To provide novel means that enables detection of platelet aggregation.SOLUTION: The above challenge is cleared by detecting platelet aggregation on the basis of first scattered light information and second scattered light information generated by irradiating blood cells in a sample with light of a wavelength in a range of 315 nm to 600 nm, inclusive, and light of a wavelength in a range of 610 nm to 750 nm, inclusive.SELECTED DRAWING: Figure 16

Description

[Technical field]

[0001] The present invention relates to a method for detecting platelet aggregation. [Background technology]

[0002] Blood contains various blood cell components such as red blood cells, white blood cells, and platelets. Among these, platelets are anucleated cells with a diameter of 2 to 3 μm, and are present at 150,000 to 350,000 pieces / μL in normal blood. However, it is known that the platelet count decreases in the blood of patients with idiopathic thrombocytopenic purpura or acute leukemia. In general, when the platelet count in blood falls below 10,000 pieces / μL, it is determined that a blood transfusion is necessary. Therefore, accurate classification and counting of platelets is important in clinical practice.

[0003] Platelet aggregation may occur in blood collection tubes due to contamination by tissue fluid, insufficient mixing, or the effects of EDTA. Platelet aggregation is different in size from individual platelets, so when blood is analyzed using an automated blood cell analyzer, platelet aggregation is not counted as platelets. Therefore, blood in which platelet aggregation has occurred will have an apparent decrease in the platelet count. This phenomenon is called pseudothrombocytopenia and can lead to misdiagnosis. If pseudothrombocytopenia is suspected, blood should be drawn again and blood cell analysis should be performed again.

[0004] Patent Document 1 describes a method for measuring platelet aggregation. In Patent Document 1, red blood cells in a blood sample are lysed with a hemolysis reagent, and the remaining blood cells are stained with a fluorescent dye and analyzed with an automatic blood cell analyzer. Then, platelet aggregation is identified and counted from a scattergram based on the scattered light intensity and the fluorescent light intensity. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] US Patent Application Publication No. 2020 / 0132589 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a novel means which allows the detection of platelet aggregation. [Means for solving the problem]

[0007] The inventors have found that platelet aggregation can be detected based on first scattered light information and second scattered light information generated by irradiating blood cells in a sample with light having a wavelength of 315 nm or more and 600 nm or less and light having a wavelength of 610 nm or more and 750 nm or less, and have completed the inventions described below in [1] to

[13] .

[0008] [1] acquiring first scattered light information and second scattered light information generated by irradiating a measurement sample containing particles with light of a first wavelength and light of a second wavelength; obtaining information indicative of the presence or absence of platelet aggregation based on the first scattered light information and the second scattered light information; wherein the first wavelength is equal to or greater than 315 nm and equal to or less than 600 nm, and the second wavelength is equal to or greater than 610 nm and equal to or less than 750 nm.

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

[0010] [3] The method according to [1] or [2] above, wherein the measurement sample is a sample prepared by mixing whole blood with a dilution reagent.

[0011] [4] The method according to any one of the above [1] to [3], wherein the measurement sample does not contain a hemolysis reagent or a fluorescent dye.

[0012] [5] The method further includes a step of preparing the measurement sample by mixing whole blood with a dilution reagent before the step of acquiring the first scattered light information and the second scattered light information; The method according to any one of [1] to [4] above, wherein the preparation step does not include a step of hemolyzing red blood cells contained in the measurement sample, and does not include a step of staining blood cells contained in the measurement sample with a fluorescent dye.

[0013] [6] The information about the side scattered light is a side scattered light intensity, and the information about the forward scattered light is a forward scattered light intensity, In the step of acquiring information indicating the presence or absence of platelet aggregation, particles in the measurement sample are classified into a group containing platelets and a group containing blood cells other than platelets based on the side scattered light intensity and the forward scattered light intensity; The method according to any one of the above [2] to [5], wherein, when the population containing platelets contains particles exhibiting a forward scattered light intensity equal to or greater than a threshold, information indicating the presence of platelet aggregation is obtained.

[0014] [7] The method according to [6] above, further comprising a step of classifying the group containing blood cells other than platelets into a group containing red blood cells and a group containing white blood cells based on the side scattered light intensity and / or the forward scattered light intensity.

[0015] [8] The method according to [6] or [7] above, wherein in the step of acquiring information indicating the presence or absence of platelet aggregation, a scattergram is created based on the side scattered light intensity and the forward scattered light intensity, and a group containing the platelets is identified in the scattergram.

[0016] [9] The information about the side scattered light is a side scattered light intensity, and the information about the forward scattered light is a forward scattered light intensity, The step of acquiring information indicating the presence or absence of platelet aggregation comprises: classifying particles in the measurement sample into a group including white blood cells and a group including blood cells other than white blood cells based on the side scattered light intensity and the forward scattered light intensity; classifying the group containing blood cells other than white blood cells into a group containing platelets and a group containing red blood cells based on the forward scattered light intensity; Including, The method according to any one of the above [2] to [5], wherein, when the population containing platelets contains particles exhibiting a forward scattered light intensity equal to or greater than a threshold, information indicating the presence of platelet aggregation is obtained.

[0017]

[10] The method according to [9] above, wherein in the step of acquiring information indicating the presence or absence of platelet aggregation, a scattergram is created based on the side scattered light intensity and the forward scattered light intensity, and a group containing the platelets is identified in the scattergram.

[0018]

[11] The information about the side scattered light is a side scattered light intensity, and the information about the forward scattered light is a forward scattered light intensity, The method according to any one of the above-mentioned [2] to [5], wherein in the step of acquiring information indicating the presence or absence of platelet aggregation, a scattergram is created based on the forward scattered light intensity and the side scattered light intensity, and a group of particles appearing in a predetermined region in the scattergram is detected as platelet aggregation.

[0019]

[12] The method according to

[11] above, wherein in the step of acquiring information indicating the presence or absence of platelet aggregation, a scattergram is created based on the forward scattered light intensity and the side scattered light intensity, and at least one selected from the group consisting of white blood cells, platelets, and red blood cells is classified in the scattergram.

[0020]

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

[12] , wherein the first wavelength is 315 nm or more and 490 nm or less. Effect of the Invention

[0021] According to the present invention, there is provided a method for detecting platelet aggregation and a reagent for blood cell analysis used in the method. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a perspective view showing an analysis system suitable for the method for detecting platelet aggregation of the present embodiment. [Diagram 2] FIG. 2 is a diagram showing a fluid circuit including a specimen aspiration section, a sample preparation section, and a flow cytometer (FCM) detection section in a measurement unit. [Diagram 3] FIG. 2 is a block diagram showing a configuration of a measurement unit. [Figure 4] 4 is a schematic diagram showing an example of the configuration of an optical system of an FCM detection unit. FIG. [Diagram 5] FIG. 2 is a schematic diagram showing that various types of light emitted when light is irradiated onto particles passing through a flow cell are received by the optical system of the FCM detection unit. [Figure 6] FIG. 2 is a block diagram showing a configuration of an analysis unit. [Figure 7] 4 is a flowchart showing the flow of operations of the analysis system. [Figure 8] 13 is a flowchart showing a procedure of a measurement process. [Figure 9] FIG. 2 is a diagram showing an example of a blood cell analysis reagent according to the present embodiment. [Figure 10] 4 is a flowchart showing a procedure of an analysis process according to the first embodiment. [Figure 11] (A) is a schematic diagram illustrating the positions where a group containing PLTs and a group containing blood cells other than PLTs appear in a scattergram with the horizontal axis representing the first side scattered light (SSC-1) intensity and the vertical axis representing the second forward scattered light (FSC-2) intensity. (B) is a schematic diagram illustrating the positions where platelet aggregates appear in the scattergram of (A). "PLTc?" is a particle suspected of being a platelet aggregate. [Figure 12] 13 is a flowchart showing a procedure of an analysis process according to a second embodiment. [Figure 13] (A) is a schematic diagram illustrating the positions where a group containing WBCs and a group containing blood cells other than WBCs appear in a scattergram with SSC-1 intensity on the horizontal axis and FSC-2 intensity on the vertical axis. (B) is a schematic diagram illustrating the positions where platelet aggregates appear in the scattergram of (A). [Figure 14] 13 is a flowchart showing a procedure of an analysis process according to a third embodiment. [Figure 15] FIG. 1 is a schematic diagram illustrating the positions at which a population containing RBCs, a population containing WBCs, a population containing PLTs, and platelet aggregates appear in a scattergram with SSC-1 intensity on the horizontal axis and FSC-2 intensity on the vertical axis. [Figure 16] 1 shows scattergrams of a platelet aggregation (PLTc) negative sample and a PLTc positive sample 1 in Example 1. [Figure 17] Scattergrams of PLTc-positive specimens 2 and 3 in Example 1. [Figure 18] Scattergrams of PLTc-positive specimens 4 and 5 in Example 1. [Figure 19] 1 shows scattergrams of a measurement sample containing no fluorescent dye and a measurement sample containing a fluorescent dye in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] (Analysis System) First, an analysis system suitable for the method for detecting platelet aggregation 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.

[0024] The measurement unit 400 is a unit for measuring a specimen, and includes a flow cytometer. In the measurement unit 400, a specimen is mixed with a reagent to prepare a measurement specimen. A dilution reagent is used as a reagent for preparing the measurement specimen. The dilution reagent includes a buffer and a solvent. The specimen and the dilution reagent will be described in detail later. In the analysis of particles in a specimen by this analysis system, particles in a measurement specimen prepared from a specimen are analyzed. "Particles in a measurement specimen" refers 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 a measurement specimen include cells, red blood cell ghosts, platelet aggregates, lipid particles, fungi, bacteria, and the like contained in the specimen. The term "cells" includes various blood cells and platelets. Red blood cell ghosts refer to remnants of hemolyzed red blood cells. Red blood cell ghosts can also be generated by hemolysis caused by something other than a hemolyzing reagent, such as physical impact on a blood collection tube, or low pH and low osmotic pressure of a measurement specimen.

[0025] The measurement sample is measured by the FCM detection section 460 (see FIG. 2) of the measurement unit 400, which will be described later. By the measurement, an optical signal related to scattered light emitted from dyed particles in the measurement sample is obtained. Although the platelet aggregation detection method of this embodiment does not use a fluorescent dye, when the particles are dyed with a fluorescent dye, an optical signal related to the fluorescence emitted from the fluorescent dye of the particles is also obtained by the measurement. The "fluorochrome of the particle" refers to a fluorescent dye that is 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 by the flow cytometer as a unit. The acquired optical signal is A / D converted to obtain digital data. The analysis unit 300 analyzes the digital data acquired by the measurement unit 400 to identify and / or classify the particles in the measurement sample.

[0026] 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 suction section 450, and an FCM detection section 460. The sample preparation section 440 includes a reagent container 410, a reagent container 411, a reaction chamber 420, and a waste liquid chamber 430. For example, the reagent container 410 contains a diluted reagent. Although the method for detecting platelet aggregation of this embodiment does not use a staining reagent, the staining reagent may be contained in the reagent container 411. Here, the staining reagent is a reagent containing a fluorescent dye for staining particles in a specimen and a solvent thereof. The staining reagent itself is known and is commercially available. The reagent container 410 and the reagent container 411 are each connected to the reaction chamber 420 by a liquid supply tube. The sample preparation section 440 injects the diluted reagent into the reaction chamber 420 through the liquid supply tube. When fluorescently staining particles, the sample preparation section 440 injects the staining reagent into the reaction chamber 420 through the 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 dispenses it into a reaction chamber 420. The reaction chamber 420 is connected to an FCM detection section 460 by a liquid delivery tube.

[0027] The reaction chamber 420 is a container for preparing a measurement sample. In the reaction chamber 420, a specimen and 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.

[0028] 3, the electrical connections of the various parts in the measuring unit 400 will be described. The sample preparing section 440, the specimen aspirating section 450 and the FCM detecting section 460 are connected to an interface (IF) section 488. The FCM detecting section 460 is connected to an analog processing section 481 and an A / D converting section 482. The analog processing section 481 processes an analog signal output from the FCM detecting section 460, and the A / D converting section 482 converts an analog signal output from the analog processing section 481 into a digital signal. The measuring unit 400 is also connected to the analyzing unit 300 via an IF section 489. The IF section 484, the IF section 488 and the IF section 489 are connected to a bus 485.

[0029] 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. Although the platelet aggregation detection method of this embodiment does not use a fluorescent dye, the example of FIG. 4 shows a configuration capable of receiving side fluorescent light corresponding to light generated by excitation of the fluorescent dye for the purpose of explaining the optical system. The first light source 411a and the second light source 411b emit light of different wavelengths. For example, the first light source 411a emits light of a first wavelength, and the second light source 411b emits light of a second wavelength. The first wavelength is preferably a wavelength absorbed by hemoglobin. Such a wavelength is, for example, 315 nm or more and 600 nm or less, preferably 400 nm or more and 490 nm or less, more preferably 400 nm or more and 450 nm or less, and even more preferably 400 nm or more and 410 nm or less. When light in the above wavelength range is irradiated to red blood cells containing hemoglobin, the light is absorbed by the hemoglobin in the blood cells, so that parameters such as the intensity (peak value), pulse width, and pulse area of ​​the scattered light generated from the red blood cells tend to be smaller than those of white blood cells not containing hemoglobin. The second wavelength is a wavelength different from the first wavelength, and is preferably a wavelength that is not absorbed by hemoglobin. Such a wavelength is, for example, 610 nm or more and 750 nm or less, preferably 620 nm or more and 700 nm or less, and more preferably 633 nm or more and 643 nm or less. It is believed that by using both light in a wavelength range that is absorbed by hemoglobin and light in a wavelength range that is not absorbed by hemoglobin, red blood cells and white blood cells can be clearly distinguished, thereby more clearly detecting platelet aggregation. As the first and second light sources, for example, semiconductor laser light sources can be used.

[0030] 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.

[0031] 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.

[0032] Although the platelet aggregation detection method of this embodiment does not use a fluorescent dye, when the particles are dyed with a fluorescent dye excited by a first wavelength, side fluorescence (first side fluorescence) corresponding to light generated by excitation of the fluorescent dye passes through the dichroic mirror 418b and is received by the side fluorescent light receiving element 422a. When the particles are dyed with a fluorescent dye excited by a second wavelength, side fluorescence (second side fluorescence) corresponding to light generated by excitation of the fluorescent dye passes through the dichroic mirror 418c and is received by the side fluorescent light receiving element 422b. The side fluorescent light receiving elements 422a and 422b are, for example, avalanche photodiodes.

[0033] 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. With reference to Fig. 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. Although the platelet aggregation detection method of this embodiment does not use a fluorescent dye, the example of Fig. 5 shows a state in which fluorescence is generated laterally with respect to the traveling direction of the light for the purpose of explaining the optical system. That is, 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 laterally with respect to the traveling direction of the light from the fluorescent dye. Also, when the particle P is stained with a fluorescent dye excited by the second wavelength, a second side fluorescence (SFL-2) excited by the light of the second wavelength is generated laterally with respect to the traveling direction of the light.

[0034] 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).

[0035] 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.

[0036] The optical information may be the above-mentioned characteristic parameters. The optical information includes first scattered light information and second scattered light information. The first scattered light information is information on side scattered light generated from particles by irradiating the measurement sample with light of a first wavelength. The second scattered light information is information on forward scattered light generated from particles by irradiating the measurement sample with light of a 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 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 referred to as the "first side scattered light intensity", and the peak value of the second forward scattered light signal is also referred to as the "second forward scattered light intensity".

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The method for detecting platelet aggregation of this embodiment includes, for example, the following first, second and third embodiments. In the "first embodiment", particles in a measurement sample are classified into platelets and blood cells other than platelets based on the first scattered light information and the second scattered light information, and information indicating whether or not platelet aggregation is included in the platelets is obtained. In the "second embodiment", particles in a measurement sample are classified into white blood cells and blood cells other than white blood cells based on the first scattered light information and the second scattered light information. Then, blood cells other than white blood cells are classified into platelets and red blood cells, and information indicating whether or not platelet aggregation is included in the platelets is obtained. In the "third embodiment", a scattergram is created based on the first scattered light information and the second scattered light information, and information indicating whether or not platelet aggregation is included in the particles in the measurement sample is obtained based on this scattergram.

[0041] (Measurement processing) An example of the measurement process in step S12 in FIG. 7 will be described with reference to FIG. 8, but is not limited to this example. The measurement process is common to all the embodiments. In step S21, the control unit 301 of the analysis unit 300 causes the measurement unit 400 to prepare a measurement sample from the specimen. Specifically, the measurement unit 400 mixes the specimen and the diluted reagent in the reaction chamber 420 to prepare the measurement sample. In step S22, the control unit 301 causes the measurement unit 400 to guide the measurement sample to the FCM detection unit 460 and perform optical measurement by the flow cytometry method. As a result, the measurement unit 400 detects optical information, for example, waveform data of SSC-1 and FSC-2, 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 in step S13 in FIG. 7. An example of the analysis process in each embodiment will be described later.

[0042] (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.

[0043] 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, red blood cells, and platelets. In this specification, the term "red blood cells" includes mature red blood cells and reticulocytes. Mature red blood cells are terminally differentiated red blood cells that do not have a nucleus and nucleic acid. Reticulocytes are young red blood cells that are released into the peripheral blood immediately after the erythroblast cells in the bone marrow are enucleated. "Abnormal cells" refer to formed elements that do not normally appear in blood.

[0044] Abnormal cells also include non-cellular particles and microorganisms. Non-cellular particles include, for example, platelet aggregates, red blood cell ghosts, and lipid particles. Microorganisms include, for example, bacteria and fungi. Platelet aggregates occur in blood collection tubes due to contamination with tissue fluid during blood collection, insufficient mixing, or the action of EDTA.

[0045] (Diluted Reagent) The dilution reagent used in the preparation of the measurement sample will be described. The dilution reagent contains a buffer and a solvent. Preferably, the dilution reagent contains a solution of a buffer. Examples of the solvent include water and physiological saline, and water is particularly preferred. The buffer preferably has a buffering effect in the range of pH 6 to 11. Such a buffer can be selected from, for example, carboxylates, phosphates, Good's buffer, taurine, triethanolamine, and the like. The pH of the dilution reagent is, for example, 6 to 11, preferably 7 to 10, and more preferably 8 to 9.5. When the pH of the dilution reagent is 6 or more, red blood cells are less likely to be hemolyzed in the measurement sample. This can suppress the generation of red blood cell ghosts. In addition, when the pH of the dilution reagent is 11 or less, nonspecific staining of red blood cells and red blood cell ghosts by a fluorescent dye can be reduced. A preferred dilution reagent is an aqueous solution of the above buffer, having a pH of 6 to 11.

[0046] The diluted reagent may further contain components such as an osmotic pressure compensator and a preservative in addition to the above-mentioned buffer. The osmotic pressure compensator is a substance that can maintain the osmotic pressure of the diluted reagent in an appropriate range. Examples of the osmotic pressure compensator include an alkali metal salt of an organic acid such as propionic acid, a sugar such as glucose or mannose, an alkali metal halide such as sodium chloride, and an alkaline earth metal halide such as magnesium chloride. One or more types of osmotic pressure compensators can be used. When an osmotic pressure compensator is used, the osmotic pressure compensator is added to the diluted reagent so that the osmotic pressure of the diluted reagent is preferably 150 mOsm / kg·H2O to 600 mOsm / kg·H2O, more preferably 200 mOsm / kg·H2O to 300 mOsm / kg·H2O.

[0047] Preservatives contained in the dilution reagent include, for example, sodium 2-pyridylthio-1-oxide, β-phenethyl alcohol, and the like.

[0048] In the method for detecting platelet aggregation according to the present embodiment, a commercially available dilution reagent may be used, such as Cellpack (registered trademark) DFL (Sysmex Corporation).

[0049] The above-mentioned diluted reagent may be provided as a blood cell analysis reagent used in the method for detecting platelet aggregation of this embodiment. The blood cell analysis reagent of this embodiment may be in the form of, for example, the above-mentioned diluted reagent housed in a container. With reference to FIG. 9, 11 indicates a container in which the blood cell analysis reagent of this embodiment is housed. When the container 11 is housed in a packaging box, it may also house an attached document describing the composition, usage method, storage method, etc. of the blood cell analysis reagent, a cushioning material for reducing external impact, etc.

[0050] (Preparation of measurement samples) The mixing ratio of the dilution reagent and the specimen can be appropriately determined depending on the type of specimen. The mixing ratio of the dilution reagent and the specimen is preferably 1000:1 or more in terms of volume ratio, for example. More preferably, it is 1000:5 or more, and even more preferably, it is 1000:10 or more. As described above, in this embodiment, platelet aggregation may be detected using a hemolytic reagent and a fluorescent dye, or may be detected without using a hemolytic reagent and a fluorescent dye. Therefore, in the method for detecting platelet aggregation of this embodiment, the measurement sample does not contain a hemolytic reagent and a fluorescent dye. That is, in the method for detecting platelet aggregation of this embodiment, the measurement sample does not include a step of hemolyzing red blood cells and a step of staining blood cells with a fluorescent dye in the preparation of the measurement sample. Here, the hemolytic reagent is a reagent for dissolving red blood cells in the specimen by mixing with the specimen. The hemolytic reagent itself is known, and contains, for example, a surfactant at a concentration capable of dissolving red blood cells.

[0051] (Analysis process of the first embodiment) With respect to step S13 in FIG. 7, an example of the analysis process of the first embodiment will be described with reference to FIGS. 10 and 11, but is not limited to this example. In this specification, platelets are also called "PLT", red blood cells are also called "RBC", and white blood cells are also called "WBC". With reference to FIG. 10, in this analysis process, particles in a measurement sample are first classified into a group containing PLT and a group containing blood cells other than PLT. In the method for detecting platelet aggregation of this embodiment, a hemolytic reagent is not used, so the measurement sample contains RBC. In addition, the measurement sample also contains WBC and PLT. In other words, blood cells other than PLT are a particle group containing RBC and WBC. When platelet aggregation is present in a specimen, the group containing PLT includes PLT and platelet aggregation. The group containing PLT is identified based on the first scattered light information and the second scattered light information. In this example, a first side scattered light intensity (also referred to as "SSC-1 intensity") is used as the first scattered light information, and a second forward scattered light intensity (also referred to as "FSC-2 intensity") is used as the second scattered light information.

[0052] Identification of a group containing PLTs can be performed, for example, by analysis using a scattergram. The scattergram is created, for example, based on SSC-1 intensity and FSC-2 intensity. Referring to FIG. 10, in step S101, the analysis unit 300 creates a scattergram with SSC-1 intensity and FSC-2 intensity as two axes based on the acquired optical information. Then, in the created scattergram, the position of a point corresponding to each particle in the measurement sample is determined. The horizontal and vertical axes of the scattergram may be displayed either logarithmically or linearly. Preferably, the horizontal and vertical axes of the scattergram are displayed logarithmically.

[0053] In step S102, the analysis unit 300 classifies the particles in the measurement sample into a group containing PLT and a group containing blood cells other than PLT based on the SSC-1 intensity and the FSC-2 intensity. The group containing blood cells other than PLT is a group that shows a higher FSC-2 intensity than the particle group containing PLT. Here, the comparison of the scattered light intensity between particle groups in the scattergram is preferably performed, for example, by a statistical representative value of the scattered light intensity of each particle group. The statistical representative value of the scattered light intensity of one particle group is a value obtained from the scattered light intensity of all particles that constitute the particle group, and examples of the value include the median, the average value, and the mode. The median is preferable.

[0054] Referring to FIG. 11(A), in the scattergram, a group containing PLT appears in an area with a low FSC-2 intensity. Also, a group containing blood cells other than PLT appears in an area with a high FSC-2 intensity. In FIG. 11(A), the group containing blood cells other than PLT is a group composed of particles in an area surrounded by a dashed line. Specifically, the group containing blood cells other than PLT includes a group containing RBC and a group containing WBC. Since PLT is the smallest size of blood cells, PLT has a lower forward scattered light intensity than RBC and WBC. Therefore, in the scattergram, the group containing PLT appears in an area with a lower FSC-2 intensity than the group containing RBC and the group containing WBC. As a result, the particles in the measurement sample are classified into a group containing PLT and a group containing blood cells other than PLT.

[0055] With reference to FIG. 11B, when platelet aggregates are contained in the sample, particles are observed on the scattergram that are distributed so as to extend from the group containing PLTs to the group containing WBCs. In FIG. 11B, particles appearing in the area surrounded by the dashed line are platelet aggregates. Platelet aggregates are an aggregate of multiple platelets, and are larger in size than normal platelets. Therefore, particles of platelet aggregates show higher forward scattered light intensity than platelets. Therefore, based on the FSC-2 intensity, it is possible to determine whether or not platelet aggregates are contained in the group containing PLTs. With reference to FIG. 10, in step S103, the analysis unit 300 determines whether or not the group containing PLTs includes particles that show an FSC-2 intensity equal to or greater than a threshold value. Specifically, the FSC-2 intensity of all particles constituting the group containing PLTs is compared with a threshold value corresponding to the FSC-2 intensity. Particles that show an FSC-2 intensity equal to or greater than this threshold value are suspected to be platelet aggregates. In other words, the group containing PLTs is suspected to include platelet aggregates. In step S103, if there are particles exhibiting an FSC-2 intensity equal to or greater than the threshold, the process proceeds to step S104. In step S103, if there are no particles exhibiting an FSC-2 intensity equal to or greater than the threshold, the process proceeds to step S105.

[0056] In a further embodiment, particles exhibiting an FSC-2 intensity equal to or greater than a threshold may be counted. The particle counting is performed by the analysis unit 300. In step S103, if the number of particles exhibiting an FSC-2 intensity equal to or greater than the threshold is equal to or greater than a predetermined value, the process proceeds to step S104. If the number of particles exhibiting an FSC-2 intensity equal to or greater than the threshold is less than a predetermined value, the process proceeds to step S105. Here, the predetermined value is a value for determining whether the particles exhibiting an FSC-2 intensity equal to or greater than the threshold are platelet aggregates.

[0057] In step S104, the analysis unit 300 outputs information indicating the presence of platelet aggregation. For example, a flag "PLTc?" may be output to the display unit 305. Not displaying the flag on the display unit 305 may be information indicating the absence of platelet aggregation. The process then proceeds to step S105. In step S105, the group including blood cells other than PLT is further analyzed. In step S105, the analysis unit 300 classifies the blood cells other than PLT into a group including RBC and a group including WBC based on the SSC-1 intensity and / or the FSC-2 intensity. Preferably, the analysis unit 300 classifies the blood cells other than PLT into a group including RBC and a group including WBC based on the SSC-1 intensity. With reference to (A) and (B) of FIG. 11, on the scattergram based on the SSC-1 intensity and the FSC-2 intensity, the group including RBC appears in an area with a lower SSC-1 intensity than the group including WBC. It is generally known that the diameter of RBC is 7-8 μm, and the diameter of WBC is 7-21 μm. Therefore, in the above scattergram, the group containing WBC tends to appear in the region with higher FSC-2 intensity than the group containing RBC. This allows the group containing blood cells other than PLT to be classified into a group containing RBC and a group containing WBC. The classification of the group containing RBC and the group containing WBC based on the SSC-1 intensity will be described below.

[0058] It is known that hemoglobin contained in RBC absorbs blue-violet light. When light of a first wavelength corresponding to blue-violet light is irradiated onto RBC, the scattered light generated from RBC becomes weaker than the scattered light generated when light of a second wavelength corresponding to red light is irradiated onto RBC. For this reason, red light has been preferably used for blood cell analysis in the past. On the other hand, when light of the first wavelength and light of the second wavelength are irradiated onto WBC, there is almost no difference in the intensity of the scattered light generated. The present inventors have conceived of discriminating between WBC and RBC by utilizing the above phenomenon. Specifically, by irradiating light of the first wavelength, a difference is generated in the first scattered light information between WBC and RBC. For example, when the first scattered light information is SSC-1 intensity, the SSC-1 intensity of RBC becomes lower due to absorption by hemoglobin. However, the SSC-1 intensity of WBC is not affected. Therefore, the SSC-1 intensity of RBC becomes lower than that of WBC. Therefore, a group including RBCs and a group including WBCs can be distinguished based on the SSC-1 intensity. After classifying blood cells other than PLTs, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG.

[0059] (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. 12 and 13, but the analysis process is not limited to this example. With reference to FIG. 12, in this analysis process, first, particles in a measurement sample are classified into a group including WBC and a group including blood cells other than WBC. As described above, in the method for detecting platelet aggregation of this embodiment, a hemolytic reagent is not used, so the measurement sample includes RBC. The measurement sample also includes WBC and PLT. That is, blood cells other than WBC are a particle group including RBC and PLT. When platelet aggregation is present in a specimen, the group including blood cells other than WBC includes RBC, PLT, and platelet aggregation. The group including blood cells other than WBC is identified based on the first scattered light information and the second scattered light information. In this example, SSC-1 intensity is used as the first scattered light information, and FSC-2 intensity is used as the second scattered light information.

[0060] Identification of a group including blood cells other than WBC can be performed, for example, by analysis using a scattergram. The scattergram is created, for example, based on SSC-1 intensity and FSC-2 intensity. Referring to FIG. 12, in step S201, the analysis unit 300 creates a scattergram with SSC-1 intensity and FSC-2 intensity as two axes based on the acquired optical information. Then, in the created scattergram, the position of a point corresponding to each particle in the measurement sample is determined. The display of the horizontal and vertical axes of the scattergram is as described above.

[0061] In step S202, the analysis unit 300 classifies the particles in the measurement sample into a group including WBCs and a group including blood cells other than WBCs based on the SSC-1 intensity and FSC-2 intensity. The group including WBCs is a group that shows higher SSC-1 intensity and FSC-2 intensity than the group including blood cells other than WBCs. The comparison of scattered light intensity between particle groups in the scattergram is as described above.

[0062] Referring to FIG. 13(A), in the scattergram, a group including WBC appears in an area with a high SSC-1 intensity. Also, a group including blood cells other than WBC appears in an area with a low SSC-1 intensity. In FIG. 13(A), the group including blood cells other than WBC is a group consisting of particles in an area surrounded by a dashed line. Specifically, the group including blood cells other than WBC includes a group including RBC and a group including PLT. As described above, since RBC contains hemoglobin, in the scattergram, the group including RBC appears on the side with a lower SSC-1 intensity than the group including WBC. Also, since PLT has the smallest size among blood cells, in the scattergram, the group including PLT appears in an area with a lower FSC-2 intensity than the group including WBC. Thus, in the scattergram based on the SSC-1 intensity and the FSC-2 intensity, the particles in the measurement sample are classified into a group including WBC and a group including blood cells other than WBC. The analysis unit 300 then identifies populations that include blood cells other than WBCs.

[0063] 12, in step S203, the analysis unit 300 classifies a group including blood cells other than WBCs into a group including PLTs and a group including RBCs based on the FSC-2 intensity. As described above, the group including PLTs appears in an area with a lower FSC-2 intensity than the group including RBCs, so the group including PLTs can be distinguished from the group including RBCs based on the FSC-2 intensity.

[0064] With reference to FIG. 13B, if platelet aggregates are contained in the sample, particles are observed on the scattergram that are distributed so as to extend from the group containing PLTs to the group containing WBCs. In FIG. 13B, particles appearing in the area surrounded by the dashed line are platelet aggregates. As described above, platelet aggregates are larger in size than normal platelets, so it is possible to determine whether or not platelet aggregates are contained in the group containing PLTs based on the FSC-2 intensity. With reference to FIG. 12, in step S204, the analysis unit 300 determines whether the group containing PLTs contains particles that exhibit an FSC-2 intensity equal to or greater than a threshold value. This determination may be made based on a comparison between the number of particles that exhibit an FSC-2 intensity equal to or greater than a threshold value and a predetermined value. This determination is similar to that described for step S103. In step S204, if particles that exhibit an FSC-2 intensity equal to or greater than the threshold value are present (or if the number of particles that exhibit an FSC-2 intensity equal to or greater than the threshold value is equal to or greater than the predetermined value), the process proceeds to step S205.

[0065] In step S204, if there are no particles exhibiting an FSC-2 intensity equal to or greater than the threshold (or if the number of particles exhibiting an FSC-2 intensity equal to or greater than the threshold is less than a predetermined value), the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG. 7. Step S205 is similar to that described for step S104. After outputting information indicating the presence of platelet aggregation, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG. 7.

[0066] (Analysis process of the third embodiment) With respect to step S13 in Fig. 7, an example of the analysis process of the third embodiment will be described with reference to Figs. 14 and 15, but is not limited to this example. With reference to Fig. 14, in this analysis process, platelet aggregation is detected based on a scattergram based on SSC-1 intensity and FSC-2 intensity. In step S301, the analysis unit 300 creates a scattergram with SSC-1 intensity and FSC-2 intensity as two axes based on the acquired optical information. Then, in the created scattergram, the position of a point corresponding to each particle in the measurement sample is determined. The display of the horizontal and vertical axes of the scattergram is as described above.

[0067] In step S302, the analysis unit 300 determines whether a particle cluster appears in a predetermined region on the scattergram. For example, when a point corresponding to each particle in the measurement sample is located within the predetermined region, the particle is determined to appear in the predetermined region. When a plurality of particles appear in the predetermined region, it is determined that a particle cluster appears in the predetermined region, and the process proceeds to step S303. The number of particles appearing in the predetermined region may be 2 or more. Preferably, the number of particles is equal to or greater than the above-mentioned predetermined value.

[0068] The predetermined region may be a region where particles that are platelet aggregates are expected to appear. In the example shown in FIG. 15, such a region is the region surrounded by a dashed line. Particles that appear in the region are platelet aggregates. As described above, when platelet aggregates are contained in a sample, particles are distributed on the scattergram so as to extend from a group containing PLTs to a group containing WBCs. As shown in FIG. 15, the predetermined region may be determined from the relative positions of a group containing WBCs, a group of RBCs, and a group containing non-aggregated PLTs. The predetermined region may also be determined, for example, by accumulating measurement data on a plurality of blood samples known to contain platelet aggregates and a plurality of normal blood samples. The samples are measured to create scattergrams, and the distribution of particles on the scattergrams is compared to accumulate data on the region where particles of platelet aggregates appear. Then, based on the data, the region where platelet aggregates are expected to appear is pre-determined as the predetermined region.

[0069] If a particle group appears in the predetermined region in step S302, the process proceeds to step S303. Step S303 is similar to that described for step S104. If no particles appear in the predetermined region or the number of particles that appear is one or less in step S302, it is determined that a particle group has not appeared in the predetermined region, and the process proceeds to step S304. Preferably, it is determined that a particle group has not appeared in the predetermined region when the number of particles that appear in the predetermined region is less than the above-mentioned predetermined value.

[0070] In step S304, the analysis unit 300 classifies at least one of a group including WBCs, a group including PLTs, and a group including RBCs from the particles in the measurement sample based on the SSC-1 intensity and the FSC-2 intensity. The group including WBCs can be identified on the scattergram as a group having the highest SSC-1 intensity and / or the highest FSC-2 intensity. The group including PLTs can be identified on the scattergram as a group having the lowest FSC-2 intensity. The group including RBCs can be identified on the scattergram as a group having a lower SSC-1 intensity than the group of WBCs and a higher FSC-2 intensity than the group including PLTs. The comparison of the scattered light intensity between the particle groups in the scattergram is as described above. After performing classification of the particles in the measurement sample, the analysis unit 300 ends the analysis process, and the process proceeds to step S14 in FIG. 7.

[0071] 7, when the above-mentioned analysis process is completed, the control unit 301 outputs the analysis result to the display unit 305 in step S14, and ends the process. As described above, the process of detecting platelet aggregation in a measurement sample and analyzing at least one of white blood cells, platelets, and red blood cells has been described using Figs. 10 to 15. However, it is sufficient that only a part of the analysis process is performed, or all of the analysis process may be performed.

[0072] For example, when a particle suspected of being a platelet aggregate is identified, the analysis result screen output to the display unit 305 may output information indicating the presence of platelet aggregates in the sample. For example, a flag "PLTc?" may be output to the display unit 305. The screen may also include the white blood cell count, the red blood cell count, and the platelet count. In addition to this information, the screen may also include a scattergram created based on the optical information.

[0073] In this way, the method for detecting platelet aggregation according to the present embodiment can provide medical personnel such as doctors and laboratory technicians with information on whether or not a specimen contains platelet aggregation as an analysis result. It can also provide analysis results on white blood cells, red blood cells, and platelets. From the obtained analysis results, medical personnel can decide, for example, whether or not to perform further testing on the specimen. The above-mentioned preparation of the measurement sample and analysis of the specimen are all performed in vitro.

[0074] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0075] In each of the following examples, the method for detecting platelet aggregation of this embodiment was carried out using the following blood samples, dilution reagents and analytical devices, the details of which are as described in the "Measurement Method" below.

[0076] [Blood sample] Whole blood (peripheral blood) was collected from multiple subjects using EDTA-2K blood collection tubes and stored at room temperature (25±2°C). When used, whole blood was taken from the blood collection tubes and used as a blood sample. Details of the blood cell components and / or subjects contained in the blood samples are described in each example.

[0077] [Diluted Reagent] As a dilution reagent, Cellpack (registered trademark) DFL (Sysmex Corporation) was used.

[0078] [Analyzer] A prototype modified from a flow cytometer XF-1600 (Sysmex Corporation) was used as the analytical device. The prototype was constructed as follows. The XF-1600 was equipped with three semiconductor laser light sources emitting blue-violet (405 nm), blue (488 nm), and red (638 nm) light, respectively, a detector for scattered light of each color, and a detector for fluorescent light excited by each light. The semiconductor laser light source 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.

[0079] [Measurement method] Except for not using staining reagent and using Cellpack DFL, 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 and 5.0 μL of whole blood. The measurement sample was measured after incubating at 41°C for 30 seconds.

[0080] Example 1: Analysis of a measurement sample containing platelet aggregation A measurement sample was prepared from a blood specimen containing platelet aggregation, and platelet aggregation in the measurement sample was detected by an analyzer.

[0081] (1) Measurement sample A normal blood sample (1 sample) and blood samples containing platelet aggregation (5 samples) were selected. Hereinafter, the normal blood sample is also referred to as a "PLTc negative sample." In addition, each blood sample containing platelet aggregation is also referred to as a "PLTc positive sample 1," "PLTc positive sample 2," "PLTc positive sample 3," "PLTc positive sample 4," and "PLTc positive sample 5." The PLTc negative sample was whole blood that was determined to be normal by analysis using a multi-item automated blood cell analyzer XN-20 (Sysmex Corporation). The PLTc positive samples 1 to 5 were whole blood that was determined to be positive for platelet aggregation by visual observation under a microscope. Measurement samples were prepared from these blood samples as described above.

[0082] (2) Analysis Each measurement sample was measured by an analyzer to obtain first scattered light information, second scattered light information, and third scattered light information. The first scattered light information was the side scattered light intensity (hereinafter also referred to as "side scattered light intensity (blue-purple)" or "V-SSC") obtained by irradiating particles in the measurement sample with a blue-purple laser. The second scattered light information was the forward scattered light intensity (hereinafter also referred to as "forward scattered light intensity (red)" or "R-FSC") obtained by irradiating particles in the measurement sample with a red laser. The third scattered light information was the side scattered light intensity (hereinafter also referred to as "side scattered light intensity (red)" or "R-SSC") obtained by irradiating particles in the measurement sample with a red laser. For each measurement sample, a scattergram (hereinafter also referred to as "Example scattergram") was created with the side scattered light intensity (blue-purple) on the horizontal axis and the forward scattered light intensity (red) on the vertical axis. For comparison, a scattergram was also created for each measurement sample, with the horizontal axis representing side scattered light intensity (red) and the vertical axis representing forward scattered light intensity (red) (hereinafter also referred to as a "comparative scattergram"). Each scattergram is shown in (A) to (D) of Figures 16 to 18. In the following figures, "WBC" stands for white blood cells, "RBC" stands for red blood cells, "PLT" stands for platelets, and "PLTc" stands for platelet aggregation.

[0083] Referring to FIG. 16(A), in the comparative scattergram of the PLTc-negative specimen, the platelet group appeared in the region with low R-FSC. Platelets are smaller in size than white blood cells and red blood cells, so the R-FSC was low. Therefore, in the comparative scattergram, the platelet group could be identified. However, the white blood cells and red blood cells appeared overlapping. Therefore, in the comparative scattergram, the white blood cells and red blood cells could not be classified. On the other hand, referring to FIG. 16(B), in the scattergram of the example of the PLTc-negative specimen, the white blood cell group, the red blood cell group, and the platelet group were clearly classified. As described above, the hemoglobin of the red blood cells absorbs light of the first wavelength (blue-purple), so it is considered that the V-SSC of the red blood cells became low. Therefore, in the scattergram of FIG. 16(B), the red blood cell group appeared in the region with lower V-SSC than the white blood cells. In this manner, in the scattergram of the embodiment, it was possible to distinguish between white blood cells, red blood cells, and platelets.

[0084] Referring to FIG. 16C, in the comparative scattergram of PLTc-positive specimen 1, a particle group extending from a platelet group to a group of overlapping white blood cells and red blood cells was observed. However, it was not possible to distinguish whether the particle group was red blood cells or platelet aggregates. Thus, it was difficult to detect platelet aggregates based on the side scattered light intensity and forward scattered light intensity obtained only by the red laser. Referring to FIG. 16D, in the scattergram of the example of PLTc-positive specimen 1, a white blood cell group, a red blood cell group, and a platelet group were clearly classified. Furthermore, in this scattergram, a particle group that was not observed in the scattergram of FIG. 16B appeared in the area surrounded by the dashed line. Platelet aggregates are larger in size than normal platelets, so the forward scattered light intensity is high. Therefore, the particle group in the area surrounded by the dashed line could be distinguished as platelet aggregates. In Fig. 16(D), "PLTc?" is displayed as a flag indicating the appearance of particles suspected to be platelet aggregates. In the scattergram of the example, the appearance of platelet aggregates could be clearly confirmed by the appearance of a group of red blood cells on the lower V-SSC side compared to the comparative scattergram. In this way, it was demonstrated that platelet aggregates can be detected based on the side scattered light intensity obtained by the blue-violet laser and the forward scattered light intensity obtained by the red laser.

[0085] With reference to FIG. 17(A), in the scattergram of the comparative example, it was not possible to determine whether or not platelet aggregates were included in the PLTc-positive specimen 2. However, with reference to FIG. 17(B), in the scattergram of the example, particles suspected to be platelet aggregates were observed in the area surrounded by the dashed line. With reference to FIG. 17(C), it was shown that the PLTc-positive specimen 3 was a blood specimen with a small number of platelets. Also, it was not possible to determine whether or not platelet aggregates were included in the PLTc-positive specimen 3. However, with reference to FIG. 17(D), particles suspected to be platelet aggregates were observed in the area surrounded by the dashed line. With reference to FIG. 18(A), in the PLTc-positive specimen 4, similar to the PLTc-positive specimen 1, a particle group was observed that could not be determined to be red blood cells or platelet aggregates. However, with reference to FIG. 18(B), in the scattergram of the example, particles suspected to be platelet aggregates were observed in the area surrounded by the dashed line. Referring to Fig. 18(C), in PLTc-positive specimen 5, it was not possible to determine whether or not platelet aggregation was present, similarly to PLTc-positive specimen 2. However, referring to Fig. 18(D), in the scattergram of the example, particles suspected to be platelet aggregation were observed in the area surrounded by the dashed line. Thus, it was demonstrated that the scattergram of the example can detect platelet aggregation in various blood specimens.

[0086] Example 2: Analysis of a measurement sample containing platelet aggregation (2) Measurement samples were prepared from blood specimens containing platelet aggregation, and platelet aggregation in each measurement sample was detected by an analyzer in the same manner as in Example 1. For comparison, measurement samples containing a fluorescent dye were prepared from the same blood specimens and analyzed in the same manner.

[0087] (1) Measurement sample A measurement sample was prepared as described above from a blood specimen that was determined to be positive for platelet aggregation by visual observation under a microscope (hereinafter, also referred to as a "measurement sample containing a fluorescent dye"). The measurement sample containing a fluorescent dye was prepared as follows. First, the fluorescent dye 2,7,9-trimethylacridine-3,6-diamine was dissolved in special grade ethylene glycol (1 L) to prepare a staining reagent. 1000 μL of Cellpack DFL, 5.0 μL of whole blood, and 20 μL of staining reagent were mixed to prepare a measurement sample containing a fluorescent dye. The dilution ratio of the staining reagent in the measurement sample was 51.25. The final concentration of the fluorescent dye in the measurement sample was 1.0 ppm.

[0088] (2) Analysis Each measurement sample was measured by an analyzer to obtain the first scattered light information and the second scattered light information. The first and second scattered light information were the same as in Example 1. For each measurement sample, a scattergram was created in the same way as in Example 1, with the horizontal axis representing the side scattered light intensity (blue-purple) and the vertical axis representing the forward scattered light intensity (red). Each scattergram is shown in (A) and (B) of FIG. 19. Referring to (A) of FIG. 19, the particles in the measurement sample not containing the fluorescent dye could be classified into a group of red blood cells, a group of white blood cells, and a group of platelets. Furthermore, particles suspected to be platelet aggregates were found in the area surrounded by the dashed line. Referring to (B) of FIG. 19, the particles in the measurement sample containing the fluorescent dye could also be classified into a group of red blood cells, a group of white blood cells, and a group of platelets, similar to the scattergram in (A). Furthermore, particles suspected to be platelet aggregates were found in the area surrounded by the dashed line. The platelet aggregation detection method of this embodiment does not use a fluorescent reagent, but it has been shown that this method is capable of classifying red blood cells, white blood cells, and platelets, and detecting platelet aggregation, even if the measurement sample contains a fluorescent dye. [Explanation of symbols]

[0089] 11 Blood cell analysis reagents 100 blood collection tube 200 Suction tube 300 analytical units 400 measurement units 410, 411 Reagent container 420 Reaction Chamber 430 Waste chamber 500 Analysis System

Claims

1. acquiring first scattered light information and second scattered light information generated by irradiating a measurement sample containing particles with light of a first wavelength and light of a second wavelength; obtaining information indicating the presence or absence of platelet aggregation based on the first scattered light information and the second scattered light information; wherein the first wavelength is equal to or greater than 315 nm and equal to or less than 600 nm, and the second wavelength is equal to or greater than 610 nm and equal to or less than 750 nm.

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

3. The method according to claim 1 , wherein the measurement sample is a sample prepared by mixing whole blood with a dilution reagent.

4. The method according to claim 1 , wherein the measurement sample does not contain a hemolysis reagent or a fluorescent dye.

5. The method further includes a step of preparing the measurement sample by mixing whole blood with a dilution reagent before the step of acquiring the first scattered light information and the second scattered light information, The method according to claim 1 , wherein the preparing step does not include a step of hemolyzing red blood cells contained in the measurement sample, and does not include a step of staining blood cells contained in the measurement sample with a fluorescent dye.

6. the information regarding the side scattered light is a side scattered light intensity, and the information regarding the forward scattered light is a forward scattered light intensity; In the step of acquiring information indicating the presence or absence of platelet aggregation, particles in the measurement sample are classified into a group containing platelets and a group containing blood cells other than platelets based on the side scattered light intensity and the forward scattered light intensity; The method according to claim 2 , wherein information indicative of the presence of platelet aggregation is obtained when the platelet-containing population contains particles exhibiting a forward scattered light intensity equal to or greater than a threshold value.

7. The method according to claim 6, further comprising a step of classifying the group containing blood cells other than platelets into a group containing red blood cells and a group containing white blood cells based on the side scattered light intensity and / or the forward scattered light intensity.

8. 7. The method according to claim 6, wherein in the step of acquiring information indicating the presence or absence of platelet aggregation, a scattergram is created based on the side scattered light intensity and the forward scattered light intensity, and a group containing the platelets is identified in the scattergram.

9. the information regarding the side scattered light is a side scattered light intensity, and the information regarding the forward scattered light is a forward scattered light intensity; The step of acquiring information indicating the presence or absence of platelet aggregation comprises: classifying particles in the measurement sample into a group including white blood cells and a group including blood cells other than white blood cells based on the side scattered light intensity and the forward scattered light intensity; classifying the group containing blood cells other than white blood cells into a group containing platelets and a group containing red blood cells based on the forward scattered light intensity; Including, The method according to claim 2 , wherein information indicative of the presence of platelet aggregation is obtained when the platelet-containing population contains particles exhibiting a forward scattered light intensity equal to or greater than a threshold value.

10. 10. The method according to claim 9, wherein in the step of acquiring information indicating the presence or absence of platelet aggregation, a scattergram is created based on the side scattered light intensity and the forward scattered light intensity, and a group containing the platelets is identified in the scattergram.

11. the information regarding the side scattered light is a side scattered light intensity, and the information regarding the forward scattered light is a forward scattered light intensity; 3. The method according to claim 2, wherein in the step of acquiring information indicating the presence or absence of platelet aggregation, a scattergram is created based on the forward scattered light intensity and the side scattered light intensity, and a group of particles appearing in a predetermined region in the scattergram is detected as platelet aggregation.

12. 12. The method according to claim 11, wherein in the step of acquiring information indicating the presence or absence of platelet aggregation, a scattergram is created based on the forward scattered light intensity and the side scattered light intensity, and at least one selected from the group consisting of white blood cells, platelets, and red blood cells is classified in the scattergram.

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

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