Measuring apparatus, reagents, and analytical methods

Abstract

The objective is to provide a measuring device, analytical method, and reagents that enable the acquisition of information regarding the mechanism of increase in abnormal cells in a sample. [Solution] The above problem is solved by a measuring device comprising: a chamber for preparing a measurement sample containing cells stained with at least one of a first fluorescent dye and a second fluorescent dye contained in a reagent by mixing a reagent supplied from a reagent container with a sample; a light source for irradiating light onto cells in the measurement sample flowing through a flow cell; a detection unit for detecting an optical signal corresponding to the cells in response to the irradiation of light; and an analysis unit for analyzing the optical signal. The detection unit detects an optical signal including at least one first fluorescent signal corresponding to a first fluorescent dye that binds to DNA and a second fluorescent signal corresponding to a second fluorescent dye that binds to RNA. The analysis unit determines whether the mechanism of leukocyte increase is neoplastic based on at least the first fluorescent signal and whether the mechanism of leukocyte increase is reactive based on at least the second fluorescent signal.

Description

[Technical Field]

[0001] This invention relates to a measuring device for analyzing cells in a sample. This invention relates to a reagent used in the measuring device. This invention relates to a method for analyzing cells in a sample. [Background technology]

[0002] In the analysis of specimens containing blood cells, such as blood and body fluids, white blood cells are classified into five subgroups, for example, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Detecting cells that may be related to a subject's health status or disease from a specimen is useful for diagnosing the subject. Patent Document 1 describes detecting abnormal lymphocytes and immature granulocytes as white blood cells that may be related to a subject's health status or disease by staining blood cells in a blood specimen with a fluorescent dye and analyzing them with a hemocytometer. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] U.S. Patent Application No. 2010 / 0151509 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] In the field of clinical testing, differentiating the mechanisms of leukocyte increase can be crucial for diagnosing a patient. Mechanisms of leukocyte increase include, for example, neoplastic increase and reactive increase. Leukocytes with neoplastic increase appear in the peripheral blood due to malignant tumors such as multiple myeloma, chronic lymphocytic leukemia, and malignant lymphoma. Leukocytes with reactive increase appear in the peripheral blood due to immune responses such as viral infections, drug allergies, and autoimmune diseases. Diagnosis and treatment methods may differ depending on the mechanism of leukocyte increase. However, there was no known technique for differentiating the mechanism of leukocyte increase through analysis of a sample using a hemocytometer. For example, differentiation between neoplastic lymphocytes and reactive lymphocytes was traditionally performed by microscopic observation of blood smears. Therefore, the present invention aims to provide a measuring device, analytical method, and reagents that enable differentiation of the mechanism of leukocyte increase in a sample. [Means for solving the problem]

[0005] The measuring device of the present invention includes a chamber for preparing a measurement sample containing cells stained with at least one of a first fluorescent dye and a second fluorescent dye contained in a reagent by mixing a reagent supplied from at least one reagent container with a sample; a light source for irradiating a plurality of cells in the measurement sample flowing through a flow cell with light; a detection unit for detecting a plurality of optical signals corresponding to each of the plurality of cells in response to the irradiation of light; and an analysis unit for analyzing the optical signals. The detection unit detects an optical signal including at least one first fluorescent signal corresponding to a first fluorescent dye that binds to the DNA of the cells and a second fluorescent signal corresponding to a second fluorescent dye that binds to the RNA of the cells. The analysis unit determines whether the mechanism of leukocyte increase is neoplastic based on at least the first fluorescent signal and whether the mechanism of leukocyte increase is reactive based on at least the second fluorescent signal. [Effects of the Invention]

[0006] The present invention provides a measuring device and an analytical method that enable differentiation of the mechanisms of leukocyte increase. The present invention also provides reagents used in the measuring device. [Brief explanation of the drawing]

[0007] [Figure 1] It is a figure showing an example of the appearance of the measuring device. [Figure 2] It is a figure showing an example of the fluid circuit in the measurement unit of the measuring device of the first embodiment. [Figure 3] It is a block diagram showing a configuration example of the measurement unit of the measuring device. [Figure 4] It is a block diagram showing a configuration example of the measurement unit of the measuring device. [Figure 5] It is a figure showing an example of the fluid circuit in the measurement unit of the measuring device. [Figure 6] It is a figure showing an example of the sample preparation unit in the measurement unit of the measuring device. [Figure 7] It is a figure showing an example of the sample preparation unit in the measurement unit. [Figure 8] It is a figure showing a configuration example of the optical system of the FCM detection unit. [Figure 9] It is a figure showing a configuration example of the FCM detection unit. [Figure 10] It is a figure showing a configuration example of the optical system of the FCM detection unit. [Figure 11] It is a block diagram showing a configuration example of the analysis unit. [Figure 12] It is a flowchart showing an example of the operation of the measuring device. [Figure 13] It is a flowchart showing an example of the analysis process. [Figure 14A] It is a schematic diagram of a scattergram. [Figure 14B] It is a schematic diagram of a scattergram. [Figure 15] It is a schematic diagram of a scattergram. [Figure 16] It is a schematic diagram of a scattergram. [Figure 17] It is a schematic diagram of a scattergram. [Figure 18] It is a schematic diagram of a scattergram. [Figure 19] It is a table showing an example of information (flag) generated by the analysis unit. [Figure 20] This is a flowchart showing an example of the analysis process. [Figure 21] This is a flowchart showing an example of the analysis process. [Figure 22] This is a schematic diagram of a scattergram. [Figure 23] This is a schematic diagram of a scattergram. [Figure 24] This is a schematic diagram of a scattergram. [Figure 25] This is a schematic diagram of a scattergram. [Figure 26] This is a flowchart showing an example of the analysis process. [Figure 27A] This figure shows an example of the analysis results screen. [Figure 27B] This figure shows an example of the analysis results screen. [Figure 28] This figure shows an example of reagents used in a measuring device. [Figure 29] This is an example of a scattergram. [Figure 30] This is an example of a scattergram. [Figure 31] This is an example of a scattergram. [Figure 32] This is an example of a scattergram. [Modes for carrying out the invention]

[0008] Referring to Figure 1, an example of the configuration of the measuring device will be described. The measuring device 500 includes, for example, a measuring unit 400 including a detection unit and an analysis unit 300 which is an analysis unit. The analysis unit 300 is, for example, a personal computer with software for analyzing the sample to be measured. The measuring unit 400 is a unit for preparing and measuring the sample to be measured and includes a flow cytometer. The analysis unit 300 and the measuring unit 400 are connected by a predetermined interface (for example, USB (Universal Serial Bus), wireless LAN (Local Area Network), wired LAN, Bluetooth, etc.). The analysis unit 300 also performs operation control of the measuring unit 400. The measuring device 500 may also be configured such that the analysis unit 300 is provided within the measuring unit 400.

[0009] The measurement unit 400 prepares a measurement sample by mixing the sample and reagents. A reagent containing a first fluorescent dye and a second fluorescent dye (also called a "staining reagent") is used to prepare the measurement sample. Preferably, a reagent containing a surfactant capable of dissolving red blood cells (also called a "hemolytic reagent") is further used to prepare the measurement sample. The particles in the measurement sample are stained by the first fluorescent dye and the second fluorescent dye.

[0010] The first and second fluorescent dyes are dyes that have different staining and fluorescence properties for cells. For example, the first component to which the first fluorescent dye binds and the second component to which the second fluorescent dye binds are different. The first and second fluorescent dyes have fluorescence emission maxima in different wavelength ranges, for example. The fluorescence emission maxima is the wavelength (peak wavelength) at which the fluorescent dye is excited by light with the highest fluorescence intensity. Also, for example, the first and second fluorescent dyes have maximum absorption maxima in different wavelength ranges. That is, the second fluorescent dye may be a fluorescent dye that emits fluorescence at a wavelength that can be detected separately from the fluorescence from the first fluorescent dye.

[0011] The first and second fluorescent dyes are dyes that have the ability to bind to nucleic acids (e.g., DNA, RNA), which are components of cells. The fluorescence signals obtained from the first and second fluorescent dyes can be used to distinguish whether the mechanism of leukocyte increase in a sample from a subject with a higher leukocyte count than a healthy subject is neoplastic or reactive. The first and second fluorescent dyes are selected such that one has a higher binding ability to DNA than the other, and the opposite is true for RNA binding. For example, the first and second fluorescent dyes are selected such that (1) the first and second fluorescent dyes have different binding abilities to DNA, (2) the first and second fluorescent dyes have different binding abilities to RNA, (3) the first fluorescent dye has different binding abilities to DNA and RNA, and (4) the second fluorescent dye has different binding abilities to DNA and RNA. By selecting such fluorescent dyes, for example, in samples containing leukocytes with a higher-than-normal amount of DNA (i.e., samples suspected of having increased leukocytes due to a neoplastic mechanism), the fluorescence signal corresponding to the first fluorescent dye will be greater than the fluorescence signal corresponding to the second fluorescent dye. On the other hand, for example, in samples containing leukocytes with a higher-than-normal amount of RNA (i.e., samples suspected of having increased leukocytes due to a reactive mechanism), the fluorescence signal corresponding to the second fluorescent dye will be greater than the fluorescence signal corresponding to the first fluorescent dye. This makes it possible to distinguish whether the mechanism of leukocyte increase in samples from subjects with a higher leukocyte count than healthy subjects is neoplastic or reactive.

[0012] For example, the first fluorescent dye has higher staining ability for neoplastic leukocytes than for neoplastic leukocytes, and the second fluorescent dye has higher staining ability for neoplastic leukocytes than for neoplastic leukocytes. The first fluorescent dye has, for example, specific binding ability to DNA, but weaker binding ability to RNA than the second fluorescent dye. The second fluorescent dye has, for example, stronger binding ability to RNA than the first fluorescent dye. The reason why the first fluorescent dye exhibits specific binding ability to DNA is, for example, the structure of the dye. For example, the first fluorescent dye has a structure that easily penetrates the gaps in the double-stranded structure of nucleic acids in DNA, and thus has specific binding ability to DNA. The first and second fluorescent dyes have the property that their fluorescence intensity increases when they bind to nucleic acids. For example, the difference in fluorescence intensity of the first fluorescent dye when bound to DNA and when not bound to DNA is about 10 times or more. As described above, both the first and second fluorescent dyes have the ability to bind to nucleic acids, but the first fluorescent dye has a higher binding ability to DNA than the second fluorescent dye, and a lower binding ability to RNA than the second fluorescent dye. Details of the fluorescent dyes and surfactants will be described later. In the analysis of particles in a sample using the measuring device, particles in the sample stained with the first and second fluorescent dyes are analyzed. In this specification, "particles in the sample" refers to formed elements contained in the sample that can be individually measured by the FCM detection unit 460 described later. Examples of particles in the sample include cells, hemolyzed red blood cell remnants (red blood cell ghosts), lipid particles, fungi, and bacteria contained in the sample. Cells include, for example, white blood cells, red blood cells, platelets (including aggregated platelets), etc.

[0013] The sample to be processed by the measurement unit 400 is contained in the sample container 100 (see Figure 2). The sample container 100 is, for example, a blood collection tube. The sample is a body fluid or its dilution collected from the subject. Examples of body fluids include blood, body cavity fluid, cerebrospinal fluid, synovial fluid, peritoneal dialysis drainage fluid, and bronchoalveolar lavage fluid. Blood is, for example, peripheral blood. Examples of body cavity fluids include ascites, pleural fluid, and pericardial fluid. Dilutions of body fluids are obtained by diluting the body fluid with a suitable aqueous solvent such as water, physiological saline, or buffer solution. The buffer solution preferably has a buffering effect at a pH near neutral (for example, pH 6 to 8). Commercially available sample diluents may also be used. Hereinafter, blood and its dilutions will also be referred to as "blood samples," and body fluids other than blood and their dilutions will also be referred to as "non-blood samples." The preferred sample is a blood sample. Blood samples may contain anticoagulants. Examples of such anticoagulants include ethylenediaminetetraacetic acid (EDTA), EDTA salts (e.g., EDTA-2K, EDTA-2Na, etc.), sodium citrate, heparin, and warfarin.

[0014] The sample is measured in the FCM detection unit 460 of the measurement unit 400. In the FCM detection unit 460, light is shone on each particle in the sample flowing through the flow cell, and the optical signal of each particle is acquired. Upon light irradiation, fluorescence is generated from the particles in the sample, originating from the first fluorescent dye and the second fluorescent dye, respectively. In addition, scattered light is emitted from the particles upon light irradiation. The scattered light includes lateral scattered light and forward scattered light. The optical signal includes the first and second fluorescent signals corresponding to each fluorescence, and the scattered light signal corresponding to the scattered light. 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 detect or classify particles in the sample.

[0015] The measuring device 500 of the first embodiment may be an automated blood cell analyzer that performs at least one of counting and classifying leukocytes in a blood sample and enables differentiation of the mechanism of leukocyte increase. The measuring device 500 can, for example, provide information on the mechanism of leukocyte increase. Referring to Figure 2, an example of the configuration of the fluid system in the measuring unit 400 of the measuring device of the first embodiment will be described. The measuring unit 400 comprises a sample preparation unit 440, a sample aspiration unit 450, and an FCM detection unit 460. The sample preparation unit 440 has a chamber 420 and a fluid delivery mechanism 430. The sample aspiration unit 450 is a mechanism for aspirating the sample T in the sample container 100 and has a sample aspiration nozzle 451. The FCM detection unit 460 is a detection unit that acquires optical signals emitted from individual particles in the measurement sample and has a light source, flow cell, dichroic mirror, and photodetector described later (see Figures 8-10).

[0016] The sample aspiration nozzle 451 is capable of penetrating the sample container 100, which is sealed by the lid 100a. The sample aspiration unit 450 is capable of moving the sample aspiration nozzle 451 in order to insert it into the sample container 100. For example, the sample aspiration unit 450 is capable of moving the sample aspiration nozzle 451 to an upper position in the chamber 420. The sample aspiration unit 450 has a quantification unit 452 (e.g., a syringe pump) for aspirating and discharging the sample T by the sample aspiration nozzle 451.

[0017] The liquid delivery mechanism 430 comprises a liquid delivery tube 431 and a liquid delivery unit 432. The liquid delivery tube 431 is provided between the reagent container 200 and the chamber 420. The liquid delivery unit 432 delivers the reagent 12 from the reagent container 200 to the chamber 420 via the liquid delivery tube 431. The reagent container 200 is mounted in a reagent container holder 60. The reagent container 200 contains the reagent 12, which includes a first fluorescent dye and a second fluorescent dye. The liquid delivery mechanism 430 is a mechanism that injects the reagent 12 from the reagent container 200 into the chamber 420 via the liquid delivery tube 431. In the chamber 420, the sample and the reagent 12 come into contact, staining the particles contained in the sample with the first fluorescent dye and the second fluorescent dye.

[0018] A suction tube 64, which forms the first end of the liquid delivery tube 431, is inserted into the reagent container 200. The second end of the liquid delivery tube 431 is connected to the chamber 420. The suction tube 64 may have a sharp tip so as to be able to penetrate the sealing film (also called a sealing member) of the reagent container 200 mounted in the reagent container holder 60. In this case, the suction tube is also called a piercer.

[0019] The liquid delivery section 432 of the liquid delivery mechanism 430 includes a pump 433. The pump 433 is a quantitative unit that generates negative pressure for drawing reagent 12 from the reagent container 200 into the liquid delivery pipe 431 and positive pressure for supplying the drawn-in reagent to the chamber 420. The pump 433 may be, for example, a syringe pump or a diaphragm pump. The liquid delivery mechanism 430 may also include a plurality of valves. In Figure 2, the liquid delivery mechanism 430 includes solenoid valves V1 and V2. For example, when the pump 433, which is composed of a syringe pump or a diaphragm pump, draws reagent 12 from the reagent container 200, solenoid valve V1 is opened and solenoid valve V2 is closed. The pump 433 generates negative pressure, filling the flow path between solenoid valves V1, V2 and the pump 433 with reagent. When supplying the filled reagent to the chamber 420, solenoid valve V1 is closed, solenoid valve V2 is opened, and the pump 433 generates positive pressure. This allows the reagent 12 in the reagent container 200 to be supplied to the chamber 420.

[0020] Chamber 420 is a container in which the measurement sample is prepared. Inside Chamber 420, the reagent 12 and the sample are mixed to prepare a measurement sample containing particles stained with the first fluorescent dye and the second fluorescent dye. One or more chambers 420 are provided in the measurement unit 400. Chamber 420 is connected to the waste liquid chamber 36 via an electromagnetic valve 37. After measurement by the FCM detection unit 460 is completed, the measurement sample remaining in Chamber 420 is discarded into the waste liquid chamber 36. In addition, before the next measurement sample is prepared, Chamber 420 is cleaned by a cleaning mechanism (not shown), and the liquid after cleaning is discarded into the waste liquid chamber.

[0021] The measurement unit 400 includes one or more reagent container holders 60. In the example shown in Figure 2, one reagent container 200 containing reagents with a first fluorescent dye and a second fluorescent dye is mounted in one reagent container holder. If the measurement unit 400 includes multiple reagent container holders, for example, a reagent container containing a reagent with a first fluorescent dye and another reagent container containing a reagent with a second fluorescent dye may be mounted in different reagent container holders. Alternatively, a reagent container containing reagents with a first fluorescent dye and a second fluorescent dye and another reagent container containing a hemolytic reagent may be mounted in different reagent container holders.

[0022] The reagent container 200 is a container that holds reagents. The reagent container 200 has an opening into which a suction tube 64, connected to the first end of the liquid delivery tube 431 of the liquid delivery mechanism 430, is inserted. Before the reagent container 200 is mounted on the reagent container holder 60, for example, the opening of the reagent container 200 is covered with a sealing film. The suction tube 64 is inserted into the opening of the reagent container 200 mounted on the reagent container holder 60. The first end of the liquid delivery tube 431 is fixed in a predetermined position inside the reagent container 200. The predetermined position may be, for example, a position where the tip of the suction tube 64 connected to the first end is close to the bottom inside the reagent container 200. The suction tube 64 inserted into the reagent container 200 remains fixed in the predetermined position as described above, for example, while the reagent container 200 is mounted on the reagent container holder 60. Furthermore, at least while the measurement of multiple samples is being performed (i.e., while multiple measurement samples corresponding to each of the multiple samples are being prepared), the first end to which the suction tube 64 is connected is fixed in the predetermined position described above.

[0023] The FCM detection unit 460 irradiates individual particles in the sample being measured flowing through the flow cell with light. As described above, when particles are irradiated with light, fluorescence originating from the first and second fluorescent dyes is generated from those particles. The FCM detection unit 460 acquires optical signals including a first fluorescence signal and a second fluorescence signal corresponding to each fluorescence. The FCM detection unit 460 acquires multiple optical signals corresponding to each of the multiple particles irradiated with light. The first fluorescence signal is the signal corresponding to the fluorescence originating from the first fluorescent dye of the stained particles. The second fluorescence signal is the signal corresponding to the fluorescence originating from the second fluorescent dye of the stained particles. In addition, scattered light is emitted from the particles irradiated with light. The FCM detection unit 460 acquires a scattered light signal corresponding to the scattered light. The scattered light signal includes a side scattered light signal corresponding to side scattered light and a forward scattered light signal corresponding to forward scattered light. The FCM detection unit 460 may be equipped with multiple light sources. For example, the FCM detection unit 460 may include a light source that emits light of a first wavelength capable of exciting the first fluorescent dye, and a light source that emits light of a second wavelength capable of exciting the second fluorescent dye. Alternatively, the FCM detection unit 460 may include a light source that emits light of a single wavelength and be configured to detect fluorescence from the first and second fluorescent dyes excited by the single wavelength light.

[0024] Referring to Figure 3, the configuration of the measurement unit 400, which includes the fluid system shown in Figure 2, will be described. The measurement unit 400 comprises a sample preparation unit 440, a sample aspiration unit 450, a device mechanism unit 455, an FCM detection unit 460, and a measurement unit control unit 480. The sample preparation unit 440 includes a chamber for mixing the sample and reagents, and a reagent container holder 60 in which the reagent containers are installed. The sample preparation unit 440 delivers reagents from the reagent containers installed in the reagent container holder 60 to the chamber via a liquid delivery tube. The sample aspiration unit 450 aspirates the sample from the sample containers and discharges the aspirated sample into the chamber of the sample preparation unit 440. The measurement sample is prepared by mixing the sample and reagents in the chamber. The device mechanism unit 455 includes motors and actuators that move each part of the measurement unit 400. The device mechanism unit 455 includes, for example, a mechanism for moving the sample container 100.

[0025] The measurement unit control unit 480 comprises an analog processing unit 481, an A / D conversion unit 481a, IF (interface) units 484, 488, and 489, and a bus 485. The analog processing unit 481 processes the analog signal output from the FCM detection unit 460. The A / D conversion unit 481a converts the analog signal output from the analog processing unit 481 into a digital signal. The IF unit 484 electrically connects the A / D conversion unit 481a and the bus 485. The IF unit 488 electrically connects the sample preparation unit 440, the device mechanism unit 455, the sample aspiration unit 450, and the FCM detection unit 460 to the bus 485. The IF unit 489 electrically connects the bus 485 to the analysis unit 300. The bus 485 is electrically connected to the IF units 484, 488, and 489.

[0026] The measuring device 500 of the second embodiment is a multi-parameter automated blood cell analyzer capable of counting and classifying leukocytes in a blood sample, outputting information on the mechanism of leukocyte increase, detecting red blood cells (RBCs) / platelets (PLTs), and measuring hemoglobin (HGB) concentration, in addition to performing at least one of these functions. Referring to Figure 4, the configuration of the measuring unit of the measuring device 500 of the second embodiment will be described. The measuring unit 400 shown in Figure 4 comprises a sample preparation unit 440, a device mechanism unit 455, a sample aspiration unit 450, an FCM detection unit 460, an RBC / PLT detection unit 461, an HGB detection unit 462, and a measuring unit control unit 480. The sample preparation unit 440, the sample aspiration unit 450, the device mechanism unit 455, and the FCM detection unit 460 are the same as those of the measuring device of the first embodiment.

[0027] The RBC / PLT detection unit 461 is an electrical resistance type detection unit that counts red blood cells and platelets by introducing a measurement sample prepared from a blood sample and diluent into an aperture and detecting the change in electrical resistance that occurs when cells pass through the aperture. The HGB detection unit 462 measures the hemoglobin concentration in a blood sample using the sodium lauryl sulfate (SLS) hemoglobin method. The HGB detection unit 462 obtains the hemoglobin concentration in the blood by irradiating a measurement sample prepared from a blood sample and SLS hemolytic agent with light at a wavelength of 555 nm, which is the absorption wavelength of SLS hemoglobin, and measuring the absorbance. Hereinafter, the FCM detection unit 460, the RBC / PLT detection unit 461, and the HGB detection unit 462 may be collectively referred to as "detection units 460-462".

[0028] The measurement unit control unit 480 comprises analog processing units 481, 482, and 483, A / D conversion units 481a, 482a, and 483a, IF units 484, 488, and 489, and a bus 485. The analog processing unit 481 processes the analog signal output from the FCM detection unit 460. The A / D conversion unit 481a converts the analog signal output from the analog processing unit 481 into a digital signal. The analog processing unit 482 processes the analog signal output from the RBC / PLT detection unit 461. The A / D conversion unit 482a converts the analog signal output from the analog processing unit 482 into a digital signal. The analog processing unit 483 processes the analog signal output from the HGB detection unit 462. The A / D conversion unit 483a converts the analog signal output from the analog processing unit 483 into a digital signal. The IF unit 484 electrically connects the A / D conversion units 481a, 482a, and 483a to the bus 485. The IF unit 488 electrically connects the sample preparation unit 440, the device mechanism unit 455, the sample aspiration unit 450, the FCM detection unit 460, the RBC / PLT detection unit 461, and the HGB detection unit 462 to the bus 485. The IF unit 489 electrically connects the bus 485 to the analysis unit 300. The bus 485 is electrically connected to the IF units 484, 488, and 489.

[0029] The sample preparation unit 440 shown in Figure 4 comprises a first sample preparation unit 440A and a second sample preparation unit 440B (see Figure 5). The first sample preparation unit 440A prepares a first measurement sample for optical measurement by the FCM detection unit 460. The second sample preparation unit 440B prepares a second measurement sample for electrical resistance measurement by the RBC / PLT detection unit, and a third measurement sample for hemoglobin measurement by the HGB detection unit 462.

[0030] Referring to Figure 5, the first sample preparation unit 440A has a first chamber 420. The first chamber 420 is connected to reagent containers R1 and R2. Reagent container R1 contains a hemolytic reagent. Reagent container R2 contains a diluent. The first chamber 420 is also connected to reagent container 200 via a liquid delivery tube 431 and a suction tube 64. Reagent container 200 contains a staining reagent. A flow path is provided between the first chamber 420 and the FCM detection unit 460.

[0031] The second sample preparation unit 440B has a second chamber 55. The second chamber 55 is connected to reagent containers R2 and R3. Reagent container R2 is provided in common with the first sample preparation unit 440A. Reagent container R3 contains an SLS hemolytic agent. The SLS hemolytic agent is a reagent used to lyse red blood cells and prepare a sample suitable for hemoglobin measurement.

[0032] A reagent container 200 containing staining reagents is mounted in a reagent container holder 60. The reagent container holder 60 is equipped with a suction tube 64 for aspirating staining reagents from the reagent container 200, and a suction tube lifting mechanism 65 for raising and lowering the suction tube 64. The tip of the suction tube 64 can penetrate (puncture) the sealing material of the reagent container 200. A cover 63 is connected to the suction tube lifting mechanism 65. When the suction tube lifting mechanism 65 is lowered and the suction tube 64 is penetrating (puncturing) the sealing material of the reagent container 200, the cover 63 also lowers and covers the reagent container 200. When the suction tube lifting mechanism 65 is raised, the cover 63 also rises, and the reagent container 200 becomes removable from the outside.

[0033] A liquid delivery mechanism 430 is provided between the suction tube 64 and the first chamber 420. The liquid delivery mechanism 430 comprises a liquid delivery tube 431 and a metering block 432. The liquid delivery tube 431 has its first end connected to the suction tube 64 and its second end connected to the first chamber 420. The metering block 432 comprises a metering unit 30 and solenoid valves V1 and V2. A syringe pump is used as the metering unit 30. A diaphragm pump, for example, can also be used instead of a syringe pump. Solenoid valves V1 and V2 open and close the flow path. When delivering the staining reagent from the reagent container 200 to the chamber 420, the metering unit 30 applies negative pressure to the liquid delivery tube 431 with solenoid valve V1 open and solenoid valve V2 closed. As a result, staining reagent is drawn from the tip of the suction tube 64 into the liquid delivery tube 431, and a fixed amount of staining reagent is filled into the flow path between the electromagnetic valves V1 and V2 and the quantitative unit 30. Next, with electromagnetic valve V1 closed and electromagnetic valve V2 open, the quantitative unit 30 applies positive pressure to the liquid delivery tube 431. As a result, the fixed amount of staining reagent filled into the flow path between electromagnetic valves V1 and V2 and the quantitative unit 30 is pushed out, and the staining reagent is supplied to the chamber 420 through the liquid delivery tube 431.

[0034] A quantitative section 22 and electromagnetic valves V3 and V4 are provided in the flow path between the reagent container R1 containing the hemolytic reagent and the first chamber 420. A syringe pump is used as the quantitative section 22. A diaphragm pump, for example, can also be used instead of a syringe pump. Electromagnetic valves V3 and V4 open and close the flow path. The quantitative section 22, electromagnetic valves V3 and V4 quantitatively deliver the hemolytic reagent from the reagent container R1 to the first chamber 420 in the same manner as the electromagnetic valves V1 and V2 and the quantitative section 30 described above.

[0035] A quantitative section 33 and electromagnetic valves V5 and V6 are provided in the flow path between the reagent container R2 containing the diluent and the first chamber 420. A syringe pump is used as the quantitative section 33. A diaphragm pump, for example, can also be used instead of a syringe pump. Electromagnetic valves V5 and V6 open and close the flow path. The quantitative section 33 and electromagnetic valves V5 and V6 quantitatively deliver the diluent from the reagent container R2 to the first chamber 420.

[0036] A waste liquid chamber 36 for containing unwanted solutions is connected to the first chamber 420. An electromagnetic valve V7 for opening and closing the flow path is provided between the first chamber 420 and the waste liquid chamber 36. The first chamber 420 is connected to a pump 56A that supplies air into the first chamber 420 to agitate the liquid inside the first chamber 420.

[0037] A quantitative section 38 and electromagnetic valves V8 and V9 are provided in the flow path between the reagent container R2 containing the diluent and the second chamber 55. A syringe pump is used as the quantitative section 38. A diaphragm pump, for example, can also be used instead of a syringe pump. Electromagnetic valves V8 and V9 open and close the flow path. The quantitative section 38 and electromagnetic valves V8 and V9 quantitatively deliver the diluent from the reagent container R2 to the second chamber 55. A waste liquid chamber 41 for containing the unused solution is connected to the second chamber 55. Between the second chamber 55 and the waste liquid chamber 41, an electromagnetic valve V10 is provided to switch the flow path between a flow path from the second chamber 55 to the waste liquid chamber 41 and a flow path from the second chamber 55 to the RBC / PLT detection unit 461 and the HGB detection unit 462. Electromagnetic valve V13 will be described later.

[0038] A quantitative section 39 and electromagnetic valves V11 and V12 are provided in the flow path between the reagent container R3 containing the SLS hemolytic agent and the second chamber 55. A syringe pump is used as the quantitative section 39. A diaphragm pump, for example, can also be used instead of a syringe pump. Electromagnetic valves V11 and V12 open and close the flow path. The quantitative section 39 and electromagnetic valves V11 and V12 quantitatively deliver the SLS hemolytic agent from the reagent container R3 to the second chamber 55. The second chamber 55 is connected to a pump 56B that supplies air into the second chamber 55 to agitate the liquid inside the second chamber 55.

[0039] The sample aspiration unit 450 has a suction tube 20 and a quantitative unit 21. The tip of the suction tube 20 is sharply formed. When the sample aspiration unit 450 lowers the suction tube 20, the suction tube 20 punctures the lid 100a that seals the sample container 100 and is inserted inside. With the suction tube 20 inserted inside the sample container 100, the quantitative unit 21 generates negative pressure, causing the blood sample T contained in the sample container 100 to be aspirated into the suction tube 20. The sample aspiration unit 450 moves the suction tube 20 upward to remove it from the sample container 100 and moves the suction tube 20 horizontally above the first chamber 420. The sample aspiration unit 450 lowers the suction tube 20 relative to the first chamber 420, and the quantitative unit 21 generates positive pressure, causing the aspirated blood sample to be discharged into the first chamber 420. The sample aspiration unit 450 moves the suction tube 20 upward and horizontally above the second chamber 55, and discharges the blood sample into the second chamber 55 in the same manner as with the first chamber 420.

[0040] The first chamber 420 is connected to the FCM detection unit 460 (see Figure 4). The blood sample discharged into the first chamber 420 is mixed with the staining reagent contained in the reagent container 200 and the hemolytic reagent contained in the reagent container R1 to prepare the measurement sample. In the measurement sample, red blood cells are lysed by the hemolytic reagent. In addition, in the measurement sample, particles containing white blood cells are stained with a first fluorescent dye and a second fluorescent dye. The measurement sample is prepared, for example, as follows: First, the hemolytic reagent is supplied to the first chamber 420, and then the blood sample is discharged into the first chamber 420. Air is supplied to the first chamber 420, and the blood sample and hemolytic reagent are mixed. This dissolves the red blood cells in the blood sample. The staining reagent is supplied to the first chamber 420, which contains the mixture of the blood sample and the hemolytic reagent. Air is supplied to the first chamber 420, and the mixture and the staining reagent are mixed. The reaction between the fluorescent dye and the particles proceeds within the first chamber 420. The reaction time is, for example, less than 1 minute, preferably less than 50 seconds, and more preferably less than 45 seconds. This stains particles containing normal leukocytes and, if present, abnormal cells in the blood sample with the first and second fluorescent dyes, thereby preparing the measurement sample. The FCM detection unit 460 is connected to a pump (not shown), and the pump drives the measurement sample in the first chamber 420 to be supplied to the FCM detection unit 460 via a flow path. The FCM detection unit 460 acquires multiple optical signals from each particle, including fluorescence corresponding to the first fluorescent dye and fluorescence corresponding to the second fluorescent dye.

[0041] The second chamber 55 is connected to the RBC / PLT detection unit 461 and the HGB detection unit 462. The electromagnetic valve V13 switches between supplying the measurement sample from the second chamber 55 to the RBC / PLT detection unit 46 and supplying it to the HGB detection unit 462. The RBC / PLT detection unit 461 and the HGB detection unit 462 are connected to a pump (not shown), and the pump drives the measurement sample in the second chamber 55 to be supplied to the RBC / PLT detection unit 461 and the HGB detection unit 462, respectively. The second chamber 55 is used to prepare both the measurement sample for RBC / PLT detection and the measurement sample for HGB detection. These measurement samples are prepared, for example, as follows: First, a diluent is supplied from the reagent container R2 to the second chamber 55. Next, blood is discharged into the second chamber 55. This yields a measurement sample containing diluted blood. A portion of this measurement sample is supplied to the RBC / PLT detection unit 461 for electrical resistance detection. Next, SLS hemolytic agent is supplied from reagent container R3 to the measurement sample remaining in the second chamber 55. This lyses the red blood cells and yields a measurement sample containing SLS hemoglobin generated from hemoglobin. This measurement sample is then delivered to the HGB detection unit 462. In the example in Figure 5, the measurement sample for RBC / PLT detection and the measurement sample for HGB detection are prepared in a common second chamber 55, but they may be prepared in separate chambers.

[0042] The measuring device 500 having the above configuration is capable of measuring the Complete Blood Count (CBC) item, which consists of at least eight parameters: red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT), hemoglobin concentration (HGB), hematocrit value (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin level (MCH), and mean corpuscular hemoglobin concentration (MCHC). The measuring device 500 may be configured to output information regarding the mechanism of white blood cell increase. In addition to the CBC item, the measuring device 500 may be configured to measure the DIFF item, which classifies white blood cells into multiple subpopulations. In addition to the CBC item and the DIFF item, the measuring device may be configured to measure other items (for example, items that measure reticulocytes (RET) and platelets (PLT) stained with fluorescent dye).

[0043] A modified example of the first sample preparation unit 440A will be described with reference to Figure 6. In Figure 6, elements similar to those in Figure 5 are omitted from the illustration. As described above, Figure 5 illustrates a first sample preparation unit 440A configured to contain reagents containing a first fluorescent dye and a second fluorescent dye in one reagent container 200, and to deliver the reagents in the one reagent container 200 into the chamber 420 by one liquid delivery mechanism 430. The first sample preparation unit 440A shown in Figure 6 is configured to contain a reagent containing a first fluorescent dye in a first reagent container 200A, a reagent containing a second fluorescent dye in a reagent container 200B, to deliver the reagents in the first reagent container 200A into the chamber 420 by a first liquid delivery mechanism 430a, and to deliver the reagents in the reagent container 200B into the chamber 420 by a second liquid delivery mechanism 430b. With this configuration as well, particles contained in the sample can be stained with two fluorescent dyes.

[0044] The first sample preparation unit 440A in Figure 6 includes one or more chambers 420 for preparing a measurement sample by mixing a reagent containing a first fluorescent dye, a reagent containing a second fluorescent dye, and a sample. The first reagent container 200A, which contains the reagent containing the first fluorescent dye, is mounted in the first holder portion of the reagent container holder 442. The second reagent container 200B, which contains the reagent containing the second fluorescent dye, is mounted in the second holder portion of the reagent container holder 442.

[0045] The first liquid delivery mechanism 430a is provided for delivering the reagent from the first reagent container 200A to the chamber 420. The configuration of the first liquid delivery mechanism 430a is the same as that of the liquid delivery unit 430 described with reference to Figure 2. The second liquid delivery mechanism 430b is provided for delivering the reagent from the second reagent container 200B to the chamber 420. The configuration of the second liquid delivery mechanism 430b is the same as that of the first liquid delivery mechanism 430a. The liquid delivery pipes 431 of the two liquid delivery mechanisms 430a and 430b merge midway through the flow path and are connected to the chamber 420. In the example in Figure 6, an example is shown where the two liquid delivery pipes merge, but the two liquid delivery pipes may also be connected to the chamber 420 individually.

[0046] Referring to Figure 7, an example of the sample preparation unit 440 will be described. In the example in Figure 7, the sample preparation unit 440 is equipped with multiple types of chambers into which the sample aspirated from the sample container 100 by the suction tube 20 is dispensed. The sample preparation unit 440 prepares the measurement sample in the reaction chamber 420 and supplies the measurement sample to the detection unit corresponding to the reaction chamber 420. The detection unit performs measurement for each measurement sample, and the analysis unit 300 provides analysis results for each measurement sample.

[0047] The RBC / PLT reaction chamber 420A is a chamber for mixing the sample and diluent to prepare the measurement sample. The measurement sample prepared in chamber 420A is measured by the RBC / PLT detection unit 461.

[0048] The HGB reaction chamber 420B is a chamber for preparing a sample for measurement by mixing the hemolytic agent, diluent, and specimen. The sample for measurement prepared in chamber 420B is measured by the HGB detection unit 462.

[0049] The leukocyte classification reaction chambers 420C and 420D are chambers for preparing measurement samples for classifying leukocytes. The leukocyte classification reaction chamber 420C is used, for example, to prepare measurement samples for classifying leukocytes into several subpopulations (e.g., subpopulations including lymphocytes, monocytes, neutrophils, and eosinophils). In the leukocyte classification reaction chamber 420C, the measurement sample is prepared by mixing, for example, a hemolytic agent, a specimen, and a staining solution (reagent) for staining the cells in the specimen. The leukocyte classification reaction chamber 420D is a chamber for preparing measurement samples for classifying, for example, a subpopulation of basophils, which are a form of leukocyte, and a subpopulation of nucleated erythrocytes. The white blood cell count (WBC) may be counted by measuring the measurement sample prepared in the leukocyte classification reaction chamber 420D. In the leukocyte classification reaction chamber 420D, the measurement sample is prepared by mixing, for example, a hemolytic agent, a specimen, and a staining solution (reagent) for staining the cells in the specimen.

[0050] The leukocyte classification reaction chambers 420C and D may be used for the same purpose. For example, the leukocyte classification reaction chambers 420C and D may be chambers for preparing samples for classifying cells in a sample into at least (1) leukocytes into at least five subpopulations (e.g., subpopulations including lymphocytes, monocytes, neutrophils, eosinophils, and basophils), and (2) subpopulations of nucleated red blood cells. The hemolytic agents used in the preparation in the leukocyte classification reaction chambers 420C and D may be hemolytic agents of different compositions in each chamber. The staining solutions (reagents) used in the preparation in the leukocyte classification reaction chambers 420C and D may be staining solutions (reagents) of different compositions in each chamber.

[0051] The RET reaction chamber 420E is a chamber for preparing a sample for, for example, the measurement of reticulocytes. In the reaction chamber 420E, the sample, a reagent containing a fluorescent dye corresponding to the reaction chamber 420E, and a diluent are mixed to prepare the sample for measurement.

[0052] The sample preparation unit 440 may include other reaction chambers in addition to the reaction chamber 420 described above. For example, the sample preparation unit 440 may include a reaction chamber 420 (PLT reaction chamber 420) for preparing a sample for measuring platelets stained with a fluorescent dye in the FCM measurement unit 460. For example, the sample preparation unit 440 may include a reaction chamber 420 (RET / PLT reaction chamber 420) for preparing a sample for measuring reticulocytes and platelets, instead of the RET reaction chamber 420E.

[0053] In the example shown in Figure 7, the relationship between each reaction chamber and the corresponding detection unit is sometimes referred to as a "measurement channel" in this specification. For example, reaction chamber 420C and FCM detection unit 460 constitute one measurement channel. In the example shown in Figure 7, the sample preparation unit 440 is equipped with five measurement channels corresponding to reaction chambers 420A to E. The number of measurement channels is not limited to this.

[0054] The sample preparation unit 440 determines which of the multiple reaction chambers 420 illustrated in Figure 7 to use to prepare the measurement sample based on the measurement order for the sample. For example, if the measuring device 500 receives a measurement order that includes a CBC measurement instruction, the sample preparation unit 440 prepares the measurement sample in the RBC / PLT reaction chamber 420A, the HGB reaction chamber 420B, and the leukocyte classification reaction chamber 420D in order to obtain measurement results for at least eight parameters: RBC, WBC, PLT, HGB, HCT, MCV, MCH, and MCHC. The measurement sample prepared in reaction chamber 420A is measured in the RBC / PLT detection unit 461. The measurement sample prepared in reaction chamber 420B is measured in the HGB detection unit 462. The measurement sample prepared in reaction chamber 420D is measured in the FCM detection unit 460 to obtain the white blood cell count (WBC). Based on the measurement data obtained by detection units 460, 461, and 462, the parameters corresponding to the above-mentioned CBC are obtained. For example, if the measuring device 500 receives a measurement order (CBC+DIFF) that includes a DIFF item in addition to the CBC item for classifying and counting leukocytes, the sample preparation unit 440 prepares a measurement sample in the RBC / PLT reaction chamber 420A, HGB reaction chamber 420B, leukocyte classification reaction chamber 420C, and leukocyte classification reaction chamber 420D in order to obtain the results of leukocyte classification (e.g., five classifications: lymphocytes, monocytes, neutrophils, eosinophils, and basophils) and counting in addition to the CBC parameters. The measurement sample prepared in reaction chamber 420A is measured in the RBC / PLT detection unit 461. The measurement sample prepared in reaction chamber 420B is measured in the HGB detection unit 462. The samples prepared in reaction chambers 420C and D are measured in the FCM detection unit 460.

[0055] The sample preparation unit 440 prepares the measurement sample in the reaction chamber 420 and supplies the measurement sample to the detection unit corresponding to the reaction chamber 420. The detection unit performs the measurement for each measurement sample, and the analysis unit 300 provides the analysis results for each measurement sample. For example, the sample preparation unit 440 prepares the measurement sample using first and second fluorescent dyes in at least one of the multiple reaction chambers 420.

[0056] The sample preparation unit 440 may prepare measurement samples using the first and second fluorescent dyes in multiple reaction chambers 420. When measurement samples using the first and second fluorescent dyes are prepared in multiple reaction chambers 420, the first and second fluorescent dyes used in each reaction chamber 420 may differ from one reaction chamber to the other. For example, the first and second fluorescent dyes used in reaction chamber 420C may be different from the first and second fluorescent dyes used in reaction chamber 420D.

[0057] Furthermore, when measurement samples using the first and second fluorescent dyes are prepared in multiple reaction chambers 420, at least one of the first and second fluorescent dyes used in each reaction chamber 420 may be common to the other reaction chambers 420. For example, at least one of the first and second fluorescent dyes used in reaction chamber 420C (e.g., the first fluorescent dye) and at least one of the first and second fluorescent dyes used in reaction chamber 420D (e.g., the first fluorescent dye) may be common to all of them.

[0058] The first and second fluorescent dyes are included, for example, in at least one of the staining reagents used in the preparation of the measurement samples in the leukocyte classification reaction chambers 420C and D. The first and second fluorescent dyes may also be included in both of the staining reagents used in the preparation of the measurement samples in the leukocyte classification reaction chambers 420C and D.

[0059] The CBC item, or the CBC item and DIFF item, as described above, are basic test items in tests that classify and / or count cells in a blood sample. For example, the need for additional tests is determined based on the CBC or CBC+DIFF test results. Therefore, many measurement orders in tests using the measuring device 500 include instructions for measuring CBC, or instructions for measuring CBC and DIFF. For example, almost all measurement orders in the first blood test (e.g., called the "initial test") for a patient visiting a medical institution include instructions for measuring CBC, or instructions for measuring CBC and DIFF. Additional tests based on the results of the initial test may not include instructions for measuring CBC, or instructions for measuring CBC and DIFF, and may only include a predetermined additional test item (e.g., reticulocytes: RET). In some cases, there are no additional tests, and the test is completed with only the initial test.

[0060] In the examples of this embodiment, for example, (1) the staining solution used in reaction chamber 420C contains the first and second fluorescent dyes, (2) the staining solution used in reaction chamber 420D contains the first and second fluorescent dyes, (3) both the staining solution used in reaction chamber 420C and the staining solution used in reaction chamber 420D contain the first and second fluorescent dyes, or (4) the staining solution used in reaction chamber 420C contains the second fluorescent dye, and the staining solution used in reaction chamber 420D contains the first fluorescent dye. In case (1) above, a measurement using the first and second fluorescent dyes is performed for a measurement order that includes a measurement instruction for CBC+DIFF. In cases (2), (3) and (4) above, a measurement using the first and second fluorescent dyes is performed for a measurement order that includes a measurement instruction for CBC or CBC+DIFF. As mentioned above, the instruction to measure CBC or CBC+DIFF is included in almost all initial examination measurement orders, so measurement results including information on the mechanism of leukocyte increase (neoplastic or reactive) can be obtained without additional tests. In other words, measurement results including information on the mechanism of leukocyte increase (neoplastic or reactive) can be obtained with a single initial examination. Furthermore, in the cases of (1), (2), and (3) above, measurement results including information on the mechanism of leukocyte increase can be obtained based on the results obtained from the measurement operation using a single measurement channel.

[0061] Referring to Figure 8, an example of the optical system of the FCM detection unit 460 will be described. The FCM detection unit 460 includes a first light source 411a, a second light source 411b, a flow cell 413, dichroic mirrors 418a, 418b, and 418c, side-scattered light receiving elements 412a and 412b, a forward-scattered light receiving element 416, and side-fluorescence receiving elements 422a and 422b. The first light source 411a and the second light source 411b emit excitation light of different wavelengths. For example, the first light source 411a emits light of a first wavelength capable of exciting a first fluorescent dye, and the second light source 411b emits light of a second wavelength capable of exciting a second fluorescent dye. The first wavelength is, for example, 315 nm to 490 nm, preferably 400 nm to 450 nm, and more preferably 400 nm to 410 nm. The second wavelength is, for example, 610 nm to 750 nm, preferably 620 nm to 700 nm, and more preferably 633 nm to 643 nm. As each light source, for example, a semiconductor laser light source, an argon laser light source, a helium-neon laser, a mercury arc lamp, etc., can be used.

[0062] The sample prepared in chamber 420 is flowed into the flow cell 413 of the FCM detection unit 460. In the example in Figure 8, the sample is flowed perpendicular to the plane of the paper. While the sample is flowing in the flow cell 413, light emitted from the first light source 411a is reflected by the dichroic mirror 418a and irradiates individual particles in the sample flowing within the flow cell 413. Light emitted from the second light source 411b passes through the dichroic mirror 418a and irradiates multiple particles in the sample flowing within the flow cell 413.

[0063] The forward-scattered light (second forward-scattered light) corresponding to the light emitted from the second light source 411b is received by the forward-scattered light receiving element 416. In the example of Figure 8, the forward-scattered light receiving element 416 is positioned to receive the second forward-scattered light. Alternatively, the forward-scattered light receiving element 416 may be positioned to receive the forward-scattered light (first forward-scattered light) corresponding to the light emitted from the first light source 411a. In this case, the first forward-scattered light is received by the forward-scattered light receiving element 416. Alternatively, a separate receiving element may be provided in addition to the forward-scattered light receiving element 416 to receive both the first and second forward-scattered light. The forward-scattered light is, for example, scattered light with a reception angle of 0 to about 20 degrees, preferably 0 to about 5 degrees. The forward-scattered light receiving element 416 is, for example, a photodiode.

[0064] The lateral scattered light (first lateral scattered light) corresponding to the light emitted from the first light source 411a is reflected by the dichroic mirror 418b and received by the lateral scattered light receiving element 412a. The lateral scattered light (second lateral scattered light) corresponding to the light emitted from the second light source 411b is reflected by the dichroic mirror 418c and received by the lateral scattered light receiving element 412b. The lateral scattered light is, for example, scattered light with a reception angle of about 45 degrees to about 135 degrees, preferably about 90 degrees. The lateral scattered light receiving elements 412a and 412b are, for example, photodiodes.

[0065] The lateral fluorescence (first lateral fluorescence) corresponding to the light generated when the first fluorescent dye is excited is transmitted through the dichroic mirror 418b and received by the lateral fluorescence photodetector 422a. The lateral fluorescence (second lateral fluorescence) corresponding to the light generated when the second fluorescent dye is excited is transmitted through the dichroic mirror 418c and received by the lateral fluorescence photodetector 422b. The lateral fluorescence photodetectors 422a and 422b are, for example, avalanche photodiodes. Alternatively, photomultiplier tubes may be used as the forward scattered light photodetector 416, the lateral scattered light photodetectors 412a and 412b, and the lateral fluorescence photodetectors 422a and 422b.

[0066] Referring to the example in Figure 9, the relationship between the various types of light emitted when light is shone on particles P passing through the flow cell 413 and the optical system of the FCM detection unit 460 will be explained. In Figure 9, the light shone from the first light source 411a is light of the first wavelength L1, and the light shone from the second light source 411b is light of the second wavelength L2. When light L1 and L2 are shone on particles P passing through the flow cell 413, forward scattered light (FSC) is generated in front of the direction of light propagation. In the example in Figure 9, the photodetector 416 receives the forward scattered light corresponding to the light shone from the second light source 411b, so only the second forward scattered light corresponding to the second wavelength is shown, and the first forward scattered light corresponding to the first wavelength is omitted. In addition, first side scattered light (SSC-1) corresponding to the first wavelength and first side fluorescence (SFL-1) excited by the first wavelength are generated to the side of the direction of light propagation. Furthermore, a second lateral scattered light (SSC-2) corresponding to the second wavelength of light and a second lateral fluorescence (SFL-2) excited by the second wavelength of light are generated laterally with respect to the direction of light propagation. As described above, FSC, SSC-1, SFL-1, SSC-2, and SFL-2 are received by photodetectors 416, 412a, 422a, 412b, and 422b, respectively. Each photodetector outputs a waveform electrical signal (also called an optical signal or analog signal) containing pulses corresponding to the received light intensity. Hereinafter, the analog signal corresponding to FSC will be called the "forward scattered light signal," the analog signal corresponding to SSC-1 will be called the "first lateral scattered light signal," the analog signal corresponding to SFL-1 will be called the "first fluorescence signal," the analog signal corresponding to SSC-2 will be called the "second lateral scattered light signal," and the analog signal corresponding to SFL-2 will be called the "second fluorescence signal." One pulse of each analog signal corresponds to one particle (for example, one cell). Among the particles in the sample being measured, the first fluorescence signal corresponding to SFL-1 from cells reflects the state and / or amount of DNA in those cells. The second fluorescence signal corresponding to SFL-2 from cells reflects the state and / or amount of RNA in the particles in the sample being measured.

[0067] Analog signals corresponding to various types of light are input to the analog processing unit 481, where processing such as noise reduction and smoothing is 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 1024 points at 10 nanosecond intervals, sampling 128 points at 80 nanosecond intervals, or sampling 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 forward scattered light data, first side scattered light data, first fluorescence data, second side scattered light data, and second fluorescence data. The forward scattered light data, first side scattered light data, first fluorescence data, second side scattered light data, and second fluorescence data are waveform data composed of multiple values ​​arranged in time series. The generated waveform data is transmitted to the analysis unit 300, which calculates feature parameters representing the morphological characteristics of individual cells from the waveform data of each signal. Examples of such feature parameters include peak value (height of the pulse peak), pulse width, pulse area, transmittance, Stokes shift, ratio, changes over time, and values ​​correlated therewith.

[0068] Optical information may be the characteristic parameters described above. Optical information includes at least first fluorescence information and second fluorescence information. First fluorescence information is not particularly limited as long as it reflects the amount of fluorescent dye used to stain DNA in nucleated cells. Second fluorescence information is not particularly limited as long as it reflects the amount of fluorescent dye used to stain RNA in nucleated cells. Preferably, the first fluorescence information and second fluorescence information are the peak value of the first fluorescence data (the largest value among the first fluorescence data, also called the "first fluorescence intensity") and the peak value of the second fluorescence data (the largest value among the second fluorescence data, also called the "second fluorescence intensity"), respectively. Optical information further includes scattered light information. Scattered light information includes forward scattered light information, first lateral scattered light information, and second lateral scattered light information. Lateral scattered light information is not particularly limited as long as it reflects internal information such as the complexity of the cell structure, granular characteristics, nuclear structure, and degree of lobulation. Preferably, the first lateral scattered light information and the second lateral scattered light information are the peak value of the first lateral scattered light data (the largest value among the first lateral scattered light data, also called the "first lateral scattered light intensity") and the peak value of the second lateral scattered light data (the largest value among the second lateral scattered light data, also called the "second lateral scattered light intensity"), respectively. The forward scattered light information is not particularly limited as long as it reflects the size of the cell. Preferably, the forward scattered light information is the peak value of the forward scattered light data (the largest value among the forward scattered light data, also called the "forward scattered light intensity").

[0069] Referring to Figure 10, an example of the optical system of an FCM detection unit equipped with a single light source will be described. The FCM detection unit 460 comprises a light source 411, a flow cell 413, a dichroic mirror 418, a side-scatter light receiving element 412, a forward-scatter light receiving element 416, and side-fluorescence receiving elements 422a and 422b. The light source 411 is a light source that emits light of a wavelength capable of exciting both the first fluorescent dye and the second fluorescent dye. The light emitted from the light source 411 irradiates individual particles in the sample being measured flowing through the flow cell 413. When the light from the light source 411 irradiates particles in the sample being measured stained with the first fluorescent dye and the second fluorescent dye, first side fluorescence and second side fluorescence are generated. In other words, the FCM detection unit 460 can acquire first and second fluorescence signals corresponding to each of multiple particles in the sample being measured (i.e., multiple particles stained with the first and second fluorescent dyes) using a single light source.

[0070] The forward scattered light corresponding to the light emitted from the light source 411 is received by the forward scattered light receiving element 416. The side scattered light corresponding to the light emitted from the light source 411 is reflected by the dichroic mirror 418 and received by the side scattered light receiving element 412. The first side fluorescence is received by the side fluorescence receiving element 422a. The second side fluorescence is transmitted through the dichroic mirror 418 and received by the side fluorescence receiving element 422b. Thus, the FCM detection unit 460 can acquire the forward scattered light signal, the first side scattered light signal, the first fluorescence signal, and the second fluorescence signal, each corresponding to multiple particles in the sample being measured.

[0071] The light emitted from the light source 411 is preferably light containing multiple wavelengths in order to excite both the first fluorescent dye and the second fluorescent dye. Examples of such light include white light. Alternatively, if the first fluorescent dye and the second fluorescent dye have maximum absorption in a wavelength range close to the point where they can be excited by a single wavelength of light, the light emitted from the light source 411 may be light of that single wavelength. For example, if one of the first fluorescent dye and the second fluorescent dye has maximum absorption in the wavelength range of 400 nm to 520 nm, and the other has maximum absorption in the wavelength range of 300 nm to 420 nm, the light emitted from the light source 411 may be light with a central wavelength of 400 nm to 420 nm, for example, light of 405 nm. Furthermore, for example, if the maximum absorption of the first fluorescent dye and the second fluorescent dye is within the wavelength range of 630 nm to 660 nm, and one of the first or second fluorescent dyes emits fluorescence with a peak in the wavelength range of 660 nm to 670 nm, while the other emits fluorescence with a peak in the wavelength range longer than 670 nm, then the light irradiated from the light source 411 may be light with a central wavelength of 630 nm to 655 nm, for example, light at 633 nm. Depending on the combination of the first and second fluorescent dyes, by irradiating the light source 411 with light of such a single wavelength, the FCM detection unit 460 can excite both the first and second fluorescent dyes and distinguish and detect the fluorescence generated from each fluorescent dye.

[0072] Referring to Figure 11, the configuration of the analysis unit 300 will be described. The analysis unit 300 is electrically connected to the measurement unit 400 via an interface unit 305. The interface unit 305 is, for example, a USB interface. The analysis unit 300 comprises a processor 301, a main memory 302, a bus 303, a storage unit 304, an interface unit 305, a display unit 306, and an operation unit 307. The analysis unit 300 is configured by, for example, a personal computer (see the analysis unit 300 in Figure 1), and controls the measurement unit 400 of the measurement device 500 by executing a program stored in the storage unit 304. The analysis unit 300 executes, for example, an analysis program and analyzes the data acquired from the measurement unit 400. The analysis unit 300 displays the analysis results on the display unit 306.

[0073] The analysis unit 300 performs at least one of particle classification and counting based on optical information including first fluorescence information corresponding to a first fluorescence signal and second fluorescence information corresponding to a second fluorescence signal. Preferably, the optical information further includes scattered light information corresponding to a scattered light signal. The scattered light information includes lateral scattered light information corresponding to a lateral scattered light signal and forward scattered light information corresponding to a forward scattered light signal. If the white blood cell count is increased compared to that of a healthy person depending on the health status or disease of the subject, the analysis unit 300 can generate information on the mechanism of the white blood cell increase based on the optical information.

[0074] The analysis unit 300 may classify cells based on the optical information of each of multiple particles. For example, the analysis unit 300 may classify a cell by inputting waveform data corresponding to a single cell (e.g., waveform data corresponding to at least one, preferably more, of forward scattered light data, first side scattered light data, first fluorescence data, second side scattered light data, and second fluorescence data) as optical information into a trained AI algorithm. Alternatively, the analysis unit 300 may classify cells based on the characteristic parameters (e.g., peak value, pulse width, and pulse area) of the waveform data corresponding to a single particle (e.g., forward scattered light data, first side scattered light data, first fluorescence data, second side scattered light data, and second fluorescence data) as optical information. One method for classifying particles into multiple types using multiple characteristic parameters is to plot the particles in a multidimensional coordinate space with multiple parameters as axes, classify at least some particles into multiple groups corresponding to multiple types, determine the degree of belonging of each particle to each group based on the distance between the centroid position of each group and the particle, and then reclassify the particles based on the degree of belonging to classify multiple particles into multiple types. Such a classification method is described, for example, in U.S. Patent No. 5,555,198, which is incorporated herein by reference. Alternatively, classification based on an AI algorithm may be performed for some particles in a single sample, while classification based on characteristic parameters may be performed for other particles.

[0075] The processor 301 is a CPU (Central Processing Unit) and executes programs deployed from the storage unit 304 to the main memory 302. The storage unit 304 is, for example, a hard disk or an SSD (Solid State Drive). The storage unit 304 stores, for example, a program for controlling the measurement unit 400 and a program for analyzing data acquired by the measurement unit 400. The display unit 306 is equipped with a computer screen. The display unit 306 is electrically connected to the processor 301 via the interface unit 305 and the bus 303. The display unit 306 displays, for example, the analysis results of data acquired by the measurement unit 400.

[0076] The control unit 307 includes a pointing device, including a keyboard, mouse, or touch panel. Users such as doctors and laboratory technicians can input measurement orders into the measuring device 500 by operating the control unit 307. Measurement instructions are input into the measuring device 500 according to the measurement order. The control unit 307 can also receive instructions from the user to display the test results. Users can operate the control unit 307 to view various information related to the test results, such as graphs, charts, and flag information assigned to the specimens. The measurement unit 400 is electrically connected to the analysis unit 300 via the interface unit 305.

[0077] Referring to Figure 12, an example of the operation of each unit of the measuring device 500 will be described, but it is not limited to this example. The analysis unit 300 performs the corresponding operation by executing a program deployed from the storage unit 304 to the memory 302 using the processor 301. In step S1, the analysis unit 300 receives a measurement execution instruction from the user via an input operation through the operation unit 307. The analysis unit 300 sends instruction data to the measurement unit 400 to instruct the start of measurement, causing the measurement unit 400 to start the preparation process of the measurement sample. In step S2, the measurement unit 400 dispenses the sample into the chamber 420. In step S3, the measurement unit 400 injects the reagent 12 into the chamber 420 via the liquid delivery tube 431 connecting the reagent container 200 and the chamber 420. The execution order of steps S2 and S3 can be changed. In step S4, the measurement unit 400 mixes the sample with reagent 12 containing first and second fluorescent dyes in the chamber 420 to prepare a measurement sample. In step S5, the measurement unit 400 sends the measurement sample prepared in the chamber 420 to the FCM detection unit 460 and irradiates multiple particles in the measurement sample with light to perform optical measurement. As a result, the measurement unit 400 acquires an optical signal including a first fluorescence signal and a second fluorescence signal corresponding to each fluorescence generated from the particles. The acquired optical signal is digitized by the A / D conversion unit 481, etc., and transmitted from the measurement unit 400 to the analysis unit 300 as waveform data, such as waveform data of the first fluorescence data and waveform data of the second fluorescence data. In step S6, the analysis unit 300 generates optical information from the received waveform data. In step S7, the analysis unit 300 analyzes the optical information. In step S8, the analysis unit 300 provides the analysis results. For example, the analysis unit 300 displays the analysis results on the display unit 306. Then, the analysis system completes the operation shown in Figure 12. The details of the analysis process in step S7 of Figure 12 will be described below for each embodiment. In the following embodiments, an example in which feature parameters are used as optical information will be described, but waveform data may also be used as optical information.

[0078] Embodiments 1 to 4 are shown as examples of the analysis process in step S7. In the analysis process of each embodiment, the analysis unit 300 performs an analysis related to the mechanism of leukocyte increase. In the analysis process of "Embodiment 1", the analysis unit 300 classifies leukocytes into subpopulations based on the first fluorescence information, the second fluorescence information, and the scattered light information, and performs an analysis related to the mechanism of leukocyte increase based on the classification. In the analysis process of "Embodiment 2", the analysis unit 300 performs an analysis related to the mechanism of leukocyte increase based on the first fluorescence information, the second fluorescence information, and the scattered light information. In the analysis process of "Embodiment 3", the analysis unit 300 performs a process of classifying leukocytes into subpopulations based on the second fluorescence information and the scattered light information, and a process of classifying leukocytes into subpopulations based on the first fluorescence information and the second fluorescence information, and performing an analysis related to the mechanism of leukocyte increase based on the classification. In the analysis process of "Embodiment 4", the analysis unit 300 performs an analysis related to the mechanism of leukocyte increase based on the first fluorescence information and the second fluorescence information.

[0079] In this specification, leukocytes that the analysis unit 300 determined to be factors in the mechanism of leukocyte increase that may be neoplastic increase are also referred to as "first-order leukocytes." In this specification, leukocytes that the analysis unit 300 determined to be factors in the mechanism of leukocyte increase that may be reactive increase are also referred to as "second-order leukocytes." First-order leukocytes may appear in the blood due to tumors such as multiple myeloma, chronic lymphocytic leukemia, and malignant lymphoma. Examples of first-order leukocytes include abnormal lymphocytes, blast cells, and immature erythroblasts. Abnormal lymphocytes are lymphocytes that have undergone neoplastic morphological changes and are clonal and homogeneous cells. Blast cells include myeloblasts and lymphoblasts. However, erythroblasts are not included in blast cells. Immature erythroblasts include proerythroblasts, basophilic erythroblasts, and polychromatic erythroblasts. Blast cells and immature erythroblasts themselves are not pathological cells, and in healthy individuals, these cells are mainly found in the bone marrow. In analytical processing, leukocytes are detected as white blood cells that contain more DNA than other white blood cells such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

[0080] Secondary leukocytes (II leukocytes) can appear in the blood due to immune responses such as viral infections, drug allergies, and autoimmune diseases. These II leukocytes are, for example, atypical lymphocytes. Also known as reactive lymphocytes, they are lymphocytes that have been activated and morphologically altered by antigen stimulation. In analytical testing, II leukocytes are detected as white blood cells containing more RNA than other white blood cells such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

[0081] Regarding step S7 in Figure 12, an example of the analytical process of Embodiment 1 will be described with reference to Figure 13, but the method is not limited to this example. This analytical process enables the classification of leukocytes into subpopulations and the analysis of the mechanism of leukocyte increase. In this example, lateral scattered light information is used as scattered light information. More specifically, the intensity of the first lateral scattered light (also called "SSC-1 intensity") is used as lateral scattered light information. The intensity of the second lateral scattered light (also called "SSC-2 intensity") may be used instead of the SSC-1 intensity. Hereinafter, the SSC-1 intensity and SSC-2 intensity may be collectively referred to as "SSC intensity". The first fluorescence intensity (also called "SFL-1 intensity") is used as the first fluorescence information, and the second fluorescence intensity (also called "SFL-2 intensity") is used as the second fluorescence information. A scattergram with SSC intensity on the horizontal axis and SFL-1 intensity on the vertical axis is also called the "first scattergram". Furthermore, a scattergram with SSC intensity on the horizontal axis and SFL-2 intensity on the vertical axis is also called a "second scattergram."

[0082] Referring to Figure 13, in step S11, the analysis unit 300 determines the position of the point corresponding to each particle on a plane with SSC intensity on the horizontal axis and SFL-1 intensity on the vertical axis, based on the acquired optical information. Based on the determined position of each point, the analysis unit 300 creates a first scattergram. In step S12, the analysis unit 300 determines the position of the point corresponding to each particle on a plane with SSC intensity on the horizontal axis and SFL-2 intensity on the vertical axis, based on the acquired optical information. Based on the determined position of each point, the analysis unit 300 creates a second scattergram.

[0083] In step S13, the analysis unit 300 classifies the leukocytes in the sample into subpopulations based on the location of each determined point. These subpopulations may include, for example, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The classification of leukocytes may also be divided into at least two, three, or four subpopulations corresponding to these subpopulations. The leukocyte subpopulations preferably include lymphocytes, more preferably lymphocytes and monocytes, and even more preferably lymphocytes, monocytes, and neutrophils. Algorithms for detecting each subpopulation of leukocytes are known. For example, a program installed in the analysis unit 300 may classify the leukocytes in the sample into subpopulations. Alternatively, the leukocytes in each subpopulation may be counted.

[0084] For example, as shown in Figures 14A and B, each subpopulation of leukocytes is distributed on each scattergram. In the figures, "Lymp" refers to the lymphocyte population, "Mono" to the monocyte population, "Neut" to the neutrophil population, "Eo" to the eosinophil population, and "Baso" to the basophil population. Figures 14A and B show only the subpopulations of leukocytes, and do not show the first and second leukocytes. In these figures, leukocytes are classified into five subpopulations: lymphocytes, monocytes, neutrophils, eosinophils, and basophils, but are not limited to these. Leukocytes may also be classified into two subpopulations: mononuclear cells and multinucleated cells. Leukocytes may also be classified into three subpopulations: lymphocytes, monocytes, and neutrophils. Alternatively, leukocytes may be classified into four subpopulations: lymphocytes, monocytes, neutrophils, and eosinophils. If necessary, the cells contained in each subpopulation of leukocytes may be counted. As can be seen from Figure 14A, the distribution range of SFL-1 intensity for each subpopulation of leukocytes is almost the same. This suggests that the amount of DNA in each subpopulation of leukocytes is approximately the same. In Figures 14A and B, leukocytes are classified using both the first and second scattergrams, but leukocytes may be classified using only one of the scattergrams. Preferably, leukocytes are classified using the second scattergram.

[0085] After the classification of leukocytes, the process proceeds to step S14 in Figure 13. In step S14, the analysis unit 300 detects first leukocytes based on the SFL-1 intensity of each detected particle. Specifically, the analysis unit 300 detects particles exhibiting an SFL-1 intensity greater than a first threshold as first leukocytes. The analysis unit 300 also counts the detected first leukocytes. The first threshold is, for example, a value greater than or equal to the SFL-1 intensity exhibited by leukocytes classified into each subpopulation: Lymp, Mono, Baso, Neut, and Eo. For example, the first threshold can be set to the maximum value or higher of the SFL-1 intensity of any subpopulation of leukocytes (preferably the lymphocyte subpopulation or the monocyte subpopulation). The maximum value of the SFL-1 intensity of a subpopulation of leukocytes refers to the highest SFL-1 intensity among the cells classified into that subpopulation. The first threshold may be a value predetermined based on the results of measuring a sample (e.g., peripheral blood) obtained from a healthy individual and classifying the leukocytes into the aforementioned subpopulations.

[0086] In step S14, the analysis unit 300 can, for example, gate the region on the first scattergram where the SFL-1 intensity is greater than or equal to a first threshold, and detect particles appearing within that region as first leukocytes. Furthermore, the analysis unit 300 counts the detected first leukocytes. Referring to Figure 15, the region where the SFL-1 intensity is greater than or equal to a first threshold is, for example, the region enclosed by the dashed line on the first scattergram. Within this dashed region, particles exhibiting an SFL-1 intensity greater than the first threshold on the first scattergram may appear. In Figure 15, the arrow indicates the maximum SFL-1 intensity of the lymphocyte population of leukocytes as an example of the first threshold. In the example in Figure 15, the analysis unit 300 performs an analysis based on the differences in staining characteristics of the first and second fluorescent dyes, and the differences in fluorescence characteristics of the first and second fluorescent dyes (such an analysis may be referred to as the "first analysis" in this specification). For example, the analysis unit 300 classifies multiple cells stained according to differences in staining characteristics into a first population (the cell population shown in Figure 15) corresponding to the first fluorescence intensity (SFL-1) from the first fluorescent dye, and a second population (the cell population shown in Figure 16, described later) corresponding to the second fluorescence intensity (SFL-2) from the second fluorescent dye. In the examples of Figures 15 and 16, the analysis unit 300 classifies the cells using a scattergram based on the first fluorescence intensity (SFL-1) (example in Figure 15) and a scattergram based on the second fluorescence intensity (SFL-2) (example in Figure 16).

[0087] As shown in the examples described later, the inventors found that in samples in which leukocytes are increased due to a neoplastic mechanism (for example, samples containing abnormal lymphocytes, blast cells, or immature erythroblasts), particles showing a higher SFL-1 intensity than leukocytes appear in the first scattergram. Since the first fluorescent dye is a dye that specifically binds to DNA, the SFL-1 intensity of the particles depends on the amount of the first fluorescent dye bound to the DNA of the particles. As can be seen from Figure 15, particles appearing in the area enclosed by the dashed line (i.e., the first leukocyte) are detected as cells containing more DNA than leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils (i.e., cells to which more of the first fluorescent dye is bound than leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils). In the example in Figure 15, the analysis unit 300 performs an analysis based on differences in a first component (DNA in the example in Figure 15) in multiple measured cells (such an analysis may be referred to herein as the "second analysis"). For example, the analysis unit 300 classifies multiple cells stained with a first fluorescent dye for a first component (DNA in the example in Figure 15) into multiple populations according to their first fluorescence intensity (SFL-1). In the example in Figure 15, the analysis unit 300 classifies cells corresponding to SFL-1 higher than a first threshold as first leukocytes, and cells corresponding to SFL-1 lower than a first threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

[0088] In step S15, the analysis unit 300 detects secondary leukocytes based on the SFL-2 intensity of each particle. Specifically, the analysis unit 300 detects particles exhibiting an SFL-2 intensity greater than a second threshold as secondary leukocytes. The analysis unit 300 also counts the detected secondary leukocytes. The second threshold is, for example, a value greater than or equal to the SFL-2 intensity exhibited by leukocytes classified into each subpopulation: Lymp, Mono, Baso, Neut, and Eo. Here, among the subpopulations of leukocytes such as Lymp, Mono, Baso, Neut, and Eo, the monocyte population usually exhibits a higher SFL-2 intensity than the other subpopulations. Therefore, the second threshold can be set to, for example, the maximum value of the SFL-2 intensity of the monocyte population of leukocytes or a higher value. The maximum value of the SFL-2 intensity of the monocyte population of leukocytes refers to the highest value among the SFL-2 intensities of cells classified into the monocyte population. The second threshold may be a value predetermined based on the results of measuring a sample (e.g., peripheral blood) obtained from a healthy individual and classifying the white blood cells.

[0089] In step S15, the analysis unit 300 can also gate the region on the second scattergram where the SFL-2 intensity is greater than or equal to the second threshold, and detect particles appearing within that region as second leukocytes. Furthermore, the analysis unit 300 counts the detected second leukocytes. Referring to Figure 16, the region where the SFL-2 intensity is greater than or equal to the second threshold is, for example, the region enclosed by the dashed line on the second scattergram. Within this dashed region, particles showing an SFL-2 intensity greater than the second threshold on the second scattergram may appear. In Figure 16, the arrow indicates the maximum SFL-2 intensity of the monocyte population of leukocytes as an example of the second threshold. In the example in Figure 16, the analysis unit 300 performs an analysis based on differences in the second component (RNA in the example in Figure 16) in multiple cells measured (such an analysis may be referred to herein as the “third analysis”). For example, the analysis unit 300 classifies multiple cells stained with a second fluorescent dye for a second component (RNA in the example in Figure 16) into multiple populations according to their second fluorescence intensity (SFL-2). In the example in Figure 16, the analysis unit 300 classifies cells corresponding to SFL-2 higher than the second threshold as second leukocytes, and cells corresponding to SFL-2 lower than the second threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

[0090] As shown in the examples described later, the inventors found that in samples containing leukocytes increased by the reactivity-enhancing mechanism (e.g., atypical lymphocytes), a large number of particles exhibiting higher SFL-2 intensity than leukocytes classified into subpopulations such as Lymp, Mono, Baso, Neut, and Eo appeared in the second scattergram. Since the second fluorescent dye is a dye with high RNA binding ability (higher RNA binding ability than the first fluorescent dye), the SFL-2 intensity of a particle depends on the amount of the second fluorescent dye bound to the RNA of that particle. As can be seen from Figure 16, particles appearing within the area enclosed by the dashed line (i.e., second leukocytes) are detected as cells containing more RNA than leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils (i.e., cells to which more of the second fluorescent dye is bound than leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils).

[0091] As a variation of step S14, the analysis unit 300 can identify first leukocytes based on the SFL-1 intensity and SSC intensity of each particle. Also, as a variation of step S15, the analysis unit 300 can identify second leukocytes based on the SFL-2 intensity and SSC intensity of each particle. The inventors have found that leukocytes increased by neoplastic or reactive mechanisms are often mononuclear cells. Here, lateral scattered light information reflects internal information of the cell structure. Therefore, by excluding particles other than leukocytes increased by neoplastic or reactive mechanisms based on SSC intensity, in addition to SFL-1 intensity or SFL-2 intensity, more accurate detection of leukocytes increased by neoplastic or reactive mechanisms becomes possible. Specifically, the analysis unit 300 detects particles that show an SFL-1 intensity greater than a first threshold and an SSC intensity within a first range as first leukocytes. Furthermore, the analysis unit 300 detects particles that exhibit an SFL-2 intensity greater than the second threshold and an SSC intensity within the second range as second leukocytes. In addition, the analysis unit 300 counts the detected first and second leukocytes.

[0092] The first range and the second range may be the same or different. In this specification, "within the first range" and "within the second range" include the lower and upper limits of each numerical range. The lower limits of the first and second ranges can be determined, for example, based on the SSC intensity of the lymphocyte population of leukocytes. For example, the lower limits of the first and second ranges may be the minimum, maximum, or representative value of the SSC intensity of the lymphocyte population of normal leukocytes. The upper limits of the first and second ranges can be determined, for example, based on the SSC intensity of the monocyte population or neutrophil population of leukocytes. For example, the upper limits of the first and second ranges may be the minimum, maximum, or representative value of the SSC intensity of the monocyte population or neutrophil population of normal leukocytes. The first and second ranges may be predetermined numerical ranges based, for example, on the results of measuring a sample (e.g., peripheral blood) obtained from a healthy person and classifying the leukocytes.

[0093] The maximum SSC intensity of a leukocyte subpopulation refers to the highest SSC intensity among the cells classified into that subpopulation. The minimum SSC intensity of a leukocyte subpopulation refers to the lowest SSC intensity among the cells classified into that subpopulation. The statistical representative value of the SSC intensity of a leukocyte subpopulation is a value obtained from the SSC intensities of the cells classified into that subpopulation. Examples of representative values ​​include the median, mean, mode, and centroid. The centroid of SSC intensity refers to the SSC intensity of the point (cell) located at the centroid of the subpopulation as displayed on the scattergram. The median is preferred as the representative value.

[0094] In a modified version of step S14 described above, the analysis unit 300 can also gate a predetermined region on the first scattergram and detect particles appearing within that region as first leukocytes. Such a predetermined region is, for example, a region where the SFL-1 intensity is above a first threshold and the SSC intensity is within a first range (also called "gate B"). Furthermore, the analysis unit 300 counts the detected first leukocytes. Referring to Figure 17, gate B is the region enclosed by the dashed line on the first scattergram. Within gate B, particles may appear that show an SFL-1 intensity greater than a first threshold on the first scattergram and an SSC intensity within a first range. In Figure 17, the arrow indicates the maximum SFL-1 intensity of the lymphocyte population of leukocytes as an example of the first threshold. Also in Figure 17, the range shown is above a representative value of the SSC intensity of the lymphocyte population and below a representative value of the SSC intensity of the neutrophil population as an example of the first range. In the example in Figure 17, the analysis unit 300 performs an analysis ("first analysis") based on the differences in staining characteristics between the first and second fluorescent dyes, and the differences in fluorescence characteristics between the first and second fluorescent dyes. For example, the analysis unit 300 classifies multiple cells stained according to the differences in staining characteristics into a first population (the population of cells shown in Figure 17) corresponding to the first fluorescence intensity (SFL-1) from the first fluorescent dye, and a second population (the population of cells shown in Figure 18, described later) corresponding to the second fluorescence intensity (SFL-2) from the second fluorescent dye. In the examples in Figures 17 and 18, the analysis unit 300 classifies the cells using a scattergram based on the first fluorescence intensity (SFL-1) (example in Figure 17) and a scattergram based on the second fluorescence intensity (SFL-2) (example in Figure 18). In the example in Figure 17, the analysis unit 300 performs an analysis (second analysis) based on the differences in a first component (DNA in the example in Figure 17) in multiple measured cells. For example, the analysis unit 300 classifies multiple cells stained with a first fluorescent dye for a first component (DNA in the example in Figure 17) into multiple populations according to their first fluorescence intensity (SFL-1).In the example shown in Figure 17, the analysis unit 300 classifies cells with an SFL-1 level higher than a first threshold and an SSC level within a first range as first leukocytes, and cells corresponding to an SFL-1 level lower than the first threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

[0095] In a modified version of step S15 described above, the analysis unit 300 can also gate a predetermined region on the second scattergram and detect particles appearing within that region as second leukocytes. Such a predetermined region is, for example, a region where the SFL-2 intensity is above a second threshold and the SSC intensity is within a second range (also called "gate A"). Furthermore, the analysis unit 300 counts the detected second leukocytes. Referring to Figure 18, gate A is the region enclosed by the dashed line on the second scattergram. Within gate A, particles may appear that show an SFL-2 intensity greater than the second threshold on the second scattergram and an SSC intensity within the second range. In Figure 18, the arrow indicates the maximum SFL-2 intensity of the monocyte population of leukocytes as an example of the second threshold. Also in Figure 18, the range shown is above the representative value of the SSC intensity of the lymphocyte population and below the representative value of the SSC intensity of the neutrophil population as an example of the second range. In the example in Figure 18, the analysis unit 300 performs a third analysis based on differences in a second component (RNA in the example in Figure 18) among multiple measured cells. For example, the analysis unit 300 classifies multiple cells stained with a second fluorescent dye for a second component (RNA in the example in Figure 18) into multiple populations according to their second fluorescence intensity (SFL-2). In the example in Figure 18, the analysis unit 300 classifies cells with an SFL-2 higher than the second threshold and an SSC within the second range as second leukocytes, and cells corresponding to an SFL-2 lower than the second threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

[0096] In step S16, the analysis unit 300 generates information regarding the mechanism of leukocyte increase based on the detection results of first and second leukocytes. The detection result of first leukocytes is, for example, the number of first leukocytes detected and counted in step S14. The detection result of second leukocytes is, for example, the number of second leukocytes detected and counted in step S15. In step S16, the analysis unit 300 determines whether the number of first leukocytes is above a third threshold. The analysis unit 300 also determines whether the number of second leukocytes is above a fourth threshold. Depending on the results of these determinations, the analysis unit 300 can generate information regarding the mechanism of leukocyte increase, including information indicating that the mechanism of increase of first leukocytes in the sample is neoplastic, and information indicating that the mechanism of increase of second leukocytes in the sample is reactive. The conditions for determination and examples of the generated information are described below with reference to Figure 19.

[0097] Condition 1 is, for example, a condition in which the number of first white blood cells is equal to or greater than the third threshold and the number of second white blood cells is less than the fourth threshold. Condition 2 is, for example, a condition in which the number of first white blood cells is less than the third threshold and the number of second white blood cells is equal to or greater than the fourth threshold. Condition 3 is, for example, a condition in which the number of first white blood cells is less than the third threshold and the number of second white blood cells is less than the fourth threshold. The analysis unit 300 determines whether the number of first white blood cells counted in step S14 and the number of second white blood cells counted in step S15 satisfy any of conditions 1 to 3.

[0098] When the number of first and second white blood cells satisfies condition 1, the analysis unit 300 generates information indicating that the mechanism of increase in white blood cells in the sample is neoplastic. The process then proceeds to step S8. In step S8, the analysis unit 300 outputs a flag "Malignant?" to the display unit 306 as information indicating that the mechanism of increase in white blood cells is neoplastic.

[0099] When the number of first and second leukocytes satisfies condition 2, the analysis unit 300 generates information indicating that the mechanism of increase in leukocytes in the sample is reactive increase. The process then proceeds to step S8. In step S8, the analysis unit 300 outputs a flag "Reactive?" to the display unit 306 as information indicating that the mechanism of increase in leukocytes is reactive increase.

[0100] When the number of first and second leukocytes satisfies condition 3, the analysis unit 300 does not generate information regarding the mechanism of leukocyte increase. The process then proceeds to step S8. In step S8, the analysis unit 300 does not output a flag regarding the mechanism of increase to the display unit 306. This indicates that the sample did not contain any leukocytes resulting from either neoplastic or reactive mechanisms of increase, for example.

[0101] It is also conceivable that the number of first white blood cells is above the third threshold and the number of second white blood cells is above the fourth threshold. In this case, for example, the analysis unit 300 may output a different flag to the display unit 306 than "Malignant?" and "Reactive?", or it may not output any flag to the display unit 306. For example, the analysis unit 300 may output a flag to the display unit 306 indicating that some abnormality is suspected in the subject. For example, the analysis unit 300 may output a flag to the display unit 306 indicating that the distinction of the mechanism of white blood cell increase was unclear (e.g., "Unknown"). The analysis unit 300 may output both the "Malignant?" and "Reactive?" flags to the display unit 306.

[0102] The third and fourth thresholds may be the same or different. The third and fourth thresholds can be determined as appropriate. For example, by accumulating optical information data obtained from measurements of samples obtained from healthy individuals, samples containing leukocytes whose increase mechanism is neoplastic, and samples containing leukocytes whose increase mechanism is reactive, values ​​can be set that can distinguish between samples obtained from healthy individuals and samples containing leukocytes whose increase is due to either neoplastic or reactive mechanisms.

[0103] In the modified version of step S16 in Figure 13, the analysis unit 300 does not detect secondary leukocytes, or does not use the detection result of secondary leukocytes, but generates information indicating that the mechanism of increase in leukocytes in the sample is neoplastic, based on the detection result of primary leukocytes. For example, the analysis unit 300 determines whether the number of primary leukocytes is above the third threshold. If the number of primary leukocytes is above the third threshold, the analysis unit 300 generates information indicating that the mechanism of increase in leukocytes in the sample is neoplastic. The process then proceeds to step S8. In step S8, the analysis unit 300 outputs a flag "Malignant?" to the display unit 306 as information indicating that the mechanism of increase in leukocytes is neoplastic. If the number of primary leukocytes is below the third threshold, the analysis unit 300 does not generate information regarding the mechanism of increase in leukocytes. The process then proceeds to step S8, and the processor 301 does not output a flag related to the mechanism of leukocyte increase to the display unit 306.

[0104] In a further modification of step S16 in Figure 13, the analysis unit 300 does not detect first leukocytes, or does not use the detection result of first leukocytes, but generates information indicating that the mechanism of increase in leukocytes in the sample is reactive, based on the detection result of second leukocytes, as information regarding the mechanism of increase in leukocytes. For example, the analysis unit 300 determines whether the number of second leukocytes is above the fourth threshold. If the number of second leukocytes is above the fourth threshold, the analysis unit 300 generates information indicating that the mechanism of increase in leukocytes in the sample is reactive. The process then proceeds to step S8. In step S8, the processor 301 outputs a flag "Reactive?" to the display unit 306 as information indicating that the mechanism of increase in leukocytes is reactive. If the number of second leukocytes is below the fourth threshold, the analysis unit 300 does not generate information regarding the mechanism of increase in leukocytes. The process then proceeds to step S8, and the processor 301 does not output a flag related to the mechanism of leukocyte increase to the display unit 306.

[0105] In Embodiment 1, in step S8 of Figure 12, the analysis unit 300 may provide information on subpopulations of leukocytes in addition to information on the mechanism of leukocyte increase. Information on subpopulations of leukocytes may be based on first fluorescence information (e.g., SFL-1 intensity) and scattered light information (e.g., SSC intensity). Alternatively, information on subpopulations of leukocytes may be based on second fluorescence information (e.g., SFL-2 intensity) and scattered light information (e.g., SSC intensity). Information on subpopulations of leukocytes may be, for example, information on the number of cells contained in each subpopulation of leukocytes. Such information may include, for example, the number of cells per unit volume (e.g., μL) for each subpopulation of leukocytes, or the ratio of the number of cells in each subpopulation to the total number of leukocytes.

[0106] Regarding step S7 in Figure 12, an example of the analytical process of Embodiment 2 will be described with reference to Figure 20, but the analysis is not limited to this example. This analytical process enables the generation of information regarding the mechanism of leukocyte increase. In this example, lateral scattered light information is used as the scattered light information. More specifically, SSC intensity is used as the lateral scattered light information. SFL-1 intensity is used as the first fluorescence information, and SFL-2 intensity is used as the second fluorescence information.

[0107] Referring to Figure 20, in step S21, the analysis unit 300 determines the position of the point corresponding to each particle on a plane with SSC intensity on the horizontal axis and SFL-1 intensity on the vertical axis, based on the acquired optical information. Based on the determined position of each point, the analysis unit 300 creates a first scattergram. In step S22, the analysis unit 300 determines the position of the point corresponding to each particle on a plane with SSC intensity on the horizontal axis and SFL-2 intensity on the vertical axis, based on the acquired optical information. Based on the determined position of each point, the analysis unit 300 creates a second scattergram.

[0108] In step S23, the analysis unit 300 detects particles exhibiting an SFL-1 intensity greater than a first threshold as first leukocytes. The analysis unit 300 also counts the detected first leukocytes. The first threshold is as described above. In a modified version of step S23, the analysis unit 300 can also gate gate B on the first scattergram and detect particles appearing within that region as first leukocytes. Furthermore, the analysis unit 300 counts the detected first leukocytes. Details of the detection of first leukocytes by gating are the same as those described in step S14 of Embodiment 1.

[0109] In step S24, the analysis unit 300 detects particles exhibiting an SFL-2 intensity greater than the second threshold as secondary leukocytes. The analysis unit 300 also counts the detected secondary leukocytes. The second threshold is as described above. In a modified version of step S24, the analysis unit 300 can also gate gate A on the second scattergram and detect particles appearing within that region as secondary leukocytes. Furthermore, the analysis unit 300 counts the detected secondary leukocytes. Details of the detection of secondary leukocytes by gating are the same as those described in step S15 of Embodiment 1.

[0110] In step S25, the analysis unit 300 generates information regarding the mechanism of leukocyte increase based on the detection results of first leukocytes and / or second leukocytes. The detection result of first leukocytes is, for example, the number of first leukocytes detected and counted in step S23. The detection result of second leukocytes is, for example, the number of second leukocytes detected and counted in step S24. Step S25 is the same as described for step S16 of Embodiment 1.

[0111] Regarding step S7 in Figure 12, an example of the analytical process of Embodiment 3 will be described with reference to Figure 21, but the analysis is not limited to this example. This analytical process enables the classification of leukocytes into subpopulations on the second scattergram and the generation of information on the mechanism of leukocyte increase. In this example, lateral scattered light information is used as the scattered light information. More specifically, SSC intensity is used as the lateral scattered light information. SFL-1 intensity is used as the first fluorescence information, and SFL-2 intensity is used as the second fluorescence information.

[0112] Referring to Figure 21, in step S31, the analysis unit 300 determines the position of a point corresponding to each particle on a plane with SSC intensity on the horizontal axis and SFL-2 intensity on the vertical axis, based on the acquired optical information. Based on the determined position of each point, the analysis unit 300 creates a second scattergram. In step S32, the analysis unit 300 classifies leukocytes into subpopulations from the particles in the sample based on the determined position of each point. The details of leukocyte classification and counting are the same as those described in step S13. In the example in Figure 21, the analysis unit 300 performs an analysis (first analysis) based on the difference in staining characteristics of the first and second fluorescent dyes, and the difference in fluorescence characteristics of the first and second fluorescent dyes. For example, the analysis unit 300 plots multiple cells stained according to differences in staining characteristics on a scattergram with the first fluorescence intensity (SFL-1) from the first fluorescent dye on the x-axis and the second fluorescence intensity (SFL-2) from the second fluorescent dye on the y-axis. Although not shown in Figure 21, if at least one of the first and second leukocytes is present in the sample, the first and second leukocytes are also plotted on the scattergram in Figure 21. Each cell on the scattergram in Figure 21 is plotted at a position corresponding to the difference in staining characteristics between the first and second fluorescent dyes, and the difference in fluorescence characteristics between the first and second fluorescent dyes.

[0113] In step S33, the analysis unit 300 determines the position of the point corresponding to each particle on a plane with SFL-1 intensity on the horizontal axis and SFL-2 intensity on the vertical axis, based on the acquired optical information. Based on the determined position of each point, the analysis unit 300 creates a third scattergram. In the third scattergram, each subpopulation of leukocytes, such as Lymp, Mono, Baso, Neut, and Eo, appears at approximately the same or close proximity to each other. Therefore, in the third scattergram, clusters corresponding to each subpopulation of leukocytes are not displayed, but are displayed as a group of leukocytes, for example, as shown in Figure 22. In the figure, "WBC" refers to leukocytes.

[0114] In step S34, the analysis unit 300 detects first leukocytes based on the SFL-1 intensity of each particle. Specifically, the analysis unit 300 detects particles exhibiting an SFL-1 intensity greater than a first threshold as first leukocytes. The analysis unit 300 also counts the detected first leukocytes. The first threshold is as described above.

[0115] In step S34, the analysis unit 300 can, for example, gate the region on the third scattergram where the SFL-1 intensity is greater than or equal to a first threshold, and detect particles appearing within that region as first leukocytes. Furthermore, the analysis unit 300 counts the detected first leukocytes. Referring to Figure 23, the region where the SFL-1 intensity is greater than or equal to a first threshold is, for example, the region enclosed by the dashed line on the third scattergram. Within this dashed region, particles showing an SFL-1 intensity greater than the first threshold on the third scattergram may appear. In the example in Figure 23, the analysis unit 300 performs an analysis (second analysis) based on differences in a first component (DNA in the example in Figure 23) in multiple measured cells. For example, the analysis unit 300 classifies multiple cells stained with a first fluorescent dye for a first component (DNA in the example in Figure 23) into multiple populations according to their first fluorescence intensity (SFL-1). In the example shown in Figure 23, the analysis unit 300 classifies cells corresponding to SFL-1 levels higher than the first threshold as primary leukocytes, and cells corresponding to SFL-1 levels lower than the first threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

[0116] In step S35, the analysis unit 300 detects secondary leukocytes based on the SFL-2 intensity of each particle. Specifically, the analysis unit 300 detects particles exhibiting an SFL-2 intensity greater than a second threshold as secondary leukocytes. The analysis unit 300 also counts the detected secondary leukocytes. The second threshold is as described above.

[0117] In step S35, the analysis unit 300 can, for example, gate the region on the third scattergram where the SFL-2 intensity is greater than or equal to the second threshold, and detect particles appearing within that region as second leukocytes. Furthermore, the analysis unit 300 counts the detected second leukocytes. Referring to Figure 24, the region where the SFL-2 intensity is greater than or equal to the second threshold is, for example, the region enclosed by the dashed line on the third scattergram. Within this dashed region, particles showing an SFL-2 intensity greater than the second threshold on the third scattergram may appear. In the example in Figure 24, the analysis unit 300 performs an analysis (third analysis) based on differences in the second component (RNA in the example of Figure 24) in multiple measured cells. For example, the analysis unit 300 classifies multiple cells stained with the second fluorescent dye for the second component (RNA in the example of Figure 24) into multiple populations according to their second fluorescence intensity (SFL-2). In the example shown in Figure 24, the analysis unit 300 classifies cells corresponding to SFL-2 levels higher than the second threshold as second leukocytes, and cells corresponding to SFL-2 levels lower than the second threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

[0118] In a modified version of step S34 above, the analysis unit 300 can also gate a predetermined region on the third scattergram and detect particles appearing within that region as first leukocytes. Such a predetermined region is a region (also called "gate D") where the SFL-1 intensity is greater than or equal to a first threshold and the SFL-2 intensity is less than or equal to a predetermined threshold. The predetermined threshold corresponding to the SFL-2 intensity is, for example, a second threshold. The predetermined threshold corresponding to the SFL-2 intensity may also be, for example, a third threshold different from the second threshold. The third threshold is, for example, an SFL-2 intensity lower than the second threshold. Furthermore, the analysis unit 300 counts the detected first leukocytes. In this example, the analysis unit 300 performs an analysis based on differences in a first component (DNA in this example) in multiple cells measured (second analysis) and an analysis based on differences in a second component (RNA in this example) in multiple cells measured (third analysis). The analysis unit 300 classifies cells appearing in a region (gate D) where the SFL-1 intensity is above a first threshold and the SFL-2 intensity is below a predetermined threshold as primary leukocytes, and classifies cells outside gate D as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, basophils, etc. (sometimes including secondary leukocytes).

[0119] In a modified version of step S35 above, the analysis unit 300 can also gate a predetermined region on the third scattergram that is different from gate D, and detect particles appearing in that region as second leukocytes. Such a predetermined region is a region (also called "gate C") where the SFL-1 intensity is above a predetermined threshold and the SFL-2 intensity is higher than the second threshold. The predetermined threshold corresponding to the SFL-1 intensity is, for example, the first threshold. The predetermined threshold corresponding to the SFL-1 intensity may also be, for example, a fourth threshold different from the first threshold. The fourth threshold is, for example, an SFL-1 intensity lower than the first threshold. Furthermore, the analysis unit 300 counts the detected second leukocytes. In this example, the analysis unit 300 performs an analysis based on differences in a first component (DNA in this example) in multiple cells measured (second analysis) and an analysis based on differences in a second component (RNA in this example) in multiple cells measured (third analysis). The analysis unit 300 classifies cells that appear in a region (gate C) where the SFL-1 intensity is above a predetermined threshold and the SFL-2 intensity is higher than a second threshold as leukocytes (secondary leukocytes), and cells outside gate C as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, basophils, etc. (sometimes including primary leukocytes).

[0120] Referring to Figure 25, gate C is the region enclosed by the solid line on the third scattergram, and gate D is the region enclosed by the dashed line on the third scattergram. Within gate C, particles may appear that exhibit an SFL-1 intensity greater than a predetermined threshold and an SFL-2 intensity greater than a second threshold on the third scattergram. In gate C, the predetermined threshold corresponding to the SFL-1 intensity is the same as in the example described above. Within gate D, particles may appear that exhibit an SFL-1 intensity greater than a first threshold and an SFL-2 intensity less than or equal to a predetermined threshold on the third scattergram. In gate D, the predetermined threshold corresponding to the SFL-2 intensity is the same as in the example described above.

[0121] In step S36, the analysis unit 300 generates information regarding the mechanism of leukocyte increase based on the detection results of first and second leukocytes. The detection result of first leukocytes is, for example, the number of first leukocytes detected and counted in step S34. The detection result of second leukocytes is, for example, the number of second leukocytes detected and counted in step S35. Step S36 is the same as described for step S16 of Embodiment 1.

[0122] In Embodiment 3, in step S8 of Figure 12, the analysis unit 300 may output information on subpopulations of leukocytes in addition to information on the mechanism of leukocyte increase. The information on subpopulations of leukocytes is as described above.

[0123] Regarding step S7 in Figure 12, an example of the analytical process of Embodiment 4 will be described with reference to Figure 26, but the analysis is not limited to this example. This analytical process enables the generation of information regarding the mechanism of leukocyte increase. In this example, SFL-1 intensity is used as the first fluorescence information, and SFL-2 intensity is used as the second fluorescence information. In step S41, the analysis unit 300 determines the position of the point corresponding to each particle on a plane with SFL-1 intensity on the horizontal axis and SFL-2 intensity on the vertical axis, based on the acquired optical information. Based on the determined position of each point, the analysis unit 300 creates a third scattergram.

[0124] In step S42, the analysis unit 300 detects particles exhibiting an SFL-1 intensity greater than a first threshold as first leukocytes. The analysis unit 300 also counts the detected first leukocytes. The first threshold is as described above. In a modified version of step S42, the analysis unit 300 can also gate gate D on the third scattergram and detect particles appearing within that region as first leukocytes. Furthermore, the analysis unit 300 counts the detected first leukocytes. Details of the detection of first leukocytes by gating are the same as those described in step S34 of Embodiment 3.

[0125] In step S43, the analysis unit 300 detects particles exhibiting an SFL-2 intensity greater than the second threshold as secondary leukocytes. The analysis unit 300 also counts the detected secondary leukocytes. The second threshold is as described above. In a modified version of step S43, the analysis unit 300 can also gate gate C on the third scattergram and detect particles appearing within that region as secondary leukocytes. Furthermore, the analysis unit 300 counts the detected secondary leukocytes. Details of the detection of secondary leukocytes by gating are the same as those described in step S35 of Embodiment 3.

[0126] In step S44, the analysis unit 300 generates information regarding the mechanism of leukocyte increase based on the detection results of first leukocytes and / or second leukocytes. The detection result of first leukocytes is, for example, the number of first leukocytes detected and counted in step S42. The detection result of second leukocytes is, for example, the number of second leukocytes detected and counted in step S43. Step S44 is the same as described for step S16 of Embodiment 1.

[0127] Referring to Figure 12, when the above analysis process is completed, the analysis unit 300 outputs the analysis results to the display unit 306 in step S8 and terminates the process. The analysis unit 300 provides, for example, information about the subject's clinical condition as analysis results. Referring to Figures 27A and 27B, an example of the analysis results displayed on the display unit 306 will be described, but it is not limited to this example. The display unit 306 displays the analysis results screen 80. The analysis results screen 80 includes a measurement item display area 81, a research item display area 82, a flag display area 83, and a scattergram display area 84. The measurement item display area 81 displays information about normal white blood cells and the number of cells included in each subpopulation. In the figure, "WBC" refers to white blood cells, "NEUT" refers to the neutrophil population, "LYMPH" refers to the lymphocyte population, "MONO" refers to the monocyte population, "EO" refers to the eosinophil population, and "BASO" refers to the basophil population. Furthermore, "#" indicates the number of cells per unit volume, and "%" indicates the ratio of the number of cells in each subpopulation to the total number of white blood cells. Research item display area 82 displays information such as the number and characteristics of cells to be displayed as supplementary information. Flag display area 83 displays information about the mechanism of white blood cell increase. The flags displayed in flag display area 83 are, for example, information that suggests the clinical condition of the subject. Scattergram display area 84 displays the scattergram created during the analysis process. Referring to Figure 27A, the flag display area 83 displays the flag "Malignant?" as information indicating that the mechanism of white blood cell increase in the sample is neoplastic increase. Also, the first scattergram is displayed in scattergram display area 84. Referring to Figure 27B, the flag display area 83 displays the flag "Reactive?" as information indicating that the mechanism of white blood cell increase in the sample is reactive increase. Also, the second scattergram is displayed in scattergram display area 84.

[0128] The reagents of this embodiment used in the measuring device 500 will now be described. The reagents of this embodiment are for preparing a measurement sample and include a first fluorescent dye and a second fluorescent dye. The first and second fluorescent dyes are dyes that have the ability to bind to nucleic acids (e.g., DNA, RNA). The first fluorescent dye is a fluorescent dye that has a high binding ability to DNA and specifically binds to DNA. The second fluorescent dye is a fluorescent dye that has the ability to bind to RNA. For example, the first fluorescent dye has a higher binding ability to neoplastic leukocytes than to neoplastic leukocytes, and the second fluorescent dye has a higher binding ability to neoplastic leukocytes than to neoplastic leukocytes. For example, the first fluorescent dye has a specific binding ability to DNA, but its binding ability to RNA is weaker than that of the second fluorescent dye. For example, the second fluorescent dye has a stronger binding ability to RNA than the first fluorescent dye. The reason why the first fluorescent dye exhibits a specific binding ability to DNA is, for example, the structure of the dye. For example, the first fluorescent dye has a structure that easily fits into the gaps in the double-stranded structure of nucleic acids in DNA, and has a specific binding ability to DNA. The first and second fluorescent dyes have the characteristic that their fluorescence intensity increases when they bind to nucleic acids. For example, the difference in fluorescence intensity of the first fluorescent dye when bound to DNA and when not bound to DNA is about 10 times or more. As described above, both the first and second fluorescent dyes have the ability to bind to nucleic acids, but the first fluorescent dye has a higher binding ability to DNA than the second fluorescent dye and a lower binding ability to RNA than the second fluorescent dye. The first and second fluorescent dyes are excited by light irradiated from a light source provided by a flow cytometer. It is preferable that the first and second fluorescent dyes have fluorescence emission maxima in different wavelength ranges. It is also preferable that the second fluorescent dye has a maximum absorption in a different wavelength range than the first fluorescent dye. That is, the second fluorescent dye may be a fluorescent dye that emits fluorescence at a wavelength that can be detected separately from the fluorescence from the first fluorescent dye.

[0129] The first and second fluorescent dyes can be appropriately selected from fluorescent dyes that have the property of binding to the nucleic acids of cells such as leukocytes. Since the first and second fluorescent dyes can directly bind to the nucleic acids of cells, it is not necessary to stain the cells in the sample using antibodies labeled with these fluorescent dyes during the preparation of the measurement sample. Therefore, the first and second fluorescent dyes do not contain antibodies.

[0130] Preferably, the first fluorescent dye is a dye that has a maximum absorption in the wavelength range of 400 nm to 490 nm, is excited and emits fluorescence by absorbing light in the same wavelength range, and has the property of having a specific binding ability to cellular DNA. Examples include fluorescent dyes having an acridine skeleton, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), and the Hoechst series Hoechst3342, Hoechst33258, and Hoechst334580.

[0131] Examples of fluorescent dyes having an acridine skeleton include proflavin, 9-aminoacridine, acridine orange, acridine yellow G, acrylflavin, basic yellow 9, ethacridine lactate, euchrysine GGNX, proflavin hemisulfate, 3,6-bis(dimethylamino)acridine (Rhoduline Orange), and 3,6-diamino-2,7,10-trimethylacridinium chloride. Among these, acridine yellow G is preferred. A commercially available fluorescent dye may be used as the first fluorescent dye.

[0132] Preferably, the second fluorescent dye has a maximum absorption in the wavelength range of 610 nm to 750 nm, is excited and emits fluorescence by absorbing light in this wavelength range, and is a dye with high binding ability to cellular RNA. For example, the second fluorescent dye has a stronger binding ability to RNA than the first fluorescent dye. Examples include fluorescent dyes represented by the following formula (I), and combinations thereof.

[0133] [ka]

[0134] In formula (I), R

[0137] , , , , ,

[0139] , , , , , ,

[0138] ,

[0136] , , , and R 4 is a hydrogen atom, a methyl group, an ethyl group or an alkyl group having 6 to 18 carbon atoms, and when one of them is an alkyl group having 6 to 18 carbon atoms, the other is a hydrogen atom, a methyl group or an ethyl group. R 2 and R 3 are the same as or different from each other, and are a methyl group, an ethyl group, a methoxy group or an ethoxy group. A is a sulfur atom, an oxygen atom, or a carbon atom having a methyl group. n is 0, 1, 2 or 3. X - is an anion.

[0135] In formula (I), the alkyl group having 6 to 18 carbon atoms may be either linear or branched. Among the alkyl groups having 6 to 18 carbon atoms, alkyl groups having 6, 8 or 10 carbon atoms are preferred. In formula (I), as the anion X - as, F - , Cl - , Br - and I - such as halogen ions, CF3SO3 - , BF4 - , ClO4 - and the like can be mentioned.

[0136] As the fluorescent dye represented by the above formula (I), the fluorescent dye represented by the following formula (II) is preferred.

[0137] [Chemical formula]

[0138] A commercially available staining reagent containing the above second fluorescent dye alone may be used. For example, FluoroCell WDF (Sysmex Corporation), Stomatrizer ˢᵘᵖᵉʳˢᶜʳᶦᵖᵗ 4DS (Sysmex Corporation) can be mentioned.

[0139] The first and second fluorescent dyes are preferably used in solution. The solvent is not particularly limited as long as it can dissolve each of the above fluorescent dyes. Examples include water, organic solvents, and mixtures thereof. As organic solvents, solvents that are miscible with water are preferred, such as C1-C6 alcohols, ethylene glycol, diethylene glycol, polyethylene glycol, and dimethyl sulfoxide (DMSO).

[0140] For the preparation of the measurement sample, it is preferable to use a reagent containing a first fluorescent dye and a second fluorescent dye. The reagent containing the first and second fluorescent dyes is in the form of a solution of these fluorescent dyes contained in a single reagent container. Alternatively, the reagent containing the first and second fluorescent dyes may be in the form of a reagent containing the first fluorescent dye and a reagent containing the second fluorescent dye, each contained in separate reagent containers. Referring to Figure 28, 200 indicates a reagent container containing the reagent of this embodiment. The reagent container 200 is mounted in the reagent container holder 60 in the measurement unit 400. The reagent container 200 may be contained in a packaging box. In that case, the packaging box may further contain an accompanying document describing the composition, usage, and storage of the reagent, as well as cushioning material to reduce external impact. The reagent container 200 may be a flexible container. A flexible container is, for example, a container in the form of a spout pouch.

[0141] The concentration of the first fluorescent dye in the reagent can be appropriately determined depending on the type of fluorescent dye. The concentration of the first fluorescent dye is, for example, 0.01 mg / L or more, preferably 0.1 mg / L or more, and more preferably 0.5 mg / L. The concentration of the first fluorescent dye is, for example, 100 mg / L or less, preferably 75 mg / L or less, and more preferably 50 mg / L or less. The concentration of the second fluorescent dye in the staining reagent is the same as that of the first fluorescent dye.

[0142] Further embodiments relate to the use of a first fluorescent dye and a second fluorescent dye for the manufacture of reagents used in a measuring device. The reagents, first fluorescent dye, and second fluorescent dye used in the measuring device are as described above.

[0143] This section describes the hemolytic reagent used in the preparation of the measurement sample. The hemolytic reagent contains a surfactant and is used in combination with a reagent containing a first fluorescent dye and a second fluorescent dye. The surfactant causes hemolysis of red blood cells in the sample and can also cause damage to the cell membranes of cells other than red blood cells to the extent that the first and second fluorescent dyes can penetrate them. Examples of surfactants include nonionic surfactants, cationic surfactants, and combinations thereof. The hemolytic reagent preferably contains a nonionic surfactant.

[0144] Examples of nonionic surfactants include those represented by the following formula (III). R 1 -R 2 -(CH2CH2O) n -H (III) (In the formula, R 1 R is an alkyl group, alkenyl group, or alkynyl group having 8 to 25 carbon atoms; 2 is an oxygen atom, -(COO)-, or the following formula (IV):

[0145] [ka] (and n is between 23 and 25 or 30.)

[0146] In formula (III), n is preferably 23 or 25, and more preferably 23. When n is between 23 and 25, the concentration of the nonionic surfactant represented by formula (III) in the hemolytic reagent is 1700 ppm or higher, preferably 1750 ppm or higher. Also, when n is between 23 and 25, the concentration of the nonionic surfactant represented by formula (III) in the measurement sample is 2300 ppm or lower, preferably 2200 ppm or lower.

[0147] When n is 30, the concentration of the nonionic surfactant represented by formula (III) in the hemolytic reagent is 1900 ppm or higher, preferably 2000 ppm or higher, and more preferably 2100 ppm or higher. Also, when n is 30, the concentration of the nonionic surfactant represented by formula (III) in the measurement sample is 2300 ppm or lower, preferably 2200 ppm.

[0148] Specific examples of nonionic surfactants represented by formula (III) include polyoxyethylene alkyl ethers, polyoxyethylene sterols, polyoxyethylene castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene polyoxypropylene alkyl ethers, and combinations thereof. Among these, polyoxyethylene alkyl ethers are preferred. The polyoxyethylene alkyl ether is preferably at least one selected from polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, polyoxyethylene (30) cetyl ether, and the group thereof. More preferably, it is polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, and combinations thereof, and even more preferably, polyoxyethylene (23) cetyl ether. The hemolytic reagent may contain one type of nonionic surfactant or two or more types. The hemolytic reagent may further contain nonionic surfactants other than the nonionic surfactant represented by formula (III).

[0149] The hemolytic reagent may further contain a cationic surfactant. Examples of cationic surfactants include quaternary ammonium salt type surfactants, pyridium salt type surfactants, and combinations thereof. As a quaternary ammonium salt type surfactant, a surfactant with a total of 9 to 30 carbon atoms, represented by the following formula (V), is preferred. The hemolytic reagent may contain one type of cationic surfactant or two or more types.

[0150] [ka]

[0151] In formula (V), R 1 R is an alkyl or alkenyl group having 6 to 18 carbon atoms; 2 and R 3 These are alkyl or alkenyl groups having 1 to 4 carbon atoms, which are either identical or different from each other; R 4 X is an alkyl group, alkenyl group, or benzyl group having 1 to 4 carbon atoms. - It is a halogen ion.

[0152] In formula (V), R 1 The R is preferably an alkyl or alkenyl group having 6, 8, 10, 12, and 14 carbon atoms, and is particularly preferably a linear alkyl group. 1 Examples include the octyl group, decyl group, and dodecyl group. 2 and R 3 These are preferably a methyl group, an ethyl group, and a propyl group, which are either the same or different from each other. 4 The group is preferably a methyl group, an ethyl group, or a propyl group.

[0153] Examples of pyridium salt-type surfactants include those represented by formula (VI).

[0154] [ka]

[0155] In formula (VI), R 1 X is an alkyl or alkenyl group having 6 to 18 carbon atoms; - It is a halogen ion.

[0156] In formula (VI), R 1 The R is preferably an alkyl or alkenyl group having 6, 8, 10, 12, and 14 carbon atoms, and is particularly preferably a linear alkyl group. 1Examples include the octyl group, decyl group, and dodecyl group.

[0157] The concentration of the cationic surfactant in the hemolytic reagent can be appropriately selected depending on the type of surfactant. The concentration of the cationic surfactant is 10 ppm or higher. The concentration of the cationic surfactant is preferably 400 ppm or higher, more preferably 500 ppm or higher, and even more preferably 600 ppm or higher. Also, the concentration of the cationic surfactant is 10,000 ppm or lower. The concentration of the cationic surfactant is preferably 1,000 ppm or lower, more preferably 800 ppm or lower, and even more preferably 700 ppm or lower.

[0158] Hemolytic reagents may contain buffering agents to maintain a constant pH. Examples of buffering agents include inorganic acid salts, organic acid salts, Good's buffers, and combinations thereof. Examples of inorganic acid salts include phosphates, borates, and combinations thereof. Examples of organic acid salts include citrates, malates, and combinations thereof. Examples of Good's buffers include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricin, Tris, Bicine, TAPS, and combinations thereof.

[0159] The hemolytic reagent may further contain an aromatic organic acid. In this specification, an aromatic organic acid means an acid having at least one aromatic ring in its molecule and its salts. Examples of aromatic organic acids include aromatic carboxylic acids and aromatic sulfonic acids. Examples of aromatic carboxylic acids include phthalic acid, benzoic acid, salicylic acid, hippuric acid, their salts, and combinations thereof. Examples of aromatic sulfonic acids include p-aminobenzenesulfonic acid, benzenesulfonic acid, their salts, and combinations thereof. The hemolytic reagent may contain one or more aromatic organic acids. Furthermore, aromatic organic acids may exhibit buffering properties. When using an aromatic organic acid that exhibits buffering properties, the addition of a buffering agent is optional and may be combined with the buffering agents mentioned above.

[0160] When the hemolytic reagent contains an aromatic organic acid, the concentration of the aromatic organic acid is not particularly limited, but from the viewpoint of the ability to classify monocytes and lymphocytes, it is preferably 20 mM or higher, and more preferably 25 mM or higher. Furthermore, the concentration of the aromatic organic acid contained in the hemolytic reagent is preferably 50 mM or lower, and more preferably 45 mM or lower.

[0161] The hemolytic reagent is preferably a liquid reagent. The solvent is not particularly limited as long as it can dissolve each component, such as the surfactant mentioned above. Examples of solvents include water, organic solvents, and mixtures thereof. As organic solvents, solvents that are miscible with water are preferred, such as C1-C6 alcohols, ethylene glycol, diethylene glycol, polyethylene glycol, and DMSO.

[0162] The pH of the hemolytic reagent is not particularly limited, but is preferably 5.5 or higher. More preferably 5.7 or higher, and even more preferably 5.9 or higher. Furthermore, a pH of 7.2 or lower is preferred. More preferably 6.9 or lower, and even more preferably 6.6 or lower. Known bases (such as sodium hydroxide) or acids (such as hydrochloric acid) can be used to adjust the pH.

[0163] In hemolytic reagents, the osmotic pressure is not particularly limited, but from the viewpoint of hemolysis efficiency of red blood cells, it is preferably 150 mOsm / kg or less, more preferably 130 mOsm / kg or less, and most preferably 110 mOsm / kg or less. Appropriate osmotic pressure adjusting agents may be added to adjust the osmotic pressure. Examples of osmotic pressure adjusting agents include sugars, amino acids, organic solvents, sodium chloride, and combinations thereof.

[0164] Commercially available hemolysis reagents for blood cell counting may be used as the hemolysis reagent. Examples include LyzaCell WDF (Sysmex Corporation) and LyzaCell WDFII (Sysmex Corporation).

[0165] In the analytical method of this embodiment, a measurement sample containing particles stained with the first fluorescent dye and the second fluorescent dye is prepared by mixing the sample with the first fluorescent dye and the second fluorescent dye. For example, a measurement sample can be prepared by mixing the sample with a staining reagent. Preferably, a measurement reagent is prepared by mixing the sample with a hemolytic reagent and a staining reagent. The first fluorescent dye and the second fluorescent dye are mixed with the sample and the hemolytic reagent so that their concentrations (final concentrations) in the measurement sample are within a predetermined range. The preferred final concentration of the first fluorescent dye in the measurement sample is 1000 ppm or less, preferably 100 ppm or less, and more preferably 10 ppm or less. The preferred final concentration of the first fluorescent dye in the measurement sample is 0.001 ppm or more, preferably 0.01 ppm or more, and more preferably 0.1 ppm or more. The preferred final concentration of the second fluorescent dye in the measurement sample is 1000 ppm or less, preferably 100 ppm or less, and more preferably 10 ppm or less. The preferred final concentration of the second fluorescent dye in the measurement sample is 0.001 ppm or higher, preferably 0.01 ppm or higher, and more preferably 0.1 ppm or higher. The measurement sample can be prepared using the measurement device 500 described above.

[0166] The mixing ratio of the hemolytic reagent, staining reagent, and sample is preferably, for example, 1000:1 or more and 1000:1 or more, expressed as a volume ratio. More preferably, it is 1000:10 or more and 1000:15 or more and 1000:15 or more. Furthermore, the mixing ratio of the hemolytic reagent, staining reagent, and sample is preferably, for example, 1000:50 or less and 50 or less, expressed as a volume ratio. More preferably, it is 1000:30 or less and 30 or less, and even more preferably, 1000:25 or less and 25 or less. The mixing ratio of the staining reagent and the sample may be the same or different.

[0167] In the analysis method of this embodiment, optical information, including first fluorescence information and second fluorescence information corresponding to each fluorescence emitted from the particles, is obtained by irradiating the particles in the sample with light. Then, information regarding the mechanism of leukocyte increase is output based on the optical information. The acquisition of optical information and the output of information regarding the mechanism of leukocyte increase can be performed by the measuring device 500 described above. The optical information and information regarding the mechanism of leukocyte increase are the same as those described for the measuring device.

[0168] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0169] (1) Reagents (1.1) Staining reagents As the first fluorescent dye, acridine yellow G (Kanto Chemical Co., Ltd.) was used. As the second fluorescent dye, dye compound A described in U.S. Patent No. 6,004,816 was used. U.S. Patent No. 6,004,816 is incorporated herein by reference. Dye compound A is a fluorescent dye represented by the above formula (II). As described in U.S. Patent No. 6,004,816, dye compound A was obtained by the following steps: 1 equivalent of 3-methyl-2-methylbenzothiazolium methane sulfate and 3 equivalents of N,N-diphenylformamidine were stirred in acetic acid on an oil bath at 90°C for 1.5 hours while heating. The reaction mixture was poured into hexane, and the red oily substance was further suspended and washed with hexane to remove the acetic acid. The crude product was recrystallized in ethyl acetate-hexane (yield 48%). 1 equivalent of 1-octyllepidinium triflate and pyridine were added to this, and the mixture was stirred on an oil bath at 90°C for 3 hours while heating. The reaction mixture was concentrated, and the remaining blue crude product was purified with methanol-chloroform by flash chromatography to obtain dye compound A as a dark blue powder (yield 62%). The results of physical property tests (TLC, 1H-NMR, MASS, etc.) of the obtained dye compound A are described in U.S. Patent No. 6,004,816. The maximum absorption spectrum of dye compound A was 629 nm. The staining reagent was prepared by dissolving acridine yellow G (25 mg) and dye compound A (27.5 mg) in special grade ethylene glycol (1 L).

[0170] (1.2) Hemolytic reagent LyzaCel WDFII (Sysmex Corporation) was used as the hemolysis agent.

[0171] (2) Measuring device A multi-parameter automated blood cell analyzer XN-1000 (Sysmex Corporation) was modified as follows and used as the measurement unit. The XN-1000 was equipped with the hardware shown in Figures 1, 4, and 5. The FCM detection unit 460 had a semiconductor laser light source emitting red (633 nm) light, a detector for red forward scattered light, a detector for red side scattered light, and a detector that receives fluorescence (light above 660 nm) excited by red light. In the modification of the XN-1000, a semiconductor laser light source emitting blue-violet (405 nm) light, a detector for blue-violet scattered light, and a fluorescence detector that detects fluorescence (450-600 nm) excited by blue-violet light were added. The FCM detection unit of the modified XN-1000 had the configuration shown in Figure 8. A computer with the flow cytometry analysis software FlowJo (trademark) (BD Biosciences) installed was used as the analysis unit. By connecting this computer with the modified XN-1000, we created a measuring device capable of analyzing optical information regarding red and blue-violet scattered light, as well as fluorescence originating from the first and second fluorescent dyes, and displaying a desired scattergram.

[0172] (3) Analysis method Except for using the staining reagent prepared in (1.1) above instead of the Fluorocell WDF (Sysmex Corporation), which is the staining reagent for XN-1000, the preparation and measurement of the measurement samples were performed according to the manual included with XN-1000 (Sysmex Corporation). The measurement sample was prepared by mixing 1000 μL of Lysacell WDFII, 17 μL of the sample, and 20 μL of the staining reagent. The dilution ratio of the staining reagent in the measurement sample was 51.85, and the final concentrations of the first and second fluorescent dyes in the measurement sample were 0.53 ppm and 0.48 ppm, respectively. Data analysis was performed using FlowJo®.

[0173] (4) Analysis of specimens containing leukocytes associated with neoplastic or reactive mechanisms (4.1) Analysis of blood samples containing abnormal lymphocytes Measurement samples were prepared from blood samples containing abnormal lymphocytes. Abnormal lymphocytes are known to appear in peripheral blood in diseases such as multiple myeloma, chronic lymphocytic leukemia, and malignant lymphoma. The prepared measurement samples were measured using the above-mentioned measuring device to obtain first fluorescence information, second fluorescence information, and scattered light information. The first fluorescence information was the peak value of fluorescence data derived from the first fluorescent dye (the largest value among the fluorescence data derived from the first fluorescent dye, hereinafter referred to as "blue-violet fluorescence intensity") generated by irradiating particles in the measurement sample with a blue-violet laser. The second fluorescence information was the peak value of fluorescence data derived from the second fluorescent dye (the largest value among the fluorescence data derived from the second fluorescent dye, hereinafter referred to as "red fluorescence intensity") generated by irradiating particles in the measurement sample with a red laser. The scattered light information was the (first) lateral scattered light intensity obtained by irradiating particles in the measurement sample with a blue-violet laser. Based on the acquired optical information, the first, second, and third scattergrams were created. The first scattergram had the horizontal axis representing the lateral scattered light intensity and the vertical axis representing the blue-violet fluorescence intensity. The second scattergram had the horizontal axis representing the lateral scattered light intensity and the vertical axis representing the red fluorescence intensity. The third scattergram had the horizontal axis representing the blue-violet fluorescence intensity and the vertical axis representing the red fluorescence intensity. Each of the created scattergrams is shown in Figure 29. In the figure, "V-SFL" represents the blue-violet fluorescence intensity, "R-SFL" represents the red fluorescence intensity, and "V-SSC" represents the lateral scattered light intensity due to blue-violet laser irradiation.

[0174] In the second scattergram shown in Figure 29A, leukocytes were classified into at least four subpopulations: lymphocytes, monocytes, neutrophils, and eosinophils. In the second scattergram, the region enclosed by the solid rectangle was the region where the red fluorescence intensity was above the second threshold and the lateral scatter intensity was within the second range (hereinafter referred to as gate A). Gate A was defined as the region where particles with a higher RNA content than leukocytes classified into the four subpopulations of lymphocytes, monocytes, neutrophils, and eosinophils appeared. There were few such particles in gate A of this second scattergram. In the first scattergram shown in Figure 29B, leukocytes were classified into at least three subpopulations: lymphocytes, monocytes, and neutrophils. In the first scattergram, the region enclosed by the solid rectangle was the region where the blue-violet fluorescence intensity was above the first threshold and the lateral scatter intensity was within the first range (hereinafter referred to as gate B). Gate B was defined as a region where particles with a higher DNA content than leukocytes, which are classified into three subpopulations—lymphocytes, monocytes, and neutrophils—appeared. A large number of such particles appeared in Gate B of this first scattergram.

[0175] In the third scattergram shown in Figure 29C, the region enclosed by the solid rectangle is called Gate C. In this example, Gate C was a region where the red fluorescence intensity was above the second threshold and the blue-violet fluorescence intensity was within a predetermined range. The region enclosed by the dashed rectangle is called Gate D. Gate D was a region where the blue-violet fluorescence intensity was above the first threshold and the red fluorescence intensity was below the second threshold. Gate C was a region where particles appeared that had a DNA amount greater than or equal to that of other leukocytes and a RNA amount greater than that of other leukocytes. Gate D was a region where particles appeared that had a DNA amount greater than that of other leukocytes and an RNA amount similar to that of other leukocytes. The group of particles that appeared in the regions other than Gates C and D of this third scattergram had a blue-violet fluorescence intensity below the first threshold and a red fluorescence intensity below the second threshold, so it was considered to be a group of leukocytes such as lymphocytes, monocytes, neutrophils, and eosinophils. Furthermore, almost no particles were observed at gate C of this third scattergram. On the other hand, a large number of particles were observed at gate D of this third scattergram.

[0176] This blood sample meets condition 2 shown in Figure 19, as it shows almost no particles at gates A and C, but many particles at gates B and D. Therefore, the "Malignant?" flag is displayed as information regarding the mechanism of leukocyte increase.

[0177] (4.2) Analysis of blood samples containing lymphoblasts A sample was prepared from a blood sample containing lymphoblasts. Lymphoblasts are undifferentiated cells normally found in the bone marrow, but they are known to appear in the peripheral blood in acute leukemias such as acute lymphoblastic leukemia. The prepared sample was measured using the above-mentioned measuring device to obtain first fluorescence information, second fluorescence information, and scattered light information. The first fluorescence information was the blue-violet fluorescence intensity, and the second fluorescence information was the red fluorescence intensity. The scattered light information was the lateral scattered light intensity obtained by irradiating particles in the sample with a blue-violet laser. Based on the acquired optical information, the first, second, and third scattergrams were created in the same manner as in Example 1. The created scattergrams are shown in Figure 30.

[0178] In the second scattergram shown in Figure 30A, leukocytes were classified into at least two subpopulations: lymphocytes and neutrophils. In the second scattergram, the area enclosed by the solid rectangle was gate A. In this second scattergram, few particles appeared within gate A. In the first scattergram shown in Figure 30B, leukocytes were classified into at least two subpopulations: lymphocytes and neutrophils. In the first scattergram, the area enclosed by the solid rectangle was gate B. In this first scattergram, a large number of particles were observed within gate B. In the third scattergram shown in Figure 30C, the area enclosed by the solid rectangle was gate C, and the area enclosed by the dashed rectangle was gate D. In this example, gates C and D are gates set under the same conditions as in the example in Figure 29. In this third scattergram, few particles appeared within gate C, but a large number of particles were observed within gate D. This blood sample meets condition 2 shown in Figure 19, as it shows almost no particles at gates A and C, but many particles at gates B and D. Therefore, the "Malignant?" flag is displayed as information regarding the mechanism of leukocyte increase.

[0179] (4.3) Analysis of blood samples containing immature erythroblasts A sample was prepared from a blood sample containing immature erythroblasts. Immature erythroblasts are undifferentiated cells normally found in the bone marrow, but they are known to appear in the peripheral blood in diseases such as bone metastases of malignant tumors, myelodysplastic syndrome, and leukemia. The prepared sample was measured using the above-mentioned measuring device to obtain first fluorescence information, second fluorescence information, and scattered light information. The first fluorescence information was the blue-violet fluorescence intensity, and the second fluorescence information was the red fluorescence intensity. The scattered light information was the lateral scattered light intensity obtained by irradiating particles in the sample with a blue-violet laser. Based on the acquired optical information, the first, second, and third scattergrams were created in the same manner as in Example 1. Each of the created scattergrams is shown in Figure 31.

[0180] In the second scattergram shown in Figure 31A, leukocytes were classified into at least four subgroups: lymphocytes, monocytes, neutrophils, and eosinophils. In the second scattergram, the area enclosed by the solid rectangle was gate A. In this second scattergram, few particles appeared within gate A. In the first scattergram shown in Figure 31B, leukocytes were classified into at least four subgroups: lymphocytes, monocytes, neutrophils, and eosinophils. In the first scattergram, the area enclosed by the solid rectangle was gate B. In this first scattergram, a large number of particles were observed within gate B. In the third scattergram shown in Figure 31C, the area enclosed by the solid rectangle was gate C, and the area enclosed by the dashed rectangle was gate D. In this example, gates C and D are gates set under the same conditions as in the example in Figure 29. In this third scattergram, few particles appeared within gate C, but many particles were observed within gate D. Since this blood sample showed almost no particles in gates A and C, and many particles in gates B and D, it meets condition 2 as shown in Figure 19, and the "Malignant?" flag is displayed as information regarding the mechanism of leukocyte increase.

[0181] (4.4) Analysis of blood samples containing atypical lymphocytes A sample was prepared from a blood sample containing atypical lymphocytes. Atypical lymphocytes are known to appear in the peripheral blood of patients with viral infections, drug allergies, autoimmune diseases, etc. The prepared sample was measured using the above-described measuring device to obtain first fluorescence information, second fluorescence information, and scattered light information. The first fluorescence information was the blue-violet fluorescence intensity, and the second fluorescence information was the red fluorescence intensity. The scattered light information was the lateral scattered light intensity obtained by irradiating particles in the sample with a red laser. Based on the acquired optical information, the first, second, and third scattergrams were created in the same manner as in Example 1. Each of the created scattergrams is shown in Figure 32. In the figure, "R-SSC" represents the lateral scattered light intensity due to red laser irradiation.

[0182] In the second scattergram shown in Figure 32A, leukocytes were classified into at least five subgroups: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. In the second scattergram, the area enclosed by the solid rectangle was gate A. In this second scattergram, a large number of particles were observed within gate A. In the first scattergram shown in Figure 32B, leukocytes were classified into at least four subgroups: lymphocytes, monocytes, neutrophils, and eosinophils. In the first scattergram, the area enclosed by the solid rectangle was gate B. In this first scattergram, a large number of particles were not observed within gate B. In the third scattergram shown in Figure 32C, the area enclosed by the solid rectangle was gate C, and the area enclosed by the dashed rectangle was gate D. In this example, gates C and D are gates set under the same conditions as in the example in Figure 29. In this third scattergram, a large number of particles were observed within gate C, but few particles appeared within gate D. Since this blood sample did not show a large number of particles in gates B and D, but did show a large number of particles in gates A and C, it meets condition 3 as shown in Figure 19, and the "Reactive?" flag is displayed as information regarding the mechanism of leukocyte increase.

[0183] The results from (4.1) to (4.4) above showed that abnormal lymphocytes, blast cells, and immature erythroblasts tended to appear within gates B and D on the scattergram, while atypical lymphocytes tended to appear within gates A and C on the scattergram. Here, abnormal lymphocytes are neoplastic cells themselves, and blast cells and immature erythroblasts are cells that increase in the peripheral blood due to malignant tumors. Atypical lymphocytes, also called reactive lymphocytes, are cells that increase in the peripheral blood when the immune system is stimulated by viral infections, etc. In other words, the mechanism of increase in abnormal lymphocytes, blast cells, and immature erythroblasts is neoplastic, and the mechanism of increase in atypical lymphocytes is reactive. Therefore, it was shown that abnormal cells in blood samples can be detected and the mechanism of increase of those abnormal cells can be differentiated based on the first fluorescence information, second fluorescence information, and scattered light information obtained by measuring the sample. [Explanation of Symbols]

[0184] 12: Reagent, 20, 64: Suction tube, 21, 22, 30, 33, 38, 39, 452: Quantitative unit, 36, 41: Waste liquid chamber, 37, V1~V13: Solenoid valve, 55: Second chamber, 56A, 56B, 433: Pump, 60, 442: Reagent container holder, 63: Cover, 65: Suction tube lifting mechanism, 100: Sample container, 100a: Lid, 200, R1, R2: Reagent container, 200A: First reagent container, 200B: Second reagent container, 300: Analysis unit, 400: Measurement unit, 411: Light source, 411a: First light source, 411b: Second light source, 412, 412a, 412 b: Side-scattered light receiving element, 416: Forward-scattered light receiving element, 422a, 422b: Side-fluorescence light receiving element, 413: Flow cell, 418, 418a, 418b, 418c: Dichroic mirror, 420: (First) chamber, 430: Fluid delivery mechanism, 430a: First fluid delivery mechanism, 430b: Second fluid delivery mechanism, 431: Fluid delivery tube, 432: Quantitative block, 440: Sample preparation unit, 440A: First sample preparation unit, 440B: Second sample preparation unit, 450: Sample aspiration unit, 451: Sample aspiration nozzle, 460: FCM detection unit, 480: Measurement unit control unit, 500: Measurement device, T: Sample

Claims

1. A measuring device for analyzing cells in a sample, A chamber for preparing a measurement sample, which includes cells stained with at least one of the first and second fluorescent dyes contained in the reagent, by mixing the reagent supplied from at least one reagent container with the sample, A light source for irradiating light onto a plurality of cells in the sample being measured that are flowing through a flow cell, A detection unit that detects multiple optical signals corresponding to each of the multiple cells in response to the irradiation of the aforementioned light, Includes an analysis unit for analyzing the optical signal, The detection unit detects the optical signal which includes at least one of a first fluorescence signal corresponding to a first fluorescence dye that binds to the DNA of the cell, and a second fluorescence signal corresponding to a second fluorescence dye that binds to the RNA of the cell. The analysis unit is a measuring device that determines, at least based on the first fluorescence signal, whether the mechanism of leukocyte increase is neoplastic, and at least based on the second fluorescence signal, whether the mechanism of leukocyte increase is reactive.

2. The aforementioned analysis unit is Based at least on the first fluorescence signal reflecting the state of the DNA, it is determined whether the mechanism of the increase in leukocytes is neoplastic. Based at least on the second fluorescence signal reflecting the state of the RNA, it is determined whether the mechanism of leukocyte increase is reactive. The measuring device according to claim 1.

3. The aforementioned analysis unit is Based at least on the first fluorescence signal reflecting the amount of DNA, it is determined whether the mechanism of increase in leukocytes is neoplastic. Based at least on the second fluorescence signal reflecting the amount of RNA, it is determined whether the mechanism of leukocyte increase is reactive. The measuring device according to claim 1.

4. The aforementioned analysis unit is Based at least on the first fluorescence signal corresponding to the first fluorescent dye whose binding amount to the cells increases in accordance with the increase in DNA amount, it is determined whether or not the mechanism of increase in leukocytes is neoplastic. The mechanism of increase in leukocytes is determined to be reactive, based at least on the second fluorescence signal corresponding to the second fluorescent dye whose binding amount to the cells increases in response to an increase in RNA quantity. The measuring device according to claim 1.

5. The aforementioned analysis unit is Based on at least the multiple first fluorescence signals corresponding to each of the multiple cells, it is determined whether or not the mechanism of leukocyte increase is neoplastic. Based on at least the plurality of second fluorescence signals corresponding to each of the plurality of cells, it is determined whether or not the mechanism of increase in leukocytes is reactive. The measuring device according to claim 1.

6. The aforementioned analysis unit is By analyzing the first fluorescence signal, which reflects the state of the DNA, for each of the plurality of cells, the cells whose DNA state is related to the mechanism of tumor growth can be detected. By analyzing the second fluorescence signal, which reflects the state of the RNA, for each of the plurality of cells, the cells whose RNA state is related to the mechanism of increased reactivity are detected. The measuring device according to claim 1.

7. The aforementioned analysis unit is By analyzing the amount of DNA based on the first fluorescence intensity obtained from the first fluorescence signal, it is determined whether or not the mechanism of increase in leukocytes is neoplastic. By analyzing the amount of RNA based on the second fluorescence intensity obtained from the second fluorescence signal, it is determined whether the mechanism of increase in leukocytes is reactive. The measuring device according to claim 1.

8. The detection unit detects the optical signal which includes at least one of the following: a first fluorescence signal corresponding to a first fluorescent dye having a higher binding ability to DNA than to RNA, and a second fluorescence signal corresponding to a second fluorescent dye having a higher binding ability to RNA than to DNA. The measuring device according to claim 1.

9. The detection unit detects the optical signal which includes at least one of the following: a first fluorescence signal corresponding to a first fluorescent dye having a higher binding ability to DNA than the second fluorescent dye, and a second fluorescence signal corresponding to a second fluorescent dye having a higher binding ability to RNA than the first fluorescent dye. The measuring device according to claim 1.

10. The measuring device according to claim 1, wherein the analysis unit provides first information corresponding to a neoplastic mechanism of increase when it is determined that the mechanism of increase of white blood cells is neoplastic, and provides second information corresponding to a reactive mechanism of increase when it is determined that the mechanism of increase of white blood cells is reactive.

11. The measuring device according to claim 1, wherein the analysis unit is capable of detecting, based on the optical signal, a first leukocyte associated with a tumorigenic increase mechanism and a second leukocyte associated with a reactivity increase mechanism.

12. The analysis unit acquires the first fluorescence intensity and the second fluorescence intensity, respectively, based on the first fluorescence signal and the second fluorescence signal. The measuring device according to claim 11, wherein the analysis unit detects the cells exhibiting a first fluorescence intensity greater than a first threshold as first leukocytes, and detects the cells exhibiting a second fluorescence intensity greater than a second threshold as second leukocytes.

13. The measuring device according to claim 11, wherein the first white blood cell comprises at least one selected from the group consisting of abnormal lymphocytes, blast cells, and immature erythroblasts.

14. The measuring device according to claim 11, wherein the second leukocyte includes atypical lymphocytes.

15. The detection unit detects the optical signal, which includes the scattered light signal corresponding to the cell. The measuring device according to claim 1.

16. The measuring device according to claim 15, wherein the scattered light signal is a side-scattered light signal.

17. The measuring device according to claim 15, wherein the analysis unit is capable of providing first information indicating that the mechanism of increase in leukocytes is neoplastic, based on the first fluorescence signal and the scattered light signal.

18. The measuring device according to claim 15, wherein the analysis unit is capable of providing second information indicating that the mechanism of increase in leukocytes is reactive increase, based on the second fluorescence signal and the scattered light signal.

19. The measuring device according to claim 15, wherein the analysis unit provides information on a subpopulation of leukocytes based on the first fluorescence signal and the scattered light signal.

20. The measuring device according to claim 15, wherein the analysis unit provides information on a subpopulation of leukocytes based on the second fluorescence signal and the scattered light signal.

21. The measuring device according to claim 19 or 20, wherein the subpopulation of leukocytes is at least two populations selected from lymphocyte population, monocyte population, neutrophil population, eosinophil population and basophil population.

22. The measuring device according to claim 19 or 20, wherein the information relating to the subpopulation of white blood cells is information relating to the number of particles contained in the subpopulation.

23. The measuring device according to claim 1, wherein the sample is a blood sample.

24. The measuring apparatus according to claim 1, wherein the first fluorescent dye and the second fluorescent dye are fluorescent dyes having fluorescence emission maxima in different wavelength ranges.

25. The measuring apparatus according to claim 1, wherein the first fluorescent dye and the second fluorescent dye are fluorescent dyes having maximum absorption in different wavelength ranges.

26. The measuring apparatus according to claim 1, wherein the light source is capable of irradiating light of a first wavelength capable of exciting the first fluorescent dye and light of a second wavelength capable of exciting the second fluorescent dye.

27. The measuring apparatus according to claim 26, wherein the first wavelength is 315 nm or more and 490 nm or less, and the second wavelength is 610 nm or more and 750 nm or less.

28. The measuring apparatus according to claim 1, further comprising a liquid delivery unit that delivers the reagent from the reagent container to the chamber via a liquid delivery pipe provided between the reagent container and the chamber.

29. The measuring apparatus according to claim 28, wherein the liquid delivery unit delivers the reagent from the reagent container to the chamber via a liquid delivery tube having a first end disposed in the reagent container and a second end connected to the chamber.

30. The measuring device according to claim 29, wherein the first end of the liquid delivery pipe is fixed in a predetermined position within the reagent container.

31. The measuring device according to claim 30, wherein the first end of the liquid delivery tube is fixed in the predetermined position while a plurality of measurement samples corresponding to each of the plurality of specimens are being prepared.

32. The measuring device according to claim 29, further comprising a mechanism for inserting the first end of the liquid delivery tube into the reagent container and positioning the first end at a predetermined location within the reagent container.

33. The measuring device according to claim 28, wherein the liquid delivery unit further comprises a quantitative unit for delivering a fixed amount of reagent from the reagent container to the chamber via the liquid delivery tube.

34. A reagent comprising the first fluorescent dye and the second fluorescent dye, used in the measuring device according to claim 1.

35. A method for analyzing cells in a sample, A measurement sample is prepared by mixing the reagent supplied from at least one reagent container with the sample, thereby preparing the cells stained with at least one of the first fluorescent dye and the second fluorescent dye contained in the reagent. Multiple cells in the sample being measured flowing through the flow cell are irradiated with light. Multiple optical signals corresponding to each of the aforementioned multiple cells are detected, The optical signal includes at least one of a first fluorescence signal corresponding to a first fluorescence dye that binds to the DNA of the cell, and a second fluorescence signal corresponding to a second fluorescence dye that binds to the RNA of the cell. Whether the mechanism of leukocyte increase is neoplastic or not is determined at least based on the first fluorescence signal, The mechanism of the increase in leukocytes is determined to be reactive or not based at least on the second fluorescence signal. Analytical methods.

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