WO2026070928A1 - Measurement device - Google Patents

Abstract

The present invention relates to a measurement device for analyzing cells, the measurement device comprising: an electric measurement unit that electrically measures cells; a first optical measurement unit that optically measures cells; a second optical measurement unit that optically measures hemoglobin; a sample preparation unit that mixes a first fluorescent dye and a second fluorescent dye with a specimen to prepare a measurement sample; and an analysis unit that provides a measurement result of the measurement sample. The first optical measurement unit measures an optical signal including at least one of a first fluorescent signal generated from the first fluorescent dye and a second fluorescent signal generated from the second fluorescent dye. The analysis unit refers to the optical signal and classifies cells by executing an analysis based on differences in staining characteristics and fluorescence characteristics of the fluorescent dyes.

Description

Measuring device

This invention relates to a measuring device for analyzing cells in a sample.

In the analysis of specimens containing blood cells, such as blood and body fluids, white blood cells are classified into five subgroups: 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 preparing a measurement sample from a blood specimen, measuring test items such as white blood cell count and white blood cell classification, and then preparing other measurement samples and performing additional measurements based on the measurement results.

U.S. Patent Application No. 2022/0334099

However, a challenge arises from the increased time and cost associated with preparing new measurement samples and performing additional measurements after the initial measurements to obtain the predetermined test items. Therefore, the present invention aims to provide a measuring device that can reduce the need to prepare new measurement samples and perform additional measurements.

The present inventors, after diligent research to solve the above problems, discovered that by using fluorescent dyes with different staining and fluorescence properties for cells in a sample, cells containing components according to the properties of the fluorescent dye can be classified, and thus completed the present invention. That is, the present invention is a measuring device for analyzing cells contained in a sample taken from a subject, comprising: an electrical measuring unit for electrically measuring cells; a first optical measuring unit for optically measuring cells; a second optical measuring unit for optically measuring hemoglobin contained in the sample; a sample preparation unit for preparing a measurement sample for measurement by at least one of the electrical measuring unit, the first optical measuring unit, and the second optical measuring unit; and an analysis unit for providing the measurement results of the measurement sample, wherein the sample preparation unit measures (1) red blood cell count, white blood cell count, hemoglobin amount, hematocrit value, mean red blood cell count. The sample preparation unit includes a plurality of chambers corresponding to a first measurement item including blood cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelet count; a second measurement item relating to the morphological classification of leukocytes; and a third measurement item different from the first and second measurement items; a plurality of reagent containers containing reagents including fluorescent dyes used for preparing the measurement sample; and a flow path for sending the measurement sample from the plurality of chambers to the first optical measurement unit. The sample preparation unit responds to a measurement order that includes a measurement instruction for at least one of the first measurement item, the second measurement item, or the third measurement item. The first optical measurement unit prepares a measurement sample using at least one chamber corresponding to a measurement instruction and at least one reagent container corresponding to at least one chamber. The sample preparation unit prepares a measurement sample by mixing a first fluorescent dye and a second fluorescent dye, which have different staining and fluorescence properties for cells, with the sample in at least one chamber corresponding to a measurement instruction. The first optical measurement unit uses a first fluorescence signal generated from a first fluorescent dye having staining properties for a first component of a cell, and a second fluorescent dye having staining properties for a second component of a cell. The device essentially measures an optical signal containing at least one of the following: a first fluorescence signal and a second fluorescence signal. The analysis unit then refers to the optical signal containing the first and second fluorescence signals and performs the following analyses to classify the measured cells: (A) a first analysis based on the differences in staining characteristics and fluorescence characteristics of the first and second fluorescence dyes; (B) a second analysis based on the differences in the first component in the measured cells; and (C) a third analysis corresponding to the differences in the second component in the measured cells.

According to the present invention, a measuring device is provided that can reduce the need to prepare new measurement samples and perform additional measurements.

This figure shows an example of the appearance of a measuring device. This figure shows an example of a fluid circuit in the measurement unit of the measuring device according to the first embodiment. This is a block diagram showing an example configuration of a measuring unit in a measuring device. This is a block diagram showing an example configuration of a measuring unit in a measuring device. This figure shows an example of a fluid circuit in the measurement unit of a measuring device. This figure shows an example of a sample preparation section in the measurement unit of a measuring device. This figure shows an example of a sample preparation section in a measurement unit. This figure shows an example of the optical system configuration for the FCM detection unit. This is a diagram showing an example of the configuration of the FCM detection unit. This figure shows an example of the optical system configuration for the FCM detection unit. This is a cylinder showing an example of the configuration of an analysis unit. This flowchart shows an example of how the measuring device works. This is a flowchart showing an example of the analysis process. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This table shows examples of information (flags) generated by the analysis unit. This is a flowchart showing an example of the analysis process. This is a flowchart showing an example of the analysis process. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a flowchart showing an example of the analysis process. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This is a schematic diagram of a scattergram. This figure shows an example of the analysis results screen. This figure shows an example of the analysis results screen.

Referring to Figure 1, an example of the configuration of the measuring device will be described. The measuring device 500 comprises, 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, and includes a flow cytometer. The analysis unit 300 and the measuring unit 400 are connected by a predetermined interface (e.g., 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 have a configuration in which the analysis unit 300 is provided within the measuring unit 400.

The measurement unit 400 prepares the 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 and second fluorescent dyes.

The first and second fluorescent dyes are dyes with 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, for example, have fluorescence emission maxima in different wavelength ranges. The fluorescence emission maxima is the wavelength (peak wavelength) at which the fluorescent dye is excited by light with the highest fluorescence intensity. Furthermore, for example, the first and second fluorescent dyes have maximum absorption maxima in different wavelength ranges. That is, the second fluorescent dye may emit fluorescence at a wavelength that can be detected separately from the fluorescence from the first fluorescent dye.

(Example 1 of fluorescent dyes)
The first and second fluorescent dyes are, for example, 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 of them has a higher binding ability to DNA than the other, and the opposite is true for its binding ability to RNA. 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 a sample containing leukocytes with a higher-than-normal amount of DNA (i.e., a sample 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 a sample containing leukocytes with a higher-than-normal amount of RNA (i.e., a sample 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 a sample from a subject with a higher leukocyte count than a healthy subject is neoplastic or reactive.

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 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, for example, 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 and a lower binding ability to RNA than the second fluorescent dye. The first fluorescent dye is, for example, a dye that emits fluorescence when excited by absorbing light in the wavelength range of 400 nm to 490 nm. The second fluorescent dye is, for example, a dye that emits fluorescence when excited by absorbing light in the wavelength range of 610 nm to 750 nm, where its maximum absorption occurs.

(Example 2 of fluorescent dyes)
One application of the first and second fluorescent dyes, which have the ability to bind to nucleic acids (e.g., DNA, RNA), which are components of cells, is the measurement of reticulocytes (sometimes referred to as "RET" herein) and platelets (sometimes referred to as "PLT" herein). For example, cells in a sample are stained using a first fluorescent dye whose ability to bind to DNA is superior to its ability to bind to RNA, and a second fluorescent dye whose ability to bind to RNA is superior to its ability to bind to DNA. The first fluorescent dye has, for example, a higher binding ability to platelets containing DNA compared to its binding ability to reticulocytes. The second fluorescent dye has, for example, a higher binding ability to reticulocytes containing RNA but not DNA compared to its binding ability to platelets containing DNA. The first and second fluorescent dyes also have different fluorescence properties. The first fluorescent dye is, for example, a dye that is excited and emits fluorescence by absorbing light in the wavelength range of 400 nm to 490 nm. The second fluorescent dye is, for example, a dye that is excited and emits fluorescence when it absorbs light in the wavelength range of 610 nm to 750 nm, where its maximum absorption occurs. By mixing such first and second fluorescent dyes with the sample to prepare a measurement sample and acquiring an optical signal with the FCM detection unit 460, reticulocytes and platelets can be measured without having to perform separate measurement operations for RET and PLT. For example, a decrease in RET may indicate that the subject is suspected of having acute leukemia or aplastic anemia. In addition, the subject's hemostatic ability and bleeding risk can be determined based on the PLT measurement results.

(Example 3 of fluorescent dyes)
For example, by using a first fluorescent dye that can bind to the nucleolus, a component of cells, and a second fluorescent dye that can bind to RNA, another component of cells, it becomes possible to classify leukocytes and differentiate between blast cells and promyelocytes. For instance, cells in a sample are stained using a first fluorescent dye whose ability to bind to the nucleolus is greater than its ability to bind to RNA, and a second fluorescent dye whose ability to bind to RNA is greater than its ability to bind to the nucleolus. Mature leukocytes, such as lymphocytes, monocytes, neutrophils, and eosinophils, which are the target of leukocyte classification, do not possess nucleoli. Therefore, the binding ability of the first fluorescent dye to mature leukocytes is lower than that of the second fluorescent dye. Blast cells and promyelocytes possess nucleoli. The amount of nucleoli in blast cells and promyelocytes differs. Therefore, the amount of the first fluorescent dye that binds to blast cells and promyelocytes depends on the amount of nucleoli in each of them. The difference in the amount of bound first fluorescent dye is measured as a difference in fluorescence intensity. Therefore, differentiation between blast cells and promyelocytes becomes possible through analysis based at least on the difference in fluorescence intensity of the first fluorescent dye. The first and second fluorescent dyes also have different fluorescence properties. The first fluorescent dye is, for example, a dye that is excited and emits fluorescence by absorbing light in the wavelength range of 400 nm to 490 nm. The second fluorescent dye is, for example, a dye that is excited and emits fluorescence by absorbing light in the wavelength range of 610 nm to 750 nm at its maximum absorption. In preparing a sample for leukocyte classification, by preparing the sample using both the first and second fluorescent dyes and acquiring the optical signal with the FCM detection unit 460, it becomes possible to perform the measurement without separating the measurement operation for leukocyte classification and the measurement operation for differentiation between blast cells and promyelocytes. The test results regarding blast cells and promyelocytes can be used, for example, to determine whether a subject is suspected of having leukemia. Prompt treatment can significantly impact the prognosis of leukemia associated with promyelocytic cells (acute promyelocytic leukemia). Effective medications exist for acute promyelocytic leukemia. Therefore, if blood sample testing can differentiate between blast cells and promyelocytic cells, it becomes possible to distinguish between patients requiring immediate treatment and those who do not.

(Example 4 of fluorescent dyes)
For example, by using a first fluorescent dye capable of binding to granules, which are components of cells, and a second fluorescent dye capable of binding to DNA, which is also a component of cells, it becomes possible to differentiate between basophils (sometimes referred to as "Baso" in this specification), a form of white blood cell, and immature granulocytes. For example, by using a first fluorescent dye whose ability to bind to granules in cells is more dominant than its ability to bind to nucleic acids (e.g., DNA, RNA), and a second fluorescent dye whose ability to bind to DNA is more dominant than its ability to bind to granules in cells, it becomes possible to differentiate between Baso and immature granulocytes even when immature granulocytes are present in the sample, and to accurately measure Baso. In white blood cell classification, reagents may be used to measure Baso, other white blood cells, and nucleated red blood cells. In this case, for example, to stain nucleated red blood cells, measurements are performed using a fluorescent dye capable of staining DNA. Baso and other white blood cells are classified based on scattered light. However, when immature granulocytes are present in a sample, it can be difficult to distinguish between baso and immature granulocytes using only a fluorescent dye capable of staining DNA. By using a first fluorescent dye capable of staining granules contained in baso (e.g., basophilic granules) in addition to a second fluorescent dye capable of staining DNA, it becomes possible to distinguish between baso and immature granulocytes. The first fluorescent dye can specifically bind to granules contained in baso, for example. By using such a first fluorescent dye, it becomes possible to prepare a sample so that, for example, the first fluorescent dye stains baso, but immature granulocytes are hardly stained. By analyzing the fluorescence signal corresponding to the first fluorescent dye, it becomes possible to distinguish and identify baso stained with the first fluorescent dye from immature granulocytes, even in samples containing immature granulocytes. The first and second fluorescent dyes also have different fluorescence properties. The first fluorescent dye is, for example, a dye that is excited and emits fluorescence by absorbing light in the wavelength range of 400 nm to 490 nm. The second fluorescent dye is, for example, a dye that is excited and emits fluorescence by absorbing light in the wavelength range of 610 nm to 750 nm, where its maximum absorption occurs. In the measurement of Baso, other white blood cells, and nucleated red blood cells, by preparing a measurement sample using the first fluorescent dye in addition to the second fluorescent dye and acquiring an optical signal with the FCM detection unit 460, it becomes possible to accurately measure Baso even in samples containing immature granulocytes, without performing remeasurement to distinguish between Baso and immature granulocytes. Chronic myeloid leukemia is a disease associated with an abnormal increase in Baso. If Baso and immature granulocytes can be distinguished and each can be accurately counted, an abnormal increase in Baso can be judged more accurately.

Details of the fluorescent dyes and surfactants will be described later. In the analysis of particles in the 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. Cells include, for example, leukocytes, red blood cells, and platelets (including aggregated platelets).

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, a pH of 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.

The sample is measured by the FCM detection unit 460 of the measurement unit 400. In the FCM detection unit 460, light is irradiated onto each particle in the sample flowing through the flow cell, and the optical signal of each particle is acquired. Upon light irradiation, fluorescence originating from the first and second fluorescent dyes is generated from the particles in the sample. Additionally, scattered light is emitted from the particles upon light irradiation. This scattered light includes lateral and forward scattered light. The optical signal includes the first and second fluorescence 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.

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 first optical measuring unit that optically measures cells and acquires optical signals emitted from individual particles in the measurement sample. The FCM detection unit 460 includes a light source, flow cell, dichroic mirror, and light-receiving element, as described later (see Figures 8-10).

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 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 in the XY direction. The sample aspiration unit 450 includes a quantitative unit 452 (e.g., a syringe pump) for aspirating and discharging the sample T using the sample aspiration nozzle 451.

The liquid delivery mechanism 430 comprises a liquid delivery pipe 431 and a liquid delivery unit 432. The liquid delivery pipe 431 is located 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 pipe 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 pipe 431. Within the chamber 420, the sample and the reagent 12 come into contact, staining the particles contained in the sample with the first and second fluorescent dyes.

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.

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 electromagnetic 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, electromagnetic valve V1 is opened and electromagnetic valve V2 is closed. The pump 433 generates negative pressure, filling the flow path between electromagnetic valves V1, V2 and the pump 433 with reagent. When supplying the filled reagent to the chamber 420, electromagnetic valve V1 is closed, electromagnetic 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.

Chamber 420 is a container in which the measurement sample is prepared. Within Chamber 420, the reagent 12 and the sample are mixed to prepare a measurement sample containing particles stained with the first and second fluorescent dyes. The measurement unit 400 is provided with one or more chambers 420. 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. Furthermore, before the next measurement sample is prepared, Chamber 420 is cleaned by a cleaning mechanism (not shown), and the resulting liquid is discarded into the waste liquid chamber 36.

The measurement unit 400 is equipped with 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 is equipped with multiple reagent container holders, for example, a reagent container containing the reagent with the first fluorescent dye and another reagent container containing the reagent with the second fluorescent dye may be mounted in different reagent container holders. Alternatively, a reagent container containing the reagents with the first and second fluorescent dyes and another reagent container containing a hemolytic reagent may be mounted in different reagent container holders.

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 above-mentioned predetermined position, for example, while the reagent container 200 is mounted on the reagent container holder 60. Furthermore, at least while the measurements of multiple specimens are being performed (i.e., while multiple measurement samples corresponding to each of the multiple specimens are being prepared), the first end to which the suction tube 64 is connected is fixed in the predetermined position described above.

The FCM detection unit 460 irradiates light onto individual particles in the sample being measured, which are flowing through the flow cell. As described above, when light is irradiated onto the particles, fluorescence is generated from the particles, each originating from the first and second fluorescent dyes, respectively. 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 the fluorescence from the first and second fluorescent dyes excited by this single-wavelength light.

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 reagent, and a reagent container holder 60 in which the reagent container is installed. The sample preparation unit 440 delivers the reagent from the reagent container 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 container and discharges the aspirated sample into the chamber of the sample preparation unit 440. The measurement sample is prepared by mixing the sample and reagent in the chamber. The device mechanism unit 455 includes motors and actuators that move various parts of the measurement unit 400. The device mechanism unit 455 includes, for example, a mechanism for moving the sample container 100.

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.

The second embodiment of the measuring device 500 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 second embodiment of the measuring device 500 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, sample aspiration unit 450, device mechanism unit 455, and FCM detection unit 460 are the same as those in the first embodiment of the measuring device.

The RBC/PLT detection unit 461 is an electrical measurement unit that electrically measures cells. It introduces a measurement sample prepared from a blood sample and diluent into an aperture and counts red blood cells and platelets by detecting the change in electrical resistance that occurs when cells pass through the aperture. The HGB detection unit 462 is a second optical measurement unit that optically measures hemoglobin contained in the sample. It measures the hemoglobin concentration in the 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".

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.

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.

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 the hemolytic reagent. Reagent container R2 contains the 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 the staining reagent. A flow path is provided between the first chamber 420 and the FCM detection unit 460.

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 the SLS hemolytic agent. The SLS hemolytic agent is a reagent used to lyse red blood cells and prepare a sample suitable for hemoglobin measurement.

The reagent container 200, which contains the staining reagent, is mounted in the reagent container holder 60. The reagent container holder 60 is equipped with a suction tube 64 for aspirating the staining reagent 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 descends and the suction tube 64 penetrates (punctures) the sealing material of the reagent container 200, the cover 63 also descends, covering the reagent container 200. When the suction tube lifting mechanism 65 rises, the cover 63 also rises, making the reagent container 200 removable from the outside.

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 electromagnetic 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. Electromagnetic 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 electromagnetic valve V1 open and electromagnetic valve V2 closed. As a result, the 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. This pushes out the fixed amount of staining reagent filled into the flow path between electromagnetic valves V1 and V2 and the quantitative unit 30, and the staining reagent is supplied to the chamber 420 through the liquid delivery tube 431.

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 the 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.

A quantitative dispensing unit 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 dispensing unit 33. Alternatively, a diaphragm pump can also be used. Electromagnetic valves V5 and V6 open and close the flow path. The quantitative dispensing unit 33 and electromagnetic valves V5 and V6 quantitatively supply the diluent from the reagent container R2 to the first chamber 420.

A waste liquid chamber 36 for containing unwanted solution 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 within it.

The flow path between the reagent container R2, which contains the diluent, and the second chamber 55 is equipped with a quantitative unit 38 and solenoid valves V8 and V9. A syringe pump is used as the quantitative unit 38. A diaphragm pump, for example, can also be used instead of the syringe pump. Solenoid valves V8 and V9 open and close the flow path. The quantitative unit 38 and solenoid valves V8 and V9 quantitatively supply the diluent from the reagent container R2 to the second chamber 55. A waste liquid chamber 41, which contains the unused solution, is connected to the second chamber 55. Between the second chamber 55 and the waste liquid chamber 41, a solenoid 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. Solenoid valve V13 will be described later.

A quantitative dispensing unit 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 dispensing unit 39. Alternatively, a diaphragm pump can also be used. Electromagnetic valves V11 and V12 open and close the flow path. The quantitative dispensing unit 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 within it.

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, and the blood sample T contained in the sample container 100 is drawn 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, thereby discharging the drawn blood sample 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.

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 hemolyzed by the hemolytic reagent. In addition, in the measurement sample, particles containing white blood cells are stained with the first fluorescent dye and the 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 the hemolytic reagent are stirred. This dissolves the red blood cells in the blood sample. The staining reagent is supplied to the first chamber 420 containing 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 stirred. 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 the particles containing normal leukocytes and, if present, abnormal cells in the blood sample with the first and second fluorescent dyes, preparing the measurement sample. The FCM detection unit 460 is connected to a pump (not shown), and the pump drives the measurement sample from the first chamber 420 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 and second fluorescent dyes.

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 461 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 measurement sample in the second chamber 55 is supplied to the RBC/PLT detection unit 461 and the HGB detection unit 462, respectively, by the drive of the pump. 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 sample is sent to the RBC/PLT detection unit 461, where electrical resistance detection is performed. Next, SLS hemolytic agent is supplied from the reagent container R3 to the sample remaining in the second chamber 55. This lyses the red blood cells, resulting in a sample containing SLS hemoglobin generated from hemoglobin. This sample is then sent to the HGB detection unit 462. In the example shown in Figure 5, the sample for RBC/PLT detection and the sample for HGB detection are prepared in a common second chamber 55; however, they may be prepared in separate chambers.

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 subgroups. In addition to the CBC item and the DIFF item, other items (for example, items measuring reticulocytes (RET) and platelets (PLT) stained with fluorescent dye) may also be configured to measure other items.

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 a single reagent container 200, and to deliver the reagents in the single reagent container 200 into the chamber 420 by a single liquid delivery mechanism 430. The first sample preparation unit 440A shown in Figure 6 is configured to contain a reagent containing the first fluorescent dye in a first reagent container 200A, a reagent containing the second fluorescent dye in a second 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 second reagent container 200B into the chamber 420 by a second liquid delivery mechanism 430b. This configuration also allows for staining of particles contained in the sample with two fluorescent dyes.

The first sample preparation unit 440A in Figure 6 comprises 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, containing 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, containing the reagent containing the second fluorescent dye, is mounted in the second holder portion of the reagent container holder 442.

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 mechanism 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 along 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.

Referring to Figure 7, an example of the sample preparation unit 440 will be described. In the example shown 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 RBC/PLT reaction chamber 420A is a chamber for preparing a measurement sample by mixing the sample and diluent. The measurement sample prepared in chamber 420A is measured by the RBC/PLT detection unit 461.

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

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 sample, and a staining solution (reagent) for staining the cells in the sample. The leukocyte classification reaction chamber 420D is a chamber for preparing measurement samples for classifying, for example, a subpopulation of basophils and a subpopulation of nucleated red blood cells, which are forms of leukocytes. 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, for example, a hemolytic agent, the specimen, and a staining solution (reagent) for staining the cells in the specimen are mixed to prepare the sample for measurement.

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) at least five subpopulations of leukocytes (e.g., subpopulations including lymphocytes, monocytes, neutrophils, eosinophils, and basophils), and (2) subpopulations of nucleated erythrocytes. The hemolytic agents used in the preparation in the leukocyte classification reaction chambers 420C and D may have different compositions in each chamber. The staining solutions (reagents) used in the preparation in the leukocyte classification reaction chambers 420C and D may have different compositions in each chamber.

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.

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) for preparing a sample for measurement of platelets stained with a fluorescent dye in the FCM measurement unit 460.

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.

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, when 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, 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 white blood cells, the sample preparation unit 440 prepares a measurement sample in the RBC/PLT reaction chamber 420A, HGB reaction chamber 420B, white blood cell classification reaction chamber 420C, and white blood cell classification reaction chamber 420D in order to obtain the results of white blood cell classification (for example, five classifications of 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 420D are measured by the FCM detection unit 460.

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. For example, the sample preparation unit 440 prepares measurement samples using first and second fluorescent dyes in at least one of the multiple reaction chambers 420.

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. For example, the first and second fluorescent dyes used in reaction chamber 420C may be different from those used in reaction chamber 420D.

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 all 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.

(Example of fluorescent dye application 1)
In Example 1 of Use, first and second fluorescent dyes (see "Example 1 of Fluorescent Dyes" above) are used to distinguish whether the mechanism of leukocyte increase is neoplastic or reactive.

The first and second fluorescent dyes are included, for example, in at least one of the staining reagents used in the preparation of measurement samples in the leukocyte classification reaction chambers 420C and D. When obtaining results for leukocyte classification measurement items (measurement items corresponding to the "DIFF" measurement order) by measurement in chambers 420C and D, the first and second fluorescent dyes are used in the sample preparation in at least one of chambers 420C and D. By preparing the sample in this manner, it becomes possible to distinguish whether the mechanism of leukocyte increase is neoplastic or reactive based on the measurement operation in response to the DIFF measurement order. In other words, it becomes possible to distinguish the mechanism of leukocyte increase without issuing an additional measurement order in addition to the DIFF measurement order. In this case, the second fluorescent dye may be used in the measurement sample prepared in chamber 420C, and the first fluorescent dye may be used in the measurement sample prepared in chamber 420D. The first and second fluorescent dyes may be included in both of the staining reagents used in the preparation of measurement samples in the leukocyte classification reaction chambers 420C and D.

The CBC (Cellular Blood Cell) and/or DIFF (Digestive Function) parameters mentioned above are fundamental 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 performed by the measuring device 500 include instructions for CBC measurement, or instructions for CBC and DIFF measurement. For example, almost all measurement orders in the first blood test (e.g., referred to as the "initial test") for a patient visiting a medical institution include instructions for CBC measurement, or instructions for CBC and DIFF measurement. Additional tests based on the results of the initial test may not include instructions for CBC measurement, or instructions for CBC and DIFF measurement, but may only include a predetermined additional test item (e.g., reticulocytes: RET). In some cases, the test may be completed with only the initial test, without any additional tests.

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 chamber C contains the second fluorescent dye and the staining solution used in chamber D contains the first fluorescent dye. In case (1) above, a measurement using the first and second fluorescent dyes is performed in response to 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 in response to a measurement order that includes a measurement instruction for CBC or CBC + DIFF. As mentioned above, the measurement instruction for CBC, or CBC + DIFF, is included in almost all initial examination measurement orders, so measurement results containing information on the mechanism of leukocyte increase (neoplastic or reactive) can be obtained without additional tests. In other words, measurement results containing 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 containing information on the mechanism of leukocyte increase can be obtained based on the results obtained from the measurement operation using a single measurement channel.

(Example of fluorescent dye applications 2)
Application example 2 is an example in which reticulocytes and platelets are measured without separating the measurement operations for each. In this example, for example, the RET reaction chamber 420E in Figure 7 is replaced with a RET/PLT reaction chamber 420F for preparing a sample for measuring RET and PLT. In preparing the sample in the RET/PLT reaction chamber 420F, the first and second fluorescent dyes (see "Example 2 of Fluorescent Dyes" above) are used.

The first and second fluorescent dyes are contained in, for example, the staining reagents used in sample preparation in the reaction chamber 420F. Sample preparation in the reaction chamber 420F and measurement by the FCM detection unit 460 are performed, for example, according to a measurement order including a RET measurement instruction. Platelets containing DNA are stained by the first fluorescent dye, which has a superior ability to bind to DNA than to RNA. Reticulocytes containing RNA but not DNA are stained by the second fluorescent dye, which has a superior ability to bind to RNA than to DNA. Since the first and second fluorescent dyes have different fluorescence properties, platelets and reticulocytes can be analyzed by analyzing the fluorescence signals corresponding to the first and second fluorescent dyes. In the reaction chamber 420F, a measurement sample is prepared using the first and second fluorescent dyes, so reticulocytes and platelets can be measured. Measurements for reticulocytes and platelets are performed together. Reticulocytes and platelets are measured simultaneously in a single measurement operation (sample preparation in reaction chamber 420F and measurement by FCM detection unit 460). In other words, reticulocytes and platelets can be measured using a single measurement channel. Since reticulocytes and platelets can be measured simultaneously without performing separate measurement operations for each, measurement time is reduced. Furthermore, since staining of reticulocytes and platelets can be performed with a single staining reagent, the number of reagents required in the measuring device is reduced, lowering the cost of performing the test. Because the reaction chamber for preparing the sample for reticulocyte measurement and the reaction chamber for preparing the sample for platelet measurement can be integrated into a single chamber, the measuring device can be made more compact.

(Example of fluorescent dye applications 3)
Example 3 of application is an example of performing leukocyte classification and distinguishing between blast cells and promyelocytes. In this example, for example, the first and second fluorescent dyes (see "Example 3 of Fluorescent Dyes" above) are used in the preparation of the measurement sample in the reaction chamber 420C shown in Figure 7.

The first and second fluorescent dyes are contained in staining reagents used, for example, in sample preparation in reaction chamber 420C. Sample preparation in reaction chamber 420C and measurement by FCM detection unit 460 are performed, for example, according to a measurement order including a DIFF measurement instruction. The first fluorescent dye, whose ability to bind to nucleoli is superior to its ability to bind to RNA, stains blast cells and promyelocytes that have nucleoli. The second fluorescent dye, whose ability to bind to RNA is superior to its ability to bind to nucleoli, stains mature leukocytes such as lymphocytes, monocytes, neutrophils, and eosinophils. The amount of nucleoli in blast cells and promyelocytes differs. Therefore, the amount of the first fluorescent dye that binds to blast cells and promyelocytes depends on the amount of nucleoli in each of the blast cells and promyelocytes. The difference in the amount of bound first fluorescent dye is measured as a difference in fluorescence intensity. Therefore, it is possible to differentiate between blast cells and promyelocytes by analysis based at least on the difference in fluorescence intensity of the first fluorescent dye. Furthermore, staining with a second fluorescent dye allows for the simultaneous classification of mature leukocytes. A single measurement operation (sample preparation in reaction chamber 420C and measurement by FCM detection unit 460) performs both leukocyte classification and differentiation of blast cells and promyelocytes. In other words, leukocyte classification and differentiation of blast cells and promyelocytes are possible with a single measurement channel. Since the measurement of blast cells and promyelocytes can be performed in a single operation without requiring separate measurements for leukocyte classification, measurement time is reduced. The amount of information obtained from the measurement results of a single measurement channel increases, improving the ability to analyze blood cells based on the measurement results.

(Example of fluorescent dye applications 4)
Application example 4 is an example in which Baso and immature granulocytes can be differentiated. In this example, for example, the first and second fluorescent dyes (see "Example 4 of Fluorescent Dyes" above) are used in the preparation of the measurement sample in the reaction chamber 420D shown in Figure 7.

The first and second fluorescent dyes are contained in, for example, the staining reagents used in sample preparation in reaction chamber 420D. Sample preparation in reaction chamber 420D and measurement by FCM detection unit 460 are performed, for example, according to a measurement order including a DIFF measurement instruction. The first fluorescent dye, whose ability to bind to granules in cells is superior to its ability to bind to nucleic acids (e.g., DNA, RNA), is used to stain Baso containing granules (e.g., basophilic granules). Staining cells with the second fluorescent dye, whose ability to bind to DNA is superior to its ability to bind to granules in cells, provides information for classifying nucleated red blood cells, Baso, and other white blood cells. When immature granulocytes are present in the sample, it was sometimes difficult to distinguish between Baso and immature granulocytes when measuring with only fluorescent dyes capable of staining DNA. By using a second fluorescent dye capable of staining DNA, as well as a first fluorescent dye capable of staining granules contained in baso (e.g., basophilic granules), it becomes possible to distinguish between baso and immature granulocytes. The first fluorescent dye can specifically bind to granules contained in baso, for example. By using such a first fluorescent dye, it becomes possible to prepare a sample so that, for example, the first fluorescent dye stains baso, but immature granulocytes are hardly stained. By analyzing the fluorescence signal corresponding to the first fluorescent dye, it becomes possible to distinguish and identify baso stained with the first fluorescent dye from immature granulocytes, even in a sample containing immature granulocytes. A single measurement operation (sample preparation in reaction chamber 420D and measurement by FCM detection unit 460) performs the classification of baso from other leukocytes and nucleated red blood cells, and the discrimination of baso from immature granulocytes all at once. In other words, a single measurement channel enables the classification of basoplasmic somatic cells (Basioplasma serotonin) and other leukocytes, as well as the differentiation of basoplasmic somatic cells (Basioplasma serotonin) and immature granulocytes. Because the amount of information obtained from a single measurement channel increases, the ability to analyze blood cells based on the measurement results is improved. The reaction chamber 420D can also be used, for example, in measurement orders that include only CBC (cardiac basin cell count). For CBC measurement orders, the results of the measurement using the reaction chamber 420D can be used, for example, to count the number of leukocytes in the CBC category. Therefore, in example 4, for measurement orders that include only CBC, a single measurement channel enables the classification of basoplasmic somatic cells (Basioplasma serotonin) and other leukocytes, as well as the differentiation of basoplasmic somatic cells (Basioplasma serotonin) and immature granulocytes.

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 the 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.

The sample prepared in chamber 420 is flowed into the flow cell 413 of the FCM detection unit 460. In the example shown 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.

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 light 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.

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.

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-scattering light detector 416, the lateral-scattering light detectors 412a and 412b, and the lateral fluorescence photodetectors 422a and 422b. The lateral-scattering light detectors 412a and 412b are also referred to as the first and second light-receiving sections, respectively.

Referring to the example in Figure 9, the relationship between the various types of light emitted when light is irradiated onto particles P passing through the flow cell 413 and the optical system of the FCM detection unit 460 will be explained. In Figure 9, the light irradiated from the first light source 411a is light of the first wavelength L1, and the light irradiated from the second light source 411b is light of the second wavelength L2. When light L1 and L2 are irradiated onto 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 irradiated 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, a first side scattered light (SSC-1) corresponding to the first wavelength and a 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 relative 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 referred to as the "forward scattered light signal," the analog signal corresponding to SSC-1 as the "first lateral scattered light signal," the analog signal corresponding to SFL-1 as the "first fluorescence signal," the analog signal corresponding to SSC-2 as the "second lateral scattered light signal," and the analog signal corresponding to SFL-2 as the "second fluorescence signal." One pulse of each analog signal corresponds to one particle (for example, one cell).

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. Such feature parameters include, for example, peak value (height of the pulse peak), pulse width, pulse area, transmittance, Stokes shift, ratio, changes over time, and values correlated therewith.

Optical information may be the characteristic parameters described above. Optical information includes at least first fluorescence information and second fluorescence information. The first fluorescence information is not particularly limited as long as it reflects the amount of fluorescent dye used to stain DNA in nucleated cells. The 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 the 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. The 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. The first and second lateral scattered light information are preferably 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. The forward scattered light information is preferably 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").

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 emits light of a wavelength capable of exciting both the first and second fluorescent dyes. The light emitted from the light source 411 irradiates individual particles in the sample being measured, flowing through the flow cell 413. The light from the light source 411 irradiates particles in the sample stained with the first and second fluorescent dyes, generating first and second side fluorescence. That is, the FCM detection unit 460 can acquire first and second fluorescence signals corresponding to multiple particles in the sample (i.e., multiple particles stained with the first and second fluorescent dyes) using a single light source.

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 passes through the dichroic mirror 418 and is received by the side fluorescence receiving element 422b. Therefore, 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 measurement sample.

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 and second fluorescent dyes 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 and second fluorescent dyes 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 and second fluorescent dyes lies within a 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 have 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 with such a single wavelength of light from the light source 411, 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.

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, for example, by 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, for example, executes 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.

The analysis unit 300 performs at least one of the classification and counting of particles (particularly cells) 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 normal levels due to the subject's health condition or disease, the analysis unit 300 can generate information regarding the mechanism of the white blood cell increase based on the optical information.

The analysis unit 300 may classify cells based on the optical information of each of the 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 multiple, 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.

The processor 301 is a CPU (Central Processing Unit) and executes programs loaded from the storage unit 304 into 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, programs for controlling the measurement unit 400 and programs 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.

The control unit 307 includes a pointing device, such as a keyboard, mouse, or touch panel. Users, such as physicians or laboratory technicians, can input measurement orders into the measuring device 500 by operating the control unit 307. Measurement instructions are then 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 specimen. The measurement unit 400 is electrically connected to the analysis unit 300 via the interface unit 305.

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 via 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 the reagent 12 containing the 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 481a, 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. The analysis system then completes the operation shown in Figure 12. The details of the analysis process in step S7 of Figure 12 will be described for each embodiment below. In the following embodiments, an example using feature parameters as optical information is described, but waveform data may also be used as optical information.

As an example of the analysis process in step S7, embodiments 1 to 4 are shown for the "Example of Use of Fluorescent Dye 1" described above. 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 subgroups 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 subgroups based on the second fluorescence information and the scattered light information, and a process of classifying leukocytes into subgroups 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 of the mechanism of leukocyte increase based on the first fluorescence information and the second fluorescence information.

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 (first-order leukocytes) are detected as leukocytes containing more DNA than other leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

Secondary leukocytes (II leukocytes) can appear in the blood as a result of immune responses, such as viral infections, drug allergies, and autoimmune diseases. These II leukocytes are, for example, atypical lymphocytes. Atypical lymphocytes, also known as reactive lymphocytes, 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.

Regarding step S7 in Figure 12, an example of the analysis process of Embodiment 1 will be described with reference to Figure 13, but the method is not limited to this example. This analysis 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". As first fluorescence information, the first fluorescence intensity ("SF
The second fluorescence intensity (also called "SFL-2 intensity") is used as the second fluorescence information. A scattergram with SSC intensity on the x-axis and SFL-1 intensity on the y-axis is also called the "first scattergram." A scattergram with SSC intensity on the x-axis and SFL-2 intensity on the y-axis is also called the "second scattergram."

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. The analysis unit 300 creates a first scattergram based on the determined position of each point. 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. The analysis unit 300 creates a second scattergram based on the determined position of each point.

In step S13, the analysis unit 300 classifies the leukocytes in the sample into subpopulations based on the determined locations of each point. These subpopulations may include, for example, lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The classification of leukocytes may also involve dividing them into at least two, three, or four subpopulations corresponding to these subpopulations. The leukocyte subpopulations preferably include lymphocytes, more preferably include lymphocytes and monocytes, and even more preferably include lymphocytes, monocytes, and neutrophils. The 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.

For example, as shown in Figures 14A and 14B, each subpopulation of leukocytes is distributed on each scattergram. In the figures, "Lymp" refers to the lymphocyte population, "Mono" refers to the monocyte population, "Neut" refers to the neutrophil population, "Eo" refers to the eosinophil population, and "Baso" refers to the basophil population. Figures 14A and 14B 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 multinuclear 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 in each subpopulation of leukocytes may be counted. As can be seen from Figure 14A, the distribution range of SFL-1 intensity in 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.

After classifying the leukocytes, the process proceeds to step S14 in Figure 13. In step S14, the analysis unit 300 detects the 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 the 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 of the subpopulations of 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 value among the SFL-1 intensities of cells classified into that subpopulation. The first threshold may be a value predetermined based on the results of measuring a sample obtained from a healthy individual (e.g., peripheral blood) and classifying the leukocytes into the aforementioned subpopulations.

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 region enclosed by the dashed line, particles showing an SFL-1 intensity greater than the first threshold on the first scattergram may appear. In Figure 15, the arrow indicates the maximum value of the SFL-1 intensity of the lymphocyte population of leukocytes as an example of the first threshold. In the example shown in Figure 15, the analysis unit 300 performs an 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 (such an analysis may be referred to as "first analysis" in this specification). For example, the analysis unit 300 classifies multiple cells stained according to the differences in staining characteristics into a first group (the group of cells shown in Figure 15) corresponding to the first fluorescence intensity (SFL-1) from the first fluorescent dye, and a second group (the group of cells 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).

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 shown in Figure 15, the analysis unit 300 performs an analysis based on differences in a first component (DNA in the example of 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 of Figure 15) into multiple populations according to a first fluorescence intensity (SFL-1). In the example of 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 the first threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

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 of Lymp, Mono, Baso, Neut, and Eo. Here, among the subpopulations of leukocytes such as Lymp, Mono, Baso, Neut, and Eo, the monocyte subpopulation 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 subpopulation of leukocytes or a higher value. The maximum SFL-2 intensity of the monocyte population of white blood cells refers to the highest SFL-2 intensity among cells classified as monocytes. The second threshold may be a predetermined value based on, for example, the results of measuring and classifying white blood cells from a sample obtained from a healthy individual (e.g., peripheral blood).

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 of Figure 16, the analysis unit 300 performs an analysis based on differences in the second component (RNA in the example of Figure 16) in multiple measured cells (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 groups according to their second fluorescence intensity (SFL-2). In the example in Figure 16, the analysis unit 300 classifies cells corresponding to an SFL-2 higher than the second threshold 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.

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 subgroups such as Lymp, Mono, Baso, Neut, and Eo appeared in the second scattergram. Since the second fluorescent dye has a high RNA-binding ability (higher 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).

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 is information that reflects the 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 exhibiting an SFL-2 intensity greater than the second threshold and an SSC intensity within the second range as second leukocytes. The analysis unit 300 also counts the detected first and second leukocytes.

The first and second ranges 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 and classifying leukocytes in a sample (e.g., peripheral blood) obtained from a healthy individual.

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.

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 (also called "gate B") where the SFL-1 intensity is above a first threshold and the SSC intensity is within a first range. 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. Furthermore, in Figure 17, as an example of the first range, a range is shown that 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. In the example in Figure 17, the analysis unit 300 performs an analysis ("first analysis") based on the difference in staining characteristics of the first fluorescent dye and the second fluorescent dye, and the difference in fluorescence characteristics of the first fluorescent dye and the second fluorescent dye. For example, the analysis unit 300 classifies multiple cells stained according to the difference in staining characteristics into a first group (the group of cells shown in Figure 17) corresponding to the first fluorescence intensity (SFL-1) by the first fluorescent dye, and a second group (the group of cells shown in Figure 18, described later) corresponding to the second fluorescence intensity (SFL-2) by the second fluorescent dye. In the examples in Figures 17 and 18, the analysis unit 300 classifies cells based on a scattergram derived from a first fluorescence intensity (SFL-1) (example in Figure 17) and a scattergram derived from a 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 differences in a first component (DNA in the example in Figure 17) among 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 the first fluorescence intensity (SFL-1). In the example in Figure 17, the analysis unit 300 classifies cells with an SFL-1 higher than a first threshold and SSC within a first range as first leukocytes, and cells corresponding to an SFL-1 lower than the first threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

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. Furthermore, in Figure 18, an example of the second range is shown, which 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. In the example in Figure 18, the analysis unit 300 performs an analysis (third analysis) based on differences in the second component (RNA in the example in Figure 18) 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 in Figure 18) into multiple groups according to the second fluorescence intensity (SFL-2). In the example in Figure 18, the analysis unit 300 classifies cells with SFL-2 higher than the second threshold and SSC within the second range as second leukocytes, and cells corresponding to SFL-2 lower than the second threshold as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

In step S16, the analysis unit 300 generates information regarding the mechanism of leukocyte increase based on the detection results of the first and second leukocytes. The detection result for the first leukocytes is, for example, the number of first leukocytes detected and counted in step S14. The detection result for the 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. Based on 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.

Condition 1 is, for example, a condition where 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 where 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 where 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.

When the number of first and second leukocytes satisfies condition 1, 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.

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. 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.

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 leukocytes resulting from either neoplastic or reactive mechanisms of increase, for example.
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 (for example, "Unknown"). The analysis unit 300 may output both the "Malignant?" and "Reactive?" flags to the display unit 306.

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 from healthy individuals, samples containing leukocytes whose increase mechanism is neoplastic, and samples containing leukocytes whose increase mechanism is reactive, values can be set to distinguish between samples from healthy individuals and samples containing leukocytes resulting from either neoplastic or reactive increase mechanisms.

In the modified version of step S16 in Figure 13, the analysis unit 300 does not detect the second leukocyte, or does not use the detection result of the second leukocyte, but generates information indicating that the mechanism of increase in leukocytes in the sample is neoplastic, based on the detection result of the first leukocyte. For example, the analysis unit 300 determines whether the number of first leukocytes is above the third threshold. When the number of first 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. When the number of first 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.

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 contained 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. When the number of second leukocytes is above the fourth threshold, the analysis unit 300 generates information indicating that the mechanism of increase in leukocytes contained 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. When 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.

In Embodiment 1, in step S8 of Figure 12, the analysis unit 300 may provide information on leukocyte subpopulations in addition to information on the mechanism of leukocyte increase. Information on leukocyte subpopulations may be based on first fluorescence information (e.g., SFL-1 intensity) and scattered light information (e.g., SSC intensity). Alternatively, information on leukocyte subpopulations may be based on second fluorescence information (e.g., SFL-2 intensity) and scattered light information (e.g., SSC intensity). Information on leukocyte subpopulations may include, 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.

Regarding step S7 in Figure 12, an example of the analysis process in Embodiment 2 will be described with reference to Figure 20, but the method is not limited to this example. This analysis 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.

Referring to Figure 20, in step S21, the analysis unit 300 determines the position of 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 positions of each point, the analysis unit 300 creates a first scattergram. In step S22, the analysis unit 300 determines the position of 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 positions of each point, the analysis unit 300 creates a second scattergram.

In step S23, the analysis unit 300 detects particles exhibiting an SFL-1 intensity greater than the 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. The details of the detection of first leukocytes by gating are the same as those described in step S14 of Embodiment 1.

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. The details of secondary leukocyte detection by gating are the same as those described in step S15 of Embodiment 1.

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 for first leukocytes is, for example, the number of first leukocytes detected and counted in step S23. The detection result for 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.

Regarding step S7 in Figure 12, an example of the analysis process of Embodiment 3 will be described with reference to Figure 21, but the method is not limited to this example. This analysis process enables the classification of leukocytes into subpopulations on the second scattergram and 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.

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 measurement 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 fluorescent dye and the second fluorescent dye, and the difference in fluorescence characteristics of the first fluorescent dye and the second fluorescent dye. 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.

In step S33, the analysis unit 300 determines the position of 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; instead, they are displayed as a group of leukocytes, as shown in Figure 22, for example. In the figure, "WBC" refers to leukocytes.

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.

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 region enclosed by the dashed line, particles showing an SFL-1 intensity greater than the first threshold on the third scattergram may appear. In the example of Figure 23, the analysis unit 300 performs an analysis (second analysis) based on differences in a first component (DNA in the example of 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 of Figure 23) into multiple groups according to the 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.

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 the second threshold as secondary leukocytes. The analysis unit 300 also counts the detected secondary leukocytes. The second threshold is as described above.

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 region enclosed by the dashed line, particles showing an SFL-2 intensity greater than the second threshold on the third scattergram may appear. In the example of 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 groups according to the 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.

In a modified version of step S34 described 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 above a first threshold and the SFL-2 intensity is below 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 measured cells (second analysis) and an analysis based on differences in a second component (RNA in this example) in multiple measured cells (third analysis). The analysis unit 300 classifies cells appearing in the 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).

In a modified version of step S35 described 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 measured cells (second analysis) and an analysis based on differences in a second component (RNA in this example) in multiple measured cells (third analysis). The analysis unit 300 classifies cells appearing in the 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 secondary leukocytes, and classifies cells outside gate C as leukocytes such as lymphocytes, monocytes, neutrophils, eosinophils, basophils, etc. (sometimes including primary leukocytes).

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.

In step S36, the analysis unit 300 generates information regarding the mechanism of leukocyte increase based on the detection results of the first and second leukocytes. The detection result for the first leukocytes is, for example, the number of first leukocytes detected and counted in step S34. The detection result for the 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.

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.

Regarding step S7 in Figure 12, an example of the analysis process of Embodiment 4 will be described with reference to Figure 26, but the process is not limited to this example. This analysis 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.

In step S42, the analysis unit 300 detects particles exhibiting an SFL-1 intensity greater than the 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. The details of the detection of first leukocytes by gating are the same as those described in step S34 of Embodiment 3.

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. The details of secondary leukocyte detection by gating are the same as those described in step S35 of Embodiment 3.

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 for first leukocytes is, for example, the number of first leukocytes detected and counted in step S42. The detection result for 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.

As an example of step S7 in Figure 12, an example of the analytical processing described in "Example 2 of Fluorescent Dye Applications" above will be explained. In the example of analytical processing described below, 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 SFL-1 intensity plotted on either the horizontal or vertical axis is also called the "first scattergram." Similarly, a scattergram with SFL-2 intensity plotted on either the horizontal or vertical axis is also called the "second scattergram."

Figures 27 and 28 show examples of the first and second scattergrams in the analytical process described in "Example 2 of Fluorescent Dye Applications" above. As shown in Figure 27, in the first scattergram with SFL-1, which corresponds to the first fluorescent dye whose binding ability to DNA is superior to its binding ability to RNA, on the horizontal axis, clusters of platelets containing DNA appear on the scattergram, but reticulocytes with little or no DNA appear as clear clusters on the scattergram. On the other hand, as shown in Figure 28, in the second scattergram with SFL-2, which corresponds to the second fluorescent dye whose binding ability to RNA is superior to its binding ability to DNA, on the horizontal axis, clusters of reticulocytes containing RNA appear on the scattergram, and clusters of platelets appear only slightly on the lower side of SFL-2 and FSC. The first and second fluorescent dyes have different staining characteristics and fluorescence characteristics for cells. By using such fluorescent dyes, as shown in Figures 27 and 28, it becomes possible to distinguish and analyze RET and PLT using a single measurement channel.

In the examples shown in Figures 27 and 28, 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 group (the group of cells shown in Figure 27) corresponding to the first fluorescence intensity (SFL-1) from the first fluorescent dye, and a second group (the group of cells shown in Figure 28) corresponding to the second fluorescence intensity (SFL-2) from the second fluorescent dye. In the examples of Figures 27 and 28, the analysis unit 300 classifies the cells using a scattergram based on the first fluorescence intensity (SFL-1) (example in Figure 27) and a scattergram based on the second fluorescence intensity (SFL-2) (example in Figure 28).

In the example shown in Figure 27, the analysis unit 300 performs a second analysis based on differences in a first component (DNA in the example of Figure 27) among 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 of Figure 27) into multiple populations according to their first fluorescence intensity (SFL-1). In the example of Figure 27, the analysis unit 300 detects platelets based on clusters of platelets stained with the first fluorescent dye that has the ability to bind to DNA, for example, in the scattergram of Figure 27. Reticulocytes (RETs), which have little or no DNA, do not appear in the scattergram of Figure 27. Thus, the analysis unit 300 classifies platelets and reticulocytes by the analysis exemplified in the scattergram of Figure 27.

In the example shown in Figure 28, the analysis unit 300 performs a third analysis based on differences in a second component (RNA in the example of Figure 28) 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 of Figure 28) into multiple populations according to the second fluorescence intensity (SFL-2). In the example of Figure 28, the analysis unit 300 detects reticulocytes based on clusters of reticulocytes stained with the second fluorescent dye that has RNA-binding ability in the scattergram of Figure 28. Platelets, which do not readily bind to the second fluorescent dye that has RNA-binding ability, appear only faintly in the scattergram of Figure 28 and do not overlap with the RET cluster. Thus, the analysis unit 300 classifies platelets and reticulocytes by the analysis exemplified in the scattergram of Figure 28.

As an example of step S7 in Figure 12, an example of the analytical processing described in "Example 3 of Fluorescent Dye Applications" above will be explained. In the example of analytical processing described below, 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 SFL-1 intensity plotted on either the horizontal or vertical axis is also called the "first scattergram." Similarly, a scattergram with SFL-2 intensity plotted on either the horizontal or vertical axis is also called the "second scattergram."

Figures 29, 30, and 31 show examples of the first and second scattergrams in the analytical process described in "Example 3 of Fluorescent Dye Applications" above. Figure 29 shows the first scattergram with SFL-1, which corresponds to the first fluorescent dye whose nucleolus binding ability is superior to its RNA binding ability, on the vertical axis. In the first scattergram, blast cells and promyelocytes, which contain nucleoli, appear at higher values than leukocytes. Since blast cells and promyelocytes contain different amounts of nucleoli, the amount of the first fluorescent dye that binds to the nucleoli of blast cells is different from the amount that binds to the nucleoli of promyelocytes. The difference in the amount of bound first fluorescent dye is reflected in the difference in fluorescence signal intensity corresponding to blast cells and promyelocytes, respectively. Therefore, clusters of blast cells and clusters of promyelocytes appear on the first scattergram based on fluorescence intensity. On the other hand, as shown in Figure 30, in the second scattergram with SFL-2, which corresponds to the second fluorescent dye whose ability to bind to RNA is superior to its ability to bind to the nucleolus, plotted on the vertical axis, clusters corresponding to mature leukocytes such as lymphocytes, monocytes, neutrophils, and eosinophils appear. However, in the second scattergram of a sample containing blast cells and other immature granulocytes (promyelocytes), as shown in Figure 31, clusters of blast cells and other immature granulocytes appear in regions overlapping with some of the mature leukocyte clusters, and in such clusters, it may be impossible to distinguish between blast cells and promyelocytes. The first and second fluorescent dyes have different staining and fluorescence properties for cells. By using such fluorescent dyes, as shown in Figure 29, it becomes possible to distinguish and analyze blast cells and promyelocytes using a single measurement channel.

In the examples in Figures 29 and 30 and 31, the analysis unit 300 performs an analysis (first analysis) based on 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. For example, the analysis unit 300 classifies multiple cells stained according to the difference in staining characteristics into a first group (the group of cells shown in Figure 29) corresponding to the first fluorescence intensity (SFL-1) from the first fluorescent dye, and a second group (the group of cells shown in Figures 30 and 31) corresponding to the second fluorescence intensity (SFL-2) from the second fluorescent dye. As shown in the example in Figure 31, if at least one of blast cells and promyelocytes appears in the sample, it is difficult for the analysis unit 300 to classify at least one of the blast cells and promyelocytes from other leukocytes such as lymphocytes and monocytes. Therefore, the analysis unit 300 classifies cells using the scattergram shown in Figure 30 to classify mature white blood cells such as lymphocytes, monocytes, neutrophils, and eosinophils, and for samples in which at least one of blast cells and promyelocytes appears, it classifies cells using the scattergram shown in Figure 29 to classify at least one of the blast cells and promyelocytes from mature white blood cells.

In the example in Figure 29, the analysis unit 300 performs a second analysis based on differences in a first component (nucleolus in the example in Figure 29) in multiple measured cells. For example, the analysis unit 300 classifies multiple cells stained with a first fluorescent dye for a first component (nucleolus in the example in Figure 29) into multiple populations according to the first fluorescence intensity (SFL-1). In the example in Figure 29, the analysis unit 300 classifies blast cells and promyelocytes based on the first fluorescence intensity, which reflects the difference in nucleolus between blast cells and promyelocytes. For example, the analysis unit 300 classifies clusters of blast cells and promyelocytes based on the fact that blast cells, which contain more nucleolus than promyelocytes, bind more of the first fluorescent dye, and their first fluorescence intensity is higher than that of promyelocytes.

In the examples of Figures 30 and 31, the analysis unit 300 performs an analysis (third analysis) based on differences in a second component (RNA in the examples of Figures 30 and 31) in 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 examples of Figures 30 and 31) into multiple populations according to the second fluorescence intensity (SFL-2). In the examples of Figures 30 and 31, the analysis unit 300 classifies mature leukocytes based on differences in RNA among mature leukocytes such as lymphocytes and monocytes. For example, the analysis unit 300 classifies mature leukocytes based on differences in the second fluorescence intensity that reflect differences in RNA among mature leukocytes.

As an example of step S7 in Figure 12, an example of the analytical processing described in "Example 4 of Fluorescent Dye Applications" above will be explained. In the example of analytical processing described below, 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 SFL-1 intensity plotted on either the horizontal or vertical axis is also called the "first scattergram." Similarly, a scattergram with SFL-2 intensity plotted on either the horizontal or vertical axis is also called the "second scattergram."

Figures 32 and 33 show examples of the first and second scattergrams when using example 4 of the fluorescent dye. As shown in Figure 32, in the first scattergram, where SFL-1 is plotted on the vertical axis, corresponding to the first fluorescent dye whose binding ability to granules in cells (e.g., basophilic granules) is superior to its binding ability to nucleic acids (e.g., DNA, RNA), Baso appears on the higher SFL-1 side. Figure 32 is an example where immature granulocytes (IG) are present in the sample. In the first scattergram of Figure 32, Baso clusters appear on the higher SFL-1 side than immature granulocyte clusters. Because the first fluorescent dye has a high binding ability to granules of Baso (e.g., basophilic granules), Baso clusters appear on the higher SFL-1 side than immature granulocyte clusters. Therefore, differentiation between Baso and immature granulocytes becomes possible. On the other hand, as shown in Figure 33, in the second scattergram with SFL-2, the second fluorescent dye whose DNA binding ability is superior to its binding ability to granules in cells, plotted on the horizontal axis, nucleated red blood cells, Baso, and other white blood cells can be differentiated. The first and second fluorescent dyes have different staining and fluorescence properties for cells. By using such fluorescent dyes, classification of white blood cells including Baso and nucleated red blood cells, and differentiation between Baso and immature granulocytes can be performed with a single measurement channel.

In the examples shown in Figures 32 and 33, 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 group (the group of cells shown in Figure 32) corresponding to the first fluorescence intensity (SFL-1) from the first fluorescent dye, and a second group (the group of cells shown in Figure 33, described later) corresponding to the second fluorescence intensity (SFL-2) from the second fluorescent dye. In the examples of Figures 32 and 33, the analysis unit 300 classifies the cells using a scattergram based on the first fluorescence intensity (SFL-1) (example in Figure 32) and a scattergram based on the second fluorescence intensity (SFL-2) (example in Figure 33).

In the example shown in Figure 32, the analysis unit 300 performs a second analysis based on differences in the first component (granules in the example of Figure 32) among the measured cells. For example, the analysis unit 300 classifies multiple cells stained with the first fluorescent dye for the first component (granules in the example of Figure 32) into multiple groups according to their first fluorescence intensity (SFL-1). The first fluorescent dye has a high binding affinity to basophilic granules, such as those contained in Baso, among other granules. The first fluorescent dye also has binding affinity to granules contained in immature granulocytes, for example, but its binding affinity is lower than that to basophilic granules contained in Baso. Therefore, the SFL-1 of Baso clusters will be higher than that of immature granulocytes.

In the example shown in Figure 33, the analysis unit 300 performs a third analysis based on differences in a second component (DNA in the example of Figure 33) among the measured cells. For example, the analysis unit 300 classifies multiple cells stained with a second fluorescent dye for a second component (DNA in the example of Figure 33) into multiple groups according to the second fluorescence intensity (SFL-2). In the example of Figure 33, the analysis unit 300 classifies Baso, non-Baso mature leukocytes, and nucleated erythrocytes based on SFL-2.

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 regarding the clinical condition of the subject as analysis results. Referring to Figures 34A and 34B, 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 regarding 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. The research item display area 82 displays supplementary information such as the number and characteristics of cells. The flag display area 83 displays information regarding the mechanism of white blood cell increase. The flags displayed in the flag display area 83 are, for example, information suggesting the clinical condition of the subject. The scattergram display area 84 displays the scattergram created during the analysis process. Referring to Figure 34A, the flag display area 83 displays the flag "Malignant?" to indicate that the mechanism of white blood cell increase in the sample is neoplastic. The first scattergram is also displayed in the scattergram display area 84. Referring to Figure 34B, the flag display area 83 displays the flag "Reactive?" to indicate that the mechanism of white blood cell increase in the sample is reactive. The second scattergram is also displayed in the scattergram display area 84.

The analysis results displayed on the display unit 306 may include results related to RET and PLT. For example, the counting results for RET and PLT, and a scattergram may be displayed. Furthermore, for example, if an abnormality is determined in at least one of the RET and PLT results, a flag corresponding to that abnormality may be displayed in the flag display area 83. For example, based on the RET counting result, if the RET count value falls below a threshold, a flag indicating that RET is low will be displayed. Similarly, based on the PLT counting result, if the PLT count value falls below a threshold, a flag indicating that PLT is low will be displayed. The displayed flags may, for example, provide information suggesting the subject's clinical condition. RE measurement results may, for example, be related to the suspected presence of acute leukemia or aplastic anemia in the subject. PLT measurement results may, for example, be related to the subject's hemostatic ability or bleeding risk.

The analysis results displayed on the display unit 306 may include results regarding blast cells and promyelocytes. For example, the analysis unit 300 can display a flag on the display unit 306 corresponding to the detection of blast cells and a flag corresponding to the detection of promyelocytes. Alternatively, a scattergram of blast cells and promyelocytes may be displayed on the display unit 306. The displayed flags may, for example, indicate information suggesting the subject's clinical condition. Measurement results regarding blast cells and promyelocytes are related, for example, to the suspected leukemia of the subject. Prompt treatment can affect the prognosis of promyelocyte-associated leukemia (acute promyelocytic leukemia). Effective medications exist for acute promyelocytic leukemia. Therefore, if blast cells and promyelocytes can be differentiated by blood sample testing, it becomes possible to distinguish between subjects requiring immediate treatment and those with less urgent need for treatment.

The analysis results displayed on the display unit 306 may include results regarding the differentiation between Baso and immature granulocytes. For example, the Baso counting result may be displayed on the display unit 306. Since the analysis unit 300 can differentiate between Baso and immature granulocytes even when they appear in the sample, it is possible to display an accurate Baso counting result on the display unit 306, excluding the influence of immature granulocytes. Furthermore, for example, a scattergram reflecting the differentiation result between Baso and immature granulocytes may be displayed on the display unit 306. The displayed flags may, for example, indicate information suggesting the clinical condition of the subject. Chronic myeloid leukemia is a disease associated with abnormal increases in Baso. If Baso and immature granulocytes can be distinguished and each can be accurately counted, abnormal increases in Baso can be judged more accurately.

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 (23)

A measuring device for analyzing cells contained in a sample taken from a subject,
An electrical measuring unit for electrically measuring the aforementioned cells,
A first optical measuring unit for optically measuring the aforementioned cells,
A second optical measuring unit for optically measuring the hemoglobin contained in the aforementioned sample,
A sample preparation unit for preparing a sample for measurement by at least one of the electrical measurement unit, the first optical measurement unit, and the second optical measurement unit,
Includes an analysis unit that provides the measurement results of the measurement sample,
The sample preparation unit is
(1) First measurement items including red blood cell count, white blood cell count, hemoglobin level, hematocrit value, mean corpuscular volume, mean corpuscular hemoglobin level, mean corpuscular hemoglobin concentration, and platelet count.
(2) The second measurement item related to the morphological classification of white blood cells, and
(3) A third measurement item different from the first and second measurement items,
Multiple chambers corresponding to,
A plurality of reagent containers for containing reagents including a fluorescent dye used in the preparation of the aforementioned measurement sample,
The system includes a flow path for sending the sample to be measured from the plurality of chambers to the first optical measuring unit,
The sample preparation unit prepares the measurement sample in accordance with a measurement order that includes at least one measurement instruction for the first measurement item, the second measurement item, or the third measurement item, using at least one chamber corresponding to the measurement instruction and at least one reagent container corresponding to the at least one chamber.
The sample preparation unit prepares the measurement sample by mixing the sample with a first fluorescent dye and a second fluorescent dye, which have different staining and fluorescence properties for the cells, in at least one chamber corresponding to the measurement instruction.
The first optical measuring unit measures an optical signal that includes at least one of a first fluorescence signal generated from a first fluorescent dye having staining properties for a first component of the cell, and a second fluorescence signal generated from a second fluorescent dye having staining properties for a second component of the cell.
The aforementioned analysis unit is
Referring to the optical signals including the first and second fluorescence signals,
(A) A first analysis based on the difference in staining characteristics between the first fluorescent dye and the second fluorescent dye, and the difference in fluorescence characteristics between the first fluorescent dye and the second fluorescent dye.
(B) A second analysis based on the differences in the first component in the multiple cells measured, and
(C) A third analysis corresponding to the differences in the second component in the multiple cells measured,
By performing this, the measured cells are classified.
Measuring device. The aforementioned analysis unit is
The classification of the cells based on the optical signal is performed for each of the measurement samples prepared in the chamber.
The measuring device according to claim 1. The aforementioned analysis unit is
In the plurality of chambers corresponding to the measurement order, in accordance with the preparation of the plurality of measurement samples corresponding to each of the plurality of chambers, the classification of the cells based on the optical signal is performed for each of the plurality of measurement samples.
The measuring device according to claim 1. The aforementioned analysis unit is
In the plurality of chambers corresponding to the measurement order, in accordance with the fact that a plurality of measurement samples corresponding to each of the plurality of chambers have been prepared, for each of the plurality of measurement samples,
The classification of the cells based on the optical signals is performed.
The sample preparation unit is
In at least one of the plurality of chambers, the measurement sample is prepared using the first fluorescent dye and the second fluorescent dye.
The measuring device according to claim 1. The first optical measuring unit measures the optical signal for each of the measurement samples,
The aforementioned analysis unit is
The measured cells are classified by performing the first, second, and third analyses with reference to the optical signals, including the first and second fluorescence signals.
The measuring device according to claim 1. The aforementioned analysis unit is
In the first analysis, the multiple cells stained according to the differences in staining characteristics are classified into a first group corresponding to the first fluorescence signal from the first fluorescent dye and a second group corresponding to the second fluorescence signal from the second fluorescent dye.
The measuring device according to claim 1. The aforementioned analysis unit is
In the second analysis, the plurality of cells stained with the first fluorescent dye for the first component are classified into a plurality of groups according to the intensity of the first fluorescence signal.
The measuring device according to claim 1. The aforementioned analysis unit is
In the third analysis, the plurality of cells stained with the second fluorescent dye are classified into a plurality of groups according to the intensity of the second fluorescence signal.
The measuring device according to claim 1. At least one of the plurality of reagent containers contains a reagent containing the first fluorescent dye and the second fluorescent dye.
The measuring device according to claim 1. At least one of the plurality of reagent containers contains a reagent containing the first fluorescent dye and the second fluorescent dye,
The sample preparation unit is
The device has at least one reagent container containing the first fluorescent dye and the second fluorescent dye, and a liquid delivery tube connecting the reagent container and the chamber corresponding to the reagent container,
The reagent is supplied to the chamber via the liquid delivery tube.
The measuring device according to claim 1. The sample preparation unit is
The system includes a liquid delivery mechanism that delivers the reagent to the chamber via a liquid delivery tube having a first end placed in the liquid reagent contained in the reagent container and a second end connected to the chamber,
The measuring device according to claim 1. The sample preparation unit is
A liquid delivery mechanism for delivering the reagent to the chamber via a liquid delivery tube having a first end placed in the liquid reagent contained in the reagent container and a second end connected to the chamber,
The mechanism includes inserting the first end of the liquid delivery tube into the reagent container and positioning the first end within the liquid reagent.
The measuring device according to claim 1. The sample preparation unit is
A liquid delivery mechanism for delivering the reagent to the chamber via a liquid delivery tube having a first end placed in the liquid reagent contained in the reagent container and a second end connected to the chamber,
A mechanism for withdrawing the first end of the liquid delivery tube from the reagent container,
The measuring device according to claim 1. The first optical measuring unit is capable of measuring the optical signal which includes at least one of the first fluorescence signal corresponding to the first fluorescent dye excited in a first wavelength range and the second fluorescence signal corresponding to the second fluorescent dye excited in a second wavelength range different from the first wavelength range.
The measuring device according to claim 1. The first optical measuring unit distinguishes and measures the first fluorescence signal and the second fluorescence signal based on the difference in fluorescence characteristics.
The measuring device according to claim 1. The first optical measuring unit is,
A first light receiving unit for detecting the first fluorescence signal,
The measuring apparatus according to claim 1, further comprising a second light-receiving unit for detecting the second fluorescence signal. The aforementioned analysis unit is
The analysis includes first information corresponding to the differences in staining characteristics and fluorescence characteristics, second information corresponding to the differences in the first component in the measured plurality of cells, and third information corresponding to the differences in the second component in the measured plurality of cells.
The measuring device according to claim 1. The sample preparation unit is
In at least one chamber corresponding to the measurement instruction, the first fluorescent dye, which has a higher binding ability to the first component than to the second component, and the second fluorescent dye, which has a higher binding ability to the second component than to the first component, are mixed with the sample to prepare the measurement sample.
The measuring device according to claim 1. The sample preparation unit is
The first fluorescent dye and the second fluorescent dye are mixed with the sample to prepare the measurement sample for measuring the second measurement item.
The measuring device according to claim 1. The sample preparation unit is
The first fluorescent dye and the second fluorescent dye are mixed with the sample to prepare the sample for measuring the second measurement item for classifying the leukocytes into at least lymphocytes, monocytes, neutrophils, and neutrophils.
The measuring device according to claim 1. The sample preparation unit is
The first fluorescent dye and the second fluorescent dye are mixed with the sample to prepare the measurement sample for measuring the first measurement item.
The measuring device according to claim 1. The sample preparation unit is
The first fluorescent dye and the second fluorescent dye are mixed with the sample to prepare the measurement sample for measuring the first measurement item corresponding to CBC (Complete Blood Count).
The measuring device according to claim 1. The aforementioned analysis unit is
Based on the classification results of the aforementioned cells, information suggesting the clinical condition of the subject is provided.
The measuring device according to claim 1.