A method for classifying white blood cells into subgroups.

Abstract

To provide a method for classifying leucocytes to a subset, capable of counting and classifying accurately basophil even using a sample for which time has passed from the sample collection.SOLUTION: A method comprises the steps of: preparing a measurement sample by mixing a sample including leucocytes, a hemolytic reagent including a surfactant, a first fluorescence dye, and a second fluorescence dye; radiating light to particles in the measurement sample; detecting optical information including first fluorescence information based on fluorescence from the first fluorescence dye, second fluorescence information based on fluorescence from the second fluorescence dye, and diffused light information; selecting a cell population including basophils from the particles in the measurement sample on the basis of the optical information including the first fluorescence information; classifying leucocytes included in the cell population to a subset on the basis of the second fluorescence information and the diffused light information; and calculating the number of cells in the subset classified to a population of the basophils. The second fluorescence dye is a fluorescence dye having a maximum absorption in a wavelength different from that of the first fluorescence dye.SELECTED DRAWING: Figure 52

Description

[Technical Field]

[0001] The present invention relates to a method for classifying leukocytes into subpopulations. [Background technology]

[0002] Normal white blood cells are classified into five subgroups: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Information such as the classification and count of white blood cells is useful for examining the health status of a subject. A method using a flow cytometer is known for classifying and counting white blood cells. For example, Patent Document 1 describes classifying white blood cells into five subgroups and counting blood cells for at least four of these subgroups.

[0003] When measuring blood samples taken some time ago using a flow cytometer, changes in cell morphology and staining by dyes can lead to poor classification of white blood cells. In particular, basophils become difficult to classify and count due to the influence of other degraded white blood cells. Patent document 2 describes a method for accurately differentiating basophils and nucleated red blood cells from other white blood cells, even in blood samples taken some time ago. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent Application Publication No. 2010 / 0151509 [Patent Document 2] U.S. Patent Application Publication No. 2010 / 0330565 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Since the method described in Patent Document 2 is a method for classifying basophils and other white blood cells, in order to classify and count white blood cells other than basophils into subpopulations, it was necessary to separately perform measurements for white blood cell classification. An object of the present invention is to provide a method capable of accurately classifying and counting basophils even when using a specimen after a lapse of time from collection in a method for classifying white blood cells into subpopulations.

Means for Solving the Problems

[0006] The present inventor has obtained the knowledge that by using fluorescent dyes of types having mutually different maximum absorption wavelengths, the cause of poor classification can be suppressed, basophils can be counted more accurately, and white blood cells including basophils can be classified into subpopulations, and thus completed the present invention.

[0007] Therefore, the present invention includes a step of preparing a measurement sample by mixing a specimen containing white blood cells, a hemolytic reagent containing a surfactant, a first fluorescent dye, and a second fluorescent dye; irradiating light onto the particles in the measurement sample, and detecting optical information including first fluorescence information based on the fluorescence from the first fluorescent dye, second fluorescence information based on the fluorescence from the second fluorescent dye, and scattered light information; a step of selecting a cell population containing basophils from the particles in the measurement sample based on the optical information including the first fluorescence information; a step of classifying the white blood cells contained in the cell population into subpopulations based on the second fluorescence information and the scattered light information; and a step of counting the cells classified into the basophil population among the subpopulations. The present invention provides a method for classifying white blood cells into subpopulations, wherein the second fluorescent dye is a fluorescent dye having a maximum absorption in a wavelength range different from that of the first fluorescent dye.

Advantages of the Invention

[0008] According to the present invention, even a specimen after a lapse of time from collection can be used to more accurately classify and count basophils. As a result, the classification of white blood cells in the specimen into subpopulations can also be performed more accurately.

Brief Description of the Drawings

[0009] [Figure 1]Perspective view showing an analysis system according to a first embodiment of the present invention. [Figure 2] Schematic diagram showing the configuration of a measurement unit according to a first embodiment of the present invention. [Figure 3] Flowchart showing the procedure of measurement sample preparation processing by the analysis system according to a first embodiment of the present invention. [Figure 4] Block diagram showing the configuration of a measurement unit of an analysis system according to a second embodiment of the present invention. [Figure 5] Diagram showing a fluid circuit including a specimen suction unit, a sample preparation unit, and a detection unit in a second embodiment of the present invention. [Figure 6] Schematic diagram showing another example of a first sample preparation unit in a second embodiment of the present invention. [Figure 7] Configuration explanatory diagram showing an example of an optical system of a FCM detection unit in a second embodiment of the present invention. [Figure 8] Configuration explanatory diagram showing another example of an optical system of a FCM detection unit in a second embodiment of the present invention. [Figure 9] Diagram showing an example in which fluorescence emitted from two different fluorescent dyes leaks into each other. [Figure 10] Block diagram showing an example of the configuration of an analysis unit in a second embodiment of the present invention. <00所0113>Perspective view showing another configuration example of an analysis system in a second embodiment of the present invention. [Figure 12] Block diagram showing the configuration of another example of a measurement unit in a second embodiment of the present invention. [Figure 13] Diagram showing a state in which the cover of a measurement unit in a second embodiment of the present invention is opened. [Figure 14] Perspective view showing a reagent container holder of a measurement unit in a second embodiment of the present invention. [Figure 15] Front view showing the reagent container holder shown in FIG. 14. [Figure 16] Schematic diagram for explaining a reagent container holding portion of the reagent container holder shown in FIG. 14. [Figure 17] Figure 14 is a schematic diagram showing the reagent container placed on the reagent container holder. [Figure 18] Figure 14 is a schematic diagram showing the reagent container placed on the reagent container holder. [Figure 19] Figure 14 is a schematic longitudinal cross-sectional view showing the internal structure of the reagent container holder. [Figure 20] Figure 19 is a cross-sectional view of the reagent container holder, illustrating the state in which the reagent containers are set. [Figure 21] This figure illustrates the state in which the cover of the reagent container holder shown in Figure 20 is lowered in a vertical cross-sectional view. [Figure 22] This is a perspective view showing a large reagent container according to a second embodiment of the present invention. [Figure 23] This is a top view showing a large reagent container according to a second embodiment of the present invention. [Figure 24] This is a longitudinal cross-sectional view showing a large reagent container according to a second embodiment of the present invention. [Figure 25] This is a perspective view showing a small reagent container according to a second embodiment of the present invention. [Figure 26] This is a top view showing a small reagent container according to a second embodiment of the present invention. [Figure 27] This is a longitudinal cross-sectional view showing a small reagent container according to a second embodiment of the present invention. [Figure 28] (A) is a diagram showing the reagent container 200 according to another embodiment of the present invention installed in the measurement unit 400. (B) is a diagram showing the reagent container 200 according to another embodiment of the present invention with the suction tube 252 inserted from above. [Figure 29] This figure shows a reagent container 200 according to another embodiment of the present invention, comprising a reagent storage section 10 and a frame 20. [Figure 30] This figure shows that the reagent storage section 10 is formed in the shape of a hollow bag, and the frame 20 is equipped with an opening 21, a mounting member 22, and a movement restricting section 23. [Figure 31] This is a top view of the movement control section 23. [Figure 32] This figure shows that the reagent suction unit 250 includes a plurality of reagent container holders, each capable of holding one reagent container 200. [Figure 33] This figure shows that the storage section 260 includes a first insertion section 261 into which the reagent storage section 10 of the reagent container 200 is inserted, and a second insertion section 262 into which the movement restricting section 23 of the reagent container 200 is inserted. [Figure 34] This figure shows a storage section 260 in which a reagent container 200 is set. [Figure 35] This is a longitudinal cross-sectional view of the reagent container holder 250a. [Figure 36] This is a conceptual diagram illustrating the analysis method according to the fourth embodiment of the present invention. [Figure 37] This is a schematic diagram illustrating the waveform data used in the analysis method of the fourth embodiment. [Figure 38] This figure schematically illustrates the conversion to a digital signal by the A / D conversion unit in the analysis method of the fourth embodiment. [Figure 39] This figure schematically shows the waveform data obtained by sampling in the analysis method of the fourth embodiment. [Figure 40] This is a schematic diagram showing an example of a method for generating training data used to train a deep learning algorithm for determining the type of component in a sample in the analysis method of the fourth embodiment. [Figure 41] This figure shows an example of label values ​​in the analysis method of the fourth embodiment. [Figure 42] This is a schematic diagram illustrating an example of a method for analyzing waveform data of components in a sample according to the analysis method of the fourth embodiment. [Figure 43] This block diagram shows an example of the configuration of another sample preparation unit, which has a different configuration from the sample preparation unit shown in Figure 12. [Figure 44] This is a flowchart showing the first example of operation of this analysis method. [Figure 45] This is a flowchart showing a second example of how this analysis method works. [Figure 46]This flowchart shows a third example of how this analysis method works. [Figure 47] This is a block diagram showing an example configuration of the fifth embodiment. [Figure 48] This is a schematic diagram showing an example configuration of a parallel processing processor. [Figure 49] The first figure shows an overview of the arithmetic processing performed on the parallel processing processor based on the control of analysis software running on the processor. [Figure 50] This is the second figure, following Figure 49. [Figure 51] This is the third figure, following Figure 50. [Figure 52] This flowchart shows the process of classifying leukocytes into subpopulations using a first fluorescent dye and a second fluorescent dye. [Figure 53] This is a flowchart showing the steps of the sixth embodiment. [Figure 54] This is a flowchart showing the process of the seventh embodiment. [Figure 55] This is a flowchart showing the steps of the eighth embodiment. [Figure 56(a)] This is a side scatter-red fluorescence scattergram obtained when measuring the sample before basophil separation in Reference Example 1. [Figure 56(b)] This is a side scatter-red fluorescence scattergram obtained when the sample was measured after basophil separation in Reference Example 1. [Figure 56(c)] This is a side scatter-blue-violet fluorescence scattergram obtained when the sample was measured before basophil separation in Reference Example 1. [Figure 56(d)] This is a side scatter-blue-violet fluorescence scattergram obtained when the sample was measured after basophil separation in Reference Example 1. [Figure 57(a)] This is a side-scatter-blue-violet fluorescence scattergram of a sample measured 4 hours after blood collection in Reference Example 2. [Figure 57(b)] This is a side-scatter-blue-violet fluorescence scattergram of a sample measured 48 hours after blood collection in Reference Example 2. [Figure 57(c)]This is a side-scatter-blue-violet fluorescence scattergram of a sample measured 72 hours after blood collection in Reference Example 2. [Figure 58(a)] In Reference Example 2, Figure 57(a) shows a side-scatter-red fluorescence scattergram plotting the cells that were gated. [Figure 58(b)] In Reference Example 2, Figure 57(b) shows a side-scatter-red fluorescence scattergram plotting the cells that were gated. [Figure 58(c)] In Reference Example 2, Figure 57(c) shows a side-scatter-red fluorescence scattergram plotting the cells that were gated. [Figure 59(a)] This is a side-scattering-blue-violet fluorescence scattergram of Example 1. [Figure 59(b)] This is a side-scattering-red fluorescence scattergram of Example 1. [Figure 60(a)] This is the blue-violet fluorescence histogram of Example 2. [Figure 60(b)] This is a side-scattering-blue-violet fluorescence scattergram of Example 2. [Figure 60(c)] This is a side-scattering-red fluorescence scattergram of Example 2. [Figure 61] This graph shows the change in basophil count over time after blood collection. [Figure 62] These are the side-scattering-red fluorescence scattergrams for Example 2 and Comparative Example 1. [Modes for carrying out the invention]

[0010] The present invention will be described in further detail below with reference to the drawings. The following description is illustrative in all respects and should not be construed as limiting the present invention.

[0011] First, an embodiment of an analytical system suitable for the leukocyte classification method according to the present invention will be described. Following the description of the embodiment of the analytical system, an embodiment of the leukocyte classification method according to the present invention will be described.

[0012] (First Embodiment) Figure 1 is a perspective view showing an analysis system according to the first embodiment of the present invention. As shown in Figure 1, the analysis system 4000 according to the first embodiment comprises a measuring device (hereinafter referred to as a measuring unit) 400 and an analysis device (hereinafter referred to as an analysis unit) 300X separately. In this embodiment, the analysis unit 300X is, for example, a PC (personal computer) into which software for analyzing a sample to be measured is installed.

[0013] The measurement unit 400 is a unit for measuring a sample and includes a flow cytometer. The measurement unit 400 mixes the sample and reagents to prepare the measurement sample. The preparation of the measurement sample uses reagents containing multiple fluorescent dyes, each corresponding to multiple wavelengths. Each of the multiple cells in the measurement sample is stained with multiple fluorescent dyes. In the analysis of cells contained in a sample using this analysis system, cells that can be stained with multiple fluorescent dyes are analyzed. In other words, the preparation of the measurement sample in this embodiment is intended to stain a single cell with multiple fluorescent dyes, and the cells to be measured are, for example, lymphocytes, monocytes, eosinophils, neutrophils, basophils, etc.

[0014] The prepared sample is measured using a flow cytometer. Multiple signals are acquired from the cells in the sample irradiated with light, corresponding to each of several fluorescent dyes, as well as signals related to forward and side scattering. Each of the acquired optical signals is A / D converted to obtain digital data. The analysis unit 300X analyzes the digital data acquired by the measurement unit 400. The analysis unit 300X performs at least one of the following: classification and counting of cells in the sample, using at least the side scattering and multiple digital data corresponding to each of the multiple wavelengths of fluorescence. The analysis unit 300X also controls the operation of the measurement unit 400.

[0015] Figure 2 is a schematic diagram showing the configuration of a measurement unit according to the first embodiment of the present invention. As shown in Figures 1 and 2, the measurement unit 400 includes a sample preparation unit 440 having a chamber 420 and a liquid delivery mechanism 430, a sample aspiration mechanism 450, and a flow cytometer detection unit (FCM detection unit) 460 as a detection unit for acquiring signals emitted from cells.

[0016] The sample aspiration unit 450 is a mechanism for aspirating a sample from the sample container T and has a sample aspiration nozzle 451. The sample aspiration nozzle 451 can penetrate the sample container sealed by the lid. The sample aspiration mechanism 450 is movable to insert the sample aspiration nozzle 451 into the sample container and is movable in the XY direction to move the sample aspiration nozzle 451 to an upper position in the chamber 420. The sample aspiration mechanism 450 has a quantitative unit 452 (e.g., a syringe pump) for aspirating and discharging the sample by the sample aspiration nozzle 451.

[0017] The liquid delivery mechanism 430 comprises a liquid delivery tube 431 and a liquid delivery unit 432 for injecting reagent 12 from the reagent container 200 into the chamber 420 via the liquid delivery tube 431. The liquid delivery mechanism 430 is a mechanism that delivers reagents from the attached reagent container 200 to the chamber 420 via the liquid delivery tube 431, which is provided between the reagent container 200, which is mounted on the reagent container holder 60 (see Figures 13 to 15), and the chamber 420. The reagent container holder 60 is mounted on the reagent container 200, which contains reagent 12 containing both the first fluorescent dye and the second fluorescent dye. The liquid delivery mechanism 430 delivers the reagents from the reagent container 200 to the chamber 420. Cells are stained by the first compound constituting the first fluorescent dye and the second compound constituting the second fluorescent dye.

[0018] A suction tube 64, which forms one end of a liquid delivery tube 431, is inserted into the reagent container 200. The other end of the liquid delivery tube 431 is connected to the chamber 420. The tip of the suction tube 64 may be formed to be sharp so that it can penetrate the sealing film (also called a sealing member) of the reagent container 200 mounted in the reagent container holder 60.

[0019] The liquid delivery unit 432 of the liquid delivery mechanism 430 includes a pump 433 as a quantitative unit that generates negative pressure to draw reagent 12 from the reagent container 200 into the liquid delivery pipe 431 and positive pressure to supply 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 V1 and V2. For example, when the quantitative unit 433, which is composed of a syringe pump or a diaphragm pump, draws reagent from the reagent container, valve V1 is opened and valve V2 is closed. The quantitative unit 433 generates negative pressure, filling the flow path between valve V1, valve V2, and the quantitative unit 433 with reagent. When supplying the filled reagent to the chamber 420, valve V1 is closed, valve V2 is opened, and the quantitative unit 433 generates positive pressure. As a result, the reagent in the reagent container 200 is supplied to the chamber 420.

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

[0021] In the measurement unit 400, one or more reagent container holders 60 are provided. The reagent container holder 60 is fitted with a reagent container 200 that contains a reagent containing both a first fluorescent dye that emits fluorescence at a first wavelength when excited by light and a second fluorescent dye that emits fluorescence at a second wavelength when excited by light. In the example shown in Figure 2, a reagent containing both the first and second fluorescent dyes is contained in one reagent container 200.

[0022] The reagent container 200 is a container that holds reagents. The reagent container 200 has an opening into which a piercer (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 piercer 64 is inserted into the opening of the reagent container 200 mounted on the reagent container holder 60. The inserted piercer 64 is fixed in a predetermined position inside the reagent container 200. The piercer 64 inserted into the reagent container 200 remains fixed in the predetermined position as described above, for example, while the reagent container 200 is mounted on the reagent container holder 60. Furthermore, it remains fixed in the predetermined position as described above at least while measurements of multiple different samples are performed (i.e., while multiple different measurement samples are prepared).

[0023] The FCM detection unit 460 acquires first and second signals corresponding to the first and second wavelength fluorescence emitted from cells stained with first and second fluorescent dyes, respectively. The FCM detection unit 460 irradiates the sample being measured flowing through the flow cell with light. The FCM detection unit 460 may, for example, have multiple light sources corresponding to each wavelength, or it may be configured to irradiate with light of a single wavelength and detect fluorescence from multiple fluorescent dyes excited by light of that single wavelength. When light is irradiated onto the sample being measured, optical signals corresponding to the first fluorescent dye and the second fluorescent dye are detected, respectively. Each optical signal is A / D converted and digital data is acquired. The acquired digital data is analyzed by the analysis unit 300X (see Figure 1).

[0024] In the first embodiment, a liquid delivery pipe 431 is provided between the reagent container 200 and the chamber 420, and a liquid delivery unit 432 delivers the reagent in the reagent container 410 to the chamber 420 via the liquid delivery pipe 431. Therefore, in the first embodiment, the process of aspirating the reagent from the reagent container with a nozzle, moving the nozzle that aspirated the reagent to the location in the chamber, and discharging the reagent into the chamber (see, for example, Non-Patent Document 1) is unnecessary.

[0025] The analysis unit 300X (see Figure 1) performs at least one of the following: cell classification and counting, based on the first and second signals, respectively, which correspond to the first and second wavelengths of fluorescence emitted from cells stained with the first and second fluorescent dyes.

[0026] Figure 3 is a flowchart showing the procedure for preparing a measurement sample using the analysis system of the first embodiment. The measurement sample preparation process using the analysis system 4000 will be described with reference to Figures 1, 2, and 3. In the measurement sample preparation process using the analysis system 4000, first, the sample is dispensed into the chamber 420 in the measurement unit 400 (step S1). Next, the reagent is injected into the chamber 420 via the liquid delivery tube 431 connecting the reagent container 410 and the chamber 420 (step S2). Next, the sample and the reagent containing the first and second fluorescent dyes are mixed in the chamber 420 to prepare the measurement sample (step S3). Next, the measurement sample prepared in the chamber 420 is delivered to the FCM detection unit 460 and irradiated with light to acquire optical signals corresponding to the side scattered light and the first and second fluorescent dyes, respectively (step S4). Next, the data generated from the acquired optical signals is analyzed by the analysis unit 300X (step S5). Then, the analysis unit 300X provides the analysis results (step S6).

[0027] According to the first embodiment, the supply of the first and second fluorescent dyes to the chamber 420 for reacting with the sample can be done through the liquid delivery pipe 431. The liquid delivery pipe 431 is a dedicated channel for supplying only the reagent 12 containing the first and second fluorescent dyes to the chamber 420. Since the inside of the liquid delivery pipe 431 can always be kept filled with reagent 12, the processing speed can be increased by enabling rapid quantification by the quantification unit (pump) 433. Furthermore, since the liquid delivery pipe 431 is a dedicated channel for supplying reagent 12, there is no need to prevent contamination between different reagents, and washing is unnecessary. The elimination of washing also contributes to the improvement in processing speed.

[0028] The advantages of the first embodiment become even clearer, for example, when compared with Non-Patent Document 1. For example, in the flow cytometer described in Non-Patent Document 1, a 96-well plate is used as the reaction vessel. The 96-well plate is a consumable item and is removed from the flow cytometer and discarded after use. Therefore, the flow cytometer described in Non-Patent Document 1 is configured such that the reagent container and the reaction vessel are connected by a liquid delivery tube, and the process for aspirating and discharging the reagent cannot be omitted.

[0029] Furthermore, the flow cytometer measurement described in Non-Patent Document 1 uses a reagent (Non-Patent Document 2: AQUIOS Tetra-1 Panel) containing four types of fluorescent dyes corresponding to four markers (CD45, CD3, CD4, CD8) in cells in a blood sample. Each of these fluorescent dyes is attached to an antibody corresponding to the above marker. In other words, the reagent in Non-Patent Document 2 stains cells in a blood sample by an antibody-antigen reaction. With such a reagent, the reaction takes time, so it takes time to measure one sample. Specifically, Non-Patent Document 1 states that it takes about 20 minutes to measure one sample, including the preparation of the measurement sample. Because the measurement time per sample (especially the time required for the antibody-antigen reaction) is long in the technique of Non-Patent Document 1, the preparation of measurement samples for multiple samples is performed in parallel using multiple wells of a consumable 96-well plate to shorten the total processing time per sample. In other words, the processing capacity (throughput) per unit time is improved. In contrast, in the first embodiment, the reagent 12 used is a reagent containing first and second fluorescent dyes, the compound itself staining the cytoplasm, nucleic acids, and DNA of cells. Such fluorescent dyes react faster for staining than antibody reagents, and for example, the time required to prepare a measurement sample for one sample is less than one minute. Therefore, according to the first embodiment, it is possible to provide an analytical instrument that can realize measurements using multiple fluorescent labels with high processing capacity. (Second Embodiment)

[0030] The analysis system 4000 of the second embodiment is a multi-parameter automated hematology analyzer that performs at least one of counting and analyzing cells in a blood sample. Figure 4 is a block diagram showing the configuration of the measurement unit 400 of the analysis system 4000 of the second embodiment (see Figure 1). The measurement unit 400 comprises a sample preparation unit 440, a device mechanism unit 455, a sample aspiration mechanism 450, an FCM detection unit 460, an RBC / PLT detection unit 461, an HGB detection unit 462, and a measurement unit control unit 480. The RBC / PLT detection unit 461 is an electrical resistance detection unit that counts red blood cells (RBCs) and platelets (PLTs) by introducing a measurement sample prepared with blood and a diluent into an aperture and detecting the change in electrical resistance that occurs when cells pass through the aperture. The HGB detection unit 462 measures the hemoglobin concentration in the blood using the SLS hemoglobin method. The HGB detection unit 462 measures the hemoglobin concentration in the blood by irradiating a sample prepared from blood and an 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".

[0031] The sample aspiration mechanism 450 aspirates a sample from the sample container and discharges the aspirated sample into the chamber of the sample preparation unit 440. The sample preparation unit 440 includes a chamber for mixing the sample and reagents, and a reagent container holder 60 in which the reagent containers are installed. The sample preparation unit 440 delivers reagents from the reagent containers installed in the reagent container holder 60 to the chamber via a liquid delivery tube, which will be described later. The sample and reagents are mixed in the chamber to prepare the measurement sample.

[0032] The device mechanism section 455 includes motors and actuators that move various parts of the measurement unit 400. The device mechanism section 455 includes, for example, a mechanism for moving the blood collection tube T (see Figure 5), which will be described later, in the vertical direction.

[0033] The measurement unit control unit 480 includes an analog processing unit 481 that processes the analog signal output from the FCM detection unit 460, an A / D conversion unit 481a that converts the analog signal output from the analog processing unit 481 into a digital signal, an analog processing unit 482 that processes the analog signal output from the RBC / PLT detection unit 461, an A / D conversion unit 482a that converts the analog signal output from the analog processing unit 482 into a digital signal, an analog processing unit 483 that processes the analog signal output from the HGB detection unit 462, an A / D conversion unit 483a that converts the analog signal output from the analog processing unit 483 into a digital signal, and an IF unit (interface unit) 484 that is electrically connected to each of the A / D conversion units 481a, 482a, and 483a. Furthermore, the measurement unit control unit 480 includes an interface (IF) unit 488 electrically connected to the sample preparation unit 440, the device mechanism unit 455, the sample aspiration mechanism 450, the FCM detection unit 460, the RBC / PLT detection unit 461, and the HGB detection unit 462, a bus 485 electrically connected to each of the IF units 484 and 488, and an IF unit 489 electrically connected to the bus 485 and the analysis unit 302X.

[0034] Figure 5 shows a fluid circuit including a sample aspiration mechanism 450, a sample preparation unit 440, and detection units 460-462. The sample preparation unit 440 shown in Figure 4 comprises a first sample preparation unit 440A for preparing a first measurement sample for optical measurement by the FCM detection unit 460, and a second sample preparation unit 440B for preparing 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 (see Figure 5).

[0035] The first sample preparation unit 440A has a first chamber 420. The first chamber 420 is connected to reagent containers R1 and R2. Reagent container R1 contains a hemolytic agent that shrinks red blood cells. Reagent container 200 contains a leukocyte staining reagent containing a fluorescent dye. Reagent container R2 contains a diluent. Reagent container R2 will be described later.

[0036] The leukocyte staining reagent contained in reagent container 200 contains a first fluorescent dye and a second fluorescent dye. The first and second fluorescent dyes will be described later.

[0037] The second sample preparation unit 400B has a second chamber 55. The second chamber 55 is connected to reagent container R2 and reagent container R3. Reagent container R2 is provided in common with the first sample preparation unit 440A. Reagent container R3 contains an SLS hemolytic agent for lysing red blood cells and preparing a sample for measurement by the SLS hemoglobin method.

[0038] A reagent container 200 containing leukocyte staining reagents is held in a reagent container holder 60. The reagent container holder 60 is equipped with a suction tube 64 for aspirating nucleic acid staining reagents from the reagent container 200, and a suction tube lifting mechanism 65 for raising and lowering the suction tube 64. The tip of the suction tube 64 can penetrate (puncture) the sealing material of the reagent container 200. A cover 63 is connected to the suction tube lifting mechanism 65. When the suction tube lifting mechanism 65 is lowered and the suction tube 64 is penetrating (puncturing) the sealing material of the reagent container 200, the cover 63 also lowers and covers the reagent container 200. When the suction tube lifting mechanism 65 is raised, the cover 63 also rises, and the reagent container 200 becomes removable from the outside.

[0039] 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. One end of the liquid delivery tube 431 is connected to the suction tube 64, and the other end is connected to the first chamber 420. The metering block 432 comprises a metering unit 30 and solenoid valves V1 and V2. A syringe pump is used as the metering unit 30. Alternatively, a diaphragm pump can also be used instead of a syringe pump. The solenoid valves V1 and V2 open and close the flow path. When delivering the leukocyte staining reagent from the reagent container 200 to the chamber 420, the metering unit 30 applies negative pressure to the liquid delivery tube 431 with solenoid valve V1 open and solenoid valve V2 closed. As a result, leukocyte staining reagent is drawn from the tip of the suction tube 64 into the liquid delivery tube 431, and a fixed amount of leukocyte staining reagent is filled into the flow path between the electromagnetic valves V1 and V2 and the quantitative unit 30. Next, with electromagnetic valve V1 closed and electromagnetic valve V2 open, the quantitative unit 30 applies positive pressure to the liquid delivery tube 431. As a result, the fixed amount of leukocyte staining reagent filled into the flow path between electromagnetic valves V1 and V2 and the quantitative unit 30 is pushed out, and the leukocyte staining reagent is supplied to the chamber 420 through the liquid delivery tube 431.

[0040] A quantitative section 22 and electromagnetic valves V3 and V4 are provided in the flow path between the reagent container R1 containing the hemolytic agent 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 and electromagnetic valves V3 and V4 quantitatively deliver the hemolytic agent 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.

[0041] A quantitative section 33 and electromagnetic valves V5 and V6 are provided in the flow path between the reagent container R2 containing the diluent and the first chamber 420. A syringe pump is used as the quantitative section 33. Alternatively, a diaphragm pump can also be used. Electromagnetic valves V5 and V6 open and close the flow path. The quantitative section 33 and electromagnetic valves V5 and V6 quantitatively supply the diluent from the reagent container R2 to the first chamber 420.

[0042] A waste liquid chamber 36 for containing unwanted solutions is connected to the first chamber 420. An electromagnetic valve V7 for opening and closing the flow path is provided between the first chamber 420 and the waste liquid chamber 36.

[0043] The first chamber 420 is connected to a pump 56A that supplies air into the first chamber 420 in order to agitate the liquid inside the first chamber 420.

[0044] A quantitative section 38 and electromagnetic valves V8 and V9 are provided in the flow path between the reagent container R2 containing the diluent and the second chamber 55. A syringe pump is used as the quantitative section 38. A diaphragm pump, for example, can also be used instead of the syringe pump. Electromagnetic valves V8 and V9 open and close the flow path. The quantitative section 38 and electromagnetic valves V8 and V9 quantitatively supply the diluent from the reagent container R2 to the second chamber 55. A waste liquid chamber 41 for containing the unused solution is connected to the second chamber 55. Between the second chamber 55 and the waste liquid chamber 41, an electromagnetic valve V10 is provided to switch the flow path between a flow path from the second chamber 55 to the waste liquid chamber 41 and a flow path from the second chamber 55 to the RBC / PLT detection unit 461 and the HGB detection unit 462. Electromagnetic valve V13 will be described later.

[0045] A quantitative section 39 and electromagnetic valves V11 and V12 are provided in the flow path between the reagent container R3 containing the SLS hemolytic agent and the second chamber 55. A syringe pump is used as the quantitative section 39. Alternatively, a diaphragm pump can also be used. Electromagnetic valves V11 and V12 open and close the flow path. The quantitative section 39 and electromagnetic valves V11 and V12 quantitatively deliver the SLS hemolytic agent from the reagent container R3 to the second chamber 55.

[0046] The second chamber 55 is connected to a pump 56B that supplies air into the second chamber 55 in order to agitate the liquid inside the second chamber 55.

[0047] The sample aspiration mechanism 450 has a suction tube 20 and a quantitative unit 21. The tip of the suction tube 20 is sharply formed. As the sample aspiration mechanism 450 lowers the blood collection tube 100, the suction tube 20 punctures the lid 100a that seals the blood collection tube 100 and is inserted inside. With the suction tube 20 inserted inside the blood collection tube 100, the quantitative unit 21 generates negative pressure, causing the blood sample contained in the blood collection tube 100 to be aspirated into the suction tube 20. The sample aspiration mechanism 450 moves the suction tube 20 upward to remove it from the blood collection tube 100 and moves the suction tube 20 horizontally above the first chamber 420. The sample aspiration mechanism 450 lowers the suction tube 20 relative to the first chamber 420, and the quantitative unit 21 generates positive pressure, causing the aspirated blood sample to be discharged into the first chamber 420. The sample aspiration mechanism 450 moves the suction tube 20 upward and horizontally above the second chamber 55, and in the same manner as with the first chamber 420, it discharges the blood sample into the second chamber 55.

[0048] 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 leukocyte staining reagent contained in the reagent container 200 and the hemolytic agent contained in the reagent container R1 to prepare the measurement sample. More specifically, a measurement sample is prepared in which red blood cells are lysed by the hemolytic agent and white blood cells are stained with a first fluorescent dye and a second fluorescent dye. Such a measurement sample is prepared, for example, as follows: First, the hemolytic agent 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 mixture is stirred. This causes the red blood cells to be lysed by the hemolytic agent. Next, the leukocyte staining reagent is supplied to the first chamber 420. Air is supplied to the first chamber 420 and the mixture is stirred. The reaction proceeds in the first chamber 420, and staining with the fluorescent dyes occurs. The reaction time is, for example, less than 1 minute, more preferably less than 50 seconds, and more preferably less than 45 seconds. This yields a measurement sample in which leukocytes contained in the blood are stained with a first fluorescent dye and a second fluorescent dye. The FCM detection unit 460 is connected to a pump (not shown), and the measurement sample in the first chamber 420 is supplied to the FCM detection unit 460 by the drive of the pump. The FCM detection unit 460 acquires multiple optical signals from the leukocytes, including fluorescence corresponding to the first fluorescent dye and fluorescence corresponding to the second fluorescent dye.

[0049] The second chamber 55 is connected to the RBC / PLT detection unit 461 and the HGB detection unit 462. The electromagnetic valve V13 switches between supplying the measurement sample from the second chamber 55 to the RBC / PLT detection unit 46 and supplying it to the HGB detection unit 462. The RBC / PLT detection unit 461 and the HGB detection unit 462 are connected to a pump (not shown), and the 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 for preparing both the measurement sample for RBC / PLT detection and the measurement sample for HGB detection. An example of the procedure for preparing such a sample will be described. First, a diluent is supplied from the reagent container R2 to the second chamber 55. Next, a blood sample is discharged into the second chamber 55. This yields a measurement sample of diluted blood. This becomes the measurement sample for RBC / PLT detection. A portion of the sample from the second chamber 55 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 causes hemolysis of red blood cells and conversion of hemoglobin into SLS hemoglobin, resulting in a sample in which the red blood cells are lysed and the hemoglobin is converted into SLS 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, but they may be prepared in separate chambers.

[0050] An analytical system 4000 having such a configuration (see Figure 1) may be configured to measure 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). Furthermore, in addition to the CBC item, the analytical system 4000 may be configured to measure the DIFF item, which classifies white blood cells into multiple subpopulations.

[0051] <Modified sample preparation section> Figure 6 is a schematic diagram showing another example of the first sample preparation unit 440A. In Figure 6, elements similar to those in Figure 5 are omitted from the illustration. In the example shown in Figure 5, the first sample preparation unit 440A is described in which reagents containing a first fluorescent dye and a second fluorescent dye are contained in one reagent container 200, and the reagents in the one reagent container 200 are delivered into the chamber 420 by one liquid delivery mechanism 430. In the modified first sample preparation unit 440A shown in Figure 6, the reagents containing the first fluorescent dye and the reagents containing the second fluorescent dye are contained in separate reagent containers 200A and 200B, respectively, and the reagents in each reagent container 200A and 200B are delivered into the chamber 420 by separate first and second liquid delivery mechanisms 430a and 430b, respectively.

[0052] In the first sample preparation unit 440A shown in Figure 6, one or more chambers 420 are provided for mixing two reagents, each containing either a first or second fluorescent dye mounted in a reagent container holder, with the sample to prepare a measurement sample in which cells have been stained with the first and second fluorescent dyes. The first reagent container 200A, containing the reagent with the first fluorescent dye, is mounted in the first holder portion of the reagent container holder 442, and the second reagent container 200B, containing the reagent with the second fluorescent dye, is mounted in the second holder portion of the reagent container holder 442.

[0053] The first liquid delivery mechanism 430a is provided for delivering the reagent from the first reagent container 200A to the chamber 54. The configuration of the first liquid delivery mechanism 430a is as described with reference to Figure 3. The second liquid delivery mechanism 430b is provided for delivering the reagent from the second reagent container 200B to the chamber 54. The configuration of the second liquid delivery mechanism 430b is the same as that of the first liquid delivery mechanism. 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. Although the example in Figure 6 shows the two liquid delivery pipes merging, the two liquid delivery pipes may also be connected to the chamber 420 individually.

[0054] In the configuration shown in Figure 6, reagents containing the first fluorescent dye and reagents containing the second fluorescent dye are housed in separate reagent containers 200A and 200B, respectively. These are supplied to the chamber 420 by two different liquid delivery mechanisms and mixed with the sample. This configuration also allows for the construction of a system that stains blood cell components in a sample with two fluorescent dyes.

[0055] Figure 7 is a diagram illustrating an example of the optical system of the FCM detection unit 460. As shown in Figure 7, the FCM detection unit 460 comprises a first light source 4111a and a second light source 4111b with different wavelengths, a flow cell 4113, dichroic mirrors 4118a, 4118b, and 4118c, side-scattered light receiving elements 4121a and 4121b, a forward-scattered light receiving element 4116, and side-fluorescence receiving elements 4122a and 4122b. In the example in Figure 7, the light source consists of a first light source 4111a for exciting a first fluorescent dye and a second light source 4111b for exciting a second fluorescent dye.

[0056] The FCM detection unit 460 acquires a first signal, which consists of multiple signals corresponding to the side-scattered light and side-fluorescence from the first light source 4111a, and a second signal, which consists of multiple signals corresponding to the forward-scattered light, side-scattered light, and side-fluorescence from the second light source 4111b. As described above, the FCM detection unit 460 is connected to the first chamber 420 of the first sample preparation unit 440A (see Figure 5). The sample prepared in the first chamber 420 is flowed into the flow cell (sheath flow cell) 4113 of the FCM detection unit 460. In the example in Figure 7, the sample is flowed perpendicular to the plane of the paper. While the sample is flowing into the flow cell 4113, the first light source 4111a and the second light source 4111b irradiate the flow cell 4113 with light. More specifically, light emitted from the first light source 4111a is reflected by the dichroic mirror 4118a and irradiates the flow cell 4113. Light emitted from the second light source 4111b passes through the dichroic mirror 4118a and irradiates the flow cell 4113.

[0057] The first light source 4111a and the second light source 4111b of the FCM detection unit 460 are not particularly limited, and light sources with wavelengths suitable for exciting fluorescent dyes are selected. The first light source 4111a can irradiate, for example, light of a first wavelength, and the second light source 4111b can irradiate light of a second wavelength, which is longer than the first wavelength. For example, the first wavelength is 315 to 490 nm, preferably 400 to 450 nm, and more preferably 400 to 410 nm. The second wavelength is 610 to 750 nm, preferably 620 to 700 nm, and more preferably 633 to 643 nm. Examples of such first and second light sources 4111a and 4111b include semiconductor laser light sources, argon laser light sources, gas laser light sources such as helium-neon lasers, and mercury arc lamps. Semiconductor laser light sources are particularly preferred because they are less expensive than gas laser light sources. As described above, by selecting first light source 4111a and second light source 4111b with divergent wavelength bands for the irradiated light, it becomes easier to select dyes with minimal overlap in the wavelength bands of fluorescence emitted from each light source. If there is a large overlap in the wavelength bands of fluorescence emitted from the fluorescent dyes, it is necessary to address the leakage described later, but by selecting fluorescent dyes with minimal (or no) overlap in wavelength bands, it becomes unnecessary to address the leakage.

[0058] The forward-scattered light receiving element 4116 is positioned to receive forward-scattered light emitted from the cells based on light irradiated from the second light source 4111b. The forward-scattered light receiving element 4116 is configured to receive only forward-scattered light corresponding to the light irradiated from the second light source 4111b, for example. The forward-scattered light receiving element 4116 is, for example, a photodiode. In this example, the receiving element 4116 receives forward-scattered light of a second wavelength irradiated from the second light source 4111b, but alternatively, it may receive forward-scattered light of a first wavelength irradiated from the first light source 4111a.

[0059] The side-scattered light receiving element 4121a is positioned to receive side-scattered light corresponding to the light from the first light source 4111a. The side-scattered light corresponding to the light irradiated from the first light source 4111a (first side-scattered light) is reflected by the dichroic mirror 4118b and received by the side-scattered light receiving element 4121a. The side-scattered light receiving element 4121a is, for example, a photodiode.

[0060] The side-scatter light receiving element 4121b is positioned to receive side-scattered light corresponding to the light from the second light source 4111b. The side-scattered light corresponding to the light irradiated from the second light source 4111b (second side-scattered light) is reflected by the dichroic mirror 4118c and received by the side-scatter light receiving element 4121b. The side-scatter light receiving element 4121b is, for example, a photodiode.

[0061] The lateral fluorescence photodetector 4122a is positioned to receive fluorescence corresponding to light from the first light source 4111a, that is, light generated when the first fluorescent dye is excited by light of a first wavelength from the first light source 4111a. The lateral fluorescence (first lateral fluorescence) corresponding to the light irradiated from the first light source 4111a is transmitted through the dichroic mirror 4118b and received by the lateral fluorescence photodetector 4122a. The lateral fluorescence photodetector 4122a is, for example, an avalanche photodiode.

[0062] The lateral fluorescence photodetector 4122b is positioned to receive fluorescence corresponding to light from the second light source 4111b, that is, light generated when the second fluorescent dye is excited by light of the second wavelength from the second light source 4111b. The lateral fluorescence (second lateral fluorescence) corresponding to the light irradiated from the second light source 4111b is transmitted through the dichroic mirror 4118c and received by the lateral fluorescence photodetector 4122b. The lateral fluorescence photodetector 4122b is, for example, an avalanche photodiode. Alternatively, photomultiplier tubes may be used as the forward scattered light photodetector 4116, the lateral scattered light photodetectors 4121a and 4121b, and the lateral fluorescence photodetectors 4122a and 4122b.

[0063] The forward-scattering light receiving element 4116, the side-scattering light receiving elements 4121a and 4121b, and the side-fluorescence light receiving elements 4122a and 4122b each output a waveform electrical signal (also called a received signal) containing pulses corresponding to the received light intensity. Typically, each pulse of the received signal corresponds to a single cell (e.g., a white blood cell). The output received signals are each input to the analog processing unit 481. The analog processing unit 481 performs noise reduction, smoothing, and other processing on the analog signal input from the FCM detection unit 460, and outputs the processed analog signal to the A / D conversion unit 481a. The other analog processing units 482 and 483 (see Figure 4) are configured similarly to the analog processing unit 481.

[0064] The A / D conversion unit 481a converts the analog signal output from the analog processing unit 481 into a digital signal. The A / D conversion unit 481a converts the analog signal from the start to the end of the measurement of the sample into a digital signal. If multiple types of analog signals (for example, analog signals corresponding to forward scattered light intensity, first side scattered light intensity, first fluorescence intensity, second side scattered light intensity, and second fluorescence intensity, respectively) are generated by measurement on a certain measurement channel, the A / D conversion unit 481a converts each analog signal from the start to the end of the measurement into a digital signal. For example, five types of analog signals (i.e., forward scattered light signal, first side scattered light signal, first fluorescence signal, second side scattered light signal, and second fluorescence signal) are input to the A / D conversion unit 481a. The A / D conversion unit 481a converts each of the input analog signals into a digital signal.

[0065] The A / D converter 481a samples the analog signal at a predetermined sampling rate (e.g., 1024 points of sampling at 10 nanosecond intervals, 128 points of sampling at 80 nanosecond intervals, or 64 points of sampling at 160 nanosecond intervals). The A / D converter 481a performs sampling on five types of analog signals corresponding to individual cells flowing through the flow cell 4113, generating waveform data for the forward scattered light signal, the first side scattered light signal, the first fluorescence signal, the second side scattered light signal, and the second fluorescence signal for each formed element. The A / D converter 481a assigns an index to each of the generated waveform data. The generated waveform data becomes digital signals, each having a signal corresponding to N cells contained in a single sample. This generates five digital signals corresponding to the five types of analog signals obtained from the N cells (forward scattered light signal, first and second side scattered light signals, and first and second fluorescence signals) (see Figure 39).

[0066] In addition to generating waveform data from the analog signal, the A / D conversion unit 481a calculates characteristic parameters representing the morphological characteristics of individual cells from the pulses of the analog signal, such as peak value, pulse width, and pulse area.

[0067] Figure 8 is a diagram illustrating another example of the optical system of the FCM detection unit. As shown in Figure 8, the light source of the FCM detection unit 460x may consist of a single light source 4111 that excites a first fluorescent dye and a second fluorescent dye. In this case, the FCM detection unit 460x acquires the first signal, which consists of a plurality of signals corresponding to the forward scattered light, side scattered light, and side fluorescence from the light source 4111, respectively, and a second signal corresponding to the side fluorescence from the light source 4111.

[0068] In this case, light of a predetermined wavelength is irradiated onto the flow cell 4113 from a single light source 4111. The cells contained in the sample flowing through the flow cell 4113 are stained with multiple types of fluorescent dyes, each with different wavelengths of fluorescence emitted from the excited fluorescent dyes, similar to the example described above. In this example, the multiple types of fluorescent dyes used are those that are excited at different wavelengths when irradiated with light of a predetermined wavelength (e.g., 488 nm). Multiple types of fluorescent dyes with different excitation wavelengths mean, for example, that each fluorescent dye is excited and emits different colors of fluorescence when irradiated with light from a single light source.

[0069] In the optical system of the FCM detection unit 460x shown in Figure 8, cells contained in the sample to be measured are stained with multiple fluorescent dyes, and light is shone onto the cells from a single light source 4111 to detect the fluorescence emitted from each of the multiple fluorescent dyes. In this case, since the light shone from a single light source 4111 has a single wavelength, when light is shone onto cells from a single light source 4111, one side-scattered light corresponding to the single wavelength of that light is emitted. Therefore, in the optical system of the FCM detection unit 460x shown in Figure 8, one dichroic mirror 4118 is provided, and the other configurations are generally the same as the optical system shown in Figure 7.

[0070] Each fluorescence emitted by a fluorescent dye has its own unique spectrum. Therefore, when separated into a single wavelength range by an optical system (for example, the dichroic mirror 4118 and the lateral fluorescence detectors 4122a and 4122b in Figure 7), fluorescence emitted from other fluorescent dyes besides the one bound to the target molecule in a single cell may leak into the lateral fluorescence detectors 4122a and 4122b. For example, as shown in the example in Figure 9, when multiple fluorescent dyes are excited by irradiating them with light of a single wavelength, the overlap of the wavelength bands of the fluorescence emitted by each excited fluorescent dye can become large. As shown in Figure 9, which illustrates an example of overlapping fluorescence wavelength bands, when multiple fluorescent dyes with overlapping fluorescence wavelength bands are selected, measures are taken to address, for example, fluorescence leakage.

[0071] Figure 9 shows an example of fluorescence leakage between two different fluorescent dyes. The example in Figure 9 shows fluorescence leakage between two dyes, fluorescent dye F1 and fluorescent dye F2. In the graph shown in Figure 9, the vertical axis represents fluorescence intensity, and the horizontal axis represents wavelength. Light emitted from fluorescent dye F1 is detected by one side-facing fluorescent photodetector in the wavelength band (a) shown in Figure 9. Light emitted from fluorescent dye F2 is detected by the other side-facing fluorescent photodetector in the wavelength band (b) shown in Figure 9. The wavelength band (a) detected by the side-facing fluorescent photodetector corresponding to fluorescent dye F1 includes the wavelength band of fluorescence emitted by fluorescent dye F2. Similarly, the wavelength band (b) detected by the other side-facing fluorescent photodetector corresponding to fluorescent dye F2 includes the wavelength band of fluorescence emitted by fluorescent dye F1. Therefore, by correcting the fluorescence in wavelength band (a) (corresponding to fluorescent dye F1) by excluding the portion corresponding to fluorescent dye F2, the fluorescence measurement value due to fluorescent dye F1 can be obtained more accurately. Similarly, by correcting the fluorescence in wavelength band (b) (corresponding to fluorescent dye F2) by excluding the portion corresponding to fluorescent dye F1, the fluorescence measurement value due to fluorescent dye F2 can be obtained more accurately.

[0072] To correct the fluorescence leakage shown in Figure 9, positive controls for fluorescent dye F1 and fluorescent dye F2 are used. For the positive control for fluorescent dye F1, for example, a control reagent containing particles to which fluorescent dye F1 has been added is used. For the positive control for fluorescent dye F2, for example, a control reagent containing particles to which fluorescent dye F2 has been added is used.

[0073] The measurement of the positive control of fluorescent dye F1 using the analytical system with the optical system illustrated in Figure 8 is performed as follows before the measurement of the sample: Light (single wavelength light) from the light source 4111 is irradiated onto the particles in the positive control flowing through the flow cell 4113. The fluorescence generated by the irradiation of the particles with light is detected by the respective side fluorescence detectors 4122a and 4122b. For example, the side fluorescence detector 4122a corresponds to the detection of fluorescence emitted from the fluorescent dye F1, and the side fluorescence detector 4122b corresponds to the detection of fluorescence generated from the fluorescent dye F2. In this case, the fluorescence emitted from the particles in the positive control of fluorescent dye F1 that is detected by the side fluorescence detector 4122b is considered leakage. For example, if the fluorescence intensity detected by the side fluorescent photodetector 4122a was "100" and the fluorescence intensity detected by the side fluorescent photodetector 4122b was "5" from the fluorescence emitted from a particle in the positive control of fluorescent dye F1, then 5% of the fluorescence from fluorescent dye F1 leaked into 4122b. For example, if the fluorescence intensity detected by the side fluorescent photodetector 4122a was "10" and the fluorescence intensity detected by the side fluorescent photodetector 4122b was "100" from the fluorescence emitted from a particle in the positive control of fluorescent dye F2, then 10% of the fluorescence from fluorescent dye F2 leaked into the side fluorescent photodetector 4122a. These results are summarized in Table 1 below. This Table 1 may hereafter be referred to as the "compensation matrix".

[0074] [Table 1]

[0075] Next, we will explain an example of correcting the measured values ​​obtained from a sample containing cells stained with the respective fluorescent dyes F1 and F2 using the compensation matrix described above. An example of measurement value correction is as follows. • Corrected fluorescence intensity of fluorescent dye F1 measured by the lateral fluorescence detector 4122a = fluorescence intensity measured by the lateral fluorescence detector 4122a - (5% of the fluorescence intensity when the positive control of fluorescent dye F2 was measured by the lateral fluorescence detector 4122b) • Corrected fluorescence intensity of fluorescent dye F2 measured by the lateral fluorescence detector 4122b = fluorescence intensity measured by the lateral fluorescence detector 4122b - (10% of the fluorescence intensity when the positive control of fluorescent dye F1 was measured by the lateral fluorescence detector 4122a)

[0076] In the above example, the fluorescence intensity when the fluorescence of the positive control of fluorescent dye F1 is detected by the side fluorescence detector 4122a is "100", and the fluorescence intensity when the fluorescence of the positive control of fluorescent dye F2 is detected by the side fluorescence detector 4122b is "100". Therefore, the corrected fluorescence intensities are as follows. Corrected fluorescence intensity of fluorescent dye F1 measured by the lateral fluorescence detector 4122a = fluorescence intensity measured by the lateral fluorescence detector 4122a 100 - 10 • Corrected fluorescence intensity of fluorescent dye F2 measured by the lateral fluorescence detector 4122b = Fluorescence intensity measured by the lateral fluorescence detector 4122b 100 - 5

[0077] In the optical systems illustrated in Figures 7 and 8, the fluorescence generated when the first and second fluorescent dyes are excited is detected as fluorescence occurring laterally with respect to the direction of light propagation (approximately 90° with respect to the direction of propagation in the examples of Figures 7 and 8) (lateral fluorescence). However, the system is not limited to this example. For example, fluorescence occurring in front of the direction of light propagation may be detected, or fluorescence occurring at a certain angle that is not perpendicular to the direction of light propagation, for example, 20 to 90°, may be detected.

[0078] Figure 10 is a block diagram showing an example configuration of the analysis unit 300X. As shown in Figure 10, the analysis unit 300X is electrically connected to the measurement unit 400 via an interface unit 3006. The interface unit 3006 is, for example, a USB interface. The analysis unit 300X comprises a processor 3001, main memory 3017, bus 3003, storage unit 3004, the interface unit 3006, display unit 3015, and operation unit 3016. The analysis unit 300X is configured by, for example, a personal computer (see analysis unit 300X in Figure 1) and controls the measurement unit 400 of the analysis system 4000 by executing a program stored in the storage unit 3004. The analysis unit 300X executes, for example, an analysis program and analyzes the data acquired by the measurement unit 400. The analysis unit 300X displays the analysis results on the display unit 3015. The analysis unit 300X may classify cells by inputting waveform data corresponding to at least one, preferably more, of the forward scattered light intensity, first lateral scattered light intensity, first fluorescence intensity, second lateral scattered light intensity, and second fluorescence intensity obtained from individual cells by the measurement unit 400 via the FCM detection unit 460 into a trained AI algorithm. Alternatively, the analysis unit 300X may classify cells based on characteristic parameters (peak value, pulse width, pulse area) of individual cells based on the forward scattered light intensity, first lateral scattered light intensity, first fluorescence intensity, second lateral scattered light intensity, and second fluorescence intensity. For example, as a method for classifying particles into multiple types using multiple characteristic parameters, a method can be employed in which particles are plotted in a multidimensional coordinate space with multiple parameters as axes, at least some particles are classified into multiple groups corresponding to multiple types, the degree of belonging of each particle to each group is determined based on the distance between the centroid position of each group and the particle, and the particles are reclassified based on the degree of belonging to classify multiple particles into multiple types. Such classification methods are 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 on some cells in a single sample, while classification based on characteristic parameters may be performed on other cells.

[0079] The processor 3001 is a CPU (Central Processing Unit) and executes programs deployed from the storage unit 3004 to the main memory 3017. The storage unit 3004 is, for example, a hard disk or an SSD (Solid State Drive). The storage unit 3004 stores, for example, a program for controlling the measurement unit 400 and a program for analyzing data acquired by the measurement unit 400. The display unit 3015 is equipped with a computer screen. The display unit 3015 is electrically connected to the processor 3001 via the interface unit 3006 and the bus 3003. The display unit 3015 displays, for example, the analysis results of data acquired by the measurement unit 400.

[0080] The control unit 3016 is equipped with a pointing device including a keyboard, mouse, or touch panel. Users such as physicians and laboratory technicians can input measurement orders into the analysis system 4000 and input measurement instructions according to the measurement orders by operating the control unit 3016. The control unit 3016 can also receive instructions from the user to display test results. Users can operate the control unit 3016 to view various information related to the test results, such as graphs, charts, and flag information assigned to samples. The measurement unit 400 described above is electrically connected to the analysis unit 300X via the interface unit 3006.

[0081] Figure 11 is a perspective view showing another configuration example of the analysis system 4000. In the configuration examples shown in Figures 1 to 10, the analysis system 4000 is illustrated in which the measurement unit 400 and the analysis unit 300X are provided as separate components. However, as shown in Figure 11, the analysis system 4001 may have the analysis unit 300X provided within the measurement unit 400. This configuration makes the entire analysis system 4001 more compact.

[0082] Figure 12 is a block diagram showing the configuration of another example of the measurement unit. In the examples in Figures 2, 5, and 6, there is one measurement system (also called a measurement channel) that can be measured by the FCM detection unit 460. In the configuration example in Figure 12, there is an example in which five measurement channels are provided.

[0083] The first sample preparation unit 440A is equipped with five chambers 54a to 54e. Chambers 54a to 54e are used for the DIFF, RET, WPC, PLT-F, and WNR measurement channels, respectively. Each chamber 54a to 54e is equipped with a hemolytic agent container containing a hemolytic agent, which is the reagent corresponding to the measurement channel, and a staining solution container containing a staining solution, which can be supplied to each chamber via a flow path. One measurement channel is composed of one chamber and the hemolytic agent container and staining solution container provided to supply liquid to the chamber. For example, the DIFF measurement channel is composed of a hemolytic agent container containing a DIFF hemolytic agent, which is the reagent for DIFF measurement, a staining solution container containing a DIFF staining solution, and a DIFF chamber 54a connected to these via a flow path. Other measurement channels are configured similarly. Note that this example illustrates a configuration in which one measurement channel is configured to supply both one type of hemolytic agent and one type of staining solution to the chamber, but the configuration is not limited to this. For example, multiple measurement channels may be configured to supply both one type of hemolytic agent and one type of staining solution to multiple different chambers. In other words, one reagent (one type of hemolytic agent and one type of staining solution) may be shared by multiple measurement channels, for example, two of the five measurement channels may be DIFF measurement channels. Furthermore, there may be only one measurement channel, or a configuration may be provided in which, for example, two DIFF measurement channels are provided.

[0084] The sample aspiration mechanism 450 (see Figures 2, 4, and 5) aspirates a blood sample from the sample container T containing the blood sample, and moves it to an upper position in the chamber of the measurement channel corresponding to the order among the chambers 54a to 54e by horizontal and vertical movement by the device mechanism unit 455 (see Figure 4), and discharges the aspirated blood sample into the chamber. The sample preparation unit 440 supplies the corresponding hemolytic agent and staining solution to the chamber from which the blood sample was discharged, and prepares the measurement sample by mixing the blood sample, hemolytic agent and staining solution in the chamber. The prepared measurement sample is supplied from the chamber to the FCM detection unit 460 via the flow path, and cell measurement is performed by flow cytometry.

[0085] The measurement channels mentioned above (DIFF, RET, WPC, PLT-F, WNR) correspond to the measurement items included in the measurement order. For example, DIFF corresponds to measurement items related to the classification of white blood cells. RET corresponds to measurement items related to reticulocytes. WPC corresponds to measurement items related to the measurement of abnormal white blood cells. PLT-F corresponds to measurement items related to the optical measurement of platelets. WNR corresponds to measurement items related to white blood cells and nucleated red blood cells. The measurement channels mentioned above (DIFF, RET, WPC, PLT-F, WNR) are measured by the FCM detection unit 460.

[0086] Figure 13 shows the measurement unit 40 with its cover 442 open. Figure 14 is a perspective view showing the reagent container holder 60 of the measurement unit 400, and Figure 15 is a front view showing the reagent container holder 60 shown in Figure 14. The measurement unit 400 of the analysis system 4000 (see Figure 1) is provided with an openable and closable front cover 442 on its front side (see Figure 13). The reagent container holder 60 is located on the upper front of the measurement unit 400, and as shown in Figure 13, opening the cover 442 exposes the reagent container holder 60, allowing user access. In other words, the user can place the reagent container 200 into the reagent container holder 60 and remove the reagent container 200 from the reagent container holder 60.

[0087] As shown in Figures 14 and 15, the reagent container holder 60 includes five holder sections 60a, 60b, 60c, 60d, and 60e, and is configured to hold a total of five (five types) reagent containers 200 (or 300). The reagent containers 200 (or 300) held in the reagent container holder 60 each contain different types of reagents (staining solutions) for measuring multiple measurement items by the FCM detection unit 460. The color of the reagent containers is, for example, black. Depending on the type of reagent, large (approximately 100 mL) reagent containers 200 (see Figure 22) and small (approximately 20 mL) reagent containers 300 (see Figure 25) are used. The capacity of the reagent containers is not limited to the above, and may be, for example, less than 20 ml, less than 100 ml, or more than 100 ml. For example, they may be approximately 10 ml, approximately 40 ml, approximately 80 ml, or approximately 300 ml. Each holder section 60a to 60e is configured to hold either a reagent container 200 or 300, for example. Therefore, the five holder sections 60a to 60e each have a similar configuration, and for example, a large reagent container 200 can be set in three holder sections 60a to 60c, while a small reagent container 300 can be set in two holder sections 60d and 60e. Each holder section 60a to 60e includes a chassis 61, a reagent container holding section 62, a cover 63 for opening and closing the reagent container holding section 62, the aforementioned suction tube 64, and a suction tube lifting mechanism 65.

[0088] The reagent container holder 62 is located at the bottom of the chassis 61 (see Figures 14 and 15). As shown in Figure 15, the reagent container holder 62 has a height H and includes a first receiving section 621 having a width W11, an intermediate receiving section 622 that is continuous with the first receiving section 621 and extends from the first receiving section 621 at a predetermined angle θ2, and a second receiving section 623 that is continuous with the intermediate receiving section 622. As shown in Figures 16 and 17, the first receiving section 621 is capable of receiving the first storage section 210 (310) of the reagent container 200 (300), which will be described later, and has a width (width W11) that prevents the user's finger from entering. Hereinafter, "finger" refers to, for example, the finger of an adult with an average thickness, and for example, the width W11 is 10 mm. The second receiving section 623 has a width W12 that is larger than the width W11. As shown in Figures 16 and 17, the first receiving section 621 is located at the innermost part (direction of arrow Y2) of the reagent container holding section 62. The reagent container 200 (300) is inserted from the entry section 212 (312), which will be described later, of the first housing section 210 (310) toward the innermost part of the reagent container holding section 62. Therefore, the reagent container holding section 62 is configured to hold the reagent container 200 (300) when it is inserted so that the entry section 212 (312) of the reagent container 200 (300) is at its innermost part (direction of arrow Y2).

[0089] Furthermore, as shown in Figures 16 to 18, the reagent container holding section 62 includes a pair of guide members 627 that guide both sides 214 (314) of the first housing section 210 (310) of the reagent container 200 (300) to the first receiving section 621. The guide member 627 has a first guide section 627a that guides the first housing section 210 (310) of the reagent container 200 (300) to the first receiving section 621, an intermediate guide section 627b corresponding to the intermediate receiving section 622, and a second guide section 627c that guides the second housing section 220 (320), which will be described later, of the reagent container 200 (300) to the second receiving section 623. The guide member 627 is formed by a part (both inner sides) of the chassis 61. The first receiving portion 621, intermediate receiving portion 622, and second receiving portion 623 are formed by the space between the corresponding pair of first guide portions 627a, the space between the pair of intermediate guide portions 627b, and the space between the pair of second guide portions 627c, respectively. Therefore, the width W11 of the first receiving portion 621 is equal to the width between the pair of first guide portions 627a, and the width W12 of the second receiving portion 623 is equal to the width between the pair of second guide portions 627c.

[0090] The pair of guide members 627 have a height H (see Figure 14) that is approximately equal to the height H1 (see Figures 24 and 27) of both sides 214 (314) of the first housing section 210 (310) of the reagent container 200 (300), and are configured to guide both sides 214 (314) of the first housing section 210 (310) of the reagent container 200 (300) from the lower end to the upper end. Furthermore, the pair of guide members 627 have a shape that reflects the external shape of the first housing section 210 (310), and are configured to guide the entire surface of both sides 214 (314) of the first housing section 210 (310) of the reagent container 200 (300). Furthermore, the intermediate guide section 627b and the second guide section 627c have shapes that reflect the external shape of the large reagent container 200 and are configured to guide both sides of the front half (first storage section 210 side) of the second storage section 220. The second storage section 220 of this large reagent container 200 has a width W22 that is larger than the width W21 of the first storage section 210, and the first guide section 627a is provided with a width W11 that is smaller than the width W22 of the second storage section 220.

[0091] Furthermore, as shown in Figure 18, the reagent container holding section 62 includes a support section 624 that supports the reagent container 200 (300) and a rotating mechanism 625 that rotatably supports the support section 624. The support section 624 is a plate-shaped member integrally having a front portion 624a that abuts against the front surface of the reagent container 200 (300) (the front end surface of the first housing section 210 (310), see Figures 16 and 17) and a lower portion 624b that abuts against the lower surface of the reagent container 200 (300). In other words, the support section 624 is formed to have a shape that corresponds to the shape of the reagent container 200 (300). The rotation mechanism 625 is configured such that a projection 624c provided on the support portion 624 is inserted into an annular bearing 625a provided on the inner surface of the chassis 61, thereby allowing the support portion 624 to rotate with the position of the projection 624c (bearing 625a) as the pivot point.

[0092] Furthermore, inside the chassis 61, there is a locking portion 626 that locks the rotating support portion 624 by contacting the front portion 624a of the support portion 624. The locking portion 626 is equipped with a magnet and is configured to hold the front portion 624a (support portion 624) when in contact with it. As a result, the support portion 624 is configured to move between a mounting position P1 (see Figure 19) where the lower portion 624b is horizontal (the bottom surface of the reagent container 200 (300) is horizontal) and a set position Q1 (see Figure 20) where the front portion 624a is vertical. As shown in Figure 20, when positioned in this set position Q1, the entry portion 212 (312) (see Figures 16 and 17) of the reagent container 200 (300), which will be described later, is configured to be horizontal (perpendicular to the suction tube 64).

[0093] As shown in Figure 19, the cover 63 is positioned to protrude forward (in the direction of arrow Y1) from each of the holder sections 60a to 60e (chassis 61) and is attached to the suction tube lifting mechanism 65. This suction tube lifting mechanism 65 allows the cover 63 to move between an elevated position P2 (see Figure 19) that opens the reagent container holding section 62 and a lowered position Q2 (see Figure 21) that covers (closes) the reagent container holding section 62. Therefore, the cover 63 is configured to allow insertion and removal of the reagent container 200 (300) in the elevated position P2, and to prohibit insertion and removal of the reagent container 200 (300) in the lowered position Q2.

[0094] Furthermore, as shown in Figure 15, a window portion 631, which is an opening, is provided at a predetermined position on the cover 63. As shown in Figure 21, when the cover 63 is in the lowered position Q2, which covers (closes) the reagent container holding portion 62, the user is able to see the label 250 (350, see Figure 25) attached to the reagent container 200 (300) through this window portion 631. A mark identifying the type of reagent container 200 (300) (type of reagent) is printed on the label 250 (350) at a position visible through the window portion 631. In addition, a label 632, which has a mark identifying the type of reagent container 200 (300) (type of reagent) to be set in the reagent container holding portion 62, is attached to the cover 63. In other words, each of the five holder sections 60a to 60e is set with a reagent container 200 (300) that contains a predetermined type of reagent. Accordingly, a label 632 identifying the type of reagent to be set is affixed to the cover 63 of each holder section 60a to 60e. This configuration allows for confirmation that the correct reagent is set in each holder section 60a to 60e by looking at the label 632 on the cover 63 and the label 250 (350) visible through the window section 631 when the reagent container 200 (300) is set in the reagent container holding section 62 (when the cover 63 is lowered to the lowered position Q2).

[0095] As shown in Figures 16 and 20, the suction tube 64 is positioned above the rear (Y2 direction) of the first receiving portion 621 of the reagent container holding portion 62, and is configured to move vertically (Z direction) by a suction tube lifting mechanism 65 that holds the suction tube 64. This allows the suction tube 64 to enter the first storage portion 210 (310) through the entry portion 212 (312) of the reagent container 200 (300) inserted into the rear side of the reagent container holding portion 62, and to aspirate the reagent inside the reagent container 200 (300). Furthermore, the tip of the suction tube 64 is formed to be able to penetrate (puncture) the sealing member 213 (313) for sealing the opening 212a (312a) (see Figures 17 and 18) formed in the entry portion 212 (312) of the reagent container 200 (300). Furthermore, as explained with reference to Figures 2, 5, and 6, the suction tube 64 constitutes one end of the liquid delivery tube 431, and the upper end of the suction tube 64 is connected to the flow path (not shown in Figures 19 to 21) leading to the liquid delivery section 430 and the chamber 420.

[0096] As shown in Figures 19 and 20, the suction tube lifting mechanism 65 is configured to hold the suction tube 26 and the cover 63. The suction tube lifting mechanism 65 is also engaged with grooves 611 and 612 provided in the chassis 61 so as to be movable in the vertical direction (Z direction). As a result, the suction tube lifting mechanism 65 is configured to move the suction tube 26 integrally in the vertical direction (Z direction) in conjunction with the opening and closing (lifting and lowering) of the cover 63. As shown in Figure 19, when the cover 63 is in the raised position P2, the suction tube 26 is positioned in the raised position P3 above the reagent container holding section 62 (outside the reagent container 200 (300) and the first receiving section 621). As shown in Figure 21, when the cover 63 is in the lowered position Q2, the suction tube 26 is configured to be positioned in the lowered position Q3, close to the inner bottom directly below the entry section 212 (312) of the reagent container 200 (300).

[0097] The tip of the suction tube 26 inserted into the reagent container becomes the first end of the liquid delivery tube. The first end of the suction tube 26 is fixed in the lowered position Q3 described above while the reagent container is in the device. In other words, the first end of the suction tube 26 remains fixed in the lowered position Q3 while multiple samples are being measured using the reagent in the reagent container.

[0098] As shown in Figures 22 and 25, a large reagent container 200 (capacity approximately 100 mL) and a small reagent container 300 (capacity approximately 20 mL) are configured to be used according to the type of reagent to be contained. Reagent containers 200 and 300 each integrally include a first storage section 210 (310) with an entry section 212 (312) into which a suction tube 26 can enter at the top, and a second storage section 220 (320) that is continuous with the first storage section 210 (310). As shown in Figures 17 and 18, the first storage section 210 (310) is configured to be placed inside the first receiving section 621 with the reagent container 200 (300) set in the reagent container holding section 62. As shown in Figures 17 and 18, the second storage section 220 (320) is configured to be located outside the first receiving section 621 with the reagent container 200 (300) set in the reagent container holding section 62. Furthermore, as shown in Figures 23 and 26, each first storage section 210 (310) is provided with a first reagent storage space 211 (311), and each second storage section 220 (320) is provided with a second reagent storage space 221 (321) that is continuous with the first reagent storage space 211 (311).

[0099] The first storage section 210 (310) is a part having a length L11, and its shape is substantially the same for reagent containers 200 and 300. Since the shapes of the first storage sections 210 and 310 are substantially the same for reagent containers 200 and 300, they can be set into the reagent container holding sections 62 (first receiving section 621) of holder sections 60a to 60e, which have the same shape.

[0100] Specifically, as shown in Figures 17 and 18, each first housing section 210 (310) has a constant width W21 that is slightly smaller than the width W11 of the first receiving section 621. In addition, each first housing section 210 (310) is provided with an entry section 212 (312) at its front end (in the direction in which the first housing section 210 (310) is inserted into the reagent container holding section 62, in the direction of arrow Y2 in Figures 17 and 18). Therefore, the reagent container 200 (300) is configured to be inserted from the entry section 212 (312) side of the first housing section 210 (310) toward the back of the reagent container holding section 62. As shown in Figures 23 and 26, this entry section 212 (312) is provided to protrude upward from the outer upper surface 200b (300b). As shown in Figures 22 and 25, the protruding entry portion 212(312) has an opening 212a(312a) that communicates with the inside of the first housing portion 210(310). Furthermore, a sealing member 213(313) made of aluminum foil or the like is provided on the entry portion 212(312) to close the opening 212a(312a), so that the reagent container 200(300) is sealed. The outer diameter of the entry portion 212(312) is equal to the width W21 of the first housing portion 210(310), and the entry portion 212(312) is formed to protrude continuously (flushly) from the front surface of the first housing portion 210(310).

[0101] Furthermore, as shown in Figures 23 and 26, the reagent container 200 (300) is configured such that its internal bottom surface 200a (300a) is non-parallel to its external top surface 200b (300b), and the distance between the internal bottom surface 200a (300a) and the external top surface 200b (300b) increases as it approaches the entry section 212 (312). In the second embodiment, the internal bottom surface 200a (300a) is configured to be an inclined surface tilted at an angle θ1 (approximately 10 degrees) with respect to the external top surface 200b (300b). Furthermore, a bottom surface 200c (300c) exists directly below the entry portion 212 (312) of the reagent container 200 (300), which is approximately parallel to the outer top surface 200b (300b), and the inclined surface 200d (300d) starts from the end of the bottom surface 200c (300c). As a result, when the reagent container 200 (300) is set in the set position Q1 shown in Figure 20, the entry portion 212 (312) is located at the highest position, and the bottom surface 200c (300c) directly below the entry portion 212 (312) is located at the lowest position.

[0102] Furthermore, the outer upper surface 200b (300b) of each reagent container 200 (300) is provided with a projection 230 (330) that protrudes upward (perpendicular to the outer upper surface 200b (300b)). Each projection 230 (330) has a plate-like shape with a length L1 extending in the longitudinal direction of the reagent container 200 (300), and is formed to have a projection amount (projection height) approximately equal to that of the entry portion 212 (312). The projection 230 (330) is located near the rear end (in the direction of arrow Y1 in Figures 17 and 18) of each second housing portion 220 (320), and functions as a handle to facilitate the user's setting or removal of the reagent container 200 (300).

[0103] On the other hand, the shape of the second storage section 220 (320) differs between the reagent container 200 and the reagent container 300. As shown in Figure 23, the second storage section 220 of the large reagent container 200 integrally includes a first section 222 that is continuous with the first storage section 210 and whose width increases as it moves away from the first storage section 210, and a second section 223 that has a constant width W22 which is larger than the width W21. Therefore, as shown in Figure 23, in the reagent container 200, the second reagent storage space 221 inside the second storage section 220 is a reagent storage space that is continuously provided across both the first section 222 and the second section 223.

[0104] As shown in Figure 23, the first section 222 is continuous with the second section 223 by widening the width of the first storage section 210 by an angle θ2 (approximately 60 degrees), thus connecting the first storage section 210 and the second section 223. Furthermore, by having a width W22 that is larger than the width W21 of the second section 223, it is possible to secure a capacity of approximately 100 mL for the second reagent storage space 221.

[0105] As shown in Figures 16 and 17, the intermediate receiving portion 622 of the reagent container holding portion 62 and the second receiving portion 623, which is continuous with the intermediate receiving portion 622 and has a width W12, have shapes corresponding to the first portion 222 and the second portion 223, which has a width W22, respectively.

[0106] As shown in Figure 26, the second storage section 320 of the small reagent container 300 is constant and has the same width W21 as the first storage section 310. In other words, in the small reagent container 300, the first storage section 310 and the second storage section 320 are formed to extend continuously in a straight line. The length L22 of this second storage section 320 is smaller than the length L21 of the second storage section 220 of the large reagent container 200. By including the second storage section 320, which has a smaller width W21 and length L22 than the second storage section 220 of the large reagent container 200, the small reagent container 300 is configured to have a total reagent capacity of approximately 20 mL.

[0107] As mentioned above, the length L11 of the first storage section 310 is common to both reagent containers 200 and 300 in order to be set in the first receiving section 621. Therefore, as shown in Figure 18, for a small reagent container 300 in which the first storage section 310 and the second storage section 320 are continuous with the same width W21, the area that is housed in the first receiving section 621 is the first storage section 310, and the area located outside the first receiving section 621 is the second storage section 320.

[0108] Furthermore, unlike the large reagent container 200, the small reagent container 300 has a recess 340 on its inclined outer bottom surface 300d that extends linearly along the longitudinal direction of the reagent container 300. This recess 340 allows the outer circumference of the recess 340 to contact the horizontal surface when the inclined surface 300d is placed on a horizontal surface, making it possible to stably stand the reagent container 300, which has a small width W21, upright.

[0109] Furthermore, as shown in Figures 22 and 25, each reagent container 200 (300) has a label 250 (350) attached to it, which is printed with the name of the reagent it contains, the reagent lot number, the expiration date, and an identification barcode. This label 250 (350) is attached to the rear surface and at least one side surface of each reagent container 200 (300). In addition, a part (corresponding to the rear surface of each reagent container 200 (300)) or all of the label 250 (350) is colored to indicate the type of reagent it contains, so that the type of reagent can be identified by the color displayed on the label 250 (350). By checking whether the colors of this label 250 (350) and the label 632 (see Figure 15) attached to the cover 63 of the reagent container holder 60 match, it is possible to confirm whether the reagent container 200 (300) is set in the correct holder section 60a to 60e.

[0110] The reagents in the reagent container 200 (300) can be stored at the ambient temperature in which the analysis system 4002 is installed. The ambient temperature in which the analysis system 4002 is installed is, for example, in the range of 20°C to 35°C. For example, at ambient temperature, the reagents in the reagent container 200 (300) can be stored in the apparatus for several months (e.g., 60 days, 90 days, 120 days) with guaranteed performance after the reagent container 200 is installed in the apparatus and the sealing member 213 of the entry section 212 is opened. The reagents contained in the reagent container 200 (300) include, for example, multiple fluorescent dyes corresponding to multiple wavelengths of light irradiated from the light source of the FCM detection unit 460. These fluorescent dyes, through the compounds that make up the dyes themselves, stain the cytoplasm, nucleic acids, and DNA of cells. One known method for staining cells in the blood is immunohistochemistry, which uses antibody reagents containing labeled antibodies that specifically bind to cell surface antigens (e.g., CD4 or CD25). However, such antibody reagents need to be refrigerated after being used for a certain period of time (e.g., 8 hours). This means that the user must remove the antibody reagent from the device at regular intervals, return it to the refrigerator, and then reinsert it into the device before use. In contrast, the reagent in this embodiment does not contain antibodies, as the compound constituting the dye (fluorescent dye) itself stains the cells and does not require refrigeration. Therefore, it can be stored at the operating temperature of the device. Consequently, in this embodiment, once the reagent container is installed in the device, it does not need to be replaced until the reagent is used up, as long as the performance is guaranteed. This reduces the frequency of reagent replacement and lowers the labor costs for the user.

[0111] (Third embodiment) In this embodiment, the reagent container 200 is configured to suppress the deterioration of the quality of the reagent containing multiple fluorescent dyes after installation in the measurement unit 400, and to allow it to be used for a longer period of time while remaining attached to the measurement unit 400. One of the main means of suppressing quality deterioration is to prevent contact between the reagent and air. To prevent contact with air, the opening of the reagent container 200 is sealed when the liquid delivery tube is inserted into the reagent container 200. To allow the reagent to be delivered by the liquid delivery tube even when contact between the reagent and air is suppressed by sealing the opening of the reagent container 200, the reagent container 200 is equipped with a flexible bag-shaped reagent storage section.

[0112] The storage period for the reagent is, for example, 75 days to 1 year from the time the reagent container 200 is installed in the measurement unit 400. The storage period is the length of time during which the measurement accuracy using the reagent by the measurement unit 400 can be guaranteed. In this embodiment, for example, the measurement accuracy is maintained for a period of 75 days to 1 year with the inside of the reagent storage section 10 sealed. Since the deterioration of the reagent can be suppressed by sealing the reagent storage section 10, the reagent quality can be maintained stably for a longer period of time. As a result, the frequency of replacing reagents (reagent containers 200) containing multiple fluorescent dyes can be reduced, thereby reducing the burden on the user involved in replacement work. The multiple fluorescent dyes may each be stored in different reagent containers 200, or they may be stored in a single reagent container 200.

[0113] Referring to Figures 28 and 29, another embodiment of the reagent container 200 will be described. The reagent container 200 is installed in the measurement unit 400 as shown in Figure 28(A). As shown in Figure 28(B), a suction tube 252 is inserted into the reagent container 200 from above in conjunction with a predetermined operation on the measurement unit 400. The suction tube 252 constitutes the first end of the liquid delivery tube described above. As shown in Figure 28(B), the reagent container 200 contains the reagent 12 that is repeatedly aspirated with the suction tube 252 provided in the measurement unit 400 inserted. The reagent container 200 comprises a reagent storage section 10 and a frame 20. Hereinafter, the vertical direction will be referred to as the Z direction. As shown in Figure 29, in the horizontal direction, the longitudinal direction of the reagent container 200 will be referred to as the first direction A, and the short direction of the reagent container 200 will be referred to as the second direction B.

[0114] The reagent container 10 contains the reagent 12 (see Figure 29). As shown in Figure 29, the reagent container 10 is a bag-shaped liquid container. The capacity of the reagent container 10 is, for example, 200 mL to 500 mL, or 20 mL to 100 mL.

[0115] The reagent storage section 10 is formed in a bag shape from a film-like material. The reagent storage section 10 is flexible and deformable. As shown in Figure 30, the reagent storage section 10 is formed in a hollow bag shape by overlapping multiple sheet-like members and joining the outer edges of the overlapping film materials. It may also be formed in a bag shape by joining the inner surfaces of the outer edges of a single folded film material.

[0116] The reagent container 10 is made of a laminated film material 11 having gas barrier properties and light-shielding properties. Gas barrier properties refer to the property of being impermeable to gases. In this specification, gas barrier properties refer to being impermeable to air, especially oxygen. Light-shielding properties refer to the property of being impermeable to light. As a result, it is possible to suppress the deterioration of the reagent 12 contained in the reagent container 200 by external air and the deterioration of the reagent 12 contained in the reagent container 200 by external light such as sunlight. As a result, the deterioration of the reagent can be effectively suppressed over a long period of time.

[0117] The laminated film material 11 may include at least one base layer and at least one gas barrier layer. The laminated film material 11 may further include a protective layer that protects the outer surface of the gas barrier layer. The laminated film material 11 may also have a light-shielding layer made of a light-shielding material. When a material having both gas barrier and light-shielding properties is used for the gas barrier layer, the gas barrier layer and the light-shielding layer may be the same layer. The number of layers in the laminated film material is two or more, but may be 3 to 9 or 10 or more, and is not particularly limited.

[0118] The laminated film material 11 used in the reagent storage section 10 may have the following configuration, for example: (outside of bag) / nylon (15 μm) / aluminum foil (9 μm) / polyethylene (9 μm) / (inside of bag). In this configuration, the reagent storage section 10 has nylon functioning as a protective layer, aluminum foil functioning as a gas barrier layer and light shielding layer, and polyethylene as the base layer. The base layers made of polyethylene in the reagent storage section 10 are joined together by heat welding. The laminated film material 11 with this configuration is a metal foil laminate film with a structure in which a gas barrier layer made of metal foil is laminated on a resin base layer.

[0119] The laminated film material 11 can also be, for example, a resin-based multilayer barrier film, a coated film, a vapor-deposited film, or an organic-inorganic composite film. A resin-based multilayer barrier film is a film with a structure in which gas barrier layers of resin material are laminated. Examples of resin materials with excellent gas barrier properties include PVDC (polyvinylidene chloride), PVA (polyvinyl alcohol), and EVOH (ethylene-vinyl alcohol copolymer). A coated film is a film with a structure in which a gas barrier material is coated (film-formed) onto a substrate layer. Examples of gas barrier materials that are film-formed include PVDC, PVA, and EVOH. A vapor-deposited film is a film with a structure in which a gas barrier material is vapor-deposited onto a substrate layer. Examples of vapor-deposited gas barrier materials include metals such as aluminum, or inorganic oxides such as alumina and silica. Organic-inorganic composite films include laminated films with a structure in which a gas barrier layer of organic material (resin material) and a gas barrier layer of inorganic material are laminated separately, and films equipped with a gas barrier layer in which an inorganic material is dispersed in an organic binder.

[0120] The frame 20 includes an opening 21a attached to the reagent storage section 10. The frame 20 is configured such that the inside of the reagent storage section 10 is sealed when the suction tube 252 is inserted from above in conjunction with a predetermined operation on the measurement unit 400.

[0121] As shown in Figure 28, the reagent container 200 is placed and held by the user in a predetermined position on the container holder 251 of the measurement unit 400. With the reagent container 200 in the set position Ps of the container holder 251, the suction tube 252, positioned above the reagent container 200, is inserted into the opening 21a from above. The set position Ps is the position where the opening 21a is positioned directly below the suction tube 252. The frame 20 is configured such that, with the suction tube 252 inserted, the opening 21a is closed, thereby sealing the inside of the reagent storage section 10.

[0122] This eliminates the need for the user to insert the suction tube 252 into the opening 21a of the reagent container 200 before installation, making it easy to insert the suction tube 252 into the reagent container 200. Furthermore, since the inside of the reagent storage section 10 is sealed when the suction tube 252 is inserted, contact between the reagent inside the reagent storage section 10 and the outside air is suppressed by the seal. Therefore, deterioration of the reagent after installation in the measurement unit 400 can be suppressed. In addition, even if the reagent storage section 10 has a flexible bag-like shape, the installation of the reagent container 200 into the measurement unit 400 can be easily performed by, for example, gripping the frame 20 or installing it in the measurement unit 400.

[0123] In the case of rigid reagent containers that do not deform, if reagents are aspirated while the container is sealed, the internal pressure of the container decreases as the amount of reagent decreases, eventually reaching equilibrium with the suction pressure and making it impossible to aspirate the reagent. Therefore, the inside of the container must be exposed to the atmosphere, and the reagents inside the container may deteriorate due to contact with air.

[0124] In contrast, this embodiment includes a deformable bag-shaped reagent container 10, so that the reagent container 10 itself can contract and deform, thereby avoiding a decrease in internal pressure, and as a result, reagent 12 can be aspirated even in a sealed state. Therefore, the reagent container 100 is configured so that the inside of the reagent container 10 is sealed by suppressing the inflow of air into the opening 21a. By suppressing the inflow of air into the opening 21a, deterioration of the reagent 12 due to contact with air inside the reagent container 10 can be suppressed. Since the deterioration of the reagent can be suppressed, the reagent in the reagent container 200 can be used for a longer period of time while the reagent container 200 is installed in the measurement unit 400. Therefore, by increasing the capacity of the reagent container 10 and enabling more reagent aspirations from a single reagent container 200, the frequency of replacing the reagent container 200 associated with the operation of the measurement unit 400 can be reduced. For the user, this has the advantage of reducing the number of times the reagent container 200 needs to be replaced over a certain period of time.

[0125] Furthermore, in a configuration where the suction tube 252 is inserted into the opening 21a by the operation of the measuring unit 400, rather than being inserted manually by the user, the positioning of the opening 21a and the prevention of displacement of the opening 21a are important in order to reliably seal the reagent container 10 without damaging it with the suction tube 252. The reagent container 200 of this embodiment is equipped with a frame structure that ensures the positioning of the opening 21a and the prevention of displacement of the opening 21a when it is installed in the measuring unit 400.

[0126] As shown in Figure 29, the frame 20 includes an opening 21 having an opening 21a, and a movement restricting portion 23 configured to contact a part of the measuring unit 400 and restrict the movement of the opening 21a.

[0127] The movement restricting section 23 contacts a part of the measuring unit 400, thereby suppressing the movement of the opening 21a. This suppresses misalignment between the suction tube 252 and the opening 21a when the suction tube 252 is inserted. Furthermore, since misalignment between the suction tube 252 and the opening 21a is suppressed when the suction tube 252 is inserted into the reagent storage section 10 through the opening 21a, the sealed state inside the reagent storage section 10 can be effectively maintained.

[0128] The opening 21 is a cylindrical portion in which the opening 21a is formed. The lower end of the opening 21a connects to the inside of the reagent storage section 10, and the upper end connects to the outside of the reagent storage section 10. The opening 21 is located at the top of the reagent storage section 10.

[0129] The opening 21 has a sealing surface 21b that contacts the sealing body 252a (see Figure 28) provided on the suction tube 252 to seal the opening 21a. As a result, the reagent container 10 can be easily and effectively sealed by the contact between the sealing body 252a and the sealing surface 21b.

[0130] The sealing surface 21b can be the upper end surface of the opening 21, the inner circumferential surface of the opening 21, or the outer circumferential surface of the opening 21. In the example of Figure 29, the sealing surface 21b is the annular upper end surface of the opening 21 surrounding the opening 21a (i.e., the edge of the opening 21a), and is in contact with the sealing body 252a (see Figure 28(B)) provided on the suction tube 252 from above and below.

[0131] Furthermore, a removable cap may be fitted to the opening 21, for example. The reagent container 200 is provided to the user with the opening 21a sealed by the cap, and the cap can be removed and the container placed in the measurement unit 400.

[0132] Furthermore, for example, a punctureable sealing film may be welded to the upper surface of the opening 21 so as to cover the opening 21a. The reagent container 200 is provided to the user with the opening 21a sealed by the sealing film, and after being placed in the measurement unit 400, it can be opened by puncturing the sealing film with the suction tube 252.

[0133] As shown in Figure 30, the opening 21 is formed in the mounting member 22 attached to the reagent container 10. The opening 21 is integrally formed with the mounting member 22 so as to protrude upward from the upper surface of the mounting member 22.

[0134] The outer periphery of the mounting member 22 is welded to the inner periphery of the reagent storage section 10. The mounting member 22 is provided at the upper end of the reagent storage section 10 so as to be sandwiched between the outer edges of the two laminated film materials 11 that constitute the reagent storage section 10, and is welded to the inner surfaces of the two laminated film materials 11. The space between the outer periphery of the mounting member 22 and the reagent storage section 10 is sealed by welding. This makes it easy to attach the mounting member 22, which has rigidity for connecting with the movement restricting section 23, to the flexible bag-shaped reagent storage section 10, and also makes it easy to achieve sealing between the mounting member 22 and the reagent storage section 10.

[0135] The mounting member 22 has a supported portion 22a that is spaced horizontally from the opening 21. The mounting member 22 has an elongated hexagonal shape, with the opening 21 located at one end in the longitudinal direction of the mounting member 22 and the supported portion 22a located at the other end. The supported portion 22a is a projection integrally formed on the mounting member 22 so as to protrude upward from the upper surface of the mounting member 22. When viewed from direction A, the supported portion 22a has a T-shape, with the base being narrower than the upper part.

[0136] Here, the opening 21 is located near the end of the reagent storage section 10 in the horizontal direction. Even when the reagent storage section 10 is expanded by accommodating the reagent 12 (see Figure 28), the opening 21 of the mounting member 22 is located relatively close to the inner surface of the reagent storage section 10. Therefore, if the suction tube 252 is inserted at an angle with respect to the central axis of the opening 21, there is a possibility that the tip of the suction tube 252 will come into contact with the inner surface of the reagent storage section 10.

[0137] Therefore, the opening 21 has a suction tube guide portion 21c that protrudes downward from the lower end of the opening 21a toward the interior of the reagent storage portion 10. The suction tube guide portion 21c is integrally formed with the mounting member 22 and has a shape that partially extends downward from the lower end of the cylindrical opening 21. The suction tube guide portion 21c is formed in a wall shape so as to separate the suction tube 252 inserted into the opening 21a from the inner surface of the reagent storage portion 10. As a result, even if the suction tube 252 is inserted into the interior of the reagent storage portion 10 at an angle with respect to the central axis of the opening 21a, the tip of the suction tube 252 can be guided so as to suppress contact between the tip of the suction tube 252 and the inner surface of the reagent storage portion 10.

[0138] Returning to Figure 29, the movement restricting unit 23 has the function of positioning the opening 21a relative to the suction pipe 252 of the measuring unit 400. The movement restricting unit 23 also has the function of preventing misalignment of the opening 21a.

[0139] The movement restricting portion 23 is fixed to the opening 21 so as to protrude horizontally from the opening 21. This allows the relative position between the movement restricting portion 23 and the opening 21a to be fixed. Furthermore, the portion of the movement restricting portion 23 that protrudes horizontally from the opening 21 can be easily used to restrict the movement of the measuring unit 400 by contacting a part of it, and the contact area necessary to prevent misalignment of the opening 21a can be easily obtained through this contact.

[0140] As shown in Figure 30, the movement restricting section 23 has a generally flat plate shape. The movement restricting section 23 has a shape that extends along a first direction A in the horizontal plane. The movement restricting section 23 has a rectangular shape including a pair of long sides extending in the first direction A and a pair of short sides extending in the second direction B. As will be described later, the reagent container 200 is installed in the container holding section 251 of the measurement unit 400 by inserting it into the container holding section 251 along the first direction A, with the end 23a of the movement restricting section 23 where the opening 21 is located as the leading edge. The movement restricting section 23 is fixed to the opening 21 between the end 23a and the central part of the movement restricting section 23 in the first direction A. In other words, the opening 21 is located closer to the end 23a of the movement restricting section 23 than to the other end 23b. This allows the user to grip the other end 23b of the movement restricting part 23 and move the reagent container 200 in the first direction A, thereby positioning the opening 21a formed in the opening 21 on one end 23a directly below the suction tube 252. In this case, since the opening 21 is separated from the other end 23b of the movement restricting part 23, it is possible to prevent the user's fingers from coming into contact with the suction tube 252 when attaching the reagent container 200.

[0141] The movement restricting section 23 is configured to restrict the horizontal movement of the opening 21a by contacting a part of the measuring unit 400 in the horizontal direction. This prevents the horizontal positional relationship between the opening 21a and the suction tube 252 from shifting. The movement restricting section 23 includes a horizontal contact surface 24 which is made up of one of the inner surface of a hole formed in the movement restricting section 23, the inner surface of a notch formed in the movement restricting section 23, or the side surface of the movement restricting section 23. This allows a horizontal contact surface 24 for restricting the horizontal movement of the opening 21a to be provided on the movement restricting section 23 with a simple configuration.

[0142] The horizontal contact surface 24 includes a first surface 24a which is the side surface (i.e., the front end surface) formed on one end 23a of the movement restricting portion 23 in the first direction A. This makes it easy to achieve both alignment of the opening 21a in the first direction A and movement restriction when attaching the reagent container 200 to the measurement unit 400, simply by advancing the reagent container 200 in the first direction A until one end 23a of the movement restricting portion 23 abuts against the inner surface of the container holding portion 251.

[0143] The horizontal contact surface 24 includes second surfaces 24b and 24c, which are sides extending along the first direction A of the movement restricting section 23. The second surfaces 24b and 24c are a pair of sides that constitute the long side of the movement restricting section 23 in a plan view, and are provided on one side and the other side of the movement restricting section 23 in the second direction B. This makes it easy to achieve both alignment and movement restriction of the opening 21a in the second direction B using the sides of the movement restricting section 23.

[0144] As shown in Figure 31, the movement restricting section 23 has a notch 23c formed on its side surface. The horizontal contact surface 24 includes a third surface 24d which is the inner surface of the notch 23c. The third surface 24d contacts a part of the measuring unit 400 that enters the notch 23c. As a result, the movement restricting section 23 and the measuring unit 400 can be engaged by the contact between the third surface 24d inside the notch 23c and a part of the measuring unit 400. This engagement effectively prevents the opening 21a from shifting. Furthermore, when attaching the reagent container 200 to the measuring unit 400, the movement restricting section 23 engages with a part of the measuring unit 400 and becomes immobile, so the user can perceive by touch that the reagent container 200 is properly positioned without having to visually confirm it.

[0145] The movement restricting section 23 has a hole 23d formed on its upper surface. The horizontal contact surface 24 includes a fourth surface 24e which is the inner surface of the hole 23d. The fourth surface 24e contacts a part of the measuring unit 400 that enters the inside of the hole 23d (for example, an entry member 281 described later, see Figure 35). As a result, the movement restricting section 23 and the measuring unit 400 can be engaged by allowing a part of the measuring unit 400 to enter the hole 23d formed in the movement restricting section 23. This engagement effectively prevents the opening 21a from shifting position. In Figure 31, two holes 23d are provided. The two holes 23d are aligned along the second direction B. There may be only one hole 23d.

[0146] The movement restricting section 23 has a guide section 23e that guides the movement of the opening 21a when it is positioned below the suction tube 252 of the measuring unit 400. The guide section 23e extends along a first direction A in the horizontal plane. The guide section 23e slides while in contact with a part of the measuring unit 400, thereby aligning the movement direction of the movement restricting section 23 with the first direction A. This makes it possible to easily and accurately position the opening 21a in the correct position for insertion of the suction tube 252 when the reagent container 200 is placed in the container holding section 251 of the measuring unit 400.

[0147] The movement restricting section 23 is configured to restrict the vertical movement of the opening 21a by contacting a part of the measurement unit 400 in the vertical direction (Z direction). As a result, even with a reagent container 200 equipped with a deformable bag-shaped reagent storage section 10, vertical movement of the opening 21a can be suppressed when inserting the suction tube 252 into the reagent storage section 10 and when withdrawing the suction tube 252 from the reagent storage section 10.

[0148] As shown in Figure 31, the movement restricting section 23 has a gripping section 25 at the other end 23b in the first direction A of the movement restricting section 23 for gripping the movement restricting section 23. This makes it easy to grip the other end 23b of the movement restricting section 23. In addition, since the bag-shaped reagent container 10 is easily deformed and difficult to grip, providing the gripping section 25 on the movement restricting section 23 makes it easier to carry the reagent container 200.

[0149] As shown in Figure 30, the movement restricting section 23 includes a fixing section 26 fixed to the opening 21 and a support section 27 that supports the upper part of the reagent container 10 at a position away from the opening 21. This allows the reagent container 10 to be supported by multiple points on the fixing section 26 and the support section 27 when the movement restricting section 23 is grasped by hand while carrying the reagent container 200, as if it were suspended from the movement restricting section 23. Because the weight of the reagent container 10 is distributed and acted upon multiple points on the movement restricting section 23, it can be stably supported even if the capacity of the reagent container 10 is large. In addition, since the user does not have to grasp the easily deformable bag-like part, handling the reagent container 200 becomes easier.

[0150] As shown in Figure 29, the movement restriction unit 23 includes an information recording medium 28 that records information about the reagent. The information recording medium 28 is an RFID (radio frequency identifier) ​​tag. This allows the measurement unit 400 to read the reagent information from the information recording medium 28 when the reagent container 200 is attached to the measurement unit 400. If the information recording medium 28 were to be placed in the easily deformable bag-shaped reagent container 10, it may be difficult to attach the information recording medium 28 or the information recording medium 28 may be difficult to read. However, by placing the information recording medium 28 in the movement restriction unit 23, the information recording medium 28 can be easily attached and read reliably.

[0151] As shown in Figure 32, the reagent suction unit 250 includes a plurality of reagent container holders, each capable of holding one reagent container 200 (or a small reagent container 300). The reagent suction unit 250 includes, for example, a total of five reagent container holders, but for convenience, only three reagent container holders 250a, 250b, and 250c are shown in Figure 32. The plurality of reagent container holders 250a to 250c are arranged side by side in the lateral direction.

[0152] Reagent container holders 250a to 250c each comprise a lower chassis 255a and an upper chassis 255b, a container holding section 251, a suction tube 252, an operating section 253, and a moving mechanism 254 (see Figure 35). Below, the structure of reagent container holder 250a will be described as a representative example of reagent container holders 250a to 250c. The container holding section 251 is provided on the lower chassis 255a. The container holding section 251 is located at the very bottom of reagent container holder 250a. The container holding section 251 is configured to hold a reagent container 200 which includes a bag-shaped reagent storage section 10 for containing reagents and a frame 20 including an opening 21a.

[0153] The container holding section 251 has a box-shaped or cylindrical housing section 260 that can accommodate the reagent container 200 inside and has an opening formed horizontally. The housing section 260 has an opening at its Y1 end and is provided to extend from the opening toward the Y2 direction.

[0154] As shown in Figure 33, the housing section 260 includes a first insertion section 261 into which the reagent housing section 10 of the reagent container 200 is inserted, and a second insertion section 262 located above the first insertion section 261 into which the movement restricting section 23 of the reagent container 200 is inserted.

[0155] The first insertion section 261 is a space partitioned by a pair of side sections 261a facing both sides of the reagent storage section 10 in the width direction (X direction), and a bottom section 261b facing the bottom surface of the reagent storage section 10 in the vertical direction (Z direction). The upper part of the first insertion section 261 and the second insertion section 262 are connected by a connecting passage 263.

[0156] The connecting passage 263 is a space for allowing the connection portion of the reagent container 200 between the movement restricting portion 23 and the reagent storage portion 10 (the portion where the mounting member 22 is located) to pass through in the Y direction.

[0157] The second insertion section 262 extends from the entrance of the housing section 260 toward the set position Ps directly below the suction tube 252 and is configured to guide the reagent container 200 to the set position Ps (see Figure 34) by contacting the movement restricting section 23. This allows the reagent container 200 to be guided from the entrance of the housing section 260 to the set position Ps using the movement restricting section 23. As a result, even a reagent container 200 equipped with a reagent housing section 10 that is easily deformed can be accurately guided to the set position Ps with a simple operation by the user, which is to insert the reagent container 200 from the entrance of the housing section 260. The second insertion section 262 has, for example, a convex cross-sectional shape. That is, the second insertion section 262 has a convex cross-sectional shape due to a wide lower part into which a flat movement restricting section 23 is inserted and a narrow upper part into which an opening 21 protruding above the movement restricting section 23 is inserted.

[0158] The reagent suction section 250 is equipped with a contact section 270. The contact section 270 contacts a movement restricting section 23 provided on the frame 20 of the reagent container 200, which is placed in the container holding section 251, thereby restricting the movement of the opening 21a. As a result, the contact section 270 contacts the movement restricting section 23, which suppresses misalignment between the suction tube 252 and the opening 21a when the suction tube 252 is inserted. Furthermore, since misalignment between the suction tube 252 and the opening 21a is suppressed when the suction tube 252 is inserted into the reagent storage section 10 through the opening 21a, the sealed state inside the reagent storage section 10 can be effectively maintained.

[0159] The contact portion 270 contacts the movement restricting portion 23 provided on the frame 20 of the reagent container 200, which is positioned on the container holding portion 251, thereby restricting the movement of the opening 21a. By the contact portion 270 contacting the movement restricting portion 23, misalignment between the suction tube 252 and the opening 21a when the suction tube 252 is inserted can be suppressed. Furthermore, since misalignment between the suction tube 252 and the opening 21a can be suppressed when the suction tube 252 is inserted into the reagent storage portion 10 through the opening 21a, the sealed state inside the reagent storage portion 10 can be effectively maintained.

[0160] The contact portion 270 is configured to restrict the movement of the opening 21a of the reagent container 200 by coming into contact with the movement restricting portion 23, which is fixed to the opening 21a of the reagent container 200. This fixes the relative position between the movement restricting portion 23 and the opening 21a, so that by bringing the contact portion 270 into contact with the movement restricting portion 23, the positioning and movement restriction of the opening 21a can be performed reliably and accurately.

[0161] At least a portion of the contact portion 270 is provided in the housing portion 260. This allows the contact portion 270 and the movement restricting portion 23 to come into contact when the reagent container 200 is inserted into the housing portion 260 from the entrance, thereby restricting the movement of the opening 21a within the housing portion 260.

[0162] The contact portion 270 is located in the second insertion portion 262. By providing the contact portion 270 in the second insertion portion 262, which corresponds to the movement restricting portion 23 of the reagent container 200, even a reagent container 200 equipped with a easily deformable reagent storage portion 10 can have the contact portion 270 and the movement restricting portion 23 come into contact in a preset relative positional relationship. As a result, the positioning and movement restriction of the opening 21a can be accurately performed.

[0163] The contact portion 270 is configured to contact either a hole formed in the movement restricting portion 23, a notch formed in the movement restricting portion 23, or a side surface of the movement restricting portion 23. This allows the contact portion 270 to contact the movement restricting portion 23 and effectively restrict the horizontal movement of the opening 21a with a simple configuration.

[0164] When the suction tube 252 moves downward, the entry member 281 also moves downward together with the suction tube 252 and enters the hole 23d of the movement restricting section 23. The entry member 281 into the hole 23d prevents the movement of the movement restricting section 23.

[0165] The contact portion 270 is configured to restrict the vertical movement of the opening 21a by contacting the movement restricting portion 23 in the vertical direction. This makes it possible to suppress the vertical movement of the opening 21a when inserting the suction tube 252 into the reagent storage portion 10 and when withdrawing the suction tube 252 from the reagent storage portion 10, even in a reagent container 200 equipped with a bag-shaped reagent storage portion 10 that is easily deformable.

[0166] As shown in Figure 35, the suction tube 252 is positioned above the innermost part (Y2 direction end) of the housing section 260 of the container holding section 251, with its tip facing downwards. The suction tube 252 is held by a moving mechanism 254 and is configured to be moved vertically (Z direction) by the moving mechanism 254. As a result, the suction tube 252 enters the inside of the reagent housing section 10 (110) through the opening 21a of the reagent container 200 (200) inserted into the inner side of the container holding section 251, and is configured to aspirate the reagent inside the reagent container 200 (200).

[0167] The suction tube 252 has a sealant 252a that seals the opening 21a by coming into contact with the opening 21a when the suction tube 252 is inserted into the opening 21a. This allows the reagent container 10 to be easily and effectively sealed by the sealant 252a.

[0168] The sealing body 252a is provided so as to surround the outer circumference of the suction tube 252 and is configured to elastically deform upon contact with the edge of the opening 21a (i.e., the sealing surface 21b of the opening 21) to seal the opening 21a. This allows the reagent container 10 to be reliably sealed by the sealing body 252a by utilizing the downward movement of the suction tube 252 when it is inserted into the opening 21a.

[0169] The sealing body 252a is made of an elastically deformable rubber material. A biasing member 252b is provided between the sealing body 252a and the suction tube holder 254a that holds the suction tube 252, for biasing the sealing body 252a toward the opening 21a. The biasing member 252b is made of a compression spring.

[0170] The moving mechanism 254 holds the suction tube 252 so that it can move vertically (in the Z direction) between an elevated position P1 and a lowered position P2. The moving mechanism 254 includes a suction tube holder 254a and a linear motion mechanism consisting of a linear rail 254b and a fixed slider 254c. The suction tube 252 is attached to the suction tube holder 254a, which is connected to the upper end of the linear rail 254b via a connecting portion 254d that extends in the Y direction. The linear rail 254b is located on the front surface (side surface in the Y1 direction) of the reagent container holder 250a and extends in the Z direction. The fixed slider 254c is fixed to the lower chassis 255a and holds the linear rail 254b so that it can move along the Z direction. This causes the linear rail 254b to move in the Z direction relative to the fixed slider 254c. As the linear rail 254b moves in the Z direction, the suction tube 252 moves integrally with the linear rail 254b in the Z direction.

[0171] Thus, the moving mechanism 254 supports the suction tube 252 so that it can move vertically, and is configured to move the suction tube 252 in conjunction with a predetermined operation on the operating unit 253, thereby allowing the suction tube 252 to enter the reagent container 200 held in the container holding unit 251 and to retract the suction tube 252 outside the reagent container 200. As a result, the suction tube 252 can be moved into the reagent container 200 and retracted outside the reagent container 200 simply by moving the suction tube 252 up and down. The less freedom of movement the suction tube 252 has in its direction, the more accurately and reproducibly the suction tube 252 can be operated, so that the operation of sealing the reagent container 10 when the suction tube 252 is inserted can be performed with greater precision by simple vertical movement.

[0172] Furthermore, as shown in Figure 35, the moving mechanism 254 holds the operating unit 253 so that the suction tube 252 and the operating unit 253 move in conjunction. The operating unit 253 is provided on the front surface (side surface in the Y1 direction) of the reagent container holder 250a, and the back surface (side surface in the Y2 direction) of the operating unit 253 is attached to the linear rail 254b. As a result, the linear rail 254b, the suction tube 252, and the operating unit 253 move together in the Z direction.

[0173] As shown in Figure 32, the operating section 253 is located on the front surface (in the direction of arrow Y1) of each of the reagent container holders 250a to 250c (lower chassis 255a). The operating section 253 is configured as a cover that can be opened and closed to cover the front surface of the container holding section 251 of the reagent container holder 250a.

[0174] The operating unit 253 is configured to be grasped and moved by the user. The operating unit 253 has an operating grip portion 253a that protrudes forward (in the Y1 direction) from the front surface of the operating unit 253. The user can move the operating unit 253 in the Z direction by grasping this operating grip portion 253a and moving it in the Z direction.

[0175] (Fourth Embodiment) In the analysis systems 4000 and 4001 described in the first and second embodiments, the analysis unit (analysis section) 300X may use a pre-learned AI algorithm in at least part of the first to third analyses, taking as input the waveforms of a first signal and a second signal corresponding to the fluorescence emitted from cells flowing in a flow cell, and classifying cells using the waveforms of signals corresponding to the fluorescence emitted from various cells to be classified. As described above, the first signal is a signal composed of multiple signals corresponding to the lateral scattered light and lateral fluorescence from the first light source, and the second signal is a signal composed of multiple signals corresponding to the forward scattered light, lateral scattered light, and lateral fluorescence from the second light source. Furthermore, the first analysis is an analysis based on the first signal, the second analysis is an analysis based on the second signal, and the third analysis is an analysis based on the first signal and the second signal.

[0176] In this embodiment, in an analysis system 4000 (see Figure 7) equipped with an FCM detection unit 460 capable of irradiating a sample with light of multiple wavelengths as described above, data acquired by the measurement unit 400 is analyzed by an AI algorithm. Since light of multiple wavelengths is irradiated onto the sample, the measurement unit 400 detects multiple types of optical signals corresponding to each wavelength. The data acquired by A / D conversion of each of the detected multiple types of optical signals is analyzed by the AI ​​algorithm. The analysis by the AI ​​algorithm is performed by the analysis unit 300X (see Figure 10). The AI ​​algorithm is stored, for example, in the storage unit 3004 of the analysis unit 300X. The processor 3001 of the analysis unit 302X performs the analysis based on the AI ​​algorithm.

[0177] As shown in the example in Figure 36, data acquired by the measurement unit 402 (hereinafter sometimes referred to as "waveform data") is input to the AI ​​algorithm 50 (or 60). The AI ​​algorithm processes the input data to classify the cell types corresponding to the input data. When a single wavelength of light is irradiated onto the measurement sample, only data corresponding to that single wavelength of light is obtained for a given cell in the measurement sample. When multiple wavelengths of light are irradiated onto the measurement sample, multiple types of data corresponding to each of the multiple wavelengths of light are obtained for a given cell in the measurement sample. Therefore, when multiple wavelengths of light are irradiated onto the measurement sample, the amount of data input to the AI ​​algorithm increases compared to when a single wavelength of light is irradiated onto the measurement sample. The increased amount of data input to the AI ​​algorithm improves the accuracy of cell classification by the AI ​​algorithm.

[0178] The method for generating training data 75 (see Figure 40) and analyzing waveform data will be explained using the examples shown in Figures 37 to 40. The following explanation will use an example where an FCM detection unit 460 with the configuration shown in Figure 7 is used.

[0179] <Waveform data> Figure 37 is a schematic diagram illustrating the waveform data used in this analysis method (fourth embodiment). As shown in Figure 37, when a sample containing cells (component) C is flowed through a flow cell FC and the cells C flowing through the flow cell FC are irradiated with light of a first wavelength L1 and light of a second wavelength L2, forward scattered light (FSC) is generated in front of the direction of light propagation. Laterally to the direction of light propagation, first lateral scattered light (SSC-1) corresponding to the first wavelength of light and first lateral fluorescence (SFL-1) excited by the first wavelength of light are generated. Also laterally to the direction of light propagation, second lateral scattered light (SSC-2) corresponding to the second wavelength of light and second lateral fluorescence (SFL-2) excited by the second wavelength of light are generated.

[0180] Forward scattered light (FSC) is received by the forward scattered light receiving element 4116, and a signal corresponding to the amount of light received is output. First side scattered light (SSC-1) is received by the side scattered light receiving element 4121a, and a signal corresponding to the amount of light received is output. First side fluorescence (SFL-1) is received by the side fluorescence receiving element 4122a, and a signal corresponding to the amount of light received is output. Second side scattered light (SSC-2) is received by the side scattered light receiving element 4121b, and a signal corresponding to the amount of light received is output. Second side fluorescence (SFL-2) is received by the side fluorescence receiving element 4122b, and a signal corresponding to the amount of light received is output. As a result, each receiving element outputs an analog signal representing the change in the signal over time. The analog signal corresponding to forward scatter light (FSC-1) is called the "forward scatter light signal," the analog signal corresponding to the first side scatter light (SSC-1) is called the "first side scatter light signal," the analog signal corresponding to the first side fluorescence (SFL-1) is called the "first fluorescence signal," the analog signal corresponding to the second side scatter light (SSC-2) is called the "second side scatter light signal," and the analog signal corresponding to the second side fluorescence (SFL-2) is called the "second fluorescence signal." Each pulse of each analog signal corresponds to one component (for example, one cell).

[0181] The analog signal is input to the A / D conversion unit 481a (see Figure 37) via the analog processing unit 480 and converted to a digital signal. Figure 38 is a schematic diagram showing the conversion to a digital signal by the A / D conversion unit. For the sake of simplicity, this diagram shows the analog signal being directly input to the A / D conversion unit 481a. As shown in Figure 38, the A / D conversion unit 481a samples the forward scattered light signal, the first side scattered light signal, the first side fluorescence signal, the second side scattered light signal, and the second side fluorescence signal from the analog signals input from each photodetector, starting from the point when the level of the forward scattered light signal reaches a predetermined threshold level. The A / D conversion unit 481a samples each analog signal 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).

[0182] Figure 39 schematically shows waveform data obtained by sampling. Through sampling, matrix data (which may also be called one-dimensional "array data") is obtained, with elements being values ​​that digitally represent the analog signal levels at multiple time points, as waveform data corresponding to one component. In this way, the A / D conversion unit 481a generates a digital signal of forward scattered light, a digital signal of first side scattered light, a digital signal of first side fluorescence, a digital signal of second side scattered light, and a digital signal of second side fluorescence corresponding to one component. The A / D conversion is repeated until the number of digitized cells reaches a predetermined number, or until a predetermined time has elapsed since the sample began to flow into the flow cell 4113. As a result, a digital signal consisting of waveform data of N components (cells C) contained in one sample is obtained, as shown in Figure 39. The set of sampling data for each component (in the example of Figure 39, a set of 1024 digital values ​​every 10 nanoseconds from t=0ns to t=10240ns) may be called waveform data, and the set of waveform data obtained from one sample may be called a digital signal. Waveform data is acquired by irradiating the flow cell 4113 with light of multiple wavelengths, so multiple waveform data corresponding to each wavelength are acquired. For example, in the example shown in Figure 37, waveform data corresponding to the first lateral scattered light and the first lateral fluorescence corresponding to the first wavelength (e.g., 405 nm) are acquired, and waveform data corresponding to the forward scattered light, the second lateral scattered light, and the second lateral fluorescence corresponding to the second wavelength (e.g., 638 nm) are acquired. The lateral fluorescence corresponding to the first wavelength is acquired based on the first fluorescent dye, and the lateral fluorescence corresponding to the second wavelength is acquired based on the second fluorescent dye.

[0183] Each waveform data generated by the A / D conversion unit 481a may be assigned an index to identify its respective component. For example, the index may be assigned an integer from 1 to N in the order of the generated waveform data, and the same index may be assigned to the waveform data of forward scattered light, the first / second side scattered light, and the first / second side fluorescence obtained from the same component. Since each waveform data corresponds to one component, the index corresponds to the measured component. By assigning the same index to waveform data corresponding to the same component, the deep learning algorithm described later can analyze the waveform data of forward scattered light, the first / second side scattered light, and the first / second fluorescence as a set and classify the type of component.

[0184] <Generating training data> Figure 40 is a schematic diagram illustrating an example of a method for generating training data used to train a deep learning algorithm for determining the types of components in a sample. As shown in Figure 40, the training data 75 is waveform data generated based on the analog signals 70a of forward scattered light (FSC), 70b of first side scattered light (SSC-1), 70c of first side fluorescence (SFL-1), 70d of second side scattered light (SSC-2), and 70e of first side fluorescence (SFL-2) obtained from measuring the sample using a flow cytometer (measurement unit 400, see Figure 7). The method for acquiring the waveform data is as described above.

[0185] The training data 75 can be obtained, for example, by measuring a sample using a flow cytometer and analyzing the components contained in the sample using computational processing, using waveform data of components that are judged to be highly likely to be of a specific type. The following explanation uses an example using the analysis system 4000 (see Figure 4) as a blood cell counter for analyzing blood samples. A blood sample is measured with a flow cytometer (measurement unit 400), and waveform data of forward scattered light, first / second lateral scattered light, and first / second lateral fluorescence of individual components contained in the sample are accumulated. Based on the first / second lateral scattered light intensity (pulse height of the lateral scattered light signal) and the first / second lateral fluorescence intensity (pulse height of the lateral fluorescence signal), the cells in the sample are classified into neutrophils, lymphocytes, monocytes, eosinophils, basophils, immature granulocytes, and abnormal cell populations. By assigning label values ​​corresponding to the classified cell types to the waveform data of those cells, the training data 75 is obtained. For example, training data 75 can be obtained by determining the mode, mean, or median of the lateral scattered light intensity and lateral fluorescence intensity of cells included in a population of neutrophils, identifying representative cells based on these values, and assigning a label value "1" corresponding to neutrophils to the waveform data of those cells. The method of generating training data is not limited to this; for example, training data may also be obtained by collecting only specific cells using a cell sorter, measuring those cells with a flow cytometer, and assigning cell label values ​​to the obtained waveform data.

[0186] Each of the analog signals 70a to 70e represents the forward scattered light signal, the first / second lateral scattered light signal, and the first / second lateral fluorescence signal, respectively, when neutrophils are measured by a flow cytometer. When these analog signals are converted using A / D as described above, waveform data 72a for the forward scattered light signal, 72b for the first lateral scattered light signal, 72c for the first lateral fluorescence signal, 72d for the second lateral scattered light signal, and 72e for the second lateral fluorescence signal are obtained. Within each of the waveform data 72a to 72e, adjacent cells store the signal level at intervals corresponding to the sampling rate, for example, 10 nanosecond intervals. Each waveform data 72a to 72e is combined with a label value 77 representing the type of cell from which the data originated, and these are input into the deep learning algorithm (neural network 50) as training data 75, so that each cell has a set of five waveform data, or in other words, five signal intensity data (signal intensity of forward scattered light, signal intensity of first / second side scattered light, and signal intensity of first / second side fluorescence). In the example in Figure 40, since the cells from which the training data 75 originated are neutrophils, each waveform data 72a to 72e is assigned the label value 77 "1" to indicate that it is a neutrophil, and the training data 75 is generated. Figure 41 shows an example of the label value 77. Since the training data 75 is generated for each cell type, different label values ​​77 are assigned depending on the cell type.

[0187] <Overview of Deep Learning> Using Figure 40 as an example, we will explain the overview of neural network training. The neural network 50 is, for example, a convolutional neural network having convolutional layers. The number of nodes in the input layer 50a of the neural network 50 corresponds to the number of elements in the array contained in the waveform data of the input training data 75. The number of elements in the array is equal to the sum of the number of elements in the waveform data 72a to 72e for forward scattered light, first / second side scattered light, and first / second side fluorescence, which correspond to one component. In the example of Figure 40, each of the waveform data 72a to 72e contains 1024 elements. Therefore, the number of nodes in the input layer 50a is 1024 × 5 = 5120. Each of the waveform data 72a to 72e is input to the input layer 50a of the neural network 50. The label value 77 of each waveform data in the training data 75 is input to the output layer 50b of the neural network to train the neural network 50. The code 50c in Figure 40 indicates the hidden layer.

[0188] <Method for analyzing waveform data> Figure 42 shows an example of a method for analyzing waveform data of components in a sample. In the waveform data analysis method illustrated in Figure 42, analysis data 85 is generated from the analog signal 80a of forward scattered light, the analog signal 80b of the first lateral scattered light, the analog signal 80c of the first lateral fluorescence, the analog signal 80d of the second lateral scattered light, and the analog signal 80e of the second lateral fluorescence, all obtained from the component to be analyzed by a flow cytometer (measurement unit 400, see Figure 7), and consists of waveform data obtained by the method described above. Since waveform data is acquired by irradiating the flow cell 4113 (see Figure 37) with light of multiple wavelengths, multiple waveform data corresponding to each wavelength are acquired. For example, waveform data corresponding to the first lateral scattered light and the first lateral fluorescence corresponding to the first wavelength (e.g., 405 nm), and waveform data corresponding to the forward scattered light, the second lateral scattered light, and the second lateral fluorescence corresponding to the second wavelength (e.g., 638 nm), are acquired. Side fluorescence corresponding to the first wavelength of light is acquired based on the first fluorescent dye, and side fluorescence corresponding to the second wavelength of light is acquired based on the second fluorescent dye.

[0189] It is preferable that the analysis data 85 and the training data 75 have at least the same acquisition conditions. The acquisition conditions include the conditions for measuring the components in the sample using a flow cytometer, such as the preparation conditions of the sample to be measured, the flow rate when the sample to be measured flows through the flow cell, the intensity of the light irradiated onto the flow cell, and the amplification factor (gain) of the photodetector that receives scattered light and fluorescence. The acquisition conditions also include the sampling rate when performing A / D conversion of the analog signal.

[0190] As the components to be analyzed flow through the flow cell 4113, analog signals 80a for forward scattered light, 80b for the first lateral scattered light, 80c for the first lateral fluorescence, 80d for the second lateral scattered light, and 80e for the second lateral fluorescence are obtained. When these analog signals 80a to 80e are A / D converted as described above, the timing of signal intensity acquisition is synchronized for each component, resulting in waveform data 82a for the forward scattered light signal, 82b for the first lateral scattered light signal, 82c for the first lateral fluorescence signal, 82d for the second lateral scattered light signal, and 82e for the second lateral fluorescence signal. Each set of waveform data 82a to 82e is combined with the signal intensity data for each component (signal intensity of forward scattered light, signal intensity of the first / second lateral scattered light, and signal intensity of the first / second lateral fluorescence) and input to the deep learning algorithm 51 as analysis data 85. In the example shown in Figure 42, a dataset consisting of five waveform data points, 82a, 82b, 82c, 82d, and 82e, is input.

[0191] When the analysis data 85 is input to the input layer 51a of the neural network 51 that constitutes the trained deep learning algorithm 51, the output layer 51b outputs the analysis result 83 as classification information regarding the type of component corresponding to the analysis data 85. In Figure 42, the symbol 51c indicates the hidden layer. The classification information regarding the type of component is, for example, the probability that each component belongs to each of several cell types. Furthermore, it is determined that the component being analyzed from which the analysis data 85 was obtained belongs to the classification with the highest value among these probabilities, and the analysis result 83 may include a label value 82, which is an identifier representing that type. The analysis result 83 may be the label value itself, or it may be data in which the label value has been replaced with information indicating the type (for example, a string). In Figure 42, an example is shown in which, based on the analysis data 85, the deep learning algorithm 51 outputs the label value "1", which had the highest probability of belonging to the component being analyzed from which the analysis data 85 was obtained, and further, the text data "neutrophil" corresponding to this label value is output as the analysis result 83. The deep learning algorithm 51 may output the label values, but another computer program may output the most preferable label values ​​based on the probabilities calculated by the deep learning algorithm 51.

[0192] Figure 43 shows an example of the configuration of another sample preparation unit 441, which has a different configuration from the sample preparation unit 440 shown in Figure 12. In Figure 43, elements that are the same as those in Figure 12 are denoted by the same reference numerals.

[0193] The example in Figure 43 shows that the configuration of the measurement channels in the sample preparation unit 440 can be changed by performing analysis using a deep learning algorithm (AI algorithm). In the example in Figure 43, the measurement channels (WNR) for counting leukocytes, nucleated red blood cells, and basophils are replaced with measurement channels (WDF) for classifying leukocytes. In other words, in the example in Figure 43, by replacing the WNR channels with WDF channels, the measurement unit 400 (see Figure 7) is configured to have multiple WDF channels. In this example, the measurement items of the WNR channels, namely the count of leukocytes, nucleated red blood cells, and basophils, are obtained by analyzing the waveform data obtained from the WDF channel measurement using the AI ​​algorithm 51. For example, by training the AI ​​algorithm 51 to classify leukocytes, nucleated red blood cells, and basophils from the waveform data obtained from the WDF channel measurement, it becomes possible to classify leukocytes, nucleated red blood cells, and basophils from the waveform data of the WDF channel. For example, by training the AI ​​algorithm 51 with waveform data corresponding to leukocytes, nucleated red blood cells, and basophils obtained from WDF channel measurements, an AI algorithm 51 capable of classifying leukocytes, nucleated red blood cells, and basophils from waveform data measured by the WDF channel is generated. In the configuration example shown in Figure 43, for example, measurements of different samples are performed in parallel in multiple WDF channels. For example, sample preparation of different samples is performed in parallel in each chamber of multiple WDF channels.

[0194] As shown in Figure 43, by replacing the analysis of a predetermined measurement channel with AI analysis of data from other measurement channels, it becomes possible to replace that predetermined measurement channel with another measurement channel. This makes it possible to increase the number of additional measurement channels without increasing the total number of measurement channels provided in the analysis system 4002 (see Figure 7). In the example in Figure 43, by replacing the WNR channel with a WDF channel, it is possible to increase the number of WDF channels without increasing the total number of measurement channels provided in the analysis system 4002. By increasing the number of WDF channels, it becomes possible to measure different samples in parallel using each of the multiple WDF channels. Parallel measurement improves the throughput of measurements using WDF channels. As shown in the example in Figure 43, a significant effect is obtained in that the throughput of sample processing can also be improved.

[0195] In an example where an AI algorithm is used to replace one measurement channel with another, it is necessary to analyze the measurement items of the measurement channel to be replaced (nucleated red blood cells and basophils in the WNR channel in the example of Figure 12) based on data measured by the other measurement channel (WDF channel in the example of Figure 12). By analyzing the data measured by the other measurement channel with the AI ​​algorithm, it becomes possible to classify the measurement items of the measurement channel to be replaced. In this embodiment, multiple types of optical signals corresponding to multiple wavelengths of light are analyzed by the AI ​​algorithm. By increasing the amount of information using data corresponding to multiple wavelengths of light, the accuracy of the AI ​​analysis is improved. Due to the improved analysis accuracy, it becomes possible to classify the measurement items of the measurement channel to be replaced (nucleated red blood cells and basophils in the WNR channel in the example of Figure 12) even with data from the other measurement channel (for example, the WDF channel).

[0196] In the analysis method shown in the example in Figure 43 (first example), for example, AI analysis of waveform data obtained from measurements using a WDF channel is performed to classify and count NRBCs (nucleated red blood cells) and BASOs (basophils), as well as classify and count other white blood cells (eosinophils, neutrophils, lymphocytes, monocytes). In this example, the AI ​​algorithm 51 is trained to classify NRBCs, BASOs, and other white blood cells (eosinophils, neutrophils, lymphocytes, monocytes) based on the waveform data. By using such an AI algorithm 51, it is possible to replace the WNR channel with a WDF channel. These analyses are performed by the analysis unit 302X (see Figure 7) as follows.

[0197] Figure 44 is a flowchart showing an example of the operation of this analysis method (first operation example). As shown in Figure 44, in the first operation example, the analysis unit 302X (see Figure 7) acquires data corresponding to the optical signal detected by the WDF channel (step S0). Next, the analysis unit 302X analyzes this data with the AI ​​algorithm 51 (step S1). By analyzing the data obtained from the measurement by the WDF channel with the AI ​​algorithm 51, the analysis unit 302X classifies monocytes, lymphocytes, neutrophils, and eosinophils, as well as basophils and nucleated red blood cells that were analyzed by the WDF channel. Next, the analysis unit 302X provides the analysis results of the WDF channel data (step S2).

[0198] In another example of the analysis method shown in Figure 45 (second operational example), for example, NRBC classification and counting and BASO classification and counting are performed by AI analysis of waveform data obtained from measurements using a WDF channel. The AI ​​algorithm 51 analyzes the waveform data corresponding to cells that were not classified as either NRBC or BASO by scattergram analysis, and performs classification and counting of eosinophils, neutrophils, lymphocytes, and monocytes. In this example, the AI ​​algorithm 51 is trained to classify, for example, the waveform data into NRBC, BASO, and others. The AI ​​algorithm 51 in this example analyzes the waveform data corresponding to cells classified as other than NRBC and BASO by scattergram analysis. For example, peak values ​​of the waveform data corresponding to cells classified as other than NRBC and BASO are extracted, and the cell type is classified based on a two-dimensional graph generated from the peak values ​​corresponding to the lateral fluorescence signal and the peak values ​​corresponding to the lateral scattered light. For example, based on the scattergram, the cells are classified as either eosinophils, neutrophils, lymphocytes, monocytes, or others. Cells classified as other than eosinophils, neutrophils, lymphocytes, and monocytes in the scattergram-based analysis are classified as, for example, "debris." These analyses are performed by analysis unit 302X (see Figure 7) as follows:

[0199] Figure 45 is another flowchart showing an example of the operation of this analysis method (second example of operation). As shown in Figure 45, the analysis unit 302X acquires data corresponding to the optical signal detected in the WDF channel (step S10). Next, the analysis unit 302X analyzes this data with the AI ​​algorithm 51 (step S11). Next, the analysis unit 302X identifies data corresponding to cells other than NRBC and BASO (step S12). Next, the analysis unit 302X analyzes the identified data using a scattergram (step S13). Next, the analysis unit 302X provides the analysis results of the WDF channel data (step S14).

[0200] In another example of the analysis method shown in Figure 46 (a third example), for example, lymphocyte classification and counting, monocyte classification and counting, eosinophil classification and counting, and neutrophil / basophil classification and counting are performed by scattergram analysis of waveform data obtained from measurements using WDF channels. In neutrophil / basophil classification and counting, for example, cells classified as either neutrophils or basophils are counted. Waveform data corresponding to cells not classified as lymphocytes, monocytes, eosinophils, or neutrophils / basophils, and cells classified as either neutrophils or basophils, are analyzed by AI algorithm 51 (see Figure 42). For example, the waveform data is classified by AI algorithm 51 into NRBCs, basophils (BASOs), and others. For example, the count of cells classified as either neutrophils or basophils by the scattergram analysis is subtracted from the count of cells classified as BASO by AI algorithm 60, and the counts of neutrophils and basophils are calculated, respectively. Cells that are not classified as either NRBC or BASO by the AI ​​analysis are classified as, for example, debris ("Debris"). These analyses are performed by analysis unit 302X (see Figure 7) as follows:

[0201] Figure 46 is a flowchart showing an example of the operation of this analysis method (third example of operation). As shown in Figure 46, the analysis unit 302X acquires data corresponding to the optical signal detected in the WDF channel (step S20). Next, the analysis unit 302X analyzes this data as a scattergram (step S21). Next, the analysis unit 302X identifies data corresponding to (1) cells that were not classified as lymphocytes, monocytes, eosinophils, or neutrophils / basophils, and (2) cells that were classified as neutrophils / basophils (step S22). Next, the analysis unit 302X analyzes the identified data with the AI ​​algorithm 60 (step S23). Next, the analysis unit 302X provides the analysis results of the WDF channel data (step S24).

[0202] (Fifth embodiment) The fifth embodiment discloses a sample analysis method in a sample analysis system (including the analysis system of the first to fourth embodiments) including a host processor and a parallel processing processor, wherein, based on control by the host processor, data relating to each component in the sample is acquired, parallel processing of the data is performed by the parallel processing processor, and information relating to each type of component is generated based on the results of the parallel processing.

[0203] According to this embodiment, even when analyzing a massive amount of data ranging from several hundred megabytes to several gigabytes per sample, the processing of the measured data can be executed in parallel by a parallel processing processor provided separately from the host processor. Therefore, even when processing a massive amount of data using a deep learning algorithm, for example, the data processing can be completed within the sample analysis system. For example, there is no need to transmit data to an analysis server storing the deep learning algorithm via the internet or intranet. Accordingly, according to this embodiment, there is no need to transmit large amounts of data from the sample analysis system to the analysis server and obtain analysis results returned from the analysis server, and the processing capacity of the sample analysis system can be maintained while improving the classification accuracy of components in the sample.

[0204] Figure 47 shows an example of the configuration of this embodiment. In Figure 47, elements similar to those in Figure 10 are denoted by the same reference numerals. The analysis unit 303X in Figure 47 is equipped with a parallel processing processor capable of processing calculations by AI algorithms in place of the master processor. By using a parallel processing processor suitable for matrix operations performed by AI algorithms (e.g., deep learning algorithms), the TAT (Turn Around Time) required for AI analysis is improved. In the following description, explanations of configurations and functions similar to those of the above-described embodiment will be omitted.

[0205] The analysis unit 302X shown in Figure 47 may be configured such that the analysis section 301X is provided within the measurement unit 401, as shown in Figure 11.

[0206] The processor 3001 uses the parallel processing processor 3002 to perform waveform data analysis processing using the deep learning algorithm 60. That is, the processor 3001 is programmed to perform waveform data analysis processing according to the deep learning algorithm 60. Analysis software 3100 for analyzing data corresponding to components in a sample based on the deep learning algorithm 60 may be stored in the storage unit 3004. In this case, the processor 3001 performs data analysis processing based on the deep learning algorithm 60 by executing the analysis software 3100 stored in the storage unit 3004. In this embodiment, for example, AI analysis is performed by the processor 3001 and the parallel processing processor 3002, while computational analysis is performed by the processor 3001 without using the parallel processing processor 3002. The processor 3001 is, for example, a CPU (Central Processing Unit). The processor 3001 may be, for example, an Intel Core i9, Core i7, Core i5, or an AMD Ryzen 9, Ryzen 7, Ryzen 5, or Ryzen 3.

[0207] Processor 3001 controls the parallel processing processor 3002. The parallel processing processor 3002 performs parallel processing, such as matrix operations, in response to control by processor 3001. In other words, processor 3001 is the master processor of the parallel processing processor 3002, and the parallel processing processor 3002 is the slave processor of processor 3001. Processor 3001 is also called the host processor or main processor.

[0208] The parallel processing processor 3002 executes multiple arithmetic operations in parallel, which are at least part of the processing related to waveform data analysis. The parallel processing processor 3002 is, for example, a GPU (Graphics Processing Unit), an FPGA (Field Programmable Gate Array), or an ASIC (Application Specific Integrated Circuit). If the parallel processing processor 3002 is an FPGA, it may have, for example, pre-programmed arithmetic operations related to a trained deep learning algorithm 60. If the parallel processing processor 3002 is an ASIC, it may have, for example, pre-built circuits for executing arithmetic operations related to a trained deep learning algorithm 60, or it may have a programmable module built in addition to such built-in circuits. The parallel processing processor 3002 may be, for example, an NVIDIA GeForce, Quadro, TITAN, Jetson, etc. If it is a Jetson series, for example, a Jetson Nano, Jetson Tx2, Jetson Xavier, or Jetson AGX Xavier can be used.

[0209] The processor 3001 performs calculations related to the control of the measurement unit 402 (see Figure 7), for example. The processor 3001 performs calculations related to control signals transmitted and received between the device mechanism unit 455, the sample preparation unit 440, and the sample aspiration mechanism 450, for example. The processor 3001 performs calculations related to the transmission and reception of information with the computer 310X, for example. The computer 310X has a function to display the analysis results transmitted from the analysis unit 303X based on the processing of the processor 3001. The computer 310X transmits a measurement order to the analysis unit 303X, for example. The measurement order is entered by the user, for example, via the input device of the computer 310X. The measurement order is transmitted from the host computer to the computer 310X, for example. The processor 3001 performs processing related to reading program data from the storage unit 3004, expanding the program into the main memory 3017, and transmitting and receiving data with the main memory 3017, for example. Each of the above processes performed by the processor 3001 is required to be executed in a predetermined order, for example. For instance, if the processes required to control the device mechanism 455, the sample preparation unit 440, and the sample aspiration mechanism 450 are A, B, and C, they may be required to be executed in the order B, A, C. Because the processor 3001 often performs sequential processes that depend on such an order, increasing the number of arithmetic units (sometimes called "processor cores," "cores," etc.) does not necessarily increase the processing speed.

[0210] On the other hand, the parallel processing processor 3002 performs routine and large-scale computational processing, such as operations on matrix data containing a large number of elements. In this embodiment, the parallel processing processor 3002 performs parallel processing of at least a portion of the process of analyzing waveform data according to the deep learning algorithm 60. The deep learning algorithm 60 includes, for example, a large number of matrix operations. The deep learning algorithm 60 may include, for example, at least 100 matrix operations, and may also include at least 1000 matrix operations. The parallel processing processor 3002 has multiple arithmetic units, each of which can perform matrix operations simultaneously. In other words, the parallel processing processor 3002 can perform matrix operations by each of the multiple arithmetic units in parallel as parallel processing. For example, the matrix operations included in the deep learning algorithm 60 can be divided into multiple operations that are not order-dependent. These divided operations can be executed in parallel by each of the multiple arithmetic units. These arithmetic units are sometimes called "processor cores," "cores," etc.

[0211] By performing such parallel processing, it is possible to speed up the overall computational processing of the measurement unit 402. Processing such as matrix operations included in the deep learning algorithm 60 is sometimes called "Single Instruction Multiple Data" (SIMD). The parallel processing processor 3002 is suitable for such SIMD operations. Such a parallel processing processor 3002 is sometimes called a vector processor.

[0212] As described above, processor 3001 is suitable for executing diverse and complex processes. On the other hand, parallel processing processor 3002 is suitable for executing a large amount of standardized processes in parallel. By executing a large amount of standardized processes in parallel, the TAT (Turn Around Time) required for computation is shortened. Note that the parallel processing performed by parallel processing processor 3002 is not limited to matrix operations. For example, when parallel processing processor 3002 performs learning processing according to the deep learning algorithm 50, differential operations related to the learning process may be subject to parallel processing.

[0213] The number of arithmetic units in processor 3001 can be, for example, dual-core (2 cores), quad-core (4 cores), or octa-core (8 cores). On the other hand, parallel processing processor 3002 has, for example, at least 10 arithmetic units (10 cores) and can perform 10 matrix operations in parallel. Some parallel processing processors 3002 have, for example, several tens of arithmetic units. Also, some parallel processing processors 3002 have, for example, at least 100 arithmetic units (100 cores) and can perform 100 matrix operations in parallel. Furthermore, some parallel processing processors 3002 have, for example, at least 1000 arithmetic units (1000 cores) and can perform 1000 matrix operations in parallel. Some parallel processing processors 3002 have, for example, several thousand arithmetic units.

[0214] Figure 48 shows an example configuration of a parallel processing processor 3002. The parallel processing processor 3002 includes multiple arithmetic units 3200 and RAM 3201. Each of the arithmetic units 3200 performs matrix data arithmetic processing in parallel. RAM 3201 stores data related to the arithmetic processing performed by the arithmetic units 3200. RAM 3201 is memory with a capacity of at least 1 gigabyte. RAM 3201 may also be memory with a capacity of 2 gigabytes, 4 gigabytes, 6 gigabytes, 8 gigabytes, or 10 gigabytes or more. The arithmetic units 3200 retrieve data from RAM 3201 and perform arithmetic processing. The arithmetic units 3200 are sometimes called "processor cores," "cores," etc.

[0215] Figures 49, 50, and 51 show an overview of the arithmetic processing performed by the parallel processing processor 3002 based on the control of the analysis software 3100 running on the processor 3001. Figure 49 shows an example configuration of the parallel processing processor 3002 that performs the arithmetic processing. The parallel processing processor 3002 has multiple arithmetic units 3200 and RAM 3201. The processor 3001, which runs the analysis software 3100, instructs the parallel processing processor 3002 to perform at least some of the arithmetic processing required when analyzing waveform data with the deep learning algorithm 60. The processor 3001 instructs the parallel processing processor 3002 to perform arithmetic processing related to the analysis of waveform data based on the deep learning algorithm 60. All or at least part of the waveform data corresponding to the signal detected by the FCM detection unit 460 (see Figure 4) is stored in the main memory 3017. The data stored in the main memory 3017 is transferred to the RAM 3201 of the parallel processing processor 3002. Data stored in main memory 3017 is transferred to RAM 3201, for example, by DMA (Direct Memory Access). Each of the multiple arithmetic units 3200 of the parallel processing processor 3002 performs arithmetic processing on the data stored in RAM 3201 in parallel. Each of the multiple arithmetic units 3200 retrieves the necessary data from RAM 3201 and performs arithmetic processing. Data corresponding to the calculation result is stored in RAM 3201 of the parallel processing processor 3002. Data corresponding to the calculation result is transferred from RAM 3201 to main memory 3017, for example, by DMA.

[0216] Figure 50 shows an overview of matrix operations performed by the parallel processing processor 3002. When analyzing waveform data according to the deep learning algorithm 60, matrix multiplication (matrix operations) is performed. The parallel processing processor 3002, for example, executes multiple matrix operations in parallel. Figure 50(a) shows the formula for matrix multiplication. In the formula shown in (a), matrix c is obtained by multiplying an n x n matrix a and an n x n matrix b. As illustrated in Figure 50, the formula is written using a multi-level loop construct. Figure 50(b) shows an example of operations executed in parallel by the parallel processing processor 3002. The formula illustrated in Figure 50(a) can be divided into n x n operations, for example, which is the number of combinations of the first-level loop variable i and the second-level loop variable j. Since each of these divided operations is independent of the others, they can be executed in parallel.

[0217] Figure 51 is a conceptual diagram illustrating how the multiple arithmetic operations exemplified in Figure 50(b) are executed in parallel by the parallel processing processor 3002. As shown in Figure 51, each of the multiple arithmetic operations is assigned to one of the multiple arithmetic units 3200 provided by the parallel processing processor 3002. Each of the arithmetic units 3200 executes its assigned arithmetic operations in parallel with the others. In other words, each of the arithmetic units 3200 executes the divided arithmetic operations simultaneously.

[0218] As illustrated in Figure 51, the parallel processing processor 3002 performs calculations to obtain, for example, information regarding the probability that a cell corresponding to waveform data belongs to each of several cell types. Based on the calculation results, the processor 3001, which executes the analysis software 3100, performs an analysis on the cell type of the cell corresponding to the waveform data. The calculation results are stored in the RAM 3201 of the parallel processing processor 3002 and transferred from the RAM 3201 to the main memory 3017. The processor 3001 retrieves the results of the analysis based on the calculation results stored in the main memory 3017 and stores them in the storage unit 3004.

[0219] The calculation of the probability that a component in a sample belongs to each of the multiple classification categories may be performed by a processor other than the parallel processing processor 3002. For example, the calculation results from the parallel processing processor 3002 may be transferred from RAM 3201 to main memory 3017, and the processor 3001 may calculate information regarding the probability that a component corresponding to each waveform data belongs to each of the multiple classification categories based on the calculation results read from main memory 3017. Alternatively, the calculation results from the parallel processing processor 3002 may be transferred from RAM 3201 to analysis unit 303X (see Figure 47), and the processor mounted on analysis unit 303X may calculate information regarding the probability that a component corresponding to each waveform data belongs to each of the multiple classification categories.

[0220] <Method of classifying white blood cells> 1. First fluorescent dye and second fluorescent dye Next, we will describe examples of the first and second fluorescent dyes. In these examples, we will describe the use of two types of fluorescent dyes, the first and second fluorescent dyes, to stain leukocytes in a sample. In particular, we will describe combinations of the first and second fluorescent dyes that are suitable for accurately counting basophils even in blood samples that have been collected some time ago.

[0221] The second fluorescent dye is a fluorescent dye that has a maximum absorption in a different wavelength range than the first fluorescent dye. That is, the second fluorescent dye is a fluorescent dye that emits fluorescence at a wavelength that can be detected separately from the fluorescence from the first fluorescent dye. The first and second fluorescent dyes can each be appropriately selected from known fluorescent dyes that have the property of binding to nucleic acids of blood cells such as leukocytes.

[0222] The first fluorescent dye and the second fluorescent dye are excited by light irradiated from a light source included in a flow cytometer. The light may be irradiated from one light source or two light sources. When the light is irradiated from one light source, the light irradiated from the light source may be light capable of exciting both the first fluorescent dye and the second fluorescent dye. Such light preferably includes light having a plurality of wavelengths, for example, white light. Alternatively, when the first fluorescent dye and the second fluorescent dye have maximum absorption in a wavelength range close enough to be excited by light of one wavelength, the light irradiated from the light source may be the light of the one wavelength. For example, when one of the first fluorescent dye or the second fluorescent dye has a maximum absorption in the wavelength range of 400 to 520 nm and the other has a maximum absorption in the wavelength range of 300 to 420 nm, both fluorescent dyes can be excited by light having a central wavelength of 400 to 420 nm, for example, light of 455 nm. Alternatively, for example, when the maximum absorption of the first fluorescent dye and the second fluorescent dye is within the wavelength range of 630 to 660 nm, one of the first fluorescent dye or the second fluorescent dye emits fluorescence having a peak in the wavelength range of 660 to 670 nm, and the other emits fluorescence having a peak in a wavelength range longer than 670 nm, both fluorescent dyes can be excited by light having a central wavelength of 630 to 655 nm, for example, light of 633 nm, and the light generated from each fluorescent dye can be detected separately.

[0223] When the light is irradiated from two light sources, the irradiated light may be two types of light, light of a first wavelength capable of exciting the first fluorescent dye and light of a second wavelength capable of exciting the second fluorescent dye. The second wavelength is different from the first wavelength. The wavelength can be appropriately determined according to the type of the fluorescent dye. For example, the first wavelength is 315 to 490 nm, preferably 400 to 450 nm, more preferably 400 to 410 nm. The second wavelength is 610 to 750 nm, preferably 620 to 700 nm, more preferably 633 to 643 nm.

[0224] When using two excitation lights, the first fluorescent dye has a maximum absorption in the wavelength range of 400 to 490 nm, and is a dye that is excited by absorbing light in this wavelength range to emit fluorescence, and a dye having a property of binding to nucleic acids (especially DNA) of blood cells is preferred. For example, fluorescent dyes having an acridine skeleton, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), Hoechst 3342, Hoechst 33258, Hoechst 334580 of the Hoechst series, etc. can be mentioned.

[0225] Examples of the fluorescent dye having an acridine skeleton include proflavine, 9-aminoacridine, acridine orange, acridine yellow G, acriflavine, basic yellow 9, ethacridine lactate, euchrysine GGNX, proflavine hemisulfate, 3,6-bis(dimethylamino)acridine (Rhoduline Orangen), 3,6-diamino-2,7,10-trimethyl-acridinium chloride. Among them, acridine yellow G is preferred. Alternatively, a commercially available fluorescent dye may be used as the second fluorescent dye.

[0226] When using two excitation lights, the second fluorescent dye is preferably a dye that has a maximum absorption in the wavelength range of 610 to 750 nm, is excited and emits fluorescence by absorbing light in this wavelength range, and has the property of binding to nucleic acids (especially RNA) of blood cells. For example, propidium iodide, ethidium bromide, ethidium acridine heterodimer, ethidium diazide, ethidium homodimer-1, ethidium homodimer-2, ethidium monoazide, trimethylene bis[[3-[[4-[[(3-methylbenzothiazole-3-ium)-2-yl]methylene]-1,4-dihydroquinoline]-1-yl]propyl]dimethylaminium]·tetraiodide (TOTO-1), 4-[(3-methylbenzothiazole-2(3H)-ylidene)methyl]-1-[3-(trimethylaminio)propyl]quinolinium·diiodide (TO Examples include -PRO-1), N,N,N',N'-tetramethyl-N,N'-bis[3-[4-[3-[(3-methylbenzothiazole-3-ium)-2-yl]-2-propenylidene]-1,4-dihydroquinoline-1-yl]propyl]-1,3-propanediaminium tetraiodide (TOTO-3) or 2-[3-[[1-[3-(trimethylamino)propyl]-1,4-dihydroquinoline]-4-ylidene]-1-propenyl]-3-methylbenzothiazole-3-ium diiodide (TOPRO-3), fluorescent dyes represented by the following formula (V), and combinations thereof.

[0227] [ka]

[0228] In formula (V), R 1 and R 4 R is a hydrogen atom, a methyl group, an ethyl group, or an alkyl group having 6 to 18 carbon atoms, where if one of them is an alkyl group having 6 to 18 carbon atoms, the other is a hydrogen atom, a methyl group, or an ethyl group. 2 and R 3are the same as or different from each other and are a methyl group, an ethyl group, a methoxy group or an ethoxy group. Z is a sulfur atom, an oxygen atom or a carbon atom having a methyl group. n is 0, 1, 2 or 3. X - is an anion.

[0229] In formula (V), the alkyl group having 6 to 18 carbon atoms may be either linear or branched. Among the alkyl groups having 6 to 18 carbon atoms, an alkyl group having 6, 8 or 10 carbon atoms is preferred.

[0230] In formula (V), R 1 and R 4 Examples of the substituent of the benzyl group include an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms or an alkynyl group having 2 to 20 carbon atoms. Among them, a methyl group or an ethyl group is particularly preferred.

[0231] In formula (V), R 2 and R 3 Examples of the alkenyl group of include an alkenyl group having 2 to 20 carbon atoms. Also, examples of the alkoxy group of R 2 and R 3 include an alkoxy group having 1 to 20 carbon atoms. Among them, a methoxy group or an ethoxy group is particularly preferred.

[0232] In formula (V), as the anion X - there may be mentioned halogen ions such as F - , Cl - , Br - and I - , CF3SO3 - , BF4 - , ClO4 - and the like. [[ID=4】7]

[0233] As the fluorescent dye represented by the above formula (V), a fluorescent dye represented by the following formula is preferred.

[0234] [[ID=】53]

Chemical formula

[0235] A commercially available staining reagent containing the above-mentioned second fluorescent dye alone may also be used. Examples include Fluorocell WDF (Sysmex Corporation) and Stomatizer 4DS (Sysmex Corporation).

[0236] When using a single excitation light, the preferred combination of the first and second fluorescent dyes may differ. For example, when using 633 nm light as the excitation light, a combination is possible in which the maximum absorption of the first and second fluorescent dyes is within the wavelength range of 630-660 nm, the second fluorescent dye emits fluorescence with a peak in the wavelength range of 660-670 nm, and the first fluorescent dye emits fluorescence with a peak in the wavelength range longer than 670 nm. As such a combination, for example, DRAQ5, DRAQ7, or DRAQ9 (BioStatus) can be used as the first fluorescent dye, and the fluorescent dye represented by the above formula (V) can be used as the second fluorescent dye.

[0237] The first and second fluorescent dyes are preferably used in solution. The solvent is not particularly limited as long as it can dissolve each of the above fluorescent dyes. Examples include water, organic solvents, and mixtures thereof. As organic solvents, solvents that can be mixed with water are preferred, such as C1-C6 alcohols, ethylene glycol, diethylene glycol, polyethylene glycol, and DMSO.

[0238] As described with reference to Figures 2 and 5, the first fluorescent dye and / or the second fluorescent dye may be contained in a single reagent container 200, or alternatively, as described with reference to Figure 6, they may be contained separately in different reagent containers 200A and 200B.

[0239] 2. Hemolytic reagent

[0240] The above-described first fluorescent dye and second fluorescent dye are used in combination with a hemolytic reagent, particularly preferably a hemolytic reagent containing a surfactant. The surfactant can hemolyze red blood cells in the specimen and cause damage to the cell membranes of white blood cells such that the first fluorescent dye and the second fluorescent dye can permeate. Examples of the surfactant include nonionic surfactants, cationic surfactants, and combinations thereof. The hemolytic reagent preferably contains a nonionic surfactant.

[0241] Examples of the nonionic surfactant include those represented by the following formula (I). R 1 -R 2 -(CH2CH2O) n -H (I) (In the formula, R 1 is an alkyl group, alkenyl group, or alkynyl group having 8 to 25 carbon atoms; R 2 is an oxygen atom, -(COO)-, or the following formula (II):

[0242]

Chemical formula

[0243] In formula (I), preferably n is 23 or 25, more preferably n is 23. When n is 23 to 25, the concentration of the nonionic surfactant represented by formula (I) in the hemolytic reagent is 1700 ppm or more, preferably 1750 ppm or more. Also, when n is 23 to 25, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 2300 ppm or less, preferably 2200 ppm or less.

[0244] When n is 30, the concentration of the nonionic surfactant represented by formula (I) in the hemolytic reagent is 1900 ppm or higher, preferably 2000 ppm or higher, and more preferably 2100 ppm or higher. Also, when n is 30, the concentration of the nonionic surfactant represented by formula (I) in the measurement sample is 2300 ppm or lower, preferably 2200 ppm.

[0245] Specific examples of nonionic surfactants represented by formula (I) include polyoxyethylene alkyl ethers, polyoxyethylene sterols, polyoxyethylene castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene polyoxypropylene alkyl ethers, and combinations thereof. Among these, polyoxyethylene alkyl ethers are preferred. The polyoxyethylene alkyl ether is preferably at least one selected from polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, polyoxyethylene (30) cetyl ether, and the group thereof. More preferably, it is polyoxyethylene (23) cetyl ether, polyoxyethylene (25) cetyl ether, and combinations thereof, and even more preferably, polyoxyethylene (23) cetyl ether. The hemolytic reagent may contain one type of nonionic surfactant or two or more types. Furthermore, the hemolytic reagent may further contain nonionic surfactants other than the nonionic surfactant represented by formula (I).

[0246] The hemolytic reagent may further contain a cationic surfactant. Examples of cationic surfactants include quaternary ammonium salt type surfactants, pyridium salt type surfactants, and combinations thereof. As a quaternary ammonium salt type surfactant, a surfactant with a total of 9 to 30 carbon atoms, represented by the following formula (III), is preferred. The hemolytic reagent may contain one type of cationic surfactant or two or more types.

[0247] [ka]

[0248] In formula (III), R 1 R is an alkyl or alkenyl group having 6 to 18 carbon atoms; 2 and R 3 These are alkyl or alkenyl groups having 1 to 4 carbon atoms, which are either identical or different from each other; R 4 X is an alkyl group, alkenyl group, or benzyl group having 1 to 4 carbon atoms. - It is a halogen ion.

[0249] In formula (III), R 1 The R is preferably an alkyl or alkenyl group having 6, 8, 10, 12, and 14 carbon atoms, and is particularly preferably a linear alkyl group. 1 Examples include the octyl group, decyl group, and dodecyl group. 2 and R 3 These are preferably a methyl group, an ethyl group, and a propyl group, which are either identical or different from each other. 4 The group is preferably a methyl group, an ethyl group, or a propyl group.

[0250] Examples of pyridium salt type surfactants include those represented by formula (IV).

[0251] [ka]

[0252] In formula (IV), R 1 X is an alkyl or alkenyl group having 6 to 18 carbon atoms; - It is a halogen ion.

[0253] In formula (IV), R 1 The R is preferably an alkyl or alkenyl group having 6, 8, 10, 12, and 14 carbon atoms, and is particularly preferably a linear alkyl group. 1Examples include the octyl group, decyl group, and dodecyl group.

[0254] The concentration of the cationic surfactant in the hemolytic reagent can be appropriately selected depending on the type of surfactant. The concentration of the cationic surfactant is 10 ppm or higher. The concentration of the cationic surfactant is preferably 400 ppm or higher, more preferably 500 ppm or higher, and even more preferably 600 ppm or higher. Also, the concentration of the cationic surfactant is 10,000 ppm or lower. The concentration of the cationic surfactant is preferably 1,000 ppm or lower, more preferably 800 ppm or lower, and even more preferably 700 ppm or lower.

[0255] Hemolytic reagents may contain buffering agents to maintain a constant pH. Examples include inorganic acid salts, organic acid salts, Good's buffers, and combinations thereof. Examples of inorganic acid salts include phosphates, borates, and combinations thereof. Examples of organic acid salts include citrates, malates, and combinations thereof. Examples of Good's buffers include MES, Bis-Tris, ADA, PIPES, Bis-Tris-Propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, Tricin, Tris, Bicine, TAPS, and combinations thereof.

[0256] The hemolytic reagent may further contain an aromatic organic acid. In this specification, an aromatic organic acid means an acid having at least one aromatic ring in its molecule and its salts. Examples of aromatic organic acids include aromatic carboxylic acids and aromatic sulfonic acids. Examples of aromatic carboxylic acids include phthalic acid, benzoic acid, salicylic acid, hippuric acid, their salts, and combinations thereof. Examples of aromatic sulfonic acids include p-aminobenzenesulfonic acid, benzenesulfonic acid, their salts, and combinations thereof. The hemolytic reagent may contain one or more aromatic organic acids. Furthermore, aromatic organic acids may exhibit buffering properties. When using an aromatic organic acid that exhibits buffering properties, the addition of a buffering agent is optional and may be combined with the buffering agents mentioned above.

[0257] When the hemolytic reagent contains an aromatic organic acid, the concentration of the aromatic organic acid is not particularly limited, but from the viewpoint of the ability to classify monocytes and lymphocytes, it is preferably 20 mM or higher, and more preferably 25 mM or higher. Furthermore, the concentration of the aromatic organic acid contained in the hemolytic reagent is preferably 50 mM or lower, and more preferably 45 mM or lower.

[0258] The hemolytic reagent is preferably a liquid reagent. The solvent is not particularly limited as long as it can dissolve each component, such as the surfactant mentioned above. Examples of solvents include water, organic solvents, and mixtures thereof. As organic solvents, solvents that are miscible with water are preferred, such as C1-C6 alcohols, ethylene glycol, diethylene glycol, polyethylene glycol, and dimethyl sulfoxide (DMSO).

[0259] The pH of the hemolytic reagent is not particularly limited, but a pH of 5.5 or higher is preferred. More preferably, it is 5.7 or higher, and even more preferably, 5.9 or higher. Furthermore, a pH of 7.2 or lower is preferred. More preferably, it is 6.9 or lower, and even more preferably, 6.6 or lower. Known bases (such as sodium hydroxide) or acids (such as hydrochloric acid) can be used to adjust the pH.

[0260] In hemolytic reagents, the osmotic pressure is not particularly limited, but from the viewpoint of hemolysis efficiency of red blood cells, it is preferably 150 mOsm / kg or less, more preferably 130 mOsm / kg or less, and most preferably 110 mOsm / kg or less. Appropriate osmotic pressure adjusting agents may be added to adjust the osmotic pressure. Examples of osmotic pressure adjusting agents include sugars, amino acids, organic solvents, sodium chloride, and combinations thereof.

[0261] Commercially available hemolysis reagents for blood cell counting may be used as the hemolysis reagent. Examples include LyzaCell WDF (Sysmex Corporation) and LyzaCell WDFII (Sysmex Corporation).

[0262] The hemolytic reagent is contained in, for example, reagent container R1 (see Figure 5) and delivered to chamber 420 by the method described above, where it is mixed with the blood sample.

[0263] 3.White blood cell classification method Next, as a sixth embodiment of the present invention, a method for classifying leukocytes into subpopulations using the first and second fluorescent dyes described above will be explained. This method is carried out, for example, by the steps shown in the flowchart of Figure 52. Each step will be described below. Figure 52 is an example of a flowchart for a method for classifying leukocytes into subpopulations, and Figure 53 is a flowchart of a preferred embodiment for realizing the method of Figure 52.

[0264] [Step S0: Step to prepare the sample for measurement] In this process, a sample for measurement is prepared by mixing a sample containing leukocytes with a hemolytic reagent containing a surfactant, a first fluorescent dye, and a second fluorescent dye. The hemolytic reagent, first fluorescent dye, and second fluorescent dye described above are preferably used.

[0265] The sample can be any sample that contains white blood cells or potentially contains blood cells, and is not limited to whole blood. Blood cells refer to cells known to be present in whole blood, such as white blood cells, red blood cells, and platelets. Samples that contain white blood cells or potentially contain blood cells are body fluid samples collected from mammals, preferably humans. Examples of samples include whole blood, ascites, synovial fluid, pleural fluid, cerebrospinal fluid, bone marrow fluid, bronchoalveolar lavage fluid, peritoneal lavage fluid, urine, and samples collected by apheresis, etc.

[0266] The first and second fluorescent dyes described above are mixed with the sample and hemolytic reagent so that their concentrations (final concentrations) in the measurement sample are within a predetermined range. The preferred upper limit for the final concentration of the first fluorescent dye in the measurement sample is 1000 ppm or less, preferably 100 ppm or less, and more preferably 10 ppm or less. The preferred lower limit for the final concentration of the first fluorescent dye in the measurement sample is 0.001 ppm or more, preferably 0.01 ppm or more, and more preferably 0.1 ppm or more. The preferred upper limit for the final concentration of the second fluorescent dye in the measurement sample is 1000 ppm or less, preferably 100 ppm or less, and more preferably 10 ppm or less. The preferred lower limit for the final concentration of the second fluorescent dye in the measurement sample is 0.001 ppm or more, preferably 0.01 ppm or more, and more preferably 0.1 ppm or more.

[0267] The mixing ratio of the hemolytic reagent, staining reagent, and sample is preferably, for example, 1000:1 or more:1 or more, expressed as a volume ratio. More preferably, it is 1000:10 or more:10 or more, and even more preferably, 1000:15 or more:15 or more. Furthermore, the mixing ratio of the hemolytic reagent, staining reagent, and sample is preferably, for example, 1000:50 or less:50 or less, expressed as a volume ratio. More preferably, it is 1000:30 or less:30 or less, and even more preferably, 1000:25 or less:25 or less. The mixing ratio of the fluorescent dye and the sample may be the same or different.

[0268] [Step S1: Step to detect optical information] In this step, light is irradiated onto the particles in the prepared sample, and optical information is detected, including first fluorescence information based on fluorescence from a first fluorescent dye, second fluorescence information based on fluorescence from a second fluorescent dye, and scattered light information. The optical information is preferably detected by the FCM detection unit 460 (see Figure 2). "Particles in the sample" refers to granular objects contained in the sample that can be individually measured by the FCM detection unit 460. Specifically, first, the sample is introduced into the flow cell 4113 of the FCM detection unit 460, and light is irradiated onto each particle in the sample as it passes through the flow cell. Then, the scattered light and fluorescence emitted from the particles are measured to detect the optical information. The particles in the sample may include not only cells such as leukocytes, but also non-cellular particles such as hemolyzed red blood cell remnants (red blood cell ghosts), platelet aggregates, and lipid particles.

[0269] The optical information detected consists of scattered light information and fluorescence information. Since this method uses two types of fluorescent dyes, fluorescence information corresponding to the fluorescence emitted from each fluorescent dye is detected. That is, first fluorescence information based on fluorescence from the first fluorescent dye and second fluorescence information based on fluorescence from the second fluorescent dye are detected. When light is irradiated from one light source, scattered light information detected by the irradiation of that light is detected. When light of the first wavelength and light of the second wavelength are irradiated from two light sources, respectively, first scattered light information detected by the irradiation of light of the first wavelength and second scattered light information detected by the irradiation of light of the second wavelength are detected as scattered light information.

[0270] Scattered light includes forward scattered light (e.g., scattered light with a reception angle of 0 to approximately 20 degrees) and side scattered light (e.g., scattered light with a reception angle of approximately 20 to approximately 90 degrees). Scattered light information and fluorescence information include the peak values ​​of scattered light and fluorescence (height of the pulse peak), pulse area, pulse width, transmittance, Stokes shift, ratio, changes over time, and values ​​correlated therewith. Forward scattered light information is not particularly limited as long as it reflects the size of the cell. Side scattered light information is not particularly limited as long as it reflects internal information such as the complexity of the cell structure, granule characteristics, nuclear structure, and degree of lobulation. Preferred scattered light information includes forward scattered light peak values ​​and side scattered light peak values, with side scattered light peak height being more preferred. Preferred fluorescence information includes fluorescence peak height.

[0271] The first scattered light information is preferably the lateral scattered light peak value detected by irradiation with light of a first wavelength (hereinafter also referred to as the "first lateral scattered light intensity"). The second scattered light information is preferably the lateral scattered light peak value detected by irradiation with light of a second wavelength (hereinafter also referred to as the "second lateral scattered light intensity"). The first fluorescence information is preferably the fluorescence peak value of the first fluorescent dye (hereinafter also referred to as the "first fluorescence intensity"). The second fluorescence information is preferably the fluorescence peak value of the second fluorescent dye (hereinafter also referred to as the "second fluorescence intensity").

[0272] For detecting optical information, for example, an FCM capable of detecting first fluorescence information based on fluorescence from a first fluorescent dye, second fluorescence information based on fluorescence from a second fluorescent dye, and scattered light information can be used. As such an FCM detection unit, for example, an FCM equipped with the optical system and detection system shown in Figures 7 and 8 can be used.

[0273] [Step S2: Selection of cell populations containing basophils] In this step, cell populations containing basophils are selected from the particles in the measurement sample based on optical information including first fluorescence information. This step is thought to allow for the removal of populations that would affect the basophil fractionation. As mentioned above, in samples collected some time ago, populations that would affect the basophil fractionation may be present. The inventors have found that there is a distinguishable difference between such populations and normal basophils in the first fluorescence information. For example, as shown in Figure 56(c) of Reference Example 1 described later, each subpopulation of normal leukocytes usually has approximately the same fluorescence intensity in a scattergram with first fluorescence intensity (blue-violet) and second lateral scattered light intensity (red) as two axes. Here, the dots on the scattergram represent individual particles measured by FCM. Note that the lateral scattered light may be the first lateral scattered light (blue-violet). On the other hand, as shown in Figures 57(a) to 57(c) of Reference Example 2, in samples collected some time ago, a group with a lower first fluorescence intensity than the subgroup of normal leukocytes appears. This group is also observed in the region where normal basophils appear on a scattergram with the second fluorescence intensity and lateral scattered light intensity as two axes, thus interfering with the fractionation of basophils. Therefore, by selecting cell populations containing basophils based on optical information including the first fluorescence information, it is possible to remove groups that affect the fractionation of basophils from the measured particles. In the sixth embodiment, it is preferable that the cell population containing basophils is a population of leukocytes containing basophils.

[0274] When normal leukocytes are stained with a hemolytic reagent and a first fluorescent dye, as described above, each subpopulation of leukocytes has approximately the same first fluorescence intensity. Therefore, cell populations containing basophils can be selected based on the first fluorescence information. Specifically, when the optical information containing the first fluorescence information is the first fluorescence intensity, a group of particles in the measurement sample whose first fluorescence intensity is greater than a predetermined threshold is selected. In this case, it is thought that cell populations containing basophils include lymphocytes, monocytes, neutrophils, eosinophils, and cell populations containing basophils.

[0275] Alternatively, as shown in steps S2-1 and S2-2 of the flowchart in Figure 53, a scattergram (also called a first scattergram) based on the first fluorescence information and scattered light information may be created, and cell populations containing basophils may be selected based on the scattergram. The scattered light information may be the first scattered light information. For example, when the optical information including the first fluorescence information is the first fluorescence intensity and the lateral scattered light intensity, a scattergram with the first fluorescence intensity and the lateral scattered light intensity as two axes can be used to select a population of particles that appear in a predetermined region where the first fluorescence intensity is greater than a predetermined threshold. Either the first lateral scattered light intensity or the second lateral scattered light intensity may be used. For example, as shown in Figure 56(c), in a scattergram with the first fluorescence intensity on the vertical axis and the second lateral scattered light intensity on the horizontal axis, each subpopulation of leukocytes is distributed in approximately a horizontal line. That is, the regions in which each subpopulation appears in the scattergram are also known. A predetermined region where the first fluorescence intensity is greater than a predetermined threshold can be pre-set as a region where a subpopulation of leukocytes, including basophils, appears. Such regions may be, for example, a region where five subpopulations of lymphocytes, monocytes, neutrophils, eosinophils, and basophils appear; a region where four subpopulations of lymphocytes, monocytes, neutrophils, and basophils appear; or a region where three subpopulations of lymphocytes, monocytes, and basophils appear. It is thought that by sorting the group of particles appearing in such regions by gating, a cell population of leukocytes, including basophils, can be selected.

[0276] The predetermined threshold corresponding to the first fluorescence intensity is not particularly limited and can be determined as appropriate. For example, blood known to contain basophils or blood collected some time ago may be measured in advance by FCM using the above-mentioned hemolytic reagent and first fluorescent dye to obtain the first fluorescence intensity for leukocytes, and the obtained value may be used as the predetermined threshold. Alternatively, the threshold and the region in which cell populations containing basophils appear may be predetermined by accumulating data on the regions in which each subpopulation of leukocytes appears from the first scattergram created based on such measurements.

[0277] [Step S3: The process of classifying the white blood cells contained in the cell population into subpopulations.] In this step, leukocytes included in a cell population containing basophils are classified into subpopulations based on the second fluorescence information and scattered light information. The scattered light information may be the second scattered light information. Since the cell population selection process described above removes populations that affect the basophil fractionation, it is considered that the leukocytes included in the selected cell population can be accurately classified into each subpopulation containing basophils. In this step, for example, as shown in S3-1 and S3-2 of the flowchart in Figure 53, a scattergram (also called a second scattergram) based on the second fluorescence information and scattered light information may be created, and the leukocytes included in a cell population containing basophils may be classified into subpopulations based on this scattergram. The scattered light information may be the second scattered light information. Specifically, a scattergram is created with the second fluorescence intensity and the lateral scattered light intensity as two axes. The lateral scattered light intensity may be the second lateral scattered light intensity. When the selected cell population is a population of leukocytes including basophils, i.e., a cell population containing lymphocytes, monocytes, neutrophils, eosinophils, and basophils, the scattergram may be divided into five subpopulations, appearing in different regions, as shown in Figure 56(a). This classification is achieved because each subpopulation differs in cell size, nucleic acid content, internal structure, etc., resulting in different secondary fluorescence intensities and lateral scattered light intensities for each subpopulation. Therefore, the selected cell population can be classified into a lymphocyte population, a monocyte population, a neutrophil population, an eosinophil population, and a basophil population.

[0278] Alternatively, when the selected cell population includes lymphocytes, monocytes, neutrophils, and basophils, the scattergram, with the second fluorescence intensity and the lateral scattered light intensity as the two axes, can be divided into four subpopulations, each appearing in a different region. That is, the selected cell population can be classified into a lymphocyte population, a monocyte population, a neutrophil population, and a basophil population.

[0279] Alternatively, when the selected cell population includes lymphocytes, monocytes, and basophils, in a scattergram with the second fluorescence intensity and lateral scattered light intensity as the two axes, each subpopulation can appear in different regions and be classified into three categories, as shown in Figure 59(b) of Example 1. That is, the selected cell population can be classified into a lymphocyte population, a monocyte population, and a basophil population.

[0280] [Step S4: Counting cells classified into basophil populations] In this step, cells classified as basophils from the leukocyte subpopulations classified as described above are counted. Since the cell populations that affect the basophil fraction are excluded in the above step of selecting the cell population containing basophils, it is considered that cells other than basophils (e.g., degraded neutrophils or lymphocytes) are substantially excluded from the cells classified as basophils. Therefore, this counting step allows for a more accurate count of basophils in the sample. It is preferable to obtain the number of cells classified as basophils by analyzing the above second scattergram with appropriate analysis software.

[0281] [Seventh Embodiment] The method for classifying leukocytes into subpopulations according to the seventh embodiment is carried out, for example, by the steps shown in the flowchart of Figure 54. The preparation of the measurement sample (step S10) and the detection of optical information (step S11) can be carried out in the same manner as in the sixth embodiment. In the seventh embodiment, between the step of detecting optical information (step S11) and the step of selecting cell populations containing basophils (steps S14, S15), a step of selecting cell populations containing nucleated cells from the particles in the measurement sample based on the first fluorescence information is performed (steps S12, S13). The first fluorescence information is preferably the first fluorescence intensity. Specifically, a histogram is created for the particles in the measurement sample, with the number of particles on the vertical axis and the first fluorescence intensity on the horizontal axis (step S12). Since the first fluorescent dye has the property of binding to the nucleic acid of cells as described above, when the particles are nucleated cells, the first fluorescence intensity is higher than that of particles that do not contain nucleic acid. Using this, particles in the above histogram whose first fluorescence intensity is above a predetermined threshold or within a predetermined range are selected as nucleated cells (step S13). By such selection, particles without a nucleus, such as red blood cell ghosts, can be removed from the particles in the measurement sample. The predetermined threshold and the lower limit of the predetermined range here may be values ​​sufficient to remove particles with a very low first fluorescence intensity, such as red blood cell ghosts. In the seventh embodiment, a cell population containing basophils can be selected from a cell population containing nucleated cells. The steps from the selection of the cell population containing basophils onward can be carried out in the same manner as in the sixth embodiment.

[0282] [Eighth Embodiment] The method for classifying leukocytes into subpopulations in the eighth embodiment is carried out, for example, by the steps shown in the flowchart of Figure 55. The preparation of the measurement sample (step S20) and the detection of optical information (step S21) can be carried out in the same manner as in the sixth embodiment. The selection of cell populations containing nucleated cells (steps S22, S23) can be carried out in the same manner as in the seventh embodiment. Whether or not to select cell populations containing nucleated cells can be arbitrarily decided. In the eighth embodiment, between the step of detecting optical information (step S21) and the step of selecting cell populations containing basophils (steps S27, S28), a step is performed to classify the particles or cell populations containing nucleated cells in the measurement sample into four subpopulations based on the second fluorescence information and scattered light information: a population of lymphocytes, a population of monocytes, a population of eosinophils, and a population containing both neutrophils and basophils (steps S24, S25). This classification itself can be carried out in the same manner as the step of classifying leukocytes contained in a cell population into subpopulations in the sixth embodiment. Specifically, a second scattergram is created based on the second fluorescence information and scattered light information (step S24), and based on this scattergram, the leukocytes contained in the particle or cell population containing nucleated cells in the sample are classified into subpopulations (step S25). If time has passed since the sample was collected, the leukocytes in the sample are poorly fractionated, and in particular, the fractionation of basophils and neutrophils becomes difficult. If the sample measured is such a poorly fractionated sample, in this classification step, the particle or cell population containing nucleated cells in the sample is classified into four subpopulations: a population of lymphocytes, a population of monocytes, a population of eosinophils, and a population containing both neutrophils and basophils. For example, particles classified into the population containing both neutrophils and basophils are counted. If the sample measured is a sample with good fractionation, in the classification step, the cell population containing particles or nucleated cells in the sample may be classified into five subpopulations: lymphocytes, monocytes, eosinophils, neutrophils, and basophils (Step S26).

[0283] In the eighth embodiment, after classifying the particle or cell population containing nucleated cells in the measurement sample into four subpopulations, the following steps are performed in the same manner as in the sixth embodiment: selecting the cell population containing basophils (steps S27, S28), classifying the leukocytes contained in the cell population into subpopulations (step S29), and counting the cells classified into the basophil population (step S30). Then, based on the results obtained from the step of classifying into four subpopulations and the results obtained from the step of counting the cells classified into the basophil population, the particle in the measurement sample is further classified into five subpopulations: lymphocyte population, monocyte population, eosinophil population, neutrophil population, and basophil population (step S31). For example, the number of particles classified into the neutrophil population can be obtained by subtracting the number of particles obtained in the step of counting cells classified into the basophil population (step S30) from the number of particles in the population containing both neutrophils and basophils obtained in the step of classifying into four subpopulations (step S25). As a result, in the eighth embodiment, the particles in the measurement sample can be classified into five subgroups: a population of lymphocytes, a population of monocytes, a population of eosinophils, a population of neutrophils, and a population of basophils. If, at the stage of classifying into four subgroups, the particles in the measurement sample can be classified into five subgroups of leukocytes, that classification result can be compared with the final classification result obtained in the eighth embodiment.

[0284] The leukocyte classification method according to the present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Examples]

[0285] The following describes the sample containing leukocytes, the hemolytic reagent, the staining dye, the analytical instrument, and the measurement method used in the example below.

[0286] [Sample containing white blood cells] (Whole blood sample) 5 mL of whole blood was collected from healthy volunteers using EDTA-2K blood collection tubes. After blood collection, the blood was stored at room temperature (25±2°C), and samples were taken from the blood collection tubes at 4 hours, 24 hours, 42 hours, and 72 hours later.

[0287] (Sample before and after basophil isolation) A 1 mL sample of a basophil-elevated sample (>2%) was prepared, and 0.5 mL of it was used as the sample before basophil isolation. The remaining sample was used to remove red blood cells using HetaSep (SCT ST-07906) and to collect white blood cells. Subsequently, basophil cells were collected using Basophil Isolation Kit II, human (Miltenyi Biotec 130-092-662), and these were used as the sample after basophil isolation.

[0288] [Hemolysis Reagent] We used LyzaCell WDFII (Sysmex Corporation).

[0289] [Staining reagents] (First fluorescent dye) Acridine Yellow G (Kanto Chemical Co., Ltd.) was used.

[0290] (Second fluorescent dye) As the second fluorescent dye, dye compound A described in U.S. Patent No. 6004816 was used. U.S. Patent No. 6004816 is incorporated herein by reference. Dye compound A is, in formula (V) above, R 1 is a methyl group, R 2 and R 3 is a hydrogen atom, and R 4 Here, n is n-octyl, n is 1, Z is a sulfur atom, and X - CF3SO3 - The compound was as follows, and its structural formula was as follows.

[0291] [ka]

[0292] As described in U.S. Patent No. 6004816, dye compound A could be obtained by the following steps: One equivalent of 3-methyl-2-methylbenzothiazolium methane sulfate and three equivalents of N,N-diphenylformamidine were heated and stirred in acetic acid on an oil bath at 90°C for 1.5 hours. The reaction mixture was poured into hexane, and the red oily substance was further suspended and washed with hexane to remove the acetic acid. The crude product was recrystallized in ethyl acetate-hexane (yield 48%). One equivalent of 1-octyllepidinium triflate and pyridine were added, and the mixture was heated and stirred on an oil bath at 90°C for 3 hours. The reaction mixture was concentrated, and the remaining blue crude product was purified with methanol-chloroform by flash chromatography to obtain dye compound A as a dark blue powder (yield 62%). The results of physical property tests (TLC, 1H-NMR, MASS, etc.) of this dark blue powder dye compound A are described in U.S. Patent No. 6004816. The maximum absorption spectrum of dye compound A was 629 nm.

[0293] A staining reagent was prepared by dissolving the above-mentioned dye compound A (27.5 mg) and acridine yellow G (25 mg) in special grade ethylene glycol (1 L).

[0294] [Analyzer] A modified version of the XN-1000 multi-parameter automated blood cell analyzer (Sysmex Corporation) was created. The XN-1000 was equipped with a semiconductor laser light source emitting red (633 nm) light, a detector for red forward scattered light, a detector for red side scattered light, and a detector that receives fluorescence (light above 660 nm) excited by red light. The XN-1000 was modified to include a semiconductor laser light source emitting blue-violet (405 nm) light, a detector for blue-violet scattered light, and a fluorescence detector that detects fluorescence (450-600 nm) excited by blue light. Furthermore, the analyzer was modified to analyze the measurement data detected for red and blue-violet scattered light information, as well as fluorescence information generated from the first and second fluorescent dyes, and to display a desired scattergram. Measurements were performed using the analyzer thus created.

[0295] [Measurement method] Except for using a staining reagent containing the first and second fluorescent dyes prepared as described above, instead of Fluorocell WDF (Sysmex Corporation), which is the staining reagent for XN-1000 (Sysmex Corporation), the preparation and measurement of the sample were performed according to the manual included with XN-1000. Data analysis was performed using FCS reanalysis software (Flowjo). The prepared sample contained 1000 μL of Lysacell WDFII as a hemolytic reagent, 17 μL of whole blood, and 20 μL of staining reagent. The dilution ratio of the staining reagent at this time was 51.85, and the final concentrations of the first and second fluorescent dyes in the sample were 0.53 ppm and 0.48 ppm, respectively.

[0296] Reference Example 1: Location of basophils Measurements were performed on the pre-basophil separation and post-basophil separation samples described above. From the obtained optical information, two scattergrams were created: a first scattergram with the horizontal axis representing the lateral scattered light intensity of the second wavelength (red wavelength) and the vertical axis representing the first wavelength (blue-violet fluorescence), and a second scattergram with the horizontal axis representing the lateral scattered light intensity of the second wavelength (red wavelength) and the vertical axis representing the second wavelength (red fluorescence).

[0297] The obtained scattergrams are shown in Figures 56(a) to 56(d). In the second scattergram (Figure 56(a)) of the sample before basophil isolation, the five types of leukocytes are distributed separately in both axial directions, indicating good fractionation. On the other hand, in the first scattergram (Figure 56(c)) of the sample before basophil isolation, the five types of leukocytes are distributed separately in the horizontal axis (lateral scattering) direction, but appear in a region with almost the same fluorescence intensity in the vertical axis (blue-violet fluorescence) direction. Furthermore, in the measurement of the sample after basophil isolation, basophils appeared in a region with lower fluorescence intensity than lymphocytes in the second scattergram (Figure 56(b)), and appeared in a position with the same fluorescence intensity as lymphocytes and monocytes in the first scattergram (Figure 56(d)), confirming the fractionation situation in the whole blood sample.

[0298] Reference Example 2: Location of clusters that change over time Measurements were performed on whole blood samples 4 hours, 48 ​​hours, and 72 hours after blood collection to detect optical information. Subsequently, the population included in the low-value region of the vertical axis (blue-violet fluorescence) that increased over time in the first scattergram was selected (gated), and this population was plotted on the second scattergram.

[0299] The results of the first scattergram are shown in Figures 57(a) to 57(d), and the results of the second scattergram are shown in Figures 58(a) to 58(c). From Figures 58(a) to 58(c), it can be seen that the number of cells included in the basophil appearance region increases with time elapsed after blood collection. It was found that the cause of this increase was the cells that were gated in the first scattergram (appearing in the area enclosed by a rectangle in Figures 57(a) to 57(c)). In other words, in the second scattergram, as time elapsed after blood collection, neutrophils shifted to the lower scattered light side, and the group thought to be derived from neutrophils that overlapped with the basophil appearance region was the group that appeared with lower blue-violet fluorescence in the first scattergram.

[0300] Example 1: Basophil counting 1 (single-step gating (no gating of nucleated cells by blue-violet fluorescence)) Measurements were performed on whole blood samples at 4 hours, 24 hours, 48 ​​hours, and 72 hours after blood collection to detect optical information. First, a first scattergram was created, and the populations included in a predetermined region (see Figure 59(a)) where the blue-violet fluorescence was greater than a specified value were selected. The populations appearing in this region include lymphocytes, monocytes, and basophils, but do not include neutrophil-derived cells, which shift to the basophil-preserving region over time after blood collection. The first scattergram for the sample collected 24 hours after blood collection is shown in Figure 59(a).

[0301] Subsequently, a second scattergram was created for the selected population. In this second scattergram, the population included in the predefined basophil appearance region was counted. The second scattergram for the samples collected 24 hours prior to blood collection is shown in Figure 59(b).

[0302] Example 2: Basophil counting 2 (2-step gating (with gating of nucleated cells using blue-violet fluorescence), corresponding to the second embodiment) and Comparative Example 1 The following analysis was performed using the optical information obtained in Example 1. First, cells (nucleated cells) whose blue-violet fluorescence exceeded a predetermined threshold were selected. The histogram for the sample 24 hours after blood collection is shown in Figure 60(a). From this point onward, the same analysis as in Example 1 was performed, and basophils were counted. The first and second scattergrams are shown in Figures 60(b) and 60(c).

[0303] On the other hand, basophils were counted based on a second scattergram created from the red side-scatter light intensity and red fluorescence intensity of the acquired optical information (Comparative Example 1).

[0304] Figure 61 shows the basophil count values ​​obtained by Example 2 and Comparative Example 1 for samples at various time intervals after blood collection. As can be seen from Figure 61, by employing the method of the present invention, the increase in basophil count values ​​was suppressed even in samples where time had elapsed since blood collection, compared to the comparative example.

[0305] Furthermore, Figure 62 shows the second scattergrams obtained using the methods of Example 2 and Comparative Example 1 for samples taken 4 hours and 48 hours after blood collection. As is clear from the scattergrams, the increase in basophils is suppressed in the method of the present invention.

[0306] Example 3: Five classifications of leukocytes Based on the optical information obtained in Example 1, leukocytes were classified into five subgroups. A second scattergram was created using the red side-scatter intensity and red fluorescence from the optical information, and the cells contained in each of the pre-defined regions—lymphocytes, monocytes, eosinophils, and neutrophils and basophils combined—were counted. In other words, at this stage, leukocytes were classified and counted into four groups.

[0307] Subsequently, similar to Example 1, a selection was made for a population containing lymphocytes, monocytes, and basophils in a first scattergram with blue-violet lateral scattered light on the horizontal axis and blue-violet fluorescence on the vertical axis. Then, for the selected population, the basophils were counted in a second scattergram with red lateral scattered light intensity on the horizontal axis and red fluorescence on the vertical axis. The basophil count obtained here was subtracted from the count of the population of neutrophils and basophils combined, which had been determined earlier, to count the neutrophil population. By combining this result with the previous result, the five subpopulations of leukocytes were counted.

[0308] Table 2 shows the counts of the five types of leukocytes at different time intervals after blood collection. Meanwhile, a second scattergram was created from the acquired optical information, specifically the red lateral scatter intensity and red fluorescence. The cells in the pre-defined regions for lymphocytes, monocytes, eosinophils, neutrophils, and basophils were counted (Comparative Example 2). In other words, the leukocytes were classified into five subgroups using the second scattergram created initially. The results obtained are also shown in Table 2.

[0309] [Table 2]

[0310] As is clear from Table 2, the present invention suppresses the increase in basophil count over time after blood collection. Furthermore, it was found that accurate 5-part leukocyte classification can be performed simultaneously with a single measurement. [Explanation of Symbols]

[0311] 54: Chamber, 60, 442: Reagent container holder, 60a, 60b, 60c, 60d, 60e: Holder part, 62: Reagent container holding part, 64: Piercer (suction tube), 100: Blood collection tube, 200, 200A, 200B, 300: Reagent container, 300X, 301X, 302X, 303X: Analytical device (analysis unit, analysis part), 310X: Computer, 400, 400X, 401, 402: Measuring device (measuring unit), 410, 410a, 410b: Reagent container, 420: Chamber, 430, 430a, 430b: Fluid delivery mechanism, 431, 431a, 431b: Fluid delivery tube, 431x, 431ax, 431bx: First end, 431y, 431ay, 431by: Second terminal, 432, 432a, 432b: Fluid delivery unit, 433: Pump (quantification unit), 440, 440A, 440B, 440X, 441: Sample preparation unit, 450: Sample aspiration mechanism, 451: Sample aspiration nozzle, 452: Aspiration discharge mechanism (pump), 460, 460x: FCM detection unit (detection unit), 4111: Light source, 4111a: First light source, 4111b: Second light source, 4113: Flow cell, 4116: Forward scattered light receiving element, 4118, 4118a, 4118b, 4118c: Dichroic mirror, 4121a, 4121b: Side scattered light receiving element 4122a,4122b: Lateral fluorescence photodetector, C: Cell (component), D1: First photodetector, D2: Second photodetector, D3: Third photodetector, F1,F2: Fluorescent dye, FC: Flow cell, FSC: Forward scattered light, SFL: Lateral fluorescence, SSC: Lateral scattered light

Claims

1. A method for classifying white blood cells into subpopulations, A step of preparing a sample for measurement by mixing a sample containing white blood cells, a hemolytic reagent containing a surfactant, a first fluorescent dye, and a second fluorescent dye, A step of irradiating the particles in the measurement sample with light and detecting optical information including first fluorescence information based on fluorescence from the first fluorescent dye, second fluorescence information based on fluorescence from the second fluorescent dye, and scattered light information, A step of selecting a cell population containing basophils from particles in the measurement sample based on optical information including the first fluorescence information, A step of classifying the leukocytes contained in the cell population into subpopulations based on the second fluorescence information and the scattered light information, A step of counting the number of cells classified as basophils from the aforementioned subpopulation, The second fluorescent dye is a fluorescent dye having a maximum absorption in a different wavelength range than the first fluorescent dye, and The aforementioned cell population is a population of white blood cells including basophils, A method for classifying the leukocytes into subpopulations, wherein the cell population is further classified into a population of lymphocytes, a population of monocytes, a population of neutrophils, a population of eosinophils, and a population of basophils.

2. A method for classifying leukocytes into subpopulations, A step of preparing a sample for measurement by mixing a sample containing white blood cells, a hemolytic reagent containing a surfactant, a first fluorescent dye, and a second fluorescent dye, A step of irradiating the particles in the measurement sample with light and detecting optical information including first fluorescence information based on fluorescence from the first fluorescent dye, second fluorescence information based on fluorescence from the second fluorescent dye, and scattered light information, A step of selecting a cell population containing basophils from particles in the measurement sample based on optical information including the first fluorescence information, A step of classifying the leukocytes contained in the cell population into subpopulations based on the second fluorescence information and the scattered light information, A step of counting the number of cells classified as basophils from the aforementioned subpopulation, The second fluorescent dye is a fluorescent dye having a maximum absorption in a different wavelength range than the first fluorescent dye, and In the step of detecting the optical information, the first fluorescent dye is irradiated with light of a first wavelength capable of exciting it, and the second fluorescent dye is irradiated with light of a second wavelength capable of exciting it. A method wherein the first wavelength is 315 to 490 nm and the second wavelength is 610 to 750 nm.

3. A method for classifying leukocytes into subpopulations, A step of preparing a sample for measurement by mixing a sample containing white blood cells, a hemolytic reagent containing a surfactant, a first fluorescent dye, and a second fluorescent dye, A step of irradiating the particles in the measurement sample with light and detecting optical information including first fluorescence information based on fluorescence from the first fluorescent dye, second fluorescence information based on fluorescence from the second fluorescent dye, and scattered light information, A step of classifying the particles in the measurement sample into four subgroups based on the second fluorescence information and the scattered light information: a group of lymphocytes, a group of monocytes, a group of eosinophils, and a group containing both neutrophils and basophils. A step of selecting a cell population containing basophils from particles in the measurement sample based on optical information including the first fluorescence information, A step of classifying the leukocytes contained in the cell population into subpopulations based on the second fluorescence information and the scattered light information, A step of counting the number of cells classified as basophils from the aforementioned subpopulation, A method comprising the above, wherein the second fluorescent dye is a fluorescent dye having a maximum absorption in a wavelength range different from that of the first fluorescent dye.

4. The optical information including the first fluorescence information is the fluorescence intensity of the first fluorescent dye. The method according to any one of claims 1 to 3, wherein in the step of selecting the cell population, a group of particles whose fluorescence intensity of the first fluorescent dye is greater than a predetermined threshold is selected.

5. The optical information including the first fluorescence information is the fluorescence intensity and lateral scattered light intensity of the first fluorescent dye. The method according to any one of claims 1 to 3, wherein, in the step of selecting the cell population, a group of particles appearing in a predetermined region where the fluorescence intensity of the first fluorescent dye is greater than a predetermined threshold is selected in a scattergram with the fluorescence intensity of the first fluorescent dye and the lateral scattered light intensity as two axes.

6. The aforementioned cell population is a population of white blood cells including basophils, The method according to any one of claims 2, 3, 4, and 5 (excluding those referenced in claim 1), wherein in the step of classifying the leukocytes into subpopulations, the cell population is classified into a population of lymphocytes, a population of monocytes, a population of neutrophils, a population of eosinophils, and a population of basophils.

7. The aforementioned cell population is a population that includes basophils, monocytes, and lymphocytes. The method according to any one of claims 2, 3, 4, and 5 (excluding those referenced from claim 1), wherein in the step of classifying the leukocytes into subpopulations, the cell population is classified into a population of lymphocytes, a population of monocytes, and a population of basophils.

8. The method according to any one of claims 1, 3, and 4 to 7 (excluding the one referred to in claim 2), wherein in the step of detecting the optical information, the first fluorescent dye is irradiated with light of a first wavelength capable of exciting the first fluorescent dye, and the second fluorescent dye is irradiated with light of a second wavelength capable of exciting the second fluorescent dye.

9. The method according to claim 8, wherein the first wavelength is 315 to 490 nm and the second wavelength is 610 to 750 nm.

10. The method according to any one of claims 1, 3, and 4 to 7 (excluding the one referenced in claim 2), wherein, in the step of detecting the optical information, light capable of exciting both the first fluorescent dye and the second fluorescent dye is irradiated.

11. The method according to any one of claims 1 to 10, wherein the first fluorescent dye is a compound having an acridine skeleton.

12. The method according to claim 11, wherein the compound having the acridine skeleton is at least one selected from the group consisting of proflavin, 9-aminoacridine, acridine orange, acridine yellow G, acrylflavin, basic yellow 9, ethacridine lactate, euchrysine GGNX, proflavin hemisulfate, 3,6-bis(dimethylamino)acridine, and 3,6-diamino-2,7,10-trimethylacridinium chloride.

13. The second fluorescent dye is given by the following formula (V): 【Chemistry 1】 (In the formula, R 1 and R 4 R is a hydrogen atom, a methyl group, an ethyl group, or an alkyl group having 6 to 18 carbon atoms, where if one is an alkyl group having 6 to 18 carbon atoms, the other is a hydrogen atom, a methyl group, or an ethyl group; 2 and R 3 are, either identical or different from each other, a methyl group, an ethyl group, a methoxy group, or an ethoxy group; Z is a sulfur atom, an oxygen atom, or a carbon atom having a methyl group; n is 0, 1, 2, or 3; X - (This is an anion.) The method according to any one of claims 1 to 12, wherein the compound is represented by the compound.

14. The method according to any one of claims 1 to 13, further comprising the step of selecting a cell population containing nucleated cells from particles in the measurement sample based on the first fluorescence information, between the step of detecting the optical information and the step of selecting the cell population.

15. The method according to any one of claims 1, 2, and 4 to 14 (excluding the one referenced in claim 3), further comprising the step of classifying the particles in the measurement sample into four subpopulations based on the second fluorescence information and the scattered light information, between the step of detecting the optical information and the step of selecting the cell population.

16. The method according to claim 15, further comprising the step of classifying the particles in the measurement sample into five subgroups: a population of lymphocytes, a population of monocytes, a population of eosinophils, a population of neutrophils, and a population of basophils, based on the results obtained by the step of classifying into the four subgroups and the results obtained by the step of counting the cells classified into the population of basophils.

17. The method according to any one of claims 1 to 16, wherein the hemolytic reagent is a reagent for hemolyzing red blood cells and causing damage to the cell membrane of white blood cells to such an extent that the first fluorescent dye and the second fluorescent dye can penetrate it.

18. The hemolytic reagent is a surfactant of the following formula (I): R 1 -R 2 -(CH 2 CH 2 O) n -H (I) (In the formula, R 1 R is an alkyl group, alkenyl group, or alkynyl group having 8 to 25 carbon atoms; 2 is an oxygen atom, -(COO)- or formula (II) below: 【Chemistry 2】 (and n is 23 or greater, 25 or less, or 30.) The method according to any one of claims 1 to 17, comprising a nonionic surfactant represented by [the specified formula].

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