Particle analysis method and particle analyzer

Abstract

This invention provides a means to obtain more clinically useful information when analyzing particles contained in blood samples using metachromatic positive dyes. In a particle analysis method for analyzing particles contained in blood samples, particles are stained with a metachromatic positive dye; the stained particles are irradiated with light; the intensity of a first fluorescence from the stacking component of the metachromatic positive dye and the intensity of a second fluorescence from the embedded component of the metachromatic positive dye are measured, the first and second fluorescence being emitted by each particle contained in the blood sample; the intensities of the first and second fluorescence emitted by each particle are normalized according to the size of each particle to obtain the fluorescence concentration of each of the first and second fluorescence in each particle; in a two-dimensional plot of the normalized fluorescence concentrations, each particle is clustered into multiple particle clusters, including at least two of erythrocyte clusters, platelet clusters, and nucleated cell clusters; and a histogram is created where the intensity of the first fluorescence is a histogram of the RNA amount of the class and the intensity of the second fluorescence is a histogram of the DNA amount of the class included in at least one of the multiple particle clusters.

Description

Technical Field

[0001] This invention relates to a particle analysis method and a particle analyzer. Background Technology

[0002] Conventionally, particles contained in blood samples are classified and counted. For example, in an optically automated hematology analyzer, flow cytometry is used to analyze particles in blood samples. Specifically, in flow cytometry, information such as the amount of intracellular nucleic acids can be obtained in addition to the number and size of cells by applying light from a light source (such as a laser) to cells stained with fluorescent dyes flowing through a cell, using information of scattered light and fluorescence. Flow cytometry has been used in fully automated hematology counters for measuring reticulocytes (RETs), where nucleic acids in reticulocytes are stained with nucleic acid-staining fluorescent dyes to detect differences from mature red blood cells by fluorescence intensity (Non-Patent Literature 1). Here, reticulocytes are enucleated immature red blood cells with RNA retained within them, which mature into mature red blood cells after 24 to 48 hours, and the RNA gradually disappears accordingly. Therefore, measuring the number (and ratio) of reticulocytes is important for assessing the hematopoietic capacity of red blood cells in the bone marrow. The degree of maturity of reticulocytes was divided into three fractions proportional to RNA content, and these fractions were collectively evaluated as the immature reticulocyte fraction (IRF). When the ratio of IRF is compared with the number of reticulocytes, it can be used clinically to differentiate hemolytic anemia, aplastic anemia, etc., and as an indicator of hematopoietic status after chemotherapy for cancer (Non-Patent Literature 2).

[0003] Furthermore, there exists an analytical parameter called the immature platelet fraction (IPF), which utilizes the detection principle of reticulocytes. Attempts to detect immature platelets freshly released from the bone marrow have been ongoing for a long time, and numerous reports exist on reticulated platelets (RP). RP is said to contain more RNA in its cytoplasm compared to mature platelets and was initially reported as platelets stained with a novel methylene blue dye, observed under a microscope. Subsequently, a method for measuring RP stained with nucleic acid dyes using flow cytometry has been developed, and it has been shown that RP increases during bone marrow recovery following idiopathic thrombocytopenic purpura (ITP) and chemotherapy or hematopoietic stem cell transplantation. RP in peripheral blood is considered to reflect the bone marrow's platelet-producing capacity and is useful for estimating bone marrow platelet-producing capacity without bone marrow aspiration, for example, in cases where bone marrow examination is difficult. For RP measurement, IPF analysis systems are used clinically, applying the principle of reticulocyte measurement functions integrated into ordinary blood analyzers. The degree of maturity of this immature platelet fraction (IPF) was also divided into three fractions, and within the divided regions, the higher the degree of immaturity, the better, based on the descending order of fluorescence intensity.

[0004] Conventionally, numerous techniques are known for measuring immature reticulocyte fraction (IRF) and immature platelet fraction (IPF) using flow cytometry-based automated hematology analyzers. However, all clinically used devices emit fluorescence of a single color as a fluorescent dye. Therefore, the nucleic acid information to be obtained is limited to information obtained from a single fluorescent wavelength band. Consequently, the nucleic acid information to be obtained includes not only information derived from RNA but also information derived from DNA, and it is impossible to extract only RNA information from this data. As a result, the measured values ​​of IRF and IPF are considered to be greater than the true values, and there is a problem that the true immaturity of reticulocytes and reticulocytes cannot be measured.

[0005] DNA information obtained as a result of blood sample analysis is considered a potential indicator of abnormalities in blood cells (e.g., the presence of various bodies such as Howell-Jolly bodies and Pappenheimer bodies, Plasmodium, Babesia, Theileria, Trypanosoma, and microflagellates of Filarial worms). However, because the nucleic acid information obtained from conventional automated blood cell analyzers must be a mixture of RNA and DNA information, it is impossible to make potentially useful DNA information readily apparent.

[0006] Incidentally, a technique has been proposed (Patent Document 1) for analyzing blood samples stained with acridine orange (AO) using a flow cytometry-based automated hematology analyzer. Acridine orange is a fluorescent dye (metachromatic positive dye) that exhibits a modulation phenomenon (metachromaticity) in which tissue components exhibit a stainability different from the original hue of the dye. Specifically, Patent Document 1 discloses a technique for normalizing the fluorescence intensity of AO-stained blood cells according to cell size and shape based on flow cytometry results, obtaining the fluorescence concentration of each cell, and classifying cells in a blood sample based on the fluorescence concentration of each cell obtained thereby. Then, according to the technique described in Patent Document 1, it is said that a parameter indicating immaturity can be calculated from reticulocytes classified by the above method based on the amount of RNA in reticulocytes. Furthermore, it is said that the number of reticulocytes that can be provided as an indicator of immaturity based on the amount of RNA in platelets can be measured from similarly classified platelets.

[0007] Reference List

[0008] Patent documents

[0009] Patent Document 1: US2009 / 0130647A1

[0010] Non-patent literature

[0011] Non-patent literature 1: Yasunori Abe et al., Journal of Thrombosis and Hemostasis, The Japanese Society on Thrombosis and Hemostasis, 18(4):289-301 (2007)

[0012] Non-patent document 2: Takayuki Takubo, The Journal of the Japanese Society of Internal Medicine, The Japanese Society of Internal Medicine, Vol. 100, No. 11: 3230-3239 (2011) Summary of the Invention

[0013] Technical issues

[0014] However, the technique described in Patent Document 1 simply classifies (clusters) cells in a blood sample based on the fluorescence concentration of each cell normalized according to the size and shape of the particles in the blood sample. Therefore, sufficient clinically useful information has not yet been obtained.

[0015] Therefore, the object of the present invention is to provide a means to obtain more clinically useful information when using metachromatic orthochromatic dyes to analyze particles contained in blood samples.

[0016] Solution to the problem

[0017] In view of the above problems, the inventors have conducted diligent research. As a result, the inventors have discovered that the above problems can be solved by classifying (clustering) particles in a blood sample into multiple particle clusters based on fluorescence concentration normalized according to the size of each particle contained in the blood sample, and then creating a first fluorescence intensity as a histogram of RNA amount and a second fluorescence intensity as a histogram of DNA amount for at least one particle cluster included in the multiple particle clusters, and thus the present invention has been completed.

[0018] In other words, according to one aspect of the present invention, a particle analysis method is provided for analyzing particles contained in a blood sample. The particle analysis method includes: staining the particles with a metachromatic positive dye; irradiating the stained particles with light; measuring the intensity of a first fluorescence from a stacking component of the metachromatic positive dye and the intensity of a second fluorescence from an embedded component of the metachromatic positive dye, the first and second fluorescence being emitted by each particle contained in the blood sample; normalizing the intensity of the first and second fluorescence emitted by each particle according to the size of each particle to obtain a fluorescence concentration of each of the first and second fluorescence in each particle; and clustering each particle into a plurality of particle clusters in a two-dimensional graph of the fluorescence concentrations obtained by normalization, the plurality of particle clusters including at least two of erythrocyte clusters, platelet clusters, and nucleated cell clusters. The particle analysis method includes creating a histogram of the intensity of the first fluorescence as a histogram of the RNA amount of the cluster and a histogram of the intensity of the second fluorescence as a histogram of the DNA amount of the cluster for at least one particle cluster included in the plurality of particle clusters.

[0019] According to another aspect of the present invention, as an apparatus capable of implementing the particle analysis method according to the above aspects of the present invention, a particle analyzer is also provided, the particle analyzer comprising: a light source that applies light to particles contained in a blood sample; a flow cell through which the blood sample flows; a photodetector comprising a plurality of fluorescence detectors that detect each of the intensities of a first fluorescence and a second fluorescence having different wavelengths; and a data processing unit that normalizes the intensities of the first fluorescence and the second fluorescence emitted by each of the particles contained in the blood sample according to the size of each of the particles to determine each fluorescence concentration of the first fluorescence and the second fluorescence in each of the particles, and in a two-dimensional graph of fluorescence concentration obtained by normalization, clusters each of the particles into a plurality of particle clusters including at least two of erythrocyte clusters, platelet clusters, and nucleated cell clusters, and creates a histogram of the intensity of the first fluorescence as the RNA amount of the cluster and a histogram of the intensity of the second fluorescence as the DNA amount of the cluster for at least one particle cluster included in the plurality of particle clusters.

[0020] According to another aspect of the present invention, as a particle analysis method similar to the above-described aspects of the present invention, replacing the features of the above-described aspects, a method is provided, the method comprising: for at least one particle cluster included in a plurality of particle clusters, calculating a fluorescence intensity ratio, the fluorescence intensity ratio being a ratio of the intensity of a first fluorescence and a second fluorescence of each particle included in the particle cluster; and creating a fluorescence intensity ratio histogram of the class wherein the fluorescence intensity ratio is a fluorescence intensity ratio histogram.

[0021] According to another aspect of the present invention, as an apparatus capable of implementing the particle analysis method according to the above aspects of the present invention, a particle analyzer is also provided, the particle analyzer comprising: a light source that applies light to particles contained in a blood sample; a flow cell through which the blood sample flows; a photodetector comprising a plurality of fluorescence detectors that detect each of the intensities of a first fluorescence and a second fluorescence having different wavelengths; and a data processing unit that normalizes the intensities of the first fluorescence and the second fluorescence emitted by each particle contained in the blood sample according to the size of each particle to determine the fluorescence concentration of the first fluorescence and the second fluorescence in each particle, and in a two-dimensional graph of fluorescence concentration obtained by normalization, clusters each particle into a plurality of particle clusters including at least two of erythrocyte clusters, platelet clusters, and nucleated cell clusters, and for at least one particle cluster included in the plurality of particle clusters, calculates a fluorescence intensity ratio, which is the ratio of the intensity of the first fluorescence and the intensity of the second fluorescence of each particle included in the particle cluster, and creates a fluorescence intensity ratio histogram of the class.

[0022] According to the present invention, more clinically useful information can be obtained when analyzing particles contained in blood samples using metachromatic orthochromatic dyes. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the preparation of the measurement sample.

[0024] Figure 2 This is a diagram illustrating the system configuration of an apparatus for performing a particle analysis method according to one aspect of the present invention.

[0025] Figure 3 This is a system diagram illustrating an outline of a flow cytometer as an embodiment of an apparatus for performing a particle analysis method according to one aspect of the present invention.

[0026] Figure 4 This is a measurement example of a two-dimensional scatter plot (FS×SS cell histogram) of forward-scattered (FS) and side-scattered (SS) light.

[0027] Figure 5 This is an example of the measurement of a two-dimensional scatter plot (FL1×FL2 cell histogram) of the first fluorescence (FL1) and the second fluorescence (FL2).

[0028] Figure 6 It is a two-dimensional graph (also referred to as the “RNP graph” in this specification) of the fluorescence concentration (CRc) of the first fluorescence (FL1) and the fluorescence concentration (CDc) of the second fluorescence (FL2) created based on the results obtained by determining CRc and CDc in each particle through a “normalization” process.

[0029] Figure 7A Through gating from Figure 6 The RNP diagram shown contains histograms of RNA and DNA quantities created by separating several particle clusters. Specifically, Figure 7A This is a histogram of RNA quantity used for erythrocyte clustering (RBCn). Figure 7A (left side of the middle) and DNA quantity histogram ( Figure 7A (The right part of the middle).

[0030] Figure 7B Through gating from Figure 6 The RNP diagram shown contains histograms of RNA and DNA quantities created by separating several particle clusters. Specifically, Figure 7B This is a histogram of RNA quantity used for platelet clustering (PLTn). Figure 7B (left side of the middle) and DNA quantity histogram ( Figure 7B (The right part of the middle).

[0031] Figure 7C Through gating from Figure 6The RNP diagram shown contains histograms of RNA and DNA quantities created by separating several particle clusters. Specifically, Figure 7C This is a histogram of RNA quantity used for clustering of nucleated cells (NCn). Figure 7C (left side of the middle) and DNA quantity histogram ( Figure 7C (The right part of the middle).

[0032] Figure 8A This is an example of a two-dimensional plot created for a particle cluster (in this case, a erythrocyte cluster (RBCn)) for which RNA and DNA quantity histograms have been created. In the two-dimensional plot, the intensity of the first fluorescence (FL1) or the intensity of the second fluorescence (FL2) for each particle included in the particle cluster is one axis, and the size of each particle included in the particle cluster is another axis.

[0033] Figure 8B This is an example of a two-dimensional plot created for a particle cluster (in this case, a platelet cluster (PLTn)) for which RNA and DNA quantity histograms have been created. In the two-dimensional plot, the intensity of the first fluorescence (FL1) or the intensity of the second fluorescence (FL2) for each particle included in the particle cluster is one axis, and the size of each particle included in the particle cluster is another axis.

[0034] Figure 8C This is an example of a two-dimensional plot created for a particle cluster (in this case, a nucleated cell population (NCn)) for which RNA and DNA quantity histograms have been created. In the two-dimensional plot, the intensity of the first fluorescence (FL1) or the intensity of the second fluorescence (FL2) for each particle included in the particle cluster is one axis, and the size of each particle included in the particle cluster is another axis.

[0035] Figure 9A The example described later illustrates an RNP plot created when the invention is applied to a blood sample with no morphological findings to measure the reticulocyte fraction.

[0036] Figure 9B The following examples illustrate an RNA quantity histogram created when the invention was applied to blood samples without morphological findings to measure reticulocyte fraction. Figure 9B (left side of the middle) and DNA quantity histogram ( Figure 9B (The right part of the middle).

[0037] Figure 9CThe two-dimensional graph created in the example described later by applying the invention to a blood sample with no morphological findings to measure the reticulocyte fraction includes the intensity of the first fluorescence (FL1) of each particle in the erythrocyte cluster or platelet cluster as the horizontal axis and the size of each particle included in the cluster as the vertical axis.

[0038] Figure 9D The two-dimensional graph created in the example described later by applying the invention to a blood sample with no morphological findings to measure the reticulocyte fraction includes the intensity of the second fluorescence (FL2) of each particle in the erythrocyte cluster or platelet cluster as the horizontal axis and the size of each particle included in the cluster as the vertical axis.

[0039] Figure 10A The example described later is an RNP plot created when the invention is applied to a blood sample with morphological findings of erythrocytes to measure the reticulocyte fraction.

[0040] Figure 10B The following embodiments illustrate an RNA quantity histogram created when the invention is applied to blood samples with morphological findings of erythrocytes to measure reticulocyte fraction. Figure 10B (left side of the middle) and DNA quantity histogram ( Figure 10B (The right part of the middle).

[0041] Figure 10C The two-dimensional graph created in the example described later by applying the invention to a blood sample with morphological findings of erythrocytes to measure the reticulocyte fraction includes the intensity of the first fluorescence (FL1) of each particle in the erythrocyte cluster or platelet cluster as the horizontal axis and the size of each particle included in the cluster as the vertical axis.

[0042] Figure 10D The two-dimensional graph created in the example described later by applying the invention to a blood sample with morphological findings of erythrocytes to measure the reticulocyte fraction includes the intensity of the second fluorescence (FL2) of each particle in the erythrocyte cluster or platelet cluster as the horizontal axis and the size of each particle included in the cluster as the vertical axis.

[0043] Figure 11A The RNP diagram is created in the example described later when the second aspect of the invention is applied to a blood sample in which no red blood cell morphology was found.

[0044] Figure 11BThe fluorescence intensity ratio histogram is created in the example described later by applying the second aspect of the invention to a blood sample in which no red blood cells were morphologically detected, and wherein the fluorescence intensity ratio (FL1 / FL2) (which is the value of the ratio of the intensity of the first fluorescence (FL1) to the intensity of the second fluorescence (FL2) of each particle included in the red blood cell cluster) is a class.

[0045] Figure 11C yes Figure 11B A magnified view of the region near the threshold (FL1 / FL2 = 2.0).

[0046] Figure 12A The RNP diagram is created in the example described later when the second aspect of the invention is applied to a blood sample with red blood cell morphological findings (HJ bodies) for measurement.

[0047] Figure 12B In the example described later, a fluorescence intensity ratio histogram is created by measuring a blood sample with morphological findings (HJ bodies) of erythrocytes, and wherein the fluorescence intensity ratio (FL1 / FL2) (which is the value of the ratio of the intensity of the first fluorescence (FL1) to the intensity of the second fluorescence (FL2) of each particle included in the erythrocyte cluster) is used as a class.

[0048] Figure 12C yes Figure 12B A magnified view of the region near the threshold (FL1 / FL2 = 2.0).

[0049] Figure 13A The RNP plot is created in the example described later when the second aspect of the invention is applied to a blood sample with red blood cell morphological findings (nucleated red blood cells (NRBC)) for measurement.

[0050] Figure 13B The fluorescence intensity ratio histogram is created in the example described later by applying the second aspect of the invention to a blood sample with morphological findings of red blood cells (nucleated red blood cells (NRBC)) for measurement, and wherein the fluorescence intensity ratio (FL1 / FL2) (which is the value of the ratio of the intensity of the first fluorescence (FL1) to the intensity of the second fluorescence (FL2) of each particle included in the red blood cell cluster) is a class.

[0051] Figure 13C yes Figure 12B A magnified view of the region near the threshold (FL1 / FL2 = 2.0). Detailed Implementation

[0052] In the following description, embodiments of the invention will be illustrated with reference to the accompanying drawings.

[0053] One aspect (first aspect) of the present invention is a particle analysis method for analyzing particles contained in a blood sample, comprising: staining the particles with a metachromatic positive dye; irradiating the stained particles with light; measuring the intensity of a first fluorescence of a stacked component derived from the metachromatic positive dye and the intensity of a second fluorescence of an embedded component derived from the metachromatic positive dye, the first fluorescence and the second fluorescence being emitted by each particle contained in the blood sample; normalizing the intensity of the first fluorescence and the second fluorescence emitted by each particle according to the size of each particle to obtain the fluorescence concentration of each of the first fluorescence and the second fluorescence in each particle; and clustering each particle into a plurality of particle clusters in a two-dimensional graph of the fluorescence concentrations obtained by normalization, the plurality of particle clusters including at least two of erythrocyte clusters, platelet clusters and nucleated cell clusters, and creating a histogram of the intensity of the first fluorescence as a histogram of the RNA amount of the cluster and the intensity of the second fluorescence as a histogram of the DNA amount of the cluster for at least one particle cluster included in the plurality of particle clusters.

[0054] In the following description, preferred embodiments for implementing the particle analysis method according to this aspect will be specifically described with reference to the use of flow cytometry for analysis. However, the scope of the invention should be determined based on the description in the claimed scope, and is not limited to the specific embodiments described below.

[0055] Figure 1 This is a schematic diagram illustrating the preparation of a measurement sample. In the particle analysis method according to the invention, firstly, a sample containing particles from blood (a blood sample) is provided, and a measurement sample is prepared using a predetermined positive dye (a heterochromatic positive dye) (typically, the dye and the blood sample are mixed). As a result, the particles contained in the blood sample are stained with the predetermined positive dye. In particle analysis performed by flow cytometry, the measurement sample prepared above is irradiated with light, and the scattered light and fluorescence generated from the particles contained in the sample are detected as electrical signals. Then, based on the detected electrical signals, the particles contained in the sample are analyzed.

[0056] (Preparation of measurement samples)

[0057] In this aspect, as described above, a sample containing particles in the blood (blood sample) is provided as the sample to be measured (measurement sample), and the measurement sample is prepared using a predetermined positive dye (metachromatic positive dye) (typically, the dye is mixed with the blood sample). In this case, for example, as... Figure 1As shown, when the required amount of primary color dye 10 is dispensed in a fixed quantity, the primary color dye is heated in the range of 20 to 50°C. A sample (blood sample) 20 containing particles from blood is added to the thus heated and dispensed primary color dye 10A, and the mixture is stirred for 5 to 10 seconds. The resulting measurement sample 30 is then kept warm in the range of 20 to 50°C for 10 to 40 seconds. As a result, in this invention, the preparation of the measurement sample 30 can be completed within 15 to 60 seconds.

[0058] In this case, for example, the metachromatic normal dye 10 is dispensed in 1 mL increments, and blood sample 20 is added to the dispensed metachromatic normal dye 10A in 2 μL increments, the blood sample being prepared such that the number of particles to be measured is approximately 1 × 10⁻⁶. 7 Particles / μL. As the metachromatic positive dye 10, for example, 0.5 to 1.5 mg / dL of acridine orange prepared using a Tris buffer solution at pH 7.4 can be used. Specifically, the dye concentration is preferably about 0.75 mg / dL. This metachromatic positive dye 10 is dispensed into 1 mL and heated to 45°C during dispensing. 2 μL of blood sample 20 is added to the heated 1 mL of positive dye 10A, and the mixture is stirred for 5 seconds. The temperature of the obtained sample is maintained at 45°C for 30 seconds, thereby preparing a suitable measurement sample 30. Alternatively, the measurement sample can be prepared by sequentially adding a buffer solution (such as a phosphate buffer solution or a Tris buffer solution having a pH of 6.4 to 8.2) and blood sample 20 to separately freeze-dried acridine orange.

[0059] "Heterochilism" is a term originally referring to the modulation phenomenon in which the components dyed with a dye exhibit a dyeability different from the original hue of the dye. In this specification, this term is used to define a "heterochilistic positive dye" as a dye that emits multiple wavelengths of fluorescence depending on the type of target to be dyed using the heterochromatic positive dye or dyeing method. Specific examples of heterochromatic positive dyes include acridine orange (AO), proline, acridine yellow, and arbutin. These heterochromatic positive dyes can be used without particular limitation, provided they are dyes in which the wavelengths of fluorescence emitted by the stacking component and the intercalating component are different, as will be described later. However, the fluorescence emitted by the stacking component of the heterochromatic positive dye is preferably orange fluorescence, and the fluorescence emitted by the intercalating component is preferably green fluorescence. From this point of view, acridine orange (AO) is particularly preferred as a heterochromatic positive dye.

[0060] (Measurement and analysis of the prepared samples)

[0061] Figure 2This is a diagram illustrating the system configuration of an apparatus for performing a particle analysis method according to one aspect of the present invention. Figure 3 This is a system diagram illustrating an outline of a flow cytometer as an embodiment of an apparatus for performing a particle analysis method according to the present invention.

[0062] like Figure 2 and Figure 3 As shown, the device is prepared by referring to the above. Figure 1 The described sample preparation unit 40 for measuring sample 30 and a flow cytometer 50 for analyzing the measuring sample 30 by flow cytometry are configured. The flow cytometer 50 has a flow cell 51 as the detection area and a laser source 52 through which the measuring sample 30 flows. The laser source is a light source that illuminates the measuring sample 30 (specifically, the particles contained in the sample) flowing through the flow cell 51. The laser source 52 is positioned relative to the flow cell 51, separated by an illumination focusing lens 53. In the flow cytometer 50, small-angle forward scattering detectors (FSs) 61 and large-angle forward scattering detectors (FLs) 62 are arranged, separated by a light-converging lens 54. These detectors detect forward scattered light generated from each particle in the measuring sample 30 due to illumination from the measuring sample 30 in the flow cell 51. The arrangement of the large-angle forward scattering detectors (FLs) 62 is not mandatory. Furthermore, in the flow cytometer 50, a side-scatter light detector (SS) 63 is provided via a beam splitter 55. This side-scatter light detector detects the side-scattered light generated from each particle in the measurement sample 30 due to light irradiation of the measurement sample 30 in the flow cell 51. Additionally, in the flow cytometer 50, a first fluorescence detector (FL1) 64 and a second fluorescence detector (FL2) 65 are arranged via beam splitters 56 and 57 and wavelength-selective filters 58 and 59, respectively, to detect two types of fluorescence generated from each particle in the measurement sample 30 due to light irradiation of the measurement sample 30 in the flow cell 51, each with different wavelengths. A dichroic mirror can be used instead of a beam splitter. Each of the detectors 61, 62, 63, 64, and 65 mentioned above functions as a photodetector (scattered light detector, fluorescence detector) to detect the intensity of scattered light and fluorescence generated from each particle contained in the light-irradiated measurement sample 30.

[0063] The flow cytometer 50 has a processor (CPU) 70. This processor (CPU) 70 also functions as a data processing unit that analyzes the particles contained in the blood sample based on the intensity of scattered light and fluorescence generated by each particle detected by the photodetector, and performs processing related to data processing in the particle analysis method according to this aspect (calculating fluorescence concentration by normalization of fluorescence intensity, clustering particles contained in the sample, and creating a nucleic acid quantity histogram).

[0064] Subsequently, the use of the present invention will be described in detail. Figure 2 and Figure 3 The particle analysis method of the configured apparatus is shown in the figure.

[0065] First, the measurement sample 30 prepared by the sample preparation unit 40 is supplied to the flow cell 51 of the flow cytometer 50 to begin analysis. When the measurement sample 30 is supplied to the flow cell 51, the laser source 52 irradiates the measurement sample 30 (specifically, the particles contained in the sample) flowing through the flow cell 51 with light. Here, the wavelength of the irradiation light is not particularly limited, but the center wavelength of the irradiation light is preferably 408 nm, 445 nm, 473 nm, or 488 nm.

[0066] When the sample 30 is illuminated with light, forward scattered light (FS) is generated from each particle contained in the sample 30, and the forward scattered light (FS) is detected by small-angle forward scattered light detectors (FSs) 61 and large-angle forward scattered light detectors (FLs) 62. When the sample 30 is illuminated with light, side scattered light (SS) is generated from each particle contained in the sample 30, and the side scattered light (SS) is detected by side scattered light detector (SS) 63. Furthermore, when the sample 30 is illuminated with light, fluorescence is generated from each particle contained in the sample 30. Here, in the method according to this aspect, the particles contained in the blood sample are stained with a heterochromatic positive dye. Therefore, when the sample 30 is illuminated with light, multiple (e.g., two) fluorescences with different wavelengths are generated from each particle contained in the sample 30. Specifically, the fluorescence includes fluorescence derived from the stacking component of the metachromatic positive dye (also referred to as "first fluorescence (FL1)" in this specification) and fluorescence derived from the intercalation component of the metachromatic positive dye (also referred to as "second fluorescence (FL2)" in this specification). The fluorescence derived from the stacking component (first fluorescence (FL1)) is generated by the electrostatic interaction of the metachromatic positive dye onto the nucleic acid, and has a center wavelength of approximately 645 to 655 nm when acridine orange (AO) is used as the dye. The fluorescence intensity of first fluorescence (FL1) is primarily related to the abundance of ribonucleic acid (RNA) in the nucleic acid. On the other hand, the fluorescence derived from the intercalated component (second fluorescence (FL2)) is fluorescence generated by intercalating a heterochromatic orthochromatic dye into the nucleic acid, and has a center wavelength of approximately 520 to 530 nm when acridine orange (AO) is used as the dye. The fluorescence intensity of the second fluorescence (FL2) is primarily correlated with the abundance of deoxyribonucleic acid (DNA) in the nucleic acid. The first fluorescence (FL1) and the second fluorescence (FL2) generated from each particle contained in the measurement sample 30 due to light irradiation of the measurement sample 30 are detected by the first fluorescence detector (FL1) 64 and the second fluorescence detector (FL2) 65, respectively.

[0067] As described above, the intensities of the scattered light (forward scattered light (FS) and side scattered light (SS)) and the fluorescence (first fluorescence (FL1) and second fluorescence (FL2)) detected by the detectors are converted into electrical signals at each detector and transmitted to the processor (CPU) 70. The processor (CPU) 70 then uses these electrical signals to perform various data processing operations. For example, the processor (CPU) 70 calculates parameters related to the size of each particle based on the intensity of the forward scattered light (FS), and calculates parameters related to the size of each particle and the amount of particles contained in each particle based on the intensity of the side scattered light (SS). The processor (CPU) 70 calculates parameters related to the amount of stacked components and embedded components in each particle, respectively, based on the intensity of the first fluorescence (FL1) and the intensity of the second fluorescence (FL2). Here, as described above, the fluorescence intensity of the first fluorescence (FL1) is primarily related to the abundance of ribonucleic acid (RNA) in the nucleic acid, and the fluorescence intensity of the second fluorescence (FL2) is primarily related to the abundance of deoxyribonucleic acid (DNA) in the nucleic acid. Therefore, the parameters related to the amount of stacked components and embedded components in each particle, calculated from the electrical signals derived from the intensity of the first fluorescence (FL1) and the intensity of the second fluorescence (FL2), can be considered as parameters related to the amount of RNA and DNA in each particle, respectively.

[0068] In this embodiment, the processor (CPU) 70 then normalizes the intensity of the first fluorescence (FL1) and the second fluorescence (FL2) emitted by each particle according to the size of each particle. Therefore, the fluorescence concentrations of the first fluorescence (FL1) and the second fluorescence (FL2) in each particle can be obtained separately. According to scattering theory, the intensity (scattering cross-section) of forward scattered light (FS) is known to be proportional to the size (diameter) of the particle emitting the forward scattered light. Therefore, when the intensity of the fluorescence (FL1, FL2) emitted by each particle is divided by a parameter related to the size of each particle (the intensity of the forward scattered light (FS) or the diameter calculated based on this intensity), the fluorescence intensity (i.e., fluorescence concentration) under the assumption that the particles have the same size can be obtained. In this specification, this process is referred to as "normalization". In this specification, for convenience, the fluorescence concentration of the first fluorescence (FL1) emitted by each particle is referred to as CRc (cellular RNA concentration; intracellular RNA concentration), and the fluorescence concentration of the second fluorescence (FL2) emitted by each particle is referred to as CDc (cellular DNA concentration; intracellular DNA concentration). The following text will first describe some information obtained by measuring blood samples until normalization is complete.

[0069] Figure 4This is an example of a measurement of a two-dimensional scatter plot (FS×SS cell histogram) of forward-scattered (FS) and side-scattered (SS) light. Figure 4 In the diagram, purple events indicate erythrocyte components, green events indicate platelet components, and blue events indicate nucleated cell components. For example... Figure 4 As shown, in the FS×SS cell histogram, the clusters of purple events (erythrocyte components) and blue events (nucleated cell components) overlap. Some of the purple events (erythrocyte components) are also present in the cluster of green events (platelet components). Therefore, when using the FS×SS cell histogram as is, regardless of the gating applied, it is impossible to separate specific blood cell components from other blood cell components.

[0070] Figure 5 This is an example of a measurement of a two-dimensional scatter plot (FL1×FL2 cell histogram) of the first fluorescence (FL1) and the second fluorescence (FL2). Figure 5 As shown, in the FL1×FL2 cell histogram, the clusters of purple events (erythrocyte components) and green events (platelet components) are shown to overlap. On the other hand, the clusters of blue events (nucleated cell components) exist independently in the upper right part of the cell histogram. Therefore, by gating the clusters of blue events (nucleated cell components), gating can be applied only to the nucleated cell components, such as... Figure 5 As shown.

[0071] Subsequently, Figure 6 This is a two-dimensional plot (also referred to as an "RNP plot" in this specification) of the fluorescence concentration (CRc) of the first fluorescence (FL1) and the fluorescence concentration (CDc) of the second fluorescence (FL2) based on the results obtained by determining the CRc and CDc in each particle through the "normalization" process described above. Figure 6 The RNP diagram shown also displays blue events (nucleated cell components) that can be deleted using the gating mechanism mentioned above, used to confirm their location. That is, based on the definitions mentioned above, the horizontal axis of the RNP diagram shows the fluorescence concentration (CRc) of the first fluorescence (FL1) of each particle, and the vertical axis shows the fluorescence concentration (CDc) of the second fluorescence (FL2) of each particle. Thus, it can be seen that nucleated cell components (nucleated cell clusters; PCn) with relatively high RNA and DNA concentrations in the particles form clusters in the upper right region of the RNP diagram. Furthermore, it can be seen that erythrocyte components (erythrocyte clusters; RBCn) with relatively low RNA and DNA concentrations in the particles form clusters in the lower left region of the RNP diagram, and platelet components (platelet clusters; PLTn) with relatively high RNA and relatively low DNA concentrations in the particles form clusters in the lower right region of the RNP diagram.

[0072] As described above, the "normalization" process is performed to obtain the fluorescence concentrations of the first fluorescence (FL1) and the second fluorescence (FL2) in each particle, and thus a two-dimensional graph with these fluorescence concentrations as two axes is created, thereby enabling the clustering of particles contained in the blood sample. In the particle analysis method according to this aspect, it is necessary to cluster each particle contained in the blood sample into multiple particle clusters including at least two of the following: erythrocyte clusters, platelet clusters, and nucleated cell clusters, and preferably to cluster each particle contained in the blood sample into multiple particle clusters including all of the following: erythrocyte clusters (RBCn), platelet clusters (PLTn), and nucleated cell clusters (NCn).

[0073] Next, in the particle analysis method according to this aspect, based on the RNP diagram created above ( Figure 6 For at least one of the multiple particle clusters generated by clustering using RNP diagrams, the intensity (CRc) of the first fluorescence (FL1) is created as a histogram of the class (also referred to as the "RNA amount histogram" in this specification) and the intensity (CDc) of the second fluorescence (FL2) is created as a histogram of the class (also referred to as the "DNA amount histogram" in this specification).

[0074] Figure 7A It is through gating that red blood cell clusters (RBCn) are separated from... Figure 6 The RNA quantity histogram for erythrocyte clustering (RBCn) created by isolating RNA from the RNP diagram shown in the figure. Figure 7A (left side) and DNA quantity histogram ( Figure 7A (The right side). Similarly, Figure 7B It uses gating to group platelet clusters (PLTn) from... Figure 6 The RNA quantity histogram for platelet clustering (PLTn) created by isolating RNP data shown in the figure is illustrated. Figure 7B (left side) and DNA quantity histogram ( Figure 7B (Right side) Figure 7C It is through gating that nucleated cell clusters (NCn) are separated from... Figure 6 The histogram of RNA quantity created from isolated RNP plots for nucleated cell clusters (NCn) is shown in the figure. Figure 7C (left side of the graph) and DNA quantity histogram ( Figure 7C (The right side of the middle).

[0075] The particle analysis method according to this aspect preferably further includes analyzing particles contained in a blood sample based on the RNA quantity histogram and DNA quantity histogram created above for at least one particle group. This analysis can be performed by a processor (CPU) 70 (data processing unit) included in the flow cytometer 50, by another computer, or by a medical professional (such as a doctor, nurse, or clinical laboratory technician).

[0076] Specifically, for example, based on Figure 7A The histogram of RNA quantity used for erythrocyte clustering (RBCn) is shown in the figure. Figure 7A The left side of the image (of the image) can measure the number or ratio of reticulocytes (Reticulocytes) in a blood sample and / or the immature reticulocyte fraction (IRF) of the blood sample. Here, Figure 7A The histogram of RNA quantity used for erythrocyte clustering (RBCn) is shown in the figure. Figure 7A The left part of the graph (RNP plot) is created using data from erythrocyte clusters separated by gating. Therefore, the FL1 (RNA quantity) data in each plot of the RNA quantity histogram reflects the amount of the packing components in each particle with high accuracy, and is highly unlikely to show false high values ​​including DNA quantity as in conventional techniques. Therefore, the particle analysis method according to this aspect has the advantage that it is based on… Figure 7A The RNA quantity histogram shown in the figure for erythrocyte clustering (RBCn) can measure various parameters (the number or ratio of reticulocytes in a blood sample and / or the fraction of immature reticulocytes (IRF) in a blood sample) with extremely high precision. In reticulocytes, the amount of RNA in blood cells is greater than in normal erythrocytes. Therefore, when in… Figure 7A When a predetermined threshold is set on the horizontal axis of the RNA quantity histogram for erythrocyte clustering (RBCn) shown in the figure, particles in erythrocyte clusters with RNA quantities equal to or greater than the threshold can be identified as reticulocytes. Furthermore, the region corresponding to reticulocytes on the horizontal axis is divided into three regions based on the fluorescence intensity (RNA quantity) of FL1: HFR (high fluorescence ratio), MFR (medium fluorescence ratio), and LFR (low fluorescence ratio), starting from the side with the highest fluorescence intensity. The immature reticulocyte fraction (IRF) can be measured as the immaturity of the reticulocytes based on the number or ratio of reticulocytes included in each region.

[0077] In the particle analysis method according to this aspect, unlike conventional techniques, for at least one particle cluster, information about the amount of DNA in each particle contained in a blood sample can be obtained as a DNA quantity histogram by separating it from information about RNA quantity. Therefore, analysis using the DNA quantity histogram described above also provides useful findings. For example, according to the particle analysis method according to this aspect, information about the amount of DNA in each particle can be obtained based on… Figure 7A The DNA quantity histogram (RBCn) for erythrocyte clustering shown in the diagram is used to determine the presence or absence of abnormalities in erythrocyte clusters. Here, "abnormality" is a concept that includes all states in which the amount of DNA in particles within the erythrocyte cluster is increased compared to the normal state. Examples of "abnormalities" include the presence of various small bodies (such as Howell-Jolly bodies and Pappenheimer bodies), microflagellates of Plasmodium, Babesia, Theileria, Trypanosoma, and filarial worms in particles included in the erythrocyte cluster. That is, in particles within erythrocyte clusters with these "abnormalities," the amount of DNA in the blood cells is greater than in normal erythrocytes. Therefore, when in Figure 7A When a predetermined threshold is set on the horizontal axis of the DNA quantity histogram for erythrocyte clustering (RBCn) shown in the figure, if there are particles in the erythrocyte cluster whose DNA quantity is equal to or greater than the threshold, it can be determined that the particle is likely to have some of the aforementioned “abnormalities” (abnormal reticulocyte fraction; ARF).

[0078] As mentioned above, [the text has already been referenced]. Figure 7A Specific embodiments of analysis based on RNA or DNA quantity histograms used for erythrocyte clustering (RBCn) are described. However, it is possible to base analysis on... Figure 7B Similar analyses can be performed using the RNA or DNA quantity histograms shown in the figure for platelet clustering (PLTn), and can be based on... Figure 7C Similar analyses can be performed using the RNA or DNA quantity histograms shown in the diagram for nucleated cell clustering (NCn). For example, there is an advantage that, based on... Figure 7B The RNA quantity histogram for platelet clustering (PLTn) shown in the image can measure various parameters (immature platelet fraction (IPF) in blood samples) with extremely high precision. Figure 7BWhen a predetermined threshold is set on the horizontal axis of the DNA quantity histogram for platelet clustering (PLTn), as shown in the diagram, if there are particles in the platelet cluster whose DNA quantity is equal to or greater than the threshold, it can be determined that the particle may have some of the aforementioned "abnormalities" (abnormal platelet fraction; APF). Similarly, based on the RNA quantity histogram and DNA quantity histogram for nucleated cell clustering (NCn), the immaturity (proportion of immature nucleated cells) and the presence or absence of abnormalities (abnormal nucleated cell fraction) of particles included in the nucleated cell cluster (NCn) can be determined, respectively.

[0079] In a preferred embodiment of the particle analysis method according to this aspect, for particle clusters in which RNA quantity histograms and DNA quantity histograms have been created as described above, a two-dimensional graph is created, including the intensity of a first fluorescence (FL1) or a second fluorescence (FL2) of each particle in the particle cluster as one axis, and the size of each particle in the particle cluster as another axis.

[0080] As an example of this two-dimensional diagram, Figure 8A This is a two-dimensional graph where, for the erythrocyte cluster (RBCn), the intensity of the first fluorescence (FL1) of each particle included in the cluster is the horizontal axis, and the size of each particle included in the cluster (in this case, the intensity of forward scattered light (FS)) is the vertical axis. Then, in the two-dimensional graph, the vertical axis (the intensity of forward scattered light (FS)) is divided into multiple regions (in this case, four regions from smallest to largest, RC0 to RC3). Therefore, the erythrocyte cluster can be reclassified into multiple sub-clusters based on the number or ratio of particles in each of the four regions. In this case, the erythrocyte cluster is reclassified into four sub-clusters starting from the largest particle size: large erythrocyte sub-cluster (RC3), normal erythrocyte sub-cluster (RC2), ruptured erythrocyte sub-cluster (RC1), and small erythrocyte sub-cluster (RC0).

[0081] Here, because Figure 8A The horizontal axis of the two-dimensional plot shown in the image represents the intensity of the first fluorescence (FL1), which is an indicator of the amount of RNA in each particle (in other words, immaturity). Therefore, based on... Figure 8A The two-dimensional diagram illustrating erythrocyte clusters (RBCn) shown in the image provides information simultaneously about immaturity and the size of each particle included within the erythrocyte cluster. Therefore, it opens the possibility of obtaining clinically useful findings that cannot be obtained from only one type of information (e.g., the value of the immature reticulocyte fraction (IRF)).

[0082] Figure 8B and Figure 8CThese are two-dimensional graphs created similarly to those for platelet clustering (PLTn) and nucleated cell clustering (NCn). Specifically, Figure 8A This is a two-dimensional graph where, for the platelet cluster (PLTn), the intensity of the first fluorescence (FL1) of each particle included in the cluster is the horizontal axis, and the size of each particle included in the cluster (in this case, the intensity of forward scattered light (FS)) is the vertical axis. Then, in the two-dimensional graph, the vertical axis (the intensity of forward scattered light (FS)) is divided into multiple regions (in this case, four regions from smallest to largest, PC0 to PC3). Therefore, the platelet cluster can be reclassified into multiple sub-clusters based on the number or ratio of particles in each of the four regions. In this case, the platelet cluster is reclassified into four sub-clusters starting from the largest particle size: giant platelet sub-cluster (PC3), large platelet sub-cluster (PC2), normal platelet sub-cluster (PC1), and small platelet sub-cluster (PC0).

[0083] Here, because Figure 8B The horizontal axis of the two-dimensional plot shown in the image represents the intensity of the first fluorescence (FL1), which is an indicator of the amount of RNA in each particle (in other words, immaturity). Therefore, based on... Figure 8B The two-dimensional diagram of platelet clusters (PLTn) shown in the image provides information simultaneously about immaturity and the size of each particle included in the platelet cluster. Therefore, it opens up the possibility of obtaining clinically useful findings that cannot be obtained from only one type of information (the value of the immature platelet fraction (IPF)).

[0084] Figure 8C This is a two-dimensional graph where, for the nucleated cell cluster (NCn), the intensity of the first fluorescence (FL1) of each particle included in the cluster is used as the horizontal axis, and the size of each particle included in the cluster (in this case, the intensity of forward scattered light (FS)) is used as the vertical axis. Then, in the two-dimensional graph, the vertical axis (the intensity of forward scattered light (FS)) is divided into multiple regions (in this case, four regions from smallest to largest, NC0 to NC3). Therefore, the nucleated cell cluster can be reclassified into multiple sub-clusters based on the number or ratio of particles in each of the four regions. In this case, the nucleated cell cluster is reclassified into four sub-clusters starting from the largest particle size: large nucleated cell sub-cluster (NC3), normal nucleated cell sub-cluster (NC2), ruptured nucleated cell sub-cluster (NC1), and small nucleated cell sub-cluster (NC0). Figure 8CThe two-dimensional diagram of the nucleated cell population (NCn) shown in the image provides information simultaneously about immaturity and the size of each particle included within the nucleated cell population. Therefore, it opens up the possibility of obtaining clinically useful findings that cannot be obtained from only one type of information.

[0085] Based on the results shown in the aforementioned two-dimensional graphs, the total number of extracellular vesicles (EVs) can be obtained from the total number of granules in subclusters such as erythrocytes (RC0), platelets (PC0), and small nucleated cells (NC0). Since information about the distribution of immaturity of the EVs obtained at that time can be obtained simultaneously, clinically useful findings can be provided. Figures 8A to 8C The two-dimensional graphs shown depict events corresponding to particles included in the erythrocyte, platelet, and nucleated cell clusters, respectively. However, as in the example described later, events corresponding to particles included in each of the multiple particle clusters can be combined and displayed in a single two-dimensional graph.

[0086] Another aspect (second aspect) of the present invention also provides a particle analysis method for analyzing particles contained in a blood sample. The particle analysis method according to the second aspect is common to the aforementioned aspect (first aspect) of the present invention in the following points. That is, the particle analysis method according to the second aspect includes: staining the particles with a metachromatic positive dye; irradiating the stained particles with light; measuring the intensity of a first fluorescence from the stacking component of the metachromatic positive dye and the intensity of a second fluorescence from the embedded component of the metachromatic positive dye, the first and second fluorescence being emitted by each particle contained in the blood sample; normalizing the intensity of the first and second fluorescence emitted by each particle according to the size of each particle to obtain the fluorescence concentration of each of the first and second fluorescence in each particle; and clustering each particle into a plurality of particle clusters in a two-dimensional graph of the fluorescence concentrations obtained by normalization, the plurality of particle clusters including at least two of erythrocyte clusters, platelet clusters, and nucleated cell clusters. On the other hand, the particle analysis method according to the second aspect is characterized in that, for at least one particle cluster included in a plurality of particle clusters, a fluorescence intensity ratio is calculated, which is the ratio of the intensity of a first fluorescence and the intensity of a second fluorescence of each particle included in the particle cluster; and a fluorescence intensity ratio histogram of the class is created. Hereinafter, preferred embodiments for implementing the particle analysis method according to the second aspect will be described with respect to points different from those in the first aspect.

[0087] As mentioned above, similarly in the particle analysis method according to the second aspect, the following is used: Figure 6The RNP diagram shown clusters the particles contained in the blood sample into multiple particle clusters, which is consistent with the particle analysis method according to the first aspect.

[0088] Subsequently, in the particle analysis method according to the second aspect, for at least one particle cluster included in a plurality of particle clusters, a fluorescence intensity ratio is calculated, which is the ratio of the intensity of a first fluorescence and the intensity of a second fluorescence of each particle included in the particle cluster. For example, from Figure 6 The RNP diagram shown in the figure is gated for erythrocyte clusters (RBCn), and for each particle included in an RBCn, the ratio of the intensity of the second fluorescence (FL2) to the intensity of the first fluorescence (FL1) is calculated (fluorescence intensity ratio (FL2 / FL1)). A histogram (fluorescence intensity ratio histogram) is then created, in which each particle in the RBCn is used as an element, and the fluorescence intensity ratio (FL2 / FL1) is used as the class.

[0089] The fluorescence intensity ratio (FL2 / FL1) of each particle calculated in this way is considered to essentially reflect the ratio of DNA to RNA in each particle (DNA / RNA). Here, in normal erythrocytes, both the amount of DNA and RNA in the cell are not very large. Therefore, for example, in particles where the value of the fluorescence intensity ratio (FL2 / FL1) calculated as described above is greater than a predetermined threshold (e.g., 2.0), it is conceivable that the amount of DNA relative to RNA in the particle is so large that it deviates from the normal range. Therefore, for such particles included in erythrocyte clusters, the presence of the "abnormality" described in the first aspect is suspected, namely various small bodies (such as Howell-Jolly bodies and Pappenheimer bodies), Plasmodium, Babesia, Theileria, Trypanosoma, and microflagellates of filarial worms, etc., is suspected. Alternatively, the particle is suspected to be a nucleated erythrocyte (NRBC). In this way, particles included in blood samples can be analyzed based on the fluorescence intensity ratio histogram created above, and this analysis also provides clinically useful findings. Needless to say, the same analysis can be performed even when the fluorescence intensity ratio is the ratio of the intensity of the first fluorescence (FL1) to the intensity of the second fluorescence (FL2) (FL1 / FL2). The fluorescence concentrations (CRc and CDc) in each particle calculated in the first aspect are each obtained by dividing the intensity of the first fluorescence (FL1) and the intensity of the second fluorescence (FL2) by the intensity of the forward scattered light (FS) or by calculating the diameter based on the intensity of the forward scattered light (FS). Therefore, when calculating the fluorescence intensity ratio in the second aspect, the same value can be obtained even when the ratio of the fluorescence concentrations (CRc) of the first fluorescence and the fluorescence concentrations (CDc) of the second fluorescence is used instead of the ratio of the intensity of the first fluorescence (FL1) and the intensity of the second fluorescence (FL2).

[0090] In summary, a preferred embodiment of the particle analysis method according to the second aspect further includes measuring the number or ratio of particles whose fluorescence intensity ratio is higher than a predetermined threshold (e.g., 2.0) when the fluorescence intensity ratio is a value of the ratio of the intensity of the second fluorescence to the intensity of the first fluorescence (FL2 / FL1), or measuring the number or ratio of particles whose fluorescence intensity ratio is less than a predetermined threshold (e.g., 0.5) when the fluorescence intensity ratio is a value of the ratio of the intensity of the first fluorescence to the intensity of the second fluorescence (FL1 / FL2), and determining the presence or absence of anomalies in particles included in the particle cluster based on the measurement results. Specific examples of "anomalies" suspected of being present in this case are as described above.

[0091] Example

[0092] In the following description, embodiments of the present invention will be specifically described with reference to examples. However, the scope of the present invention is not limited to the following examples.

[0093] <<Example of measurement of the first aspect of the invention>>

[0094] [Example of measuring the reticulocyte ratio in blood samples without morphological findings]

[0095] The reticulocyte fraction was measured in blood samples collected from adult males. No morphological findings were observed in the blood morphology tests performed by microscopic examination of the blood samples regarding red blood cells and platelets. The standard range for the reticulocyte fraction in males was 0.76% to 2.18%.

[0096] First, as a control, a commercially available multi-item automated hematology analyzer (XN series manufactured by Sysmex Corporation) was used, and the reticulocyte fraction was measured according to the accompanying manual. The result showed a reticulocyte fraction of 2.7% in the blood sample.

[0097] On the other hand, as a measurement example of applying the first aspect of the present invention, firstly, the same blood sample as described above is provided, and 5 μL of the blood sample is added to 2 mL of 0.006 g / L acridine orange (AO) (which is a metachromatic positive dye) solution and mixed to prepare a measurement sample. Then, measurements are performed using a fully automated hematology counter (manufactured by Nihon Kohden Corporation, MEK-9000 series, Celltac G+ prototype). Next, using the obtained data of FS, SS, FL1 (fluorescence wavelength of 525 nm) and FL2 (fluorescence wavelength of 650 nm), a measurement is created as follows. Figure 6 The RNP diagram shown here. The RNP diagram actually created using the blood sample described above is... Figure 9A As shown in the diagram. Then, by setting (gating) a gate to the erythrocyte clusters, the data is separated from the RNP plot, and particles included in the separated erythrocyte clusters are created, such as... Figure 7A The histograms showing RNA and DNA quantities are shown here. Figure 9B This shows a histogram of RNA levels actually created using blood samples. Figure 9B (left side) and DNA quantity histogram ( Figure 9B (The right side). Then, a threshold is set for the horizontal axis (the intensity of the first fluorescence (FL1) reflecting the RNA amount) based on the peak value of the RNA quantity histogram, and particles with values ​​equal to or greater than the threshold on the horizontal axis are identified as reticulocytes. Then, when the reticulocyte fraction is calculated as the ratio of reticulocytes to particles included in the erythrocyte cluster, the reticulocyte fraction in the blood sample is 2.97%. As described above, in blood samples in which no morphological findings of erythrocytes and platelets were found, when the first aspect of the invention is applied, a reticulocyte fraction value that is almost equal to the reticulocyte fraction in the control portion is obtained.

[0098] Based on RNP graph ( Figure 9A ), for which an RNA quantity histogram was created above ( Figure 9B (left side of the middle) and DNA quantity histogram ( Figure 9B The right side of the image shows a cluster of erythrocytes, and the intensity of the first fluorescence (FL1) of each particle in the erythrocyte cluster is shown on the horizontal axis, and the size of each particle in the erythrocyte cluster is shown on the vertical axis. Figure 9C A two-dimensional plot of the fluorescence (FL2) was created, comprising the intensity of the second fluorescence (FL2) of each particle in the erythrocyte cluster as the horizontal axis and the size of each particle in the erythrocyte cluster as the vertical axis. Figure 9D A two-dimensional diagram. In Figure 9C and Figure 9D In the two-dimensional plots shown, the intensity (FS) of forward scattered light is used as the vertical axis (size of each particle). In these two-dimensional plots, in addition to events of particles included in the erythrocyte cluster, events of particles gated from the RNP plot data in the same manner as those of particles included in the platelet cluster are also displayed together in different colors.

[0099] (Example of measuring the reticulocyte fraction in a blood sample with morphological findings of red blood cells)

[0100] The reticulocyte fraction was measured in blood samples collected from adult males. Morphological findings regarding erythrocytes were discovered during hemomorphological testing of the blood samples by microscopic observation. Specifically, 17 nucleated erythrocytes (NRBCs) were counted out of 100 erythrocytes. The presence of Howell-Jolly bodies (HJ bodies) was confirmed in more than half of the erythrocytes, and Pappenheimer bodies (PH bodies) were also confirmed in some erythrocytes.

[0101] First, as a control, a commercially available multi-item automated hematology analyzer (XN series manufactured by Sysmex Corporation) was used, and the reticulocyte fraction was measured according to the accompanying manual. The result showed a reticulocyte fraction of 7.97% in the blood sample.

[0102] On the other hand, as a measurement example of the first aspect of the present invention, firstly, the same blood sample as described above is provided, and 5 μL of the blood sample is added to 2 mL of 0.006 g / L acridine orange (AO) (which is a metachromatic positive dye) solution and mixed to prepare a measurement sample. Then, measurements are performed using a fully automated hematology counter (manufactured by Nihon Kohden Corporation, MEK-9000 series, Celltac G+ prototype). Next, using the obtained data of FS, SS, FL1 (fluorescence wavelength of 525 nm) and FL2 (fluorescence wavelength of 650 nm), a sample is created in the same manner as described above. Figure 6 The RNP diagram shown here. The RNP diagram actually created using the blood sample described above is... Figure 10A As shown in the diagram. Then, by setting (gating) a gate to the erythrocyte clusters, the data is separated from the RNP plot, and particles included in the separated erythrocyte clusters are created, such as... Figure 7A The histograms showing RNA and DNA quantities are shown here. Figure 10B This shows a histogram of RNA levels actually created using blood samples. Figure 10B (left side) and DNA quantity histogram ( Figure 10B(The right side). Then, a threshold is set for the horizontal axis (the intensity of the first fluorescence (FL1) reflecting the amount of RNA) based on the peak value of the RNA quantity histogram, and particles with values ​​equal to or greater than the threshold on the horizontal axis are identified as reticulocytes. Then, when the reticulocyte fraction is calculated as the ratio of reticulocytes to particles included in the erythrocyte cluster, the reticulocyte fraction in the blood sample is 2.97%. As described above, as a result of the measurement of the reticulocyte fraction in the blood sample in which morphological findings about erythrocytes are found, when the first aspect of the invention is applied, a value close to the standard value is obtained, and on the other hand, in the control part, a value with a large deviation is obtained. Here, in the measurement method of the control part, the fluorescence emitted by the particles is a single color without the use of metachromatic positive dyes. Therefore, it is not possible to distinguish and detect the stacked components derived from RNA and the embedded components derived from DNA. As a result, in the application example of the invention, in Figure 10B The presence of various small bodies and nucleated red blood cells (which can be detected to distinguish them from reticulocytes) shown in the upper right part of the diagram is incorrectly included in and counted among the reticulocytes in the control section, causing the measured reticulocyte fraction to deviate significantly from the actual value. When the measured reticulocyte fraction deviates significantly from the actual value, there is a concern about the presence of diseases with high reticulocyte fractions (e.g., hemolytic anemia, iron deficiency anemia, pernicious anemia).

[0103] Based on RNP graph ( Figure 10A ), for which an RNA quantity histogram was created above ( Figure 10B (left side of the middle) and DNA quantity histogram ( Figure 10B The right side of the image shows clusters of erythrocytes, creating a two-dimensional graph where the intensity of the first fluorescence (FL1) of each particle in the erythrocyte cluster is on the horizontal axis and the size of each particle in the erythrocyte cluster is on the vertical axis. Figure 10C Similarly, a two-dimensional graph was created, in which the intensity of the second fluorescence (FL2) of each particle in the erythrocyte cluster is represented by the horizontal axis and the size of each particle included in the erythrocyte cluster is represented by the vertical axis. Figure 10D ).exist Figure 10C and Figure 10D In the two-dimensional plots shown, the intensity of forward scattered light (FS) is used as the vertical axis (size of each particle). In these two-dimensional plots, in addition to events for particles included in the erythrocyte clusters, events for particles gated from the RNP plot data in the same manner as those for particles included in the platelet clusters are also displayed together in different colors. Sequentially, for example, in Figure 10D In the two-dimensional diagram of FS×FL2 shown in the figure, and Figure 9DCompared to the two-dimensional FS×FL2 plot shown, the events of erythrocyte clustering are distributed to expand along the horizontal axis. This corresponds to their existence already being... Figure 10B The DNA quantity histogram shown in the upper right corner of the image confirms the presence of various small bodies and nucleated red blood cells. (Comparison) Figure 9D and Figure 10D The two-dimensional diagram shown in the figure shows that, in blood samples with morphological findings of red blood cells, the particles included in the red blood cell clusters are relatively small in size, and it can also be seen that, for example, the number of particles included in the platelet clusters is relatively large.

[0104] <<Example of measurement of the second aspect of the invention>>

[0105] (Example of measurements on blood samples with no morphological findings)

[0106] The second aspect of the invention was applied to measure blood samples collected from adult males. No morphological findings were observed regarding red blood cells and platelets during blood morphology tests performed on the blood samples under a microscope.

[0107] First, the 5 μL blood sample provided above was added to 2 mL of 0.006 g / L acridine orange (AO) solution (a metachromatic positive dye) and mixed to prepare the measurement sample. Then, measurements were performed using a fully automated hematology counter (manufactured by Nihon Kohden Corporation, MEK-9000 series, Celltac G+ prototype). Next, using the obtained FS, SS, FL1 (fluorescence wavelength of 525 nm), and FL2 (fluorescence wavelength of 650 nm) data, a statistical analysis was created as follows: Figure 6 The RNP diagram shown here. The RNP diagram actually created using the blood sample described above is... Figure 11A As shown in the diagram. Then, data is separated from the RNP plot by setting (gating) a gate to the erythrocyte clusters. For particles included in the separated erythrocyte clusters, a fluorescence intensity ratio is calculated, which is the ratio (FL1 / FL2) of the intensity of the first fluorescence (FL1) to the intensity of the second fluorescence (FL2) for each particle, and a fluorescence intensity ratio histogram is created where the fluorescence intensity ratio is used as the class's fluorescence intensity ratio histogram. Here, the fluorescence intensity ratio histogram actually created using the blood sample described above is used in... Figure 11B As shown in [the image]. Figure 11B In the fluorescence intensity ratio histogram shown, the threshold for an indicator of outliers in the fluorescence intensity ratio is set to FL1 / FL2 = 2.0. Figure 11C yes Figure 11B A magnified view of the region near the threshold (FL1 / FL2 = 2.0). Based on... Figure 11B and Figure 11CThe fluorescence intensity ratio histogram shown in the figure indicates that the number of particles with a fluorescence intensity ratio higher than the threshold of 2.0 is three. This result is consistent with the absence of morphological findings on erythrocytes in blood morphology tests.

[0108] (Example of measurement of blood samples with morphological findings of red blood cells (1))

[0109] The second aspect of the invention was applied to measure blood samples collected from adult males. Morphological findings regarding erythrocytes were discovered during blood morphology testing of the blood samples by microscopic observation. Specifically, the presence of Howell-Jolly bodies (HJ bodies) was confirmed in the erythrocytes.

[0110] Using this blood sample, the measurement sample was prepared using the same method described above, and measurements were performed using a fully automated hematology counter (manufactured by Nihon Kohden Corporation, MEK-9000 series, Celltac G+ prototype). Next, using the obtained data for FS, SS, FL1 (fluorescence wavelength of 525 nm), and FL2 (fluorescence wavelength of 650 nm), a model was created as follows: Figure 6 The RNP diagram shown here. The RNP diagram actually created using the blood sample described above is... Figure 12A As shown in the diagram. Then, data is separated from the RNP plot by setting (gating) gates to the erythrocyte clusters. For particles included in the separated erythrocyte clusters, a fluorescence intensity ratio is calculated, which is the ratio (FL1 / FL2) of the intensity of the first fluorescence (FL1) to the intensity of the second fluorescence (FL2) for each particle, and a fluorescence intensity ratio histogram is created where the fluorescence intensity ratio is the class. Here, the fluorescence intensity ratio histogram actually created using the blood sample described above is used in... Figure 12B As shown in [the image]. Figure 12B In the fluorescence intensity ratio histogram shown, the threshold for an indicator of outliers in the fluorescence intensity ratio is set to FL1 / FL2 = 2.0. Figure 12C yes Figure 12B A magnified view of the region near the threshold (FL1 / FL2 = 2.0). Based on... Figure 12B and Figure 12C The fluorescence intensity ratio histogram shown in the figure counts 57 particles with fluorescence intensity ratios higher than the threshold of 2.0, and their fluorescence intensity ratios are concentrated in the region slightly above 2.0. This result is consistent with the presence of HJ bodies observed in blood morphology tests.

[0111] (Example of measurement of blood samples with morphological findings of red blood cells (2))

[0112] The second aspect of the invention was applied to measure blood samples collected from adult males. Morphological findings regarding red blood cells were discovered during blood morphology testing of the blood samples performed under a microscope. Specifically, the presence of nucleated red blood cells (NRBCs) was confirmed in 100 white blood cell counts.

[0113] Using this blood sample, the measurement sample was prepared using the same method described above, and measurements were performed using a fully automated hematology counter (manufactured by Nihon Kohden Corporation, MEK-9000 series, Celltac G+ prototype). Next, using the obtained data for FS, SS, FL1 (fluorescence wavelength of 525 nm), and FL2 (fluorescence wavelength of 650 nm), a model was created as follows: Figure 6 The RNP diagram shown here. The RNP diagram actually created using the blood sample described above is... Figure 13A As shown in the diagram. Then, data is separated from the RNP plot by setting (gating) a gate to the erythrocyte clusters. For particles included in the separated erythrocyte clusters, a fluorescence intensity ratio is calculated, which is the ratio (FL1 / FL2) of the intensity of the first fluorescence (FL1) to the intensity of the second fluorescence (FL2) for each particle, and a fluorescence intensity ratio histogram is created where the fluorescence intensity ratio is used as the class's fluorescence intensity ratio histogram. Here, the fluorescence intensity ratio histogram actually created using the blood sample described above is used in... Figure 13B As shown in [the image]. Figure 13B In the fluorescence intensity ratio histogram shown, the threshold for an indicator of outliers in the fluorescence intensity ratio is set to FL1 / FL2 = 2.0. Figure 13C yes Figure 13B A magnified view of the region near the threshold (FL1 / FL2 = 2.0). Based on... Figure 13B and Figure 13C The fluorescence intensity ratio histogram shown in the figure counted 25 particles with fluorescence intensity ratios above the threshold of 2.0, and their fluorescence intensity ratios were widely distributed in the region of 2.0 to 3.3. This result is consistent with the presence of nucleated erythrocytes (NRBCs) observed in blood morphology tests.

[0114] As described above, by using the present invention with metachromatic orthochromatic dyes to analyze particles in blood samples, more clinically useful information can be obtained.

[0115] This application is based on Japanese Patent Application No. 2020-018264, filed on February 5, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0116] List of reference numerals

[0117] 10. Metachromatic Orthochromatic Dyes

[0118] 10A is a heterochromatic positive dye.

[0119] 20 blood samples

[0120] 30. Measurement Sample

[0121] 40 Sample preparation units

[0122] 50 flow cytometer

[0123] 51 Flow cell

[0124] 52 Laser source

[0125] 53. Converging lens for illumination light

[0126] 54. Scattered light converging lens

[0127] 55, 56, 57 beam splitters

[0128] Wavelength selective filters 58 and 59

[0129] 61 Small-angle forward-scattering light detectors (FSs)

[0130] 62 large-angle forward-scattering light detectors (FLs)

[0131] 63 Side-scattered light detector (SS)

[0132] 64 First fluorescence detector (FL1)

[0133] 65. Second fluorescence detector (FL2)

[0134] 70 Processor (CPU)

Claims

1. A particle analysis method for analyzing particles contained in a blood sample, comprising: The particles were dyed using a heterochromatic positive dye; The stained particles are irradiated with light; The intensity of a first fluorescence originating from the stacked component of the heterochromatic positive dye and the intensity of a second fluorescence originating from the embedded component of the heterochromatic positive dye are measured, the first fluorescence and the second fluorescence being emitted by each particle contained in the blood sample; The intensity of the first fluorescence and the intensity of the second fluorescence emitted by each of the particles are normalized according to the size of each of the particles to obtain the fluorescence concentration of each of the first fluorescence and the second fluorescence in each of the particles. In the two-dimensional graph of the fluorescence concentration obtained through the normalization, each particle is clustered into multiple particle clusters, wherein the multiple particle clusters include at least two of the following: erythrocyte clusters, platelet clusters, and nucleated cell clusters; and For at least one of the plurality of particle clusters, a histogram of the first fluorescence intensity as the RNA amount of the cluster and a histogram of the second fluorescence intensity as the DNA amount of the cluster are created. The particle analysis method further includes, for at least one particle cluster in which the RNA quantity histogram and the DNA quantity histogram are created, creating a two-dimensional graph with the intensity of the first fluorescence or the intensity of the second fluorescence of each particle included in the particle cluster as one axis and the size of each particle included in the particle cluster as another axis, and dividing the axis in the two-dimensional graph indicating the size of each of the particles into multiple regions, and reclassifying the at least one particle cluster into multiple sub-clusters based on the number or ratio of particles in each of the multiple regions formed by the division.

2. The particle analysis method according to claim 1 further includes analyzing the particles contained in the blood sample based on the RNA quantity histogram and the DNA quantity histogram.

3. The particle analysis method according to claim 2, comprising creating the RNA quantity histogram and the DNA quantity histogram for the red blood cell clusters. in, The analysis includes measuring the number or ratio of reticulocytes in the blood sample and / or the immature reticulocyte fraction (IRF) of the blood sample based on the RNA amount histogram of the red blood cell clusters.

4. The particle analysis method according to claim 2 or 3, comprising creating the RNA quantity histogram and the DNA quantity histogram for the erythrocyte clusters. in, The analysis includes determining the presence or absence of anomalies in the red blood cell clusters based on the DNA quantity histogram of the red blood cell clusters.

5. The particle analysis method according to claim 2 or 3, comprising creating the RNA quantity histogram and the DNA quantity histogram based on the platelet clustering. in, The analysis includes measuring the immature platelet fraction (IPF) in the blood sample based on the RNA quantity histogram of the platelet clusters.

6. The particle analysis method according to claim 2 or 3, comprising creating the RNA quantity histogram and the DNA quantity histogram based on the platelet clustering. in, The analysis includes determining the presence or absence of anomalies in the platelet clusters based on the DNA quantity histogram of the platelet clusters.

7. The particle analysis method according to claim 4, wherein, The anomaly includes the presence of microflagellates of small bodies, Plasmodium, Babesia, Theileria, Trypanosoma, and filarial worms in the particles within the particle cluster.

8. The particle analysis method according to claim 6, wherein, The anomaly includes the presence of microflagellates of small bodies, Plasmodium, Babesia, Theileria, Trypanosoma, and filarial worms in the particles within the particle cluster.

9. The particle analysis method according to claim 1, wherein, The two-dimensional graph includes a two-dimensional graph for red blood cell clustering. The particle analysis method further includes reclassifying the red blood cell clusters into multiple sub-clusters, including sub-clusters of normal red blood cells, large red blood cells, fragmented red blood cells, and / or small red blood cells.

10. The particle analysis method according to claim 1 or 9, wherein, The two-dimensional graph includes a two-dimensional graph for platelet clustering. The particle analysis method further includes reclassifying the platelet clusters into multiple sub-clusters, including sub-clusters of normal platelets, giant platelets, large platelets, and / or small platelets.

11. The particle analysis method according to claim 1 or 9, wherein, The two-dimensional graph includes a two-dimensional graph for clustering nucleated cells. The particle analysis method further includes reclassifying the nucleated cell clusters into multiple sub-clusters, including sub-clusters of normal nucleated cells, large nucleated cells, ruptured nucleated cells, and / or small nucleated cells.

12. The particle analysis method according to any one of claims 1 to 3 and 9, wherein, The first fluorescence is orange fluorescence, and the second fluorescence is green fluorescence.

13. The particle analysis method according to any one of claims 1 to 3 and 9, wherein, The light irradiated onto the particles contained in the blood sample has a central wavelength of 408 nm, 445 nm, 473 nm or 488 nm.

14. A particle analysis method for analyzing particles contained in a blood sample, comprising: The particles were dyed using a heterochromatic positive dye; The stained particles are irradiated with light; The intensity of a first fluorescence originating from the stacked component of the heterochromatic positive dye and the intensity of a second fluorescence originating from the embedded component of the heterochromatic positive dye are measured, the first fluorescence and the second fluorescence being emitted by each particle contained in the blood sample; The intensity of the first fluorescence and the intensity of the second fluorescence emitted by each of the particles are normalized according to the size of each of the particles to obtain the fluorescence concentration of each of the first fluorescence and the second fluorescence in each of the particles. In the two-dimensional graph of the fluorescence concentration obtained by the normalization, each of the particles is clustered into multiple particle clusters, the multiple particle clusters including at least two of the following: red blood cell cluster, platelet cluster, and nucleated cell cluster; and For at least one particle cluster included in the plurality of particle clusters, a fluorescence intensity ratio is calculated and a fluorescence intensity ratio histogram is created using the fluorescence intensity ratio as the class, wherein the fluorescence intensity ratio is the ratio of the intensity of the first fluorescence to the intensity of the second fluorescence for each particle included in the particle cluster. The particle analysis method further includes: When the fluorescence intensity ratio is the ratio of the intensity of the second fluorescence to the intensity of the first fluorescence, the number or ratio of particles in which the fluorescence intensity ratio is higher than a predetermined threshold is measured. When the fluorescence intensity ratio is the ratio of the intensity of the first fluorescence to the intensity of the second fluorescence, the number or ratio of particles in which the fluorescence intensity ratio is less than a predetermined threshold is measured; and, The presence or absence of anomalies in the particles included in the particle cluster is determined based on the measurement results.

15. The particle analysis method according to claim 14, further comprising analyzing particles contained in the blood sample based on the fluorescence intensity ratio histogram.

16. The particle analysis method according to claim 14, wherein, The anomaly includes the presence of microflagellates of small bodies, Plasmodium, Babesia, Theileria, Trypanosoma, and filarial worms in the particles within the particle cluster, or the presence of nucleated red blood cells in the particle cluster.

17. The particle analysis method according to claim 14 or 15, wherein, The first fluorescence is orange fluorescence, and the second fluorescence is green fluorescence.

18. The particle analysis method according to claim 17, wherein, The heterochromatic positive dye is acridine orange (AO).

19. The particle analysis method according to claim 14 or 15, wherein, The light irradiated onto the particles contained in the blood sample has a central wavelength of 408 nm, 445 nm, 473 nm or 488 nm.

20. A particle analyzer, comprising: A light source that illuminates particles contained in a blood sample; A flow cell through which the blood sample flows; A photodetector comprising a plurality of fluorescence detectors for detecting each of the intensities of a first fluorescence and a second fluorescence having different wavelengths; as well as The data processing unit normalizes the intensity of the first fluorescence and the second fluorescence emitted by each of the particles contained in the blood sample according to the size of each particle to determine the fluorescence concentration of each of the first and second fluorescence in each particle. Furthermore, in a two-dimensional graph of the fluorescence concentrations obtained through the normalization, the data processing unit clusters each of the particles into multiple particle clusters including at least two of the following: erythrocyte clusters, platelet clusters, and nucleated cell clusters. It also creates a histogram of the intensity of the first fluorescence as a class RNA amount for at least one of the multiple particle clusters. The intensity of the second fluorescence is used as a DNA quantity histogram for the class. The data processing unit also creates a two-dimensional graph for at least one particle cluster in which the RNA quantity histogram and the DNA quantity histogram are created, with the intensity of the first fluorescence or the intensity of the second fluorescence of each particle included in the particle cluster as one axis and the size of each particle included in the particle cluster as another axis. The data processing unit also divides the axis indicating the size of each particle in the two-dimensional graph into multiple regions, and reclassifies the at least one particle cluster into multiple sub-clusters based on the number or ratio of particles in each of the multiple regions formed by the division.

21. The particle analyzer according to claim 20, wherein, The data processing unit analyzes the particles contained in the blood sample based on the RNA quantity histogram and the DNA quantity histogram.

22. A particle analyzer, comprising: A light source that illuminates particles contained in a blood sample; A flow cell through which the blood sample flows; A photodetector comprising a plurality of fluorescence detectors for detecting each of the intensities of a first fluorescence and a second fluorescence having different wavelengths; as well as The data processing unit normalizes the intensity of a first fluorescence and a second fluorescence emitted by each particle contained in the blood sample according to the size of each particle to determine the fluorescence concentration of the first fluorescence and the second fluorescence in each particle. Furthermore, in a two-dimensional graph of fluorescence concentration obtained through the normalization, the data processing unit clusters each particle into multiple particle clusters including at least two of the following: erythrocyte clusters, platelet clusters, and nucleated cell clusters. It also calculates a fluorescence intensity ratio for at least one particle cluster included in the multiple particle clusters and creates the fluorescence intensity ratio as a class fluorescence intensity ratio histogram. The intensity ratio is the ratio of the intensity of the first fluorescence to the intensity of the second fluorescence for each particle in the particle cluster. The data processing unit further performs the following processing: when the fluorescence intensity ratio is a value of the ratio of the intensity of the second fluorescence to the intensity of the first fluorescence, it measures the number or ratio of particles whose fluorescence intensity ratio is higher than a predetermined threshold of the fluorescence intensity ratio; or when the fluorescence intensity ratio is a value of the ratio of the intensity of the first fluorescence to the intensity of the second fluorescence, it measures the number or ratio of particles whose fluorescence intensity ratio is lower than a predetermined threshold of the fluorescence intensity ratio; and, based on the measurement results, it determines whether anomalies exist in the particles included in the particle cluster.

23. The particle analyzer of claim 22, wherein the data processing unit analyzes particles contained in the blood sample based on the fluorescence intensity ratio histogram.

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