EP4100716A1

EP4100716A1 - Particle analysis method and particle analyzer

Info

Publication number: EP4100716A1
Application number: EP21707394.9A
Authority: EP
Inventors: Yutaka Nagai
Original assignee: Nihon Kohden Corp
Current assignee: Nihon Kohden Corp
Priority date: 2020-02-05
Filing date: 2021-02-05
Publication date: 2022-12-14
Also published as: JP7491703B2; WO2021157711A1; CN114641680A; JP2021124398A

Abstract

Provided is a means capable of obtaining more clinically useful information when particles contained in a blood sample are analyzed using a metachromatic orthochromatic dye. In a particle analysis method of analyzing particles contained in a blood sample, the particles are stained with a metachromatic orthochromatic dye, the stained particles are irradiated with light, intensity of a first fluorescence derived from a stacking component of the metachromatic orthochromatic dye and intensity of a second fluorescence derived from an intercalation component of the metachromatic orthochromatic 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 by the size of each of the particles to obtain a fluorescence concentration of each of the first fluorescence and the second fluorescence in each of the particles, each of the particles is clustered into a plurality of particle clusters including at least two of an erythrocyte cluster, a platelet cluster and a nucleated cell cluster, in a two-dimensional plot of the fluorescence concentration obtained by the normalization, and a size histogram in which the size of each particle included in the particle cluster is a class is created for at least one particle cluster included in the plurality of particle clusters.

Description

PARTICLE ANALYSIS METHOD AND PARTICLE ANALYZER

The present invention relates to a particle analysis method and a particle analyzer.

Conventionally, particles contained in a blood sample have been classified and counted. For example, in an optical automatic blood cell analyzer, a flow cytometer is used to analyze the particles in the blood sample. In a flow cytometry method, specifically, using information of scattered light and fluorescence obtained by applying light from a light source such as a laser to a cell stained with a fluorescent dye or the like flowing through the cell, in addition to the number and size of cells, information such as an amount of nucleic acid inside the cell is also obtained.

By the way, there has been proposed a technique for analyzing a blood sample stained with acridine orange (AO), which is a fluorescent dye (metachromatic orthochromatic dye) showing a modulation phenomenon (metachromasia) in which a tissue component exhibits a dyeability different from an original color tone of the dye, by a flow cytometry type automatic blood cell analyzer (Patent Literature 1). Specifically, Patent Literature 1 discloses a technique for normalizing the fluorescence intensities of blood cells stained with AO by the sizes and shapes of the cells according to each scattered light, based on the result of the flow cytometry to obtain a fluorescence concentration of each cell and classifying the cells in the blood sample based on the fluorescence concentration of each cell thus obtained.

Patent Literature 1: US2009/0130647A1

SUMMARY OF THE INVENTION

Technical Problem
However, the technique described in Patent Literature 1 simply classifies the cells in the blood sample based on the fluorescence concentration of each cell normalized by the sizes and shapes of particles in the blood sample. Thus, it is said that sufficient clinically useful information has not yet been obtained.
Thus, an object of the present invention is to provide a means capable of obtaining more clinically useful information when particles contained in a blood sample are analyzed using a metachromatic orthochromatic dye.
Solution to Problem
The present inventor has made diligent studies in view of the above problem. As a result, the present inventor has found that the above problem can be solved by classifying (clustering) particles in a blood sample into a plurality of particle clusters based on a fluorescence concentration normalized by the size of each particle contained in the blood sample and then creating a histogram (in the present specification, also referred to as “size histogram”) in which the size of each particle included in at least one particle cluster included in the plurality of particle clusters is a class, and the present invention is completed accordingly.
That is, according to one aspect of the present invention, there is provided a particle analysis method for analyzing particles contained in a blood sample. The particle analysis method is characterized by including staining the particles with a metachromatic orthochromatic dye, irradiating the stained particles with light, measuring intensity of a first fluorescence derived from a stacking component of the metachromatic orthochromatic dye and intensity of a second fluorescence derived from an intercalation component of the metachromatic orthochromatic 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 intensity of the second fluorescence, emitted by each of the particles, by the size of each of the particles to obtain a fluorescence concentration of each of the first fluorescence and the second fluorescence in each of the particles, clustering each of the particles into a plurality of particle clusters including at least two of an erythrocyte cluster, a platelet cluster and a nucleated cell cluster, in a two-dimensional plot of the fluorescence concentration obtained by the normalization, and creating a size histogram in which the size of each particle included in at least one particle cluster included in the plurality of particle clusters is a class.
According to another aspect of the present invention, as an apparatus capable of carrying out the particle analysis method according to the above-described aspect of the present invention, there is also provided a particle analyzer including a light source that applies light to particles contained in a blood sample, a flow cell through which the blood sample flows, a light detector including a plurality of fluorescence detectors that detect each of intensity of a first fluorescence and intensity of a second fluorescence having different wavelengths, and a data processing part that normalizes the intensities of the first fluorescence and the second fluorescence emitted by each of the particles contained in the blood sample by 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, clusters each of the particles into a plurality of particle clusters including at least two of an erythrocyte cluster, a platelet cluster and a nucleated cell cluster, in a two-dimensional plot of the fluorescence concentration obtained by the normalization, and creates a size histogram in which the size of each particle included in at least one particle cluster included in the plurality of particle clusters is a class.
According to the present invention, it is possible to obtain more clinically useful information when the particles contained in the blood sample are analyzed using the metachromatic orthochromatic dye.

FIG. 1 is a schematic diagram showing preparation of a measurement sample. FIG. 2 is a diagram showing a system configuration of an apparatus for carrying out a particle analysis method according to an aspect of the present invention. FIG. 3 is a system diagram showing an outline of a flow cytometer as an embodiment of the apparatus for carrying out the particle analysis method according to an aspect of the present invention. FIG. 4 is a measurement example of a two-dimensional scattergram (FS × SS cytogram) of forward-scattered light (FS) and lateral-scattered light (SS). FIG. 5 is a measurement example of a two-dimensional scattergram (FL1 × FL2 cytogram) of a first fluorescence (FL1) and a second fluorescence (FL2). FIG. 6 is a two-dimensional plot diagram (also referred to as “RNP diagram” in the present specification) of a fluorescence concentration (CRc) of a first fluorescence (FL1) and a fluorescence concentration (CDc) of a second fluorescence (FL2) created based on a result obtained by determining the CRc and the CDc in each particle by processing of “normalization”. FIG. 7 is an example of a size histogram for an erythrocyte cluster (RBCn), which is created by separating the erythrocyte cluster (RBCn) from the RNP diagram shown in FIG. 6 by gating (created when measurement is performed by applying the present invention to a blood sample with morphological findings of erythrocyte in an Example described later). FIG. 8A is a two-dimensional plot diagram in which, 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 (the intensity of the forward-scattered light (FS)) of each particle included in the cluster is the vertical axis. FIG. 8B is a two-dimensional plot diagram in which, 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 (the intensity of the forward-scattered light (FS)) of each particle included in the cluster is the vertical axis. FIG. 8C is a two-dimensional plot diagram in which, for a nucleated cell cluster (NCn), the intensity of the first fluorescence (FL1) of each particle included in the cluster is the horizontal axis and the size (the intensity of the forward-scattered light (FS)) of each particle included in the cluster is the vertical axis. FIG. 9A is an RNP diagram created when measurement is performed by applying the present invention to the blood sample with morphological findings of erythrocyte in an Example described later. FIG. 9B is a size histogram of the erythrocyte cluster, which is created when measurement is performed by applying the present invention to the blood sample without morphological findings of erythrocyte in an Example described later. FIG. 10A is an RNP diagram created when measurement is performed by applying the present invention to the blood sample with morphological findings of platelet in an Example described later. FIG. 10B is a size histogram of the platelet cluster, which is created when measurement is performed by applying the present invention to the blood sample with morphological findings of platelet in an Example described later. FIG. 10C is a size histogram of the platelet cluster, which is created when measurement is performed by applying the present invention to the blood sample without morphological findings of platelet in an Example described later.

DETAILED DESCRIPTION

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings.
An aspect (first aspect) of the present invention is a particle analysis method of analyzing particles contained in a blood sample, including staining the particles with a metachromatic orthochromatic dye, irradiating the stained particles with light, measuring intensity of a first fluorescence derived from a stacking component of the metachromatic orthochromatic dye and intensity of a second fluorescence derived from an intercalation component of the metachromatic orthochromatic 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 intensity of the second fluorescence, emitted by each of the particles, by the size of each of the particles to obtain a fluorescence concentration of each of the first fluorescence and the second fluorescence in each of the particles, clustering each of the particles into a plurality of particle clusters including at least two of an erythrocyte cluster, a platelet cluster and a nucleated cell cluster, in a two-dimensional plot of the fluorescence concentration obtained by the normalization, and creating a size histogram in which the size of each particle included in at least one particle cluster included in the plurality of particle clusters is a class.
Hereinafter, a preferred embodiment for carrying out the particle analysis method according to the present aspect will be specifically described with reference to a case where analysis is performed by a flow cytometry method using a flow cytometer as an example. However, the technical scope of the present invention should be determined based on the description in the claims, and is not limited only to the following specific embodiments.
FIG. 1 is a schematic diagram showing preparation of a measurement sample. In the particle analysis method according to the present invention, first, a sample (blood sample) containing particles in blood is provided, and a predetermined orthochromatic dye (metachromatic orthochromatic dye) is used (usually, the dye and the blood sample are mixed) to prepare the measurement sample. As a result, the particles contained in the blood sample are stained with the predetermined orthochromatic dye. In the analysis of particles by the flow cytometry method, the measurement sample prepared above is irradiated with light, whereby scattered light and a fluorescence generated from the particles contained in the sample are detected as electrical signals. Then, based on the detected electrical signal, the particles contained in the sample are analyzed.
(Preparation of measurement sample)
In the present aspect, as described above, as a sample to be measured (measurement sample), a sample (blood sample) containing particles in blood is provided, and a predetermined orthochromatic dye (metachromatic orthochromatic dye) is used (usually, the dye and the blood sample are mixed) to prepare the measurement sample. In this case, for example, as shown in FIG. 1, when a required amount of orthochromatic dye 10 is dispensed in a fixed amount, the orthochromatic dye is warmed in a range of 20 to 50°C. To an orthochromatic dye 10A thus warmed and dispensed, a sample (blood sample) 20 containing particles in blood is added, and the mixture is stirred for 5 to 10 seconds. A measurement sample 30 thus obtained is kept warm in the range of 20 to 50°C and held for 10 to 40 seconds. As a result, in the present invention, the preparation of the measurement sample 30 can be completed in 15 to 60 seconds.
In this case, for example, 1 mL each of the metachromatic orthochromatic dye 10 is dispensed, and to the dispensed metachromatic orthochromatic dye 10A, 2 μL each of the blood sample 20 prepared so that the number of particles to be measured is about 1 × 10⁷particles/μL is added. As the metachromatic orthochromatic dye 10, for example, 0.5 to 1.5 mg/dL of acridine orange prepared using a tris buffer solution having a pH of 7.4 can be used. In particular, a dye concentration is preferably about 0.75 mg/dL. This metachromatic orthochromatic dye 10 is dispensed into 1 mL and warmed to 45°C during the dispensing, 2 μL of the blood sample 20 is added to 1 mL of the orthochromatic dye 10A warmed, and the mixture is stirred for 5 seconds. The temperature of the obtained sample is kept at 45°C and held for 30 seconds, whereby the measurement sample 30 as a proper sample can be prepared. Alternatively, a measurement sample may 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 pH 8.2, and the blood sample 20 to separately freeze-dried acridine orange.
“Metachromasia” is a term originally meant for a modulation phenomenon in which a component stained with a dye exhibits a dyeability different from an original color tone of the dye. In the present specification, this term is used to define a “metachromatic orthochromatic dye” as a dye having a property that emits a plurality of fluorescences having different wavelengths depending on the type of target to be stained with the metachromatic orthochromatic dye or a dyeing method. Specific examples of the metachromatic orthochromatic dye include acridine orange (AO), proflavine, acriflavine, atebrin, and the like. These metachromatic orthochromatic dyes can be used without particular limitation as long as they are dyes in which the wavelengths of fluorescences emitted by a stacking component and an intercalation component, which will be described later, are different. However, the fluorescence emitted by the stacking component of the metachromatic orthochromatic dye is preferably an orange fluorescence, and the fluorescence emitted by the intercalation component is preferably a green fluorescence. From this point of view, acridine orange (AO) is particularly preferably used as the metachromatic orthochromatic dye.
FIG. 2 is a diagram showing a system configuration of an apparatus for carrying out the particle analysis method according to an embodiment of the present invention. FIG. 3 is a system diagram showing an outline of a flow cytometer as an embodiment of the apparatus for carrying out the particle analysis method according to the present aspect.
As shown in FIG. 2 and FIG. 3, the apparatus is constituted of a sample preparation unit 40 that prepares the measurement sample 30 described above with reference to FIG. 1 and a flow cytometer 50 that analyzes the measurement sample 30 by the flow cytometry method. The flow cytometer 50 has a flow cell 51 as a detection region through which the measurement sample 30 flows and a laser light source 52 that is a light source that irradiates the measurement sample 30 (specifically, particles contained in the sample) flowing through the flow cell 51 with light. The laser light source 52 is disposed with respect to the flow cell 51 through an irradiation-light condensing lens 53. In the flow cytometer 50, a detector for small-angled forward-scattered light (FSs) 61 and a detector for large-angled forward-scattered light (FLs) 62 that detect forward-scattered light generated from each particle in the measurement sample 30 by light irradiation of the measurement sample 30 in the flow cell 51 are arranged through a scattered-light condensing lens 54. The arrangement of the detector for large-angled forward-scattered light (FLs) 62 is not essential. Further, in the flow cytometer 50, a lateral-scattered light detector (SS) 63 that detects scattered light in the lateral direction generated from each particle in the measurement sample 30 by light irradiation of the measurement sample 30 in the flow cell 51 is disposed through a beam splitter 55. Furthermore, in the flow cytometer 50, a first fluorescence detector (FL1) 64 and a second fluorescence detector (FL2) 65 for detecting the respective two fluorescences having different wavelengths and generated from each particle in the measurement sample 30 by light irradiation of the measurement sample 30 in the flow cell 51 are arranged through beam splitters 56 and 57 and wavelength-selective filters 58 and 59, respectively. A dichroic mirror may be used instead of the beam splitter. Each of the above-mentioned detectors 61, 62, 63, 64, and 65 functions as a light detector (scattered-light detector, fluorescence detector) that detects intensities of the scattered light and fluorescence generated from each particle contained in the measurement sample 30 irradiated with light.
The flow cytometer 50 has a processor (CPU) 70. This processor (CPU) 70 also functions as a data processing part that analyzes particles contained in a blood sample based on the intensity of scattered light and fluorescence generated from each particle detected by the light detector and carries out a process related to data processing (calculation of fluorescence concentration by normalization of fluorescence intensity, clustering of particles contained in a sample, and creation of a size histogram) in the particle analysis method according to the present aspect.
Subsequently, the particle analysis method according to the present invention using the apparatus having the configurations shown in FIG. 2 and FIG. 3 will be described in detail.
First, the measurement sample 30 prepared by the sample preparation unit 40 described above is supplied to the flow cell 51 of the flow cytometer 50 to start the analysis. When the measurement sample 30 is supplied to the flow cell 51, the laser light 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 a central wavelength of the irradiation light is preferably 408 nm, 445 nm, 473 nm or 488 nm.
When the measurement sample 30 is irradiated with light, forward-scattered light (forward-scattered light (FS)) is generated from each particle contained in the measurement sample 30, and the forward-scattered light (FS) is detected by the detector for small-angled forward-scattered light (FSs) 61 and the detector for large-angled forward-scattered light (FLs) 62. When the measurement sample 30 is irradiated with light, scattered light in the lateral direction (lateral-scattered light (SS)) is generated from each particle contained in the measurement sample 30, and the lateral-scattered light (SS) is detected by the lateral-scattered light detector (SS) 63. In addition, when the measurement sample 30 is irradiated with light, fluorescence is generated from each particle contained in the measurement sample 30. Here, in the method according to the present aspect, the particles contained in the blood sample are stained with the metachromatic orthochromatic dye. Therefore, when the measurement sample 30 is irradiated with light, a plurality of (for example, two) fluorescences having different wavelengths from each other are generated from each particle contained in the measurement sample 30. Specifically, the plurality of fluorescences include a fluorescence (also referred to as “first fluorescence (FL1)” in the present specification) derived from the stacking component of the metachromatic orthochromatic dye and a fluorescence (also referred to as “second fluorescence (FL2)” in the present specification) derived from the intercalation component of the metachromatic orthochromatic dye. The fluorescence (first fluorescence (FL1)) derived from the stacking component is the fluorescence generated by stacking the metachromatic orthochromatic dye to nucleic acid by electrostatic interaction, and is the fluorescence having a central wavelength of about 645 to 655 nm when acridine orange (AO) is used as the dye. The fluorescence intensity of the first fluorescence (FL1) is mainly correlated with an abundance of ribonucleic acid (RNA) among the nucleic acids. On the other hand, the fluorescence (second fluorescence (FL2)) derived from the intercalation component is the fluorescence generated by intercalating the metachromatic orthochromatic dye to nucleic acid, and is the fluorescence having a central wavelength of about 520 to 530 nm when acridine orange (AO) is used as the dye. The fluorescence intensity of the second fluorescence (FL2) is mainly correlated with an abundance of deoxyribonucleic acid (DNA) among the nucleic acids. The first fluorescence (FL1) and the second fluorescence (FL2) generated from each particle contained in the measurement sample 30 by light irradiation of the measurement sample 30 are detected by the first fluorescence detector (FL1) 64 and the second fluorescence detector (FL2) 65, respectively.
As described above, the intensity of the scattered light (forward-scattered light (FS) and lateral-scattered light (SS)) and the intensity of the fluorescence (first fluorescence (FL1) and second fluorescence (FL2)) detected by the detectors are each converted into an electrical signal at the detector and transmitted to the processor (CPU) 70. Then, the processor (CPU) 70 performs various data processing using the electrical signal thus obtained. For example, the processor (CPU) 70 calculates a parameter related to the size of each particle based on the intensity of the forward-scattered light (FS), and calculates a parameter related to the size of each particle and an amount of granules contained in each particle based on the intensity of the lateral-scattered light (SS). The processor (CPU) 70 calculates parameters related to an amount of the stacking component and an amount of the intercalation component 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 mainly correlated with the abundance of ribonucleic acid (RNA) among the nucleic acids, and the fluorescence intensity of the second fluorescence (FL2) is mainly correlated with the abundance of deoxyribonucleic acid (DNA) among the nucleic acids. Therefore, the parameters related to the amount of the stacking component and the amount of the intercalation component 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 regarded as parameters related to an amount of RNA and an amount of DNA in each particle, respectively.
In the present embodimenet, the processor (CPU) 70 then normalizes the intensity of the first fluorescence (FL1) and the intensity of the second fluorescence (FL2) emitted by each particle by the size of each particle. Consequently, the respective fluorescence concentrations of the first fluorescence (FL1) and the second fluorescence (FL2) in each particle can be obtained. According to scattering theory, it is known that the intensity (scattering cross section) of the forward-scattered light (FS) is proportional to the size (diameter) of the particles that emit the forward-scattered light. Therefore, when the intensity of the fluorescence (FL1, FL2) emitted by each particle is divided by the parameter related to the size of each particle (the intensity of the forward-scattered light (FS) or diameter calculated based on this intensity), the fluorescence intensity when it is assumed that the particles have the same size (that is, the fluorescence concentration) can be obtained. In the present specification, this processing is referred to as “normalization”. In the following, some information obtained up to the time of completion of normalization by measuring blood samples will be described first.
FIG. 4 is a measurement example of a two-dimensional scattergram (FS × SS cytogram) of the forward-scattered light (FS) and the lateral-scattered light (SS). In FIG. 4, a purple event indicates an erythrocyte component, a green event indicates a platelet component, and a blue event indicates a nucleated cell component. As shown in FIG. 4, in the FS × SS cytogram, a cluster of the purple events (erythrocyte components) and a cluster of the blue events (nucleated cell components) are displayed overlapping. Some of the purple events (erythrocyte components) are present in a cluster of the green events (platelet components). Therefore, when the FS × SS cytogram is used as it is, no specific blood cell component can be separated from other blood cell components no matter how gating is applied.
FIG. 5 is a measurement example of a two-dimensional scattergram (FL1 × FL2 cytogram) of the first fluorescence (FL1) and the second fluorescence (FL2). As shown in FIG. 5, also in the FL1 × FL2 cytogram, the cluster of the purple events (erythrocyte components) and the cluster of the green events (platelet components) are displayed overlapping. On the other hand, the cluster of the blue events (nucleated cell components) is present independently in the upper right of the cytogram. Therefore, it is possible to gate only the nucleated cell component by setting a gate for the cluster of the blue events (nucleated cell component) as shown in FIG. 5.
Subsequently, FIG. 6 is a two-dimensional plot diagram (also referred to as “RNP diagram” in the present 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 result obtained by determining the CRc and the CDc in each particle by the processing of “normalization” described above. In the RNP diagram shown in FIG. 6, the blue event (nucleated cell component) that can be deleted by the above-mentioned gating is also displayed for confirming the position of presence. That is, based on the above-mentioned definition, in the RNP diagram, the horizontal axis 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. Reflecting this, it can be seen that the nucleated cell components (nucleated cell cluster; PCn) in which both the RNA concentration and the DNA concentration in the particles are relatively high form a cluster in an upper right region of the RNP diagram. In addition, it can also be seen that the erythrocyte components (erythrocyte cluster; RBCn) in which both the RNA concentration and the DNA concentration in the particles are relatively low form a cluster in a lower left region of the RNP diagram, and the platelet components (platelet cluster; PLTn) in which while the RNA concentration in the particles is relatively high, the DNA concentration is relatively low, form a cluster in a lower right region of the RNP diagram.
As described above, the processing of “normalization” described above is performed to obtain the respective fluorescence concentrations of the first fluorescence (FL1) and the second fluorescence (FL2) in each particle, and thus to create a two-dimensional plot diagram in which these fluorescence concentrations are two axes, whereby the particles contained in the blood sample can be clustered. In the particle analysis method according to the present aspect, it is essential to cluster each particle contained in the blood sample into a plurality of particle clusters including at least two of the erythrocyte cluster, the platelet cluster and the nucleated cell cluster, and it is preferable to cluster each particle contained in the blood sample into a plurality of particle clusters including all of the erythrocyte cluster (RBCn), the platelet cluster (PLTn) and the nucleated cell cluster (NCn).
Next, in the particle analysis method according to the present aspect, from the RNP diagram (FIG. 6) created above, for at least one particle cluster included in the plurality of particle clusters generated by clustering using the RNP diagram, a histogram (size histogram) in which the size of each particle included in the particle cluster is a class is created.
FIG. 7 is an example of the size histogram for an erythrocyte cluster (RBCn), which is created by separating the erythrocyte cluster (RBCn) from the RNP diagram shown in FIG. 6 by gating. This size histogram is a histogram in which the size is a class for each particle included in the erythrocyte cluster (RBCn). Regarding information on the size of each particle used when creating the size histogram, the intensity of the forward-scattered light (FS) is adopted in FIG. 7. However, the information is not limited to this intensity, and in some cases, the intensity of the lateral-scattered light (SS) or the like may be adopted.
The particle analysis method according to the present aspect preferably further includes analyzing each particle included in the particle cluster based on the size histogram for at least one particle cluster created above. This analysis may be performed by the processor (CPU) 70 (data processing part) included in the flow cytometer 50, may be performed by another computer, or may be performed by a medical professional such as a doctor, a nurse, or a clinical laboratory technician.
Specifically, for example, an axis indicating the size of each particle in the size histogram created above can be divided into a plurality of regions, and the at least one particle cluster can be reclassified into a plurality of subclusters based on the number or ratio of the particles in each of the plurality of regions obtained by the division. Regarding the size histogram for the erythrocyte cluster (RBCn) shown in FIG. 7, the axis (that is, the horizontal axis) indicating the size of each particle is divided into two regions with a predetermined threshold value as a boundary. Here, the threshold value shown as the boundary in FIG. 7 is a value of the intensity of the forward-scattered light (FS) corresponding to a lower limit of the size of normal erythrocyte. This makes it possible to measure the number or ratio of particles (disrupted erythrocytes, small erythrocytes) smaller in size than normal erythrocytes among the particles included in the erythrocyte cluster (RBCn). However, the division is not limited to the division into two regions, and may be a division into three or more regions. For example, when the particles included in the erythrocyte cluster (RBCn) are reclassified, for example, the particles are also preferably reclassified into a plurality of subclusters including a subcluster of normal erythrocytes, and a large erythrocyte subcluster, a disrupted erythrocyte subcluster and/or a small erythrocyte subcluster.
When the size histogram created above includes a histogram for a platelet cluster (PLTn), it is also preferable that, based on the size histogram, the platelet cluster (PLTn) be reclassified into a plurality of subclusters including a normal platelet subcluster, and a giant platelet subcluster, a large platelet subcluster and/or a small platelet subcluster. In addition, when the size histogram created above includes a histogram for the nucleated cell cluster (NCn), it is also preferable that the nucleated cell cluster (NCn) be reclassified into a plurality of subclusters including a normal nucleated cell subcluster, and a large nucleated cell subcluster, a disrupted nucleated cell subcluster and/or a small nucleated cell subcluster.
In a preferred embodiment of the particle analysis method according to the present aspect, for the particle cluster in which the size histogram is created as described above, a two-dimensional plot diagram is created in which the intensity of the first fluorescence (FL1) or the intensity of the second fluorescence (FL2) of each particle included in the particle cluster is one axis and the size of each particle included in the particle cluster is the other axis.
As an example of such a two-dimensional plot diagram, FIG. 8A is a two-dimensional plot diagram in which, 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 (in this case, the intensity of the forward-scattered light (FS)) of each particle included in the cluster is the vertical axis. Then, in the two-dimensional plot diagram, the vertical axis (intensity of forward-scattered light (FS)) is divided into a plurality of regions (in this case, four regions RC0 to RC3 from the smallest to the largest). In this case, the erythrocyte cluster is reclassified into four subclusters from the largest particle size: a large erythrocyte subcluster (RC3), a normal erythrocyte subcluster (RC2), a disrupted erythrocyte subcluster (RC1), and a small erythrocyte subcluster (RC0). It is preferable that the plurality of regions in this two-dimensional plot diagram correspond to the plurality of regions obtained by dividing the size histogram in the analysis based on the size histogram described above. However, these may be different from each other.
Here, since the horizontal axis of the two-dimensional plot diagram shown in FIG. 8A shows the intensity of the first fluorescence (FL1), which is an indicator of the RNA amount (in other words, immaturity) in each particle, information on the immaturity and information on the size of each particle included in the erythrocyte cluster can be simultaneously obtained based on the two-dimensional plot diagram showing the erythrocyte cluster (RBCn) shown in FIG. 8A. As a result, it opens up the possibility of obtaining clinically useful findings (for example, the immaturity of reticulocytes, such as an immature reticulocyte fraction (IRF)) that cannot be grasped from only one of the information (for example, size distribution of particles included in the erythrocyte cluster).
FIG. 8B and FIG. 8C are two-dimensional plot diagrams similarly created for the platelet cluster (PLTn) and the nucleated cell cluster (NCn) , respectively. Specifically, FIG. 8B is a two-dimensional plot diagram in which, 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 (in this case, the intensity of the forward-scattered light (FS)) of each particle included in the cluster is the vertical axis. Then, in the two-dimensional plot diagram, the vertical axis (intensity of forward-scattered light (FS)) is divided into a plurality of regions (in this case, four regions PC0 to PC3 from the smallest to the largest). In this case, the platelet cluster is reclassified into four subclusters from the largest particle size: a giant platelet subcluster (PC3), a large platelet subcluster (PC2), a normal platelet subcluster (PC1), and a small platelet subcluster (PC0).
Here, since the horizontal axis of the two-dimensional plot diagram shown in FIG. 8B shows the intensity of the first fluorescence (FL1), which is an indicator of the RNA amount (in other words, immaturity) in each particle, information on the immaturity and information on the size of each particle included in the platelet cluster can be simultaneously obtained based on the two-dimensional plot diagram showing the platelet cluster (PLTn) shown in FIG. 8B. As a result, it opens up the possibility of obtaining clinically useful findings (for example, the immaturity of platelet, such as an immature platelet fraction (IPF)) that cannot be grasped from only one of the information (for example, size distribution of particles included in the platelet cluster).
FIG. 8C is a two-dimensional plot diagram in which, for the nucleated cell cluster (NCn), the intensity of the first fluorescence (FL1) of each particle included in the cluster is the horizontal axis and the size (in this case, the intensity of the forward-scattered light (FS)) of each particle included in the cluster is the vertical axis. Then, in the two-dimensional plot diagram, the vertical axis (intensity of forward-scattered light (FS)) is divided into a plurality of regions (in this case, four regions NC0 to NC3 from the smallest to the largest). In this case, the nucleated cell cluster is reclassified into four subclusters from the largest particle size: a large nucleated cell subcluster (NC3), a normal nucleated cell subcluster (NC2), a disrupted nucleated cell subcluster (NC1), and a small nucleated cell subcluster (NC0). The information on the immaturity and the information on the size of each particle included in the nucleated cell cluster can be simultaneously obtained based on the two-dimensional plot diagram showing the nucleated cell cluster (NCn) shown in FIG. 8C. As a result, it opens up the possibility of obtaining clinically useful findings that cannot be grasped from only one of the information.
A total number of extracellular vesicles (EV) can be grasped from, for example, a total number of particles in the small erythrocyte subcluster (RC0), the small platelet subcluster (PC0), and the small nucleated cell subcluster (NC0), based on the results shown in the above-mentioned two-dimensional plot diagram. Since information on a distribution of immaturity of EV grasped at that time can be obtained at the same time, clinically useful findings can be provided. The two-dimensional plot diagrams shown in FIG. 8A to FIG. 8C display the events corresponding to the particles included in the erythrocyte cluster, the platelet cluster, and the nucleated cell cluster, respectively. However, as in the Examples described later, the events corresponding to the particles included in each of the plurality of particle clusters may be combined and displayed in one two-dimensional plot diagram.
In FIG. 8A to FIG. 8C, the two-dimensional plot diagram in which the intensity of the first fluorescence (FL1) of each particle is the horizontal axis and the size (intensity of the forward-scattered light (FS)) of each particle included in at least one particle cluster is the vertical axis has been described as an example. However, another embodiment may be adopted. For example, the intensity of the lateral-scattered light (SS) may be used as an indicator of the size of each particle. A two-dimensional plot diagram similarly created so that the intensity of the second fluorescence (FL2) of each particle is the horizontal axis may also provide clinically useful findings. For example, the intensity of the second fluorescence (FL2) of each particle reflects the DNA amount in each particle. Therefore, the presence or absence of an abnormality in each particle cluster can be determined based on the two-dimensional plot diagram similarly created so that the intensity of the second fluorescence (FL2) of each particle is the horizontal axis. Here, the “abnormality” is a concept including all states in which the DNA amount in the particles included in the particle cluster increases as compared with the normal state. Examples of the “abnormality” include the presence of various bodies (such as Howell-Jolly body and Pappenheimer body), malaria parasite, Babesia, Theileria, Trypanosoma, and microphilia of filaria, and the like in the particles included in each particle cluster. That is, in the particles having these “abnormalities”, the DNA amount in the blood cells is large as compared with normal erythrocytes. Therefore, in the above two-dimensional plot diagram, when the intensity of the second fluorescence (FL2) of each particle is set as one axis and a predetermined threshold value is set on the axis, if there is a particle in which the DNA amount is equal to or more than the threshold value, it can be determined that the particle is likely to have some of the above “abnormalities”.
Examples
Hereinafter, embodiments of the present invention will be specifically described with reference to Examples. However, the technical scope of the present invention is not limited to the following Examples.
(Measurement example of blood sample with morphological findings of erythrocytes)
A blood sample collected from an adult male was measured by applying the present invention. In the blood morphology test of the blood sample by microscopic observation, morphological findings regarding erythrocytes were found. Specifically, it was confirmed that the presence of disrupted erythrocytes and small erythrocytes significantly increased.
5 μL of the blood sample was added to 2 mL of a 0.006 g/L solution of acridine orange (AO), which is a metachromatic orthochromatic dye, and mixed to prepare a measurement sample. Then, measurement was performed using a fully automatic blood cell counter (manufactured by Nihon Kohden Corporation, MEK-9000 series, Celltac G + prototype). Next, using data of obtained FS, SS, FL1 (fluorescence wavelength of 525 nm) and FL2 (fluorescence wavelength of 650 nm), an RNP diagram as shown in FIG. 6 was created. Here, the RNP diagram actually created using the above blood sample is shown in FIG. 9A. Then, the data was separated from the RNP diagram by setting (gating) a gate to the erythrocyte cluster (RBCn), and the size histogram shown in FIG. 7 was created for the particles included in the separated erythrocyte cluster. Then, a threshold value was set for the horizontal axis (intensity of the forward-scattered light (FS)) based on the lower limit of the size of normal erythrocyte, and particles in which the value of the horizontal axis was less than the threshold value were determined as disrupted erythrocytes or small erythrocytes. This result shown in FIG. 7 is consistent with the observation of the presence of disrupted erythrocytes and small erythrocytes in the blood morphology test.
FIG. 9B shows an example in which a size histogram for the erythrocyte cluster is created by the same method as above for a blood sample in which no morphological findings regarding erythrocyte are found in the blood morphology test by microscopic observation. In the size histogram shown in FIG. 9B, among the particles included in the erythrocyte cluster, almost no particles having a size less than the threshold value were observed. This result is consistent with the absence of morphological findings regarding erythrocytes in the blood morphology test.
(Measurement example of blood sample with morphological findings of platelet)
A blood sample collected from an adult male was measured by applying the present invention. In the blood morphology test of the blood sample by microscopic observation, morphological findings regarding platelet were found. Specifically, it was confirmed that the presence of giant platelets significantly increased.
Using this blood sample provided above, a measurement sample was prepared by the same method as above, and measurement was performed using a fully automatic blood cell counter (manufactured by Nihon Kohden Corporation, MEK-9000 series, Celltac G + prototype). Next, using data of obtained FS, SS, FL1 (fluorescence wavelength of 525 nm) and FL2 (fluorescence wavelength of 650 nm), an RNP diagram as shown in FIG. 6 was created. Here, the RNP diagram actually created using the above blood sample is shown in FIG. 10A. Then, the data was separated from the RNP diagram by setting (gating) a gate to the erythrocyte cluster, and the size histogram shown in FIG. 10B was created for the particles included in the separated erythrocyte cluster. Then, a threshold value was set for the horizontal axis (intensity of the forward-scattered light (FS)) based on a lower limit of the size of the giant platelet, and particles in which the value of the horizontal axis was equal to or more than the threshold value were determined as giant platelets. The result shown in FIG. 10B is consistent with the observation of the presence of the giant platelet in the blood morphology test.
In the meantime, an example in which the size histogram for the platelet cluster was created by the same method as above for a blood sample in which no morphological findings regarding platelet are found in the blood morphology test by microscopic observation is shown in FIG. 10C. In the size histogram shown in FIG. 10C, among the particles included in the platelet cluster, almost no particles having a size equal to or more than the threshold value were observed. This result is consistent with the absence of morphological findings regarding platelet in the blood morphology test.
As described above, the particles in the blood sample are analyzed by applying the present invention using the metachromatic orthochromatic dye, so that more clinically useful information can be obtained.
This application is based on Japanese Patent Application No. 2020-018279, filed on February 5, 2020, and the disclosed contents thereof are incorporated herein by reference in its entirety.

Reference Signs List
10 Metachromatic orthochromatic dye
10A Dispensed metachromatic orthochromatic dye
20 Blood sample
30 Measurement sample
40 Sample preparation unit
50 Flow cytometer
51 Flow cell
52 Laser light source
53 Irradiation-light condensing lens
54 Scattered-light condensing lens
55, 56, 57 Beam splitter
58, 59 Wavelength selection filter
61 Detector for small-angled forward-scattered light (FSs)
62 Detector for large-angled forward-scattered light (FLs)
63 Lateral-scattered light detector (SS)
64 First fluorescence detector (FL1)
65 Second fluorescence detector (FL2)
70 Processor (CPU)

Claims

A particle analysis method of analyzing particles contained in a blood sample, comprising:
staining the particles with a metachromatic orthochromatic dye;
irradiating the stained particles with light;
measuring intensity of a first fluorescence derived from a stacking component of the metachromatic orthochromatic dye and intensity of a second fluorescence derived from an intercalation component of the metachromatic orthochromatic 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 intensity of the second fluorescence, emitted by each of the particles, by the size of each of the particles to obtain a fluorescence concentration of each of the first fluorescence and the second fluorescence in each of the particles;
clustering each of the particles into a plurality of particle clusters including at least two of an erythrocyte cluster, a platelet cluster and a nucleated cell cluster, in a two-dimensional plot of the fluorescence concentration obtained by the normalization; and
creating a size histogram in which the size of each particle included in at least one particle cluster included in the plurality of particle clusters is a class.
The particle analysis method according to claim 1, further comprising analyzing the particles included in the particle cluster based on the size histogram.
The particle analysis method according to claim 1 or 2, further comprising dividing an axis indicating the size of each of the particles in the size histogram into a plurality of regions and reclassifying the at least one particle cluster into a plurality of subclusters based on the number or ratio of the particles in each of the plurality of regions formed by the division.
The particle analysis method according to claim 3, wherein the size histogram includes a size histogram for an erythrocyte cluster, the particle analysis method comprising reclassifying the erythrocyte cluster into a plurality of subclusters including a subcluster of normal erythrocytes, and a large erythrocyte subcluster, a disrupted erythrocyte subcluster and/or a small erythrocyte subcluster.
The particle analysis method according to claim 3 or 4, wherein the size histogram includes a size histogram for a platelet cluster, the particle analysis method comprising reclassifying the platelet cluster into a plurality of subclusters including a subcluster of normal platelets, and a giant platelet subcluster, a large platelet subcluster and/or a small platelet subcluster.
The particle analysis method according to any one of claims 3 to 5, wherein the size histogram includes a size histogram for a nucleated cell cluster, the particle analysis method comprising reclassifying the nucleated cell cluster into a plurality of subclusters including a subcluster of normal nucleated cells, and a large nucleated cell subcluster, a disrupted nucleated cell subcluster and/or a small nucleated cell subcluster.
The particle analysis method according to any one of claims 1 to 6, further comprising, for the at least one particle cluster for which the size histogram is created, creating a two-dimensional plot diagram in which the intensity of the first fluorescence or the intensity of the second fluorescence of each particle included in the particle cluster is one axis and the size of each particle included in the particle cluster is the other axis.
The particle analysis method according to any one of claims 1 to 7, wherein the first fluorescence is an orange fluorescence, and the second fluorescence is a green fluorescence.
The particle analysis method according to claim 8, wherein the metachromatic orthochromatic dye is acridine orange (AO).
The particle analysis method according to any one of claims 1 to 9, wherein a central wavelength of the light applied to the particles contained in the blood sample is 408 nm, 445 nm, 473 nm or 488 nm.
A 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 light detector including a plurality of fluorescence detectors that detect each of a first fluorescence and a second fluorescence having different wavelengths; and
a data processing part that normalizes the intensities of the first fluorescence and the second fluorescence emitted by each of the particles contained in the blood sample by 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, clusters each of the particles into a plurality of particle clusters including at least two of an erythrocyte cluster, a platelet cluster and a nucleated cell cluster, in a two-dimensional plot of the fluorescence concentration obtained by the normalization, and creates a size histogram in which the size of each particle included in at least one particle cluster included in the plurality of particle clusters is a class.
The particle analyzer according to claim 11, wherein the data processing part analyzes the particles included in the particle cluster based on the size histogram.