Biological sample analysis system, analysis method, and information processing device

Abstract

One objective of the present disclosure is to improve a S / N ratio in a different-axis illumination system.　The present disclosure provides a biological sample analysis system including: a light emitting unit configured to emit light at least at two positions onto particles flowing through a flow passage; and an information processing unit configured to calculate average signal intensity data of the particles from a signal intensity of light generated by the emission of light at each of the at least two positions, and to execute analysis processing using the average signal intensity data. The present disclosure also provides an information processing device and an analysis method that employ the average signal intensity data.

Description

Biological sample analysis system, analysis method, and information processing device

[0001] The present disclosure relates to a biological sample analysis system, an analysis method, and an information processing device. More specifically, the present disclosure relates to a biological sample analysis system configured to irradiate particles flowing through a flow path with light at at least two positions, an analysis method including performing analysis using the signal intensity of light generated by irradiating particles flowing through the flow path with light at at least two positions, and an information processing device that executes the analysis process.

[0002] For example, particle characteristics are measured by labeling a particle population, such as cells, microorganisms, and liposomes, with a fluorescent dye, irradiating each particle in the particle population with a laser beam, and measuring the intensity and / or pattern of fluorescence emitted from the excited fluorescent dye. An example of a biological sample analyzer that performs such measurements is a flow cytometer. Another example of a biological sample analyzer configured to separate cells is a cell sorter.

[0003] Flow cytometers and cell sorters can be configured to analyze multiple particles one by one by irradiating particles flowing in a single file through a flow channel with laser light (excitation light) of a specific wavelength and detecting the fluorescence and / or scattered light emitted from each particle. These devices convert the light detected by a photodetector into an electrical signal, which is then digitized and subjected to statistical analysis to determine the properties of each particle, such as type, size, and structure.

[0004] Such biological sample analyzers may be configured to irradiate each particle with light at one position, or may be configured to irradiate each particle with light at two or more positions. For example, Patent Document 1 below discloses a biological sample analyzer including a light irradiator that irradiates biological particles contained in a biological sample with light, a detector that detects light generated by the light irradiation, and an information processor that controls the light irradiator. The information processor determines whether the fluorescence detection result by the detector satisfies a predetermined condition and adjusts the output of light irradiation by the light irradiator based on the determination result (claim 1). This document also describes that the light irradiator may include two or more laser light sources that irradiate coaxially, or two or more laser light sources that irradiate at different axes (claims 5 and 6).

[0005] International Publication No. 2022 / 270009

[0006] Biological sample analysis systems such as flow cytometers and cell separation systems can be configured to acquire scattered light and / or fluorescent light signals and perform analytical evaluation of cells based on the acquired signals. For example, in biological sample analysis systems such as spectral flow cytometers, an axis and a fluorescent wavelength band can be set in the setting of a fluorescent dye channel, and data obtained by averaging signals acquired in the set channel can be used for analysis.

[0007] The light irradiation unit that irradiates light to generate the scattered light and / or fluorescence may be configured to irradiate one spot with laser light of two or more different wavelengths, and this light irradiation method is also called a coaxial irradiation method. The light irradiation unit may also be configured to irradiate two or more spots with laser light of one or more different wavelengths, and this light irradiation method is also called a heteroaxial irradiation method. In the case of the coaxial irradiation method, increasing the number of irradiated lasers per spot reduces the effect of shot noise and increases the S / N ratio for some fluorescent dyes, but reduces the number of dyes that can be separated. On the other hand, in the case of the heteroaxial irradiation method, the number of dyes that can be separated increases, but as the number of lasers per spot decreases, some fluorescent dyes experience a decrease in the S / N ratio due to shot noise.

[0008] When an off-axis illumination system is employed, it is considered desirable to improve the S / N ratio. Therefore, an object of the present disclosure is to address such problems in the off-axis illumination system.

[0009] The present disclosure provides a biological sample analysis system including: a light irradiation unit configured to irradiate particles flowing through a flow path with light at at least two positions; and an information processing unit configured to calculate average signal intensity data for the particles from the signal intensities of light generated by the light irradiation at each of the at least two positions, and to perform an analytical process using the average signal intensity data. In one embodiment, the average signal intensity data may be average signal intensity data of fluorescence generated by the light irradiation of the particles. The information processing unit may be configured to perform a verification process in the analytical process, using the average signal intensity data of fluorescence, to verify the state of a device included in the system. The information processing unit may be configured to perform an analytical process of the biological sample using the average signal intensity data of fluorescence. The biological sample analysis system may further include a detection unit having at least two detector arrays that detect fluorescence generated by the light irradiation, and the at least two detector arrays may be configured to detect fluorescence generated by the light irradiation at the at least two positions, respectively. The average signal intensity data of fluorescence may be average signal intensity data of fluorescence generated by the light irradiation of one type of fluorescent dye. The light irradiated onto the at least two positions may be light of different wavelengths. In another embodiment, the average signal intensity data may be average signal intensity data of scattered light generated by irradiating the particles with light. The information processing unit may use the average signal intensity data of scattered light as a trigger in the analysis process. The biological sample analysis system may further include a detection unit including at least two detectors that detect scattered light generated by the light irradiation, and the at least two detectors may be configured to detect scattered light generated by irradiating the at least two positions with light, respectively. The biological sample analysis system may be configured to allow a user to select whether to perform the analysis process using the average signal intensity data or to perform the analysis process without using the average signal intensity data. The biological sample analysis system may be configured to display a screen prompting the user to make the selection.The biological sample analysis system may have a display unit that displays a screen for making the selection. The biological sample analysis system may be configured to display a screen for selecting the type of light from which the average signal intensity data is generated. The biological sample analysis system may be configured to allow selection of at least two light irradiation positions from which signal intensity data used to calculate the average signal intensity data is derived. The biological sample analysis system may be configured to display an area that prompts the user to select a fluorescence channel to be used to calculate the average signal intensity data. The biological sample analysis system may be a flow cytometer. The present disclosure also provides an analysis method including: calculating average signal intensity data for particles flowing through a flow path from signal intensities of light generated by irradiating the particles flowing through a flow path with light at at least two positions; and performing an analytical process using the average signal intensity data. The present disclosure also provides an information processing device configured to calculate average signal intensity data for particles flowing through a flow path from signal intensities of light generated by irradiating the particles flowing through a flow path with light at each of the at least two positions, and to perform an analytical process using the average signal intensity data.

[0010] 1 is a schematic diagram showing an example configuration of a biological sample analysis system according to the present disclosure; FIG. 1 is a schematic diagram illustrating a plurality of irradiation points where light intersects with particles in a biological sample; FIG. 1 is a schematic diagram illustrating an example configuration of a light irradiation unit and detection unit using a different axis irradiation method; FIG. 2 is a schematic diagram illustrating a detector array; FIG. 3 is a diagram illustrating a histogram generated by flow cytometry of 8-peak beads; FIG. 4 is a table showing evaluation values ​​calculated by flow cytometry of 8-peak beads; FIG. 4 is a diagram illustrating a histogram generated by flow cytometry of a bead sample; FIG. 5 is a table showing coefficients of variation calculated by flow cytometry of a bead sample; FIG. 6 is an example of a screen prompting a user to select whether to use average signal intensity data; FIG. 7 is an example of an area prompting a user to select a fluorescence channel to be used for calculating average signal intensity data; FIG. 8 is an example of a screen prompting a user to select whether to use average signal intensity data; FIG. 9 is an example of a screen prompting a user to select a type of light to calculate average signal intensity data; FIG. 10 is an example of a screen prompting a user to select a type of light to calculate average signal intensity data; FIG. 11 is an example of an area prompting a user to select a fluorescence channel to be used for calculating average signal intensity data. 10 is an example of an area that prompts the user to select a fluorescence channel to be used for calculating average signal intensity data. 11 is an example of an area that prompts the user to select a fluorescence channel to be used for calculating average signal intensity data.

[0011] Preferred embodiments for carrying out the present disclosure will be described below. Note that the embodiments described below are representative embodiments of the present disclosure, and the scope of the present disclosure is not limited to these embodiments. Note that the present disclosure will be described in the following order. 1. First Embodiment (Biological Sample Analysis System) (1) Basic Concept (1-1) Application Example 1 of the Present Disclosure (1-2) Application Example 2 of the Present Disclosure (1-3) Application Example 3 of the Present Disclosure (2) Configuration Examples (2-1) Configuration Examples of the Device (2-2) Example of Different Axis Irradiation Method (2-3) Examples of Light Irradiation Unit and Detection Unit (2-4) Examples of Particles (3) Verification Example 1 (4) Verification Example 2 (5) Reference Examples and Examples (6) Examples Related to User Interface 2. Second Embodiment (Information Processing Device) 3. Third Embodiment (Analysis Method and Program for Executing the Analysis Method)

[0012] 1. First embodiment (biological sample analysis system)

[0013] (1) Basic Concept The inventors discovered that when light irradiation is performed using a different-axis irradiation method, the S / N ratio can be improved by using the average of the same type of scattering signal or the average of fluorescence signals in the same fluorescence wavelength band obtained by light irradiation at each spot. In other words, by using this average, the S / N ratio can be improved compared to when signal data obtained by light irradiation at each spot are used as separate data in analysis. For example, by using this average, the detection ability of microparticles can be improved. Furthermore, by using this average, fluorescence separation performance can also be improved.

[0014] That is, a biological sample analysis system according to the present disclosure may include a light irradiation unit configured to irradiate particles flowing through a flow path with light at at least two positions, and an information processing unit configured to calculate average signal intensity data for the particles from the signal intensity of the light generated by the light irradiation at each of the at least two positions, and to perform analysis processing using the average signal intensity data.

[0015] The light irradiation unit is configured to irradiate particles flowing through the flow path with light at at least two positions. That is, as each particle flows through the flow path, each particle is irradiated with light at each of the at least two positions. That is, the light irradiation unit may be configured to perform light irradiation using a different-axis irradiation method. Furthermore, the light irradiated at the at least two positions may be light of different wavelengths. Furthermore, the information processing unit may be configured to calculate average signal intensity data for each particle from the signal intensity of light generated by light irradiation at each of the at least two positions, and to perform analytical processing using the average signal intensity data. That is, the information processing unit may be configured to calculate average signal intensity data for each particle from the signal intensity of light generated at each position by irradiating each particle with light using the different-axis irradiation method. The information processing unit may then perform analytical processing using the average signal intensity data. By utilizing the average signal intensity data, the S / N ratio can be improved in a biological sample analysis system configured to perform light irradiation using the different-axis irradiation method, such as a flow cytometer or a system configured to perform flow cytometry.

[0016] The analytical process may be a verification process for verifying the state of an apparatus or an analytical process for analyzing a biological sample. The verification process may be a process for verifying the state of an apparatus included in the system, for example, a process for verifying the state of a light irradiation unit and / or a detection unit included in the system, or a process for verifying the state of a biological sample analyzer including the light irradiation unit and the detection unit. The verification process may be a verification process using beads as particles. The verification process may include calculating an evaluation value for the verification. As the evaluation value, for example, one or more of a linearity evaluation value (Linearity), a precision evaluation value (MESF), a sensitivity evaluation value (Q value and B value), and a coefficient of variation (CV) may be calculated in the verification process. Furthermore, the process for analyzing the biological sample may be a process for analyzing biological particles such as cells or exosomes as particles. The process for analyzing the biological sample may be an analytical process including, for example, an unmixing process or a fluorescence compensation process, or may include both of these processes. In this manner, the average signal intensity data can be utilized in a variety of analyses.

[0017] Furthermore, the average signal intensity data may be average signal intensity data of fluorescent light generated by irradiating the particles with light, or average signal intensity data of scattered light generated by irradiating the particles with light. In this manner, average signal intensity data for various types of light may be acquired.

[0018] Furthermore, the signal intensity data may be, for example, height data or area data, or both of these. That is, the average signal intensity data may be average signal intensity data calculated from height data of each particle (or each event), or may be average signal intensity data calculated from area data of each particle (or each event), or may be both of these two types of average signal intensity data.

[0019] According to the present disclosure, the average signal intensity data for each particle (or each event) is calculated, and various analytical processes can be performed using the calculated average signal intensity data (particularly, a dataset of the average signal intensity data). The calculation of the average signal intensity data may be, for example, a simple average (arithmetic mean), or may be a calculation of another average value (weighted mean, geometric mean, or harmonic mean). For example, a coefficient used in calculating the other average value may be appropriately set by a person skilled in the art. That is, the average signal intensity data may be calculated by averaging using a predetermined coefficient. The predetermined coefficient can be appropriately set by a person skilled in the art depending on the axis to be averaged (particularly, the light irradiation position or the wavelength or intensity of light irradiated at the light irradiation position, etc.).

[0020] (1-1) Application Example 1 of the Present Disclosure An example of the use of average signal intensity data according to the present disclosure is when a fluorescent dye with a wide range of excitation wavelengths is used. That is, a fluorescent dye excited by two or more light beams (particularly laser beams) with different wavelengths is assumed to be used in the analysis. In this case, average fluorescence signal intensity data is used. An example of such a fluorescent dye is PE. PE generates fluorescence when irradiated with, for example, 488 nm light and 561 nm light. When a light irradiation unit using a different axis irradiation method is used, configured to irradiate a particle with 488 nm laser beam at one position and irradiate the particle with 561 nm laser beam at another position, fluorescence is generated by the light irradiation of the particle at each position, and the fluorescence generated at each position is detected by separate photodetectors. In this way, signal intensity data of fluorescence generated by light irradiation at the one position and signal intensity data of fluorescence generated by light irradiation at the other position are obtained. According to the present disclosure, these two signal intensity data may be averaged to generate average signal intensity data. In this manner, in the present disclosure, two or more signal intensity data are averaged to generate average signal intensity data. The two or more signal intensity data to be averaged may be signal intensity data in a predetermined wavelength band. The wavelength band may be appropriately set by a person skilled in the art, for example, depending on the type of fluorescent dye, or the system according to the present disclosure (particularly, the information processing unit) may set the wavelength band depending on the type of fluorescent dye. For example, when a photodetector having multiple fluorescence channels is used, one or more specific fluorescence channels may be set as the fluorescence channels to be averaged depending on the fluorescence wavelength band of the fluorescent dye used. The information processing unit may average the signal intensity data acquired by the one or more fluorescence channels set in this manner to obtain average signal intensity data. Furthermore, the average fluorescence signal intensity data may include average signal intensity data of fluorescence generated by light irradiation of one type of fluorescent dye. The fluorescent dye may be, for example, a fluorescent dye that generates fluorescence when irradiated with two or more light beams (particularly two or more laser beams) having different wavelengths.Examples of such fluorescent dyes include, but are not limited to, PE, which will be described later. The present disclosure may be applied to such fluorescent dyes with a relatively wide excitation wavelength range. Furthermore, when two or more such fluorescent dyes are used in an analysis, average signal intensity data may be calculated for each of the two or more fluorescent dyes. The information processing unit may be configured to calculate the average signal intensity data as described above.

[0021] Furthermore, a fluorescent dye used in an analysis may produce fluorescence only when irradiated with light at one of multiple light irradiation positions. In this case, average signal intensity data may not be acquired, and only the signal intensity data may be used. That is, the signal intensity data output from the photodetector that detected the fluorescence may be used in the analysis process without being subjected to averaging. In this manner, the information processing unit may use the signal intensity data of the fluorescence produced by light irradiation at one position for analysis without performing averaging. That is, the system may be configured to allow a user to select whether to use the average signal intensity data when performing the analysis. Furthermore, the information processing unit may display a screen prompting the user to make the selection. The system may also have a display unit (e.g., a display device such as a monitor) that displays the screen.

[0022] For example, the system may be configured to allow a user to select whether to use average signal intensity data or non-averaged signal intensity data for a fluorescent dye that fluoresces when irradiated with light of two or more different wavelengths, such as the above-mentioned PE, and the information processing unit may be configured to display a screen prompting the user to make the selection.

[0023] (1-2) Application Example 2 of the Present Disclosure Another example of using average signal intensity data according to the present disclosure is when the average signal intensity of scattered light or fluorescence is used as a trigger. That is, the information processing unit may use the average signal intensity data of scattered light as a trigger in the analysis process. In this case, the average signal intensity data of scattered light or fluorescence may be used, and in particular, the average signal intensity data of scattered light is used. In biological sample analysis such as flow cytometry, not all signals output by a photodetector are attributable to light generated from target particles. For example, light or objects unrelated to target particles may be detected. Therefore, in order to analyze only signals related to target particles, a channel to be used as a trigger is set. Then, a predetermined threshold is set for determining whether the signal is related to a target particle. That is, only events whose signal intensity acquired by that channel is equal to or greater than the predetermined threshold are considered to be events related to target particles and are analyzed. According to the present disclosure, the average signal intensity data of scattered light or fluorescence may be used as a trigger. For example, when a light irradiation unit of a different axis type is used, which is configured to irradiate a particle with laser light at one position and to irradiate the particle with laser light at another position, scattered light (e.g., FSC or SSC) is generated by the light irradiation of the particle at each position, and the scattered light (e.g., FSC or SSC) generated at each position is detected by a photodetector. In this manner, signal intensity data of the scattered light (e.g., FSC or SSC) generated by the light irradiation at the one position and signal intensity data of the scattered light (e.g., FSC or SSC) generated by the light irradiation at the other position are obtained. According to the present disclosure, these two signal intensity data are averaged to generate average signal intensity data of the scattered light (e.g., FSC or SSC). The channel used for triggering may be a scattered light channel or a fluorescence channel, particularly a scattered light channel, and more particularly an FSC (forward scattered light) channel or an SSC (side scattered light) channel. In this manner, the information processing unit may be configured to use the average signal intensity data as a trigger. The information processing unit may be configured to allow a user to select whether or not to use average signal strength data as a trigger.By using the average signal intensity data as a trigger, i.e., by analyzing only particles whose average signal intensity data is equal to or greater than a predetermined threshold, unwanted signals can be more accurately excluded.

[0024] (1-3) Application Example 3 of the Present Disclosure Yet another example of the use of average signal intensity data according to the present disclosure is when an unmixing process or a fluorescence compensation process is performed using the average signal intensity. For example, average fluorescence signal intensity data is used in the unmixing process or the fluorescence compensation process. In this case, multiple fluorescence channels to be combined to obtain average signal intensity data may first be determined, and single stain data may be created from the signal intensity data obtained in the multiple fluorescence channels. The unmixing process or the fluorescence compensation process may then be performed using the single stain data. Alternatively, the unmixing process or the fluorescence compensation process may first be performed as usual without obtaining average signal intensity data, and then average signal intensity data for a specified channel may be calculated. Analysis results may then be displayed based on the average signal intensity data. In this way, the information processing unit may be configured to perform the unmixing process, the fluorescence compensation process, or both using the average signal intensity data. Furthermore, the information processing unit may be configured to allow a user to select whether to use average signal intensity data when performing these processes.

[0025] As described above, by using average signal intensity data according to the present disclosure, the S / N ratio can be improved compared to single-axis data, and separation performance can be improved. Furthermore, by using average signal intensity data for triggering according to the present disclosure, target particles can be more accurately identified, which contributes to, for example, obtaining better analytical results and more accurate particle sorting.

[0026] The average signal intensity data may be used in various analytical processes. Examples of the analytical processes include an analytical process for biological particles and a verification process for verifying the status of a device included in the system (e.g., a device equipped with a light irradiation unit and / or a detection unit, particularly a biological sample analysis device, such as a flow cytometer or a cell sorter). The verification process may include, for example, a QC (Quality Control) process. The verification process may include, for example, a determination of changes or abnormalities in output (fluorescence output or scattered light output).

[0027] Below, an example configuration of a system according to the present disclosure will be described first. A system according to the present disclosure may be configured like the biological sample analyzer described in (2-1) below, i.e., it may include a light irradiation unit, a detection unit, and an information processing unit (and optionally a fractionation unit) included in the device. A system according to the present disclosure may be configured as a biological sample analyzer in which the light irradiation unit, the detection unit, and the information processing unit (and optionally the fractionation unit) are incorporated into a single device, or as a biological sample analysis system in which these components are distributed across multiple devices. In the latter case, for example, the light irradiation unit and the detection unit (and the fractionation unit) may be incorporated into a single device (e.g., constituting a biological sample analyzer), and the information processing unit may be configured as another device (e.g., an information processing device). For example, the biological sample analysis system may include an information processing device configured as the information processing unit. In other words, the biological sample analysis system may be configured as a system including an analyzer having the light irradiation unit and the detection unit (and the fractionation unit) in addition to the information processing device. These devices may be connected to each other via wire or wirelessly. These devices may be connected via a network. More specific examples of the light irradiation unit and the detection unit are described in (2-2) and (2-3) below. Furthermore, a specific demonstration example is described in (3) below.

[0028] (2) Configuration example

[0029] (2-1) Example Device Configuration An example configuration of a biological sample analyzer according to the present disclosure is shown in FIG. 1. The biological sample analyzer 6100 shown in FIG. 1 includes a light irradiation unit 6101 that irradiates light onto a biological sample S flowing through a flow path C, a detection unit 6102 that detects light generated by irradiating the biological sample S with light, and an information processing unit 6103 that processes information about the light detected by the detection unit. Examples of the biological sample analyzer 6100 include a flow cytometer and an imaging cytometer. The biological sample analyzer 6100 may also include a fractionation unit 6104 that separates specific biological particles P from within the biological sample. An example of a biological sample analyzer 6100 that includes the fractionation unit is a cell sorter.

[0030] (Biological Sample) The biological sample S may be a liquid sample containing biological particles. The biological particles may be, for example, cells or non-cellular biological particles. The cells may be living cells, and more specific examples include blood cells such as red blood cells and white blood cells, and reproductive cells such as sperm and fertilized eggs. The cells may be directly collected from a specimen such as whole blood, or may be cultured cells obtained after culturing. Examples of the non-cellular biological particles include extracellular vesicles, particularly exosomes and microvesicles. The biological particles may be labeled with one or more labeling substances (e.g., dyes (particularly fluorescent dyes) and fluorescent dye-labeled antibodies). Note that the biological sample analyzer of the present disclosure may also analyze particles other than biological particles, such as beads for calibration purposes.

[0031] (Flow Channel) The flow channel C is configured to allow the biological sample S to flow. In particular, the flow channel C can be configured to form a flow in which biological particles contained in the biological sample are aligned in a substantially straight line. The flow channel structure including the flow channel C may be designed to form a laminar flow. In particular, the flow channel structure is designed to form a laminar flow in which the flow of the biological sample (sample flow) is surrounded by the flow of sheath liquid. The design of the flow channel structure may be appropriately selected by those skilled in the art, and a known design may be adopted. The flow channel C may be formed in a flow channel structure such as a microchip (a chip having flow channels on the order of micrometers) or a flow cell. The width of the flow channel C may be 1 mm or less, particularly 10 μm or more and 1 mm or less. The flow channel C and the flow channel structure including it may be formed from a material such as plastic or glass.

[0032] The biological sample analyzer of the present disclosure is configured so that light from light irradiation unit 6101 is irradiated onto the biological sample flowing within flow path C, and particularly onto biological particles within the biological sample. The biological sample analyzer of the present disclosure may be configured so that the interrogation point of light on the biological sample is within the flow path structure in which flow path C is formed, or so that the interrogation point of light is outside the flow path structure. An example of the former is a configuration in which the light is irradiated onto flow path C within a microchip or flow cell. In the latter, the light may be irradiated onto biological particles after they have left the flow path structure (particularly its nozzle portion), and an example of this is a jet-in-air flow cytometer.

[0033] (Light Irradiation Unit) The light irradiation unit 6101 includes a light source unit that emits light and a light-guiding optical system that guides the light to an irradiation point. The light source unit includes one or more light sources. The type of light source is, for example, a laser light source or an LED. The wavelength of the light emitted from each light source may be any of ultraviolet light, visible light, and infrared light. The light-guiding optical system includes optical components such as a beam splitter group, a mirror group, or an optical fiber. The light-guiding optical system may also include a lens group for focusing light, such as an objective lens. There may be one or more irradiation points where the light intersects with the biological sample. The light irradiation unit 6101 may be configured to focus light irradiated from one or more different light sources onto one irradiation point.

[0034] (Detection Unit) The detection unit 6102 includes at least one photodetector that detects light generated by irradiating the bioparticles with light. The detected light is, for example, fluorescence or scattered light (e.g., one or more of forward scattered light, back scattered light, and side scattered light). Each photodetector includes one or more light-receiving elements, for example, a photodetector array. Each photodetector may include one or more PMTs (photomultiplier tubes) and / or photodiodes such as APDs and MPPCs as light-receiving elements. The photodetector includes, for example, a PMT array in which multiple PMTs are arranged in a one-dimensional direction. The detection unit 6102 may also include an imaging element such as a CCD or CMOS. The detection unit 6102 can acquire images of the bioparticles (e.g., bright-field images, dark-field images, and fluorescence images) using the imaging element.

[0035] The detection unit 6102 includes a detection optical system that allows light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system includes a spectroscopic unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or an optical filter. The detection optical system is configured to, for example, disperse light generated by irradiating bioparticles with light, and detect the dispersed light using a plurality of photodetectors, the number of which is greater than the number of fluorescent dyes with which the bioparticles are labeled. A flow cytometer that includes such a detection optical system is called a spectral flow cytometer. The detection optical system is also configured to, for example, separate light corresponding to the fluorescent wavelength range of a specific fluorescent dye from the light generated by irradiating bioparticles with light, and detect the separated light using a corresponding photodetector.

[0036] The detection unit 6102 may also include a signal processing unit that converts the electrical signal obtained by the photodetector into a digital signal. The signal processing unit may include an A / D converter as a device that performs the conversion. The digital signal obtained by the conversion by the signal processing unit may be transmitted to the information processing unit 6103. The digital signal may be handled by the information processing unit 6103 as data related to light (hereinafter also referred to as "light data"). The light data may be light data including, for example, fluorescent light data. More specifically, the light data may be light intensity data, and the light intensity may be light intensity data of light including fluorescent light (which may include feature quantities such as area, height, and width).

[0037] (Information Processing Unit) The information processing unit 6103 includes, for example, a processing unit that processes various data (e.g., optical data) and a storage unit that stores various data. When the processing unit acquires optical data corresponding to a fluorescent dye from the detection unit 6102, the processing unit may perform fluorescence spillover correction (compensation processing) on ​​the light intensity data. Furthermore, in the case of a spectral flow cytometer, the processing unit performs fluorescence separation processing on the optical data to acquire light intensity data corresponding to the fluorescent dye. The fluorescence separation processing may be performed, for example, according to the unmixing method described in Japanese Patent Application Laid-Open No. 2011-232259. When the detection unit 6102 includes an image sensor, the processing unit may acquire morphological information of bioparticles based on images acquired by the image sensor. The storage unit may be configured to store the acquired optical data. The storage unit may further be configured to store spectral reference data used in the unmixing processing.

[0038] If the biological sample analyzer 6100 includes a fractionating unit 6104 (described below), the information processing unit 6103 can determine whether to fractionate bioparticles based on the optical data and / or morphological information. The information processing unit 6103 can then control the fractionating unit 6104 based on the result of this determination, allowing the fractionating unit 6104 to fractionate the bioparticles.

[0039] The information processing unit 6103 may be configured to output various data (e.g., optical data and images). For example, the information processing unit 6103 may output various data (e.g., two-dimensional plots, spectral plots, etc.) generated based on the optical data. The information processing unit 6103 may also be configured to accept input of various data, such as accepting gating processing on a plot by a user. The information processing unit 6103 may include an output unit (e.g., a display, etc.) or an input unit (e.g., a keyboard, etc.) for executing the output or input.

[0040] The information processing unit 6103 may be configured as a general-purpose computer, for example, as an information processing device including a CPU, RAM, and ROM. The information processing unit 6103 may be included in a housing that includes the light irradiation unit 6101 and the detection unit 6102, or may be located outside the housing. Furthermore, various processes or functions performed by the information processing unit 6103 may be realized by a server computer or a cloud connected via a network.

[0041] (Sorting unit) The sorting unit 6104 sorts the bioparticles according to the determination result by the information processing unit 6103. The sorting method may be a method of generating droplets containing bioparticles by vibration, applying an electric charge to the droplets to be sorted, and controlling the direction of travel of the droplets using electrodes. The sorting method may also be a method of controlling the direction of travel of the bioparticles within the flow channel structure to perform sorting. The flow channel structure is provided with, for example, a control mechanism using pressure (spray or suction) or electric charge. An example of such a flow channel structure is a chip (for example, the chip described in JP 2020-76736 A) having a flow channel structure in which a flow channel C branches downstream into a recovery flow channel and a waste flow channel, and specific bioparticles are recovered into the recovery flow channel.

[0042] (2-2) Example of Different-Axis Illumination Method In the present disclosure, the biological sample analysis system may be configured to perform light irradiation using a different-axis illumination method. For example, with respect to the light irradiation unit, there are multiple illumination points where the biological sample and the light intersect. This will be described with reference to FIG. 2. The figure shows a flow path C through which particles P to be analyzed flow. As shown in the figure, flow path C has three positions S1, S2, and S3 where laser light is irradiated. These positions are also called light irradiation spots. That is, particles P flowing through flow path C in the direction of the arrows in the figure first pass through spot S1 to generate light, then pass through spot S2 to generate more light, and finally pass through spot S3 to generate more light. In this way, a biological sample analysis system employing a different-axis illumination method may be configured to irradiate each particle with light at multiple spots. The number of spot positions is not limited to the three shown in the figure, and may be two or more, for example, 2 to 20, particularly 2 to 15, more particularly 2 to 10, 2 to 9, or 2 to 8, for example, 2 to 7. Furthermore, it is not necessary to use signal intensity data for all of these multiple spots to calculate the average signal intensity data, and the average signal intensity may be calculated from the signal intensities of, for example, two, three, or four of these multiple spots, particularly the average signal intensity data may be calculated from the signal intensities generated by light irradiation at two or three spots, more particularly the average signal intensity data may be calculated from the signal intensities generated by light irradiation at two spots.

[0043] (2-3) Examples of Light Irradiation Unit and Detection Unit Next, a more specific configuration example of the light irradiation unit and detection unit will be described with reference to FIG. 3A. This figure shows a schematic diagram of the light irradiation unit and detection unit of a biological sample analysis system using a different-axis illumination method. As shown in the figure, the biological sample analysis system 10 includes a light irradiation unit 20 configured to irradiate light onto a flow path through which particles to be analyzed flow, and a detection unit 30 that detects the light generated by the light irradiation. The system is configured to irradiate light onto each particle at seven positions within the flow path, i.e., it is a system using a different-axis illumination method.

[0044] The light irradiation unit has a plurality of light sources 21 (21a to 21g) that irradiate a plurality of types of laser light with different wavelengths. The light irradiation unit further has a light irradiation unit-side light guiding optical system 22 configured to irradiate the laser light emitted from each light source to a predetermined irradiation position. The light guiding optical system 22 has, for example, a group of mirrors 22a to 22g as shown in the figure. The group of mirrors may include, for example, a half mirror or a mirror. The light guiding optical system 22 may be designed appropriately by a person skilled in the art depending on, for example, the structure of the system, and may include other optical elements. The light guiding optical system 22 may include, for example, a group of lenses. The group of lenses may include, for example, one or more of a condenser lens, a collimator lens, and an objective lens.

[0045] The light irradiation unit is configured to irradiate light onto a predetermined position, such as a flow cell 40 (or chip) provided with a flow path through which particles flow. While three light irradiation positions are shown in FIG. 2 described in (2-2) above, the biological sample analysis system (or) 10 shown in FIG. 3A is configured to irradiate particles with light at, for example, seven light irradiation positions. The seven light irradiation positions may be aligned in a line along the particle flow direction. As mentioned above, the number of light irradiation positions may be two or more, and may be, for example, 2 to 20, particularly 2 to 15, more particularly 2 to 10, 2 to 9, or 2 to 8, or may be, for example, 2 to 7.

[0046] The detection unit is configured to detect light generated by the light irradiation unit irradiating particles with light. The detection unit includes multiple detector arrays 34 (34a to 34g). Each detector array may have multiple fluorescence channels. Each fluorescence channel may be assigned to detect light of a predetermined wavelength. Each detector array may be configured as a module housed in a housing 42 having a window through which the detector array 41 is exposed, as shown in FIG. 3B, for example. The detector array 41 may be configured so that photodetector units 43 are arranged in a row, and each photodetector unit may be assigned to a corresponding fluorescence channel. The module may be connected to, for example, an information processing unit via any cable. Signal intensity data may be transmitted from the module to the information processing unit, and the information processing unit may calculate average signal intensity data using the signal intensity data. Note that, although the detector array in FIG. 3B has 10 photodetector units, the number of photodetector units in each detector array is not limited thereto and may be, for example, 2 or more, particularly 5 or more, or 10 or more. The number of photodetector units may be, for example, 100 or less, particularly 50 or less, 40 or less, or 30 or less. This number may be appropriately selected by one skilled in the art. Although the module includes one detector array in the figure, the module may include multiple detector arrays. Light generated by light irradiation from light sources 21a to 21g is detected by detector arrays 34a to 34g, respectively. That is, light generated by light irradiation of particles by light source 21a is detected by detector array 34a. A pair of light source 21a and detector array 34a constitutes one axis. The same applies to light sources 21b to 21g and detector arrays 34b to 34g. In this manner, system 10 is configured as a different-axis illumination system having seven axes. Thus, the biological sample analysis system according to the present disclosure may have a detection unit including at least two detector arrays that detect fluorescence generated by the light irradiation. The at least two detector arrays may be configured to detect fluorescence generated by light irradiation at the at least two positions, respectively.

[0047] The detection unit may have, for example, a detection unit-side light-guiding optical system 31 on the optical path of the light generated by the light irradiation. The light-guiding optical system 31 may be configured, for example, like the detection optical system described in Japanese Patent Application Laid-Open No. 2021-76451, but is not limited to this configuration and other configurations may be adopted. The detection unit further includes an optical fiber 32 into which the light emitted from the light-guiding optical system 31 is incident, and a plurality of focusing lenses 33 (33a to 33g) that focus the light emitted from each optical fiber 32 onto each detection element 34 (34a to 34g).

[0048] The multiple light sources 21 (21a-21g) may be configured to emit laser light having wavelengths (excitation wavelengths), for example, 320 nm, 355 nm, 405 nm, 488 nm, 561 nm, 637 nm, and 808 nm. Each detection element 34 (34a-34g) may be configured to detect at least light having a wavelength equal to or greater than the wavelength of each light source 21. For example, corresponding to light sources 21a and 21b emitting laser light having wavelengths of 320 nm and 355 nm, detection elements 34a and 34b detecting scattered light and fluorescence from particles excited by these laser beams may be configured to detect wavelengths of approximately 360.5 nm to 843.8 nm. Corresponding to light source 21c emitting laser light having a wavelength of 405 nm, detection element 34c detecting scattered light and fluorescence from particles excited by these laser beams may be configured to detect wavelengths of approximately 413.6 nm to 843.8 nm. Corresponding to the light source 21d emitting a laser beam with a wavelength of 488 nm, the detection element 34d detecting scattered light and fluorescence from particles excited by this laser beam may be configured to be capable of detecting wavelengths of approximately 492.9 nm to 843.4 nm. Corresponding to the light source 21e emitting a laser beam with a wavelength of 561 nm, the detection element 34e detecting scattered light and fluorescence from particles excited by this laser beam may be configured to be capable of detecting wavelengths of approximately 555.3 nm to 843.8 nm. Corresponding to the light source 21f emitting a laser beam with a wavelength of 638 nm, the detection element 34f detecting scattered light and fluorescence from particles excited by this laser beam may be configured to be capable of detecting wavelengths of approximately 643.3 nm to 843.8 nm. Corresponding to the light source 21g that emits laser light with a wavelength of 808 nm, the detection element 34g that detects scattered light and fluorescence from particles excited by this laser light may be configured to be able to detect wavelengths of approximately 823.5 nm to 920.0 nm.

[0049] The detection unit may further include at least two detectors for detecting scattered light generated by the light irradiation. The at least two detectors may be configured to respectively detect scattered light generated by light irradiation at the at least two positions. By detecting scattered light generated by light irradiation at each of the at least two positions in this manner, the average signal intensity data of the scattered light described above can be calculated.

[0050] As in the configuration examples described above, the biological sample analysis system according to the present disclosure may have a configuration using an out-of-axis illumination method and may be configured to irradiate particles flowing through a flow path with light at at least two positions. In particular, the system may have a light irradiation unit configured in this manner. Furthermore, the system may have a detection unit that detects light generated by the light irradiation at the at least two positions. The detection unit may have the same number of detection elements as the number of positions.

[0051] (2-4) Examples of Particles The particles used in the present disclosure may be beads, such as fluorescent beads, or biological particles, such as cells or exosomes. The beads may be selected appropriately by those skilled in the art, but may be, for example, particles that emit high levels of fluorescence over a wide wavelength range. In one embodiment, the fluorescent beads emit fluorescence over the entire range of wavelengths detected by the multiple fluorescence channels. Examples of such fluorescent beads include the Automatic Setup Beads and Align Check Beads (both manufactured by Sony Group Corporation) mentioned above. In another embodiment, the fluorescent beads may include fluorescent beads containing multiple particle groups with gradually varying fluorescence intensity levels, such as 8-peak beads and 6-peak beads. The beads may have a substantially uniform particle size. The particle size may be selected appropriately by those skilled in the art depending on, for example, the size of the flow channel, but may be, for example, 100 μm or less, 50 μm or less, or 30 μm or less. The particle size may be, for example, 0.1 μm or more, 0.3 μm or more, or 0.5 μm or more. The fluorescent beads may include one type or two or more types of beads.

[0052] (3) Verification Example 1: The condition of a spectral flow cytometer (ID7000, Sony Group Corporation) was verified using commercially available fluorescent beads (8-peak beads, Sony Group Corporation). The fluorescent beads were 8-peak beads, consisting of one type of non-fluorescent bead and seven types of fluorescent beads. The fluorescent beads were configured to form eight peaks when flow cytometry was performed on the fluorescent beads. As described in (2) above, the flow cytometer had a light irradiation unit, a detection unit, and an information processing unit.

[0053] In the verification process, each particle constituting the fluorescent bead was irradiated at one position with a 488 nm excitation laser beam and at another position with a 561 nm excitation laser beam. For each event, signal intensity data (area data) in the PE fluorescence wavelength band of the fluorescence generated by the laser beam irradiation at these two positions was acquired. A histogram was generated using the signal intensity data of the fluorescence generated by the irradiation with the 488 nm excitation laser beam. Similarly, a histogram was generated using the signal intensity data of the fluorescence generated by the irradiation with the 561 nm excitation laser beam.

[0054] Furthermore, for each event, average signal intensity data was calculated from the signal intensity data (area data) of the PE fluorescence wavelength band of the fluorescence generated by laser light irradiation at these two positions. The average signal intensity data was the simple average of the values ​​of the two area data. A histogram was generated using the average signal intensity data obtained in this way.

[0055] Furthermore, using the two signal intensity data and the one average signal intensity data, evaluation values ​​for verifying the condition were calculated, including a linearity evaluation value (Linearity), a precision evaluation value (MESF), sensitivity evaluation values ​​(Q value and B value), and a coefficient of variation (CV).

[0056] Details of these evaluation values ​​and calculation methods will be explained below for the case of 8-peak beads. For other verification samples, those skilled in the art can also calculate each evaluation value as appropriate.

[0057] (a) Definitions Particle group Dim1: a particle group belonging to the peak with the lowest fluorescence intensity Particle group Dim2: a particle group belonging to the second peak from the bottom in fluorescence intensity Particle group Dim3: a particle group belonging to the third peak from the bottom in fluorescence intensity Particle group Dim4: a particle group belonging to the fourth peak from the bottom in fluorescence intensity Particle group Dim5: a particle group belonging to the fifth peak from the bottom in fluorescence intensity Particle group Dim6: a particle group belonging to the sixth peak from the bottom Particle group Dim7: a particle group belonging to the seventh peak from the bottom Particle group Dim8: a particle group belonging to the peak with the highest fluorescence intensity (eighth from the bottom) (Dim1 to Dim8 are also called Peak1 to Peak8, respectively.) MFI: mean fluorescence intensity (value obtained by converting the highest and lowest values ​​of the mean fluorescence signal intensity to 1 and 0) MEF2 to MEF8: reference MESF values ​​of Dim2 to Dim8 MESF: Molecules of Equivalent Soluble Fluorochromes (number of fluorescent molecules per particle) Average values ​​of Dim1 to Dim8: MFI1 to MFI8 Log values ​​of MFI1 to MFI8: LogMFI1 to LogMFI8 Reference values ​​of Dim1 to Dim8: MEF1 to MEF8 Log values ​​of MEF2 to MEF8: LogMEF2 to LogMEF8 Calculated MESF value of Dim1: MESF MESF of Dim2 to Dim4: MESF2 to MESF4 Coefficient of variation of Dim2 to Dim8: CV2 to CV8 Standard deviation of Dim2 to Dim8: SD2 to SD8

[0058] (b) Linearity evaluation value (Linearity) R is calculated using LogMFI2 to LogMFI8 and LogMEF2 to LogMEF8. 2 The coefficient of determination (COD) is calculated, and the square root of this is calculated as R, which is the linearity evaluation value (also called linearity). The closer the linearity evaluation value is to 100%, the better.

[0059] (c) Precision evaluation value (MESF: Molecules of Equivalent Soluble Fluorochromes, also known as fluorescence detection sensitivity) [1] A regression line is calculated using LogMFI2 to LogMFI8 and LogMEF2 to LogMEF8. [2] The fluorescence detection sensitivity (MESF) is calculated from the regression line using MFI1. The closer the fluorescence detection sensitivity (MESF) is to the Dim1 reference value (usually 0), the better.

[0060] (d) Sensitivity (Q value and B value) Calculate MESF2 to MESF4 for Dim2 to Dim4 (SD2). 2 = ((CV2) 2 - (CV8) 2 ) × (MESF2) 2 Similarly, (SD3) 2 , (SD4) 2 Also calculate. MESF2 to MESF4 on the horizontal axis, (SD2) on the vertical axis 2 ~(SD4) 2 Plot the above and determine the regression line, with the slope a and intercept b. The Q value is the reciprocal of the slope of the regression line (1 / a). The B value is the ratio of the slope to the intercept of the regression line (b / a).

[0061] (e) Coefficient of Variation (CV) The coefficient of variation is the degree of variation in signal intensity or average signal intensity expressed as a percentage, and is expressed by the following formula: CV (%) = SD / average value × 100 (SD is the standard deviation of the signal intensity or average signal intensity. The average value is the arithmetic mean value of the signal intensity or average signal intensity.)

[0062] The three generated histograms are shown in Fig. 4. The calculated evaluation values ​​are shown in Fig. 5. Among the evaluation values, three coefficients of variation P2 CV, P3 CV, and P8 CV refer to the coefficients of variation of the second, third, and eighth peaks from the left in the histograms, respectively.

[0063] Comparing the three histograms, the histogram generated based on the average signal intensity data has sharper peaks, for example, the second peak P2 and the third peak P3, than the histogram generated based only on the signal intensity data generated by irradiation with the 488 nm laser beam and the histogram generated based only on the signal intensity data generated by irradiation with the 561 nm laser beam. These results demonstrate that the use of average signal intensity data reduces the variability of the data.

[0064] Among the evaluation values, for example, the sensitivity evaluation value Q value calculated based on the average signal intensity data is significantly higher than the individual Q values ​​calculated based on the signal intensity data at each position. In particular, the sum of the Q value calculated based on the signal intensity data for 488 nm laser light irradiation and the Q value calculated based on the signal intensity data for 561 nm laser light irradiation is 0.4770 (= 0.2820 + 0.1950), while the Q value calculated based on the average signal intensity data is 0.5078, which is higher than the sum. A higher Q value indicates an improved S / N ratio. An increase in the Q value indicates, for example, a reduction in the impact of shot noise. These results demonstrate that quantum efficiency was improved by using the average signal intensity data. Furthermore, the fact that the Q value based on the average signal intensity data is higher than the sum indicates that sensitivity was particularly improved.

[0065] Among the evaluation values, the coefficient of variation P2 CV calculated based on the average signal intensity data was smaller than the coefficient of variation calculated based on the signal intensity data at each position. Similar results were obtained for the other coefficients of variation P3 CV and P8 CV. The smaller the coefficient of variation, the smaller the data variability, indicating better results. These results demonstrate that the use of average signal intensity data reduced data variability. This is thought to be due to an improved S / N ratio, which may have reduced the effects of shot noise, for example.

[0066] Among the evaluation values, the linearity evaluation value obtained excellent results in both cases. The precision evaluation value calculated using the average signal intensity data was between the value calculated from the signal intensity data generated by irradiation with 488 nm laser light and the value calculated from the signal intensity data generated by irradiation with 561 nm laser light. This is thought to directly reflect the relationship of the autofluorescence component of P1 (the first peak from the left). Regarding the B value, the quantum noise was slightly larger when using the average signal intensity data compared to the other two cases, but the results were almost the same.

[0067] From the above results, it can be seen that for a flow cytometer using a cross-axis illumination method, the S / N ratio can be improved by using average signal intensity data from two or more axes, thereby improving the fluorescence separation performance.

[0068] (4) Verification Example 2 Flow cytometry was performed using a commercially available 0.1 μm bead sample with a spectral flow cytometer (ID7000, Sony Group Corporation). In the flow cytometry, each particle constituting the fluorescent beads was irradiated at one position with a 355 nm laser beam and at another position with a 405 nm excitation laser beam. In the flow cytometry, 405 nm SSC was used as a trigger to acquire signal intensity data (height data and area data) of 355 nm SSC and signal intensity data (height data and area data) of 405 nm SSC. These signal intensity data were used to generate a histogram.

[0069] Furthermore, for each event, average signal intensity data was calculated from the signal intensity data at 355 nm SSC and the signal intensity data at 405 nm. The average signal intensity data was a simple average of the two signal intensity data. That is, for each event, a simple average of the height data value at 355 nm SSC and the height data value at 405 nm SSC was calculated. Also, for each event, a simple average of the area data value at 355 nm SSC and the area data value at 405 nm SSC was calculated. Histograms were generated using the average signal intensity data for area and height calculated in this way.

[0070] Furthermore, the coefficient of variation (CV) was calculated using the signal intensity data and average signal intensity data thus obtained.

[0071] The six generated histograms are shown in Figure 6. The calculated coefficient of variation (CV) is shown in Figure 7.

[0072] For height data, the coefficient of variation for the average signal intensity data was lower than that for the signal intensity data at 355 nm SSC and 405 nm SSC. Therefore, an improvement in the coefficient of variation was confirmed by using the average signal intensity data. Similarly, for area data, an improvement in the coefficient of variation was confirmed by using the average signal intensity data. These results demonstrate that, even for scattered light, data variability can be reduced by using average signal intensity data obtained by averaging two or more signal intensity data from different axes. This indicates that the S / N ratio can be improved. Furthermore, it is believed that the use of average signal intensity data can also reduce the influence of white noise. In addition, 405 nm SSC was used as the trigger in the above flow cytometry. From the above results, the coefficients of variation for the average signal intensity data for heights at 355 nm and 405 nm were the smallest, i.e., the average signal intensity data was the best. Therefore, it is believed that using the average signal intensity data for heights as the trigger improves the quality of the data obtained.

[0073] (5) Reference Examples and Examples Below, we describe a reference example in which signal intensity data for each axis is used without using average signal intensity data, and an example in which average signal intensity data is used in accordance with the present disclosure. A biological sample analysis system to which these examples are applied has a light irradiation unit configured to irradiate, for example, laser light with a wavelength of 320 nm, laser light with a wavelength of 355 nm, laser light with a wavelength of 405 nm, laser light with a wavelength of 488 nm, laser light with a wavelength of 561 nm, laser light with a wavelength of 638 nm, and laser light with a wavelength of 808 nm. The seven laser light irradiation positions are aligned in a line along the flow path, i.e., the light irradiation unit may be configured as a light irradiation unit using a different axis irradiation method, irradiating seven excitation spots with the seven laser lights, respectively. The light irradiation unit has seven axes. The biological sample analysis system also has a detection unit that detects light generated by irradiating particles with light at each irradiation position. The detection unit has multiple fluorescence channels, and two specific channels among these multiple fluorescence channels are configured to detect fluorescence generated by PE.

[0074] (A1: Reference example for setting fluorescence channels in fluorescence detection) One axis is set for the fluorescent dye to be detected, and a fluorescence channel is set to detect fluorescence generated by light irradiation along that axis. The fluorescence generated by light irradiation along the set axis is detected by the set fluorescence channel, and the average value of the signal intensity data of the detected fluorescence is used in various analyses as signal intensity data of the fluorescence generated by light irradiation of the fluorescent dye. For example, for the PE described above, an axis is set for irradiating laser light with a wavelength of 488 nm, and the average value of the signal intensity data of two fluorescence channels (e.g., Ch10 and Ch11) is used in various analyses as signal intensity data of the fluorescence generated by irradiating the PE with laser light along that axis.

[0075] (B1: Reference example for setting scattered light channels in scattered light detection) One scattered light detection channel is set for each axis. For example, for an axis that irradiates laser light with a wavelength of 488 nm, a scattered light channel that detects 488 nm side scattered light is set. Furthermore, for an axis that irradiates laser light with a wavelength of 405 nm, a scattered light channel that detects 405 nm side scattered light is set, and for an axis that irradiates laser light with a wavelength of 355 nm, a scattered light channel that detects 355 nm side scattered light is set. Furthermore, for one axis that irradiates laser light with a different wavelength, a scattered light channel that detects forward scattered light is set, and for another axis that irradiates laser light with yet another wavelength, a scattered light channel that detects side scattered light is set.

[0076] The fluorescence channel settings and scattered light channel settings of the reference example described above are changed in accordance with the present disclosure, for example, as follows.

[0077] (A2: Setting of Fluorescence Channels in Fluorescence Detection According to the Present Disclosure) For the fluorescent dye to be detected, multiple axes are set, and fluorescence channels are set to detect fluorescence generated by light irradiation along the multiple axes, or an additional axis is set in addition to the multiple axes, and a fluorescence channel is set to detect fluorescence generated by light irradiation along the additional axis. Fluorescence generated by light irradiation along the multiple set axes is detected by the set fluorescence channels, and the average value of the signal intensity data of the fluorescence detected by the fluorescence channels is used in various analyses as signal intensity data of the fluorescence generated by light irradiation of the fluorescent dye. For example, for the PE described above, an axis for irradiating with laser light having a wavelength of 488 nm and an axis for irradiating with laser light having a wavelength of 561 nm are set, and two fluorescence channels (e.g., Ch10 and Ch11) are set as channels for detecting fluorescence generated by light irradiation along the axis for irradiating with laser light having a wavelength of 488 nm, and two fluorescence channels (e.g., Ch10 and Ch11) are set as channels for detecting fluorescence generated by light irradiation along the axis for irradiating with laser light having a wavelength of 561 nm. The average value of the signal intensity data of the fluorescence detected in these four fluorescence channels is used as the average signal intensity data of the fluorescence of PE in various analysis processes.

[0078] (B2: Setting of scattered light channels for scattered light detection according to the present disclosure) One scattered light detection channel is set for each axis, and additional scattered light detection channels that detect the same type of scattered light are set for multiple axes. These additional scattered light detection channels may detect scattered light of different wavelengths. For example, as described in B1 above, a scattered light channel that detects 488 nm side scattered light is set for an axis that irradiates laser light with a wavelength of 488 nm, a scattered light channel that detects 405 nm side scattered light is set for an axis that irradiates laser light with a wavelength of 405 nm, and a scattered light channel that detects 355 nm side scattered light is set for an axis that irradiates laser light with a wavelength of 355 nm, a scattered light channel that detects forward scattered light is set for one axis that irradiates laser light with a different wavelength, and a scattered light channel that detects side scattered light is set for another axis that irradiates laser light with yet another wavelength. In addition to these five scattered light channels, a scattered light channel is set to detect side scattered light of 355 nm relative to the axis irradiating laser light of 355 nm wavelength, and a scattered light channel is set to detect side scattered light of 405 nm relative to the axis irradiating laser light of 405 nm wavelength, and the average value of the signal intensity of the side scattered light detected in these two scattered light channels is used in various analysis processes as additional average signal intensity data for scattered light.

[0079] The S / N ratio can be improved by setting channels to detect fluorescence or scattered light as described above in A2 and B2, and using average signal intensity data.

[0080] (6) Examples of User Interface In one embodiment, the biological sample analyzing system may be configured to allow a user to select whether to perform an analytical process using the average signal intensity data or to perform an analytical process without using the average signal intensity data. For example, the biological sample analyzing system may be configured to display a screen prompting the user to make the selection. The biological sample analyzing system may further include a display unit that displays the screen. The display unit may be, for example, a display or monitor, or may be a terminal such as a tablet or smartphone. In particular, an information processing unit (or information processing) included in the system may display the screen on the display unit. This configuration allows analytical processes to be performed according to the user's needs or preferences.

[0081] The embodiment will be described below with reference to the drawings. For example, the system may prompt the user to select a fluorescent dye for which average signal intensity data is to be used. An example of such a screen is shown in FIG. 8A . The screen 200 shown in FIG. 8A may include a list 201 of one or more fluorescent dyes to be used in the analysis process. The screen 200 also includes a field 202 for inputting the fluorescent dye for which average signal intensity data is to be used. The field 202 may be configured, for example, to allow the user to select a fluorescent dye for which the user wishes to use average signal intensity data, or to allow the user to input the fluorescent dye for which the user wishes to use average signal intensity data. After the fluorescent dye for which the user wishes to use average signal intensity data is selected or input, the system may add the fluorescent dye for which average signal intensity data is to be used to the fluorescent dye list 201, as shown in the row labeled 204. The figure indicates that average signal intensity data for the 488 nm axis and the 561 nm axis is calculated for PE. In the figure, only PE is shown as the fluorescent dye used in the analysis process, but two or more types of fluorescent dyes may of course be displayed on the screen.

[0082] The system may be configured to allow the user to select or change the fluorescence channel used to calculate the average signal intensity data for each fluorescent dye included in the list. For example, in response to selecting (e.g., clicking or double-clicking) a fluorescent dye button 205 in the row of the added symbol 204 on the screen 200, the system may display the screen 210 shown in FIG. 8B. The screen 210 has an area 211 prompting the user to select a fluorescence channel. Each horizontal block in the area corresponds to a fluorescence channel. When the user selects a block, the fluorescence channel corresponding to the selected block is selected as the fluorescence channel used to calculate the average signal intensity data. In the figure, three blocks are selected, indicating that three fluorescence channels have been selected as the fluorescence channels used to calculate the average signal intensity data. When selected, the fluorescence channel used to calculate the average signal intensity data can be confirmed by pressing a predetermined confirm button. Note that in the figure, each block is used as an indication corresponding to each fluorescence channel, but the indication corresponding to the fluorescence channel is not limited to a block and may be an indication of another shape, character, or the like.

[0083] The system may also display a screen (or window) that prompts the user to select whether to use average signal strength data before, during, or after the analysis process. Another example of such a screen is shown in FIG. 8C . Screen 300 shown in FIG. 8C may include a display 301 that asks the user whether to use average signal strength data. The display may include, for example, a button 302 for selecting whether to use average signal strength data. The display may also include a button 303 for selecting not to use average signal strength data.

[0084] In response to a user selection to use average signal intensity data, the system displays the screen shown in FIG. 8D . Screen 310 shown in FIG. 8D may have an area 311 for allowing the user to select which light signal intensity data to average. In FIG. 8D , FSC (forward scattered light), SSC (side scattered light), and fluorochrome (fluorescence) are displayed as selectable types of light for which average signal intensity data is calculated. In addition to these three types, BSC (backscattered light) may also be displayed as selectable. In this manner, the biological sample analysis system may be configured to display a screen for selecting the type of light for which average signal intensity data is generated.

[0085] As shown in the figure, the screen may include an area 312 for selecting an axis to be used to calculate average signal intensity data. In this area, the axes of the system are indicated by the wavelength of the irradiated light. This screen relates to a system having seven light irradiation spots, each irradiated with light of a different wavelength. Therefore, a total of seven check boxes corresponding to each wavelength are displayed in this area. The number of check boxes displayed in this area may be changed depending on the number of axes (number of light irradiation spots). That is, this area may display items (e.g., check boxes) corresponding to two or more axes that may be used for averaging. Note that this screen may also include other items, such as a reset button to cancel the selection or a cancel button to cancel the selection. In this way, the biological sample analysis system may be configured to allow the user to select at least two light irradiation positions from which signal intensity data used to calculate average signal intensity data is derived. Furthermore, the biological sample analysis system may be configured to display a screen prompting the user to make the selection.

[0086] For example, as shown in FIG. 8E , SSC is selected as the type of light for calculating average signal intensity data, and items (e.g., check boxes) corresponding to the 355 nm wavelength axis and the 405 nm wavelength axis are selected as the axes used to calculate the average signal intensity data. In this selected state, when the user presses the confirm button (OK button in FIG. 8E ), the system determines that the average signal intensity data calculated from the SSC signal intensity on the 355 nm wavelength axis and the SSC signal intensity on the 405 nm wavelength axis will be used in the analysis process. For example, the system may start the process of calculating the average signal intensity data in response to the confirmation button being pressed. The system may then use the calculated average signal intensity data in any analysis process. The same applies to FSC (or BSC), where items (e.g., check boxes) corresponding to two or more axes for which FSC is selected and averaged may be selected. Then, when the user presses the decision button with the selection made in this manner, average signal intensity data is calculated from the FSC signal intensities of the axes of the two or more selected wavelengths in the analysis process, and this average signal intensity data is used in the analysis process.

[0087] When using average fluorescence signal intensity data, an area (or screen) may be displayed that prompts the user to select a fluorescence channel to be used to calculate the average signal intensity data. This area (or screen) will be described with reference to FIG. 8F. For example, in FIG. 8D described above, in response to the user's selection of average signal intensity data, the system displays an area 314 prompting the user to select the type of scattering or fluorescent dye for which average signal intensity data is to be calculated, as shown in FIG. 8E or 8F. This area may also be displayed on the screen in advance. Even in this case, as described above, an area 312 for selecting the axis to be used to calculate the average signal intensity data may be displayed. That is, the biological sample analysis system may be configured to allow the user to select at least two light irradiation positions from which the signal intensity data used to calculate the average signal intensity data is derived. Furthermore, the biological sample analysis system may be configured to display a screen prompting the user to make this selection. In FIG. 8D, items (checkboxes in FIG. 8F) corresponding to the 488 nm wavelength axis and the 561 nm wavelength axis are selected as the axes to be used to calculate the average signal intensity data. Note that the system may automatically select an axis (or an item corresponding to the axis) to be used for calculating average signal intensity data in response to the selection of a fluorescent dye from the fluorescent dye list described below. This is because the wavelength at which the fluorescent dye is excited is known, and therefore it is possible to associate the fluorescent dye with an axis in advance. Therefore, in response to the selection of a fluorescent dye, the system can select the axis (or an item corresponding to the axis) associated with that fluorescent dye.

[0088] When the user selects a fluorescent dye from, for example, a fluorescent dye list (e.g., a pull-down list) in the area, the system displays an area 315 on the screen (or in another screen or window) prompting the user to select a fluorescent channel to be used to calculate average signal intensity data, as shown in FIG. 8G. For example, as shown in FIG. 8H, fluorescent channels 10 and 11 (Ch10 and Ch11) are selected from among multiple fluorescent channels in the area, and the selected channel column 316 is grayed out. Furthermore, the selected channel numbers (CH10-CH11) and their wavelength ranges (566.1-589.5 nm) are displayed at the bottom of the area. In this manner, the area may be configured so that the user can select the fluorescent channels to be used to generate average signal intensity data. As described above, the area may be displayed as, for example, a row of selection boxes for the fluorescent channels. The display of the selection boxes may change depending on the user's selection, such as by changing the color as described above, or by displaying any symbol, such as a check mark. Furthermore, as shown in the figure, the area may display a spectrum 317 of the selected fluorescent dye. The spectrum may be displayed so as to correspond to the wavelength detected by the fluorescence channel, as shown in the figure. In this way, by displaying the fluorescence channel and the spectrum in the area so that they correspond to each other, the user can easily select the fluorescence channel to be averaged.

[0089] Although the figure shows that the user selects the fluorescence channels to be averaged, the fluorescence channels to be used for averaging may be pre-selected when the area is displayed, depending on the selected fluorescent dye. Since the spectrum of a fluorescent dye is often known, displaying the area in this pre-selected state can improve user convenience. Even when an area is displayed in this manner, the area may be configured to allow additional fluorescent channels to be selected after the area is displayed, or to allow the selection of a previously selected fluorescent channel to be deselected.

[0090] With the above selections made, when the user presses the OK button, average signal intensity data is calculated from the fluorescence signal intensities of the two or more selected wavelength axes in the analysis process, and this average signal intensity data is used in the analysis process.

[0091] 2. Second embodiment (information processing device)

[0092] The present disclosure also provides an information processing device configured to perform the analytical process described in 1. above. That is, the information processing device may be configured to calculate average signal intensity data of particles from the signal intensity of light generated by irradiating particles flowing through a flow path with light at at least two positions, and perform the analytical process using the average signal intensity data. The information processing device may be a device including the information processing unit described in 1. above. The information processing device may be configured to perform the analytical process using the signal intensity data acquired by the light irradiation unit and detection unit described in 1. above.

[0093] The analysis process may be an analysis method using the average signal intensity data. As described in 1. above, the analysis method may be the analysis of a biological sample or a verification process for the state of the device.

[0094] 3. Third embodiment (analysis method and program for executing the analysis method)

[0095] The present disclosure also provides an analytical method including performing the analytical process described in 1. above. That is, the analytical method may be an analytical method including performing the analytical process using the average signal intensity data described in 1. above. The analytical method may be performed by the information processing unit described in 1. above. The average signal intensity data is as described in 1. above, and the description thereof also applies to this embodiment. The analytical process is also as described in 1. above, and the description thereof also applies to this embodiment. That is, the analytical process may be an analytical process of a biological sample, or a process of verifying the state of an apparatus.

[0096] The present disclosure also provides a program for causing a biological sample analysis system or an information processing device to execute the analysis method. The program may be stored in, for example, the biological sample analysis system or the information processing device, or in an information storage medium. The information storage medium may be, for example, an SD card, a microSD card, a CD, a DVD, a flash memory, or a magnetic recording medium.

[0097] The present disclosure may be configured as follows: [1] A biological sample analysis system including: a light irradiation unit configured to irradiate particles flowing through a flow path with light at at least two positions; and an information processing unit configured to calculate average signal intensity data of the particles from the signal intensity of light generated by the light irradiation at each of the at least two positions, and to perform an analytical process using the average signal intensity data. [2] The biological sample analysis system according to [1], wherein the average signal intensity data is average signal intensity data of fluorescence generated by the light irradiation of the particles. [3] The biological sample analysis system according to [2], wherein the information processing unit is configured to perform a verification process in the analytical process that verifies the state of a device included in the system, using the average signal intensity data of fluorescence. [4] The biological sample analysis system according to [2] or [3], wherein the information processing unit is configured to perform an analytical process of the biological sample using the average signal intensity data of fluorescence in the analytical process. [5] The biological sample analyzing system according to any one of [2] to [4], further comprising a detection unit including at least two detector arrays that detect fluorescence generated by the light irradiation, and the at least two detector arrays are configured to detect fluorescence generated by light irradiation at the at least two positions, respectively. [6] The biological sample analyzing system according to any one of [2] to [5], wherein the average signal intensity data of fluorescence is average signal intensity data of fluorescence generated by light irradiation of one type of fluorescent dye. [7] The biological sample analyzing system according to any one of [2] to [6], wherein the light irradiated at the at least two positions is light of mutually different wavelengths. [8] The biological sample analyzing system according to [1], wherein the average signal intensity data is average signal intensity data of scattered light generated by light irradiation of the particles. [9] The biological sample analyzing system according to [8], wherein the information processing unit uses the average signal intensity data of scattered light as a trigger in the analysis process.

[10] The biological sample analyzing system according to [8] or [9], further comprising a detection unit equipped with at least two detectors that detect scattered light generated by the light irradiation, wherein the at least two detectors are configured to detect scattered light generated by the light irradiation at the at least two positions, respectively.

[11] The biological sample analyzing system according to any one of [1] to

[10] , configured to allow a user to select whether to perform an analytical process using the average signal intensity data or to perform an analytical process without using the average signal intensity data.

[12] The biological sample analyzing system according to

[11] , configured to display a screen that prompts the user to make the selection.

[13] The biological sample analyzing system according to

[11] or

[12] , comprising a display unit that displays the screen for making the selection.

[14] The biological sample analyzing system according to any one of [1] to

[13] , configured to display a screen for selecting the type of light for which the average signal intensity data is generated.

[15] The biological sample analyzing system according to any one of [1] to

[14] , wherein the biological sample analyzing system is configured to enable selection of at least two light irradiation positions from which signal intensity data used to calculate the average signal intensity data is derived.

[16] The biological sample analyzing system according to any one of [1] to

[15] , wherein the biological sample analyzing system is configured to enable display of an area that prompts the user to select a fluorescence channel to be used to calculate the average signal intensity data.

[17] The biological sample analyzing system according to any one of [1] to

[16] , wherein the biological sample analyzing system is a flow cytometer.

[18] An analysis method comprising: calculating average signal intensity data of particles flowing through a flow path from the signal intensity of light generated by irradiating the particles with light at at least two positions; and performing an analysis process using the average signal intensity data.

[19] An information processing device configured to calculate average signal intensity data of particles flowing through a flow path from the signal intensity of light generated by irradiating the particles with light at at least two positions, and to perform analytical processing using the average signal intensity data.

[0098] 6100 Biological sample analyzer 6101 Light irradiation unit 6102 Detection unit 6103 Information processing unit

Claims

1. A biological sample analysis system comprising: a light irradiation unit configured to irradiate light onto particles flowing through a flow path at at least two positions; and an information processing unit configured to calculate average signal intensity data for the particles from the signal intensity of light generated by the light irradiation at each of the at least two positions, and to perform analytical processing using the average signal intensity data.

2. The biological sample analyzing system according to claim 1, wherein the average signal intensity data is average signal intensity data of fluorescence generated by irradiating the particles with light.

3. The biological sample analysis system of claim 2, wherein the information processing unit is configured to execute a verification process in the analysis process, using the average fluorescent signal intensity data, to verify the state of an apparatus included in the system.

4. The biological sample analyzing system according to claim 2, wherein the information processing unit is configured to execute an analytical process of the biological sample using the average fluorescent signal intensity data in the analytical process.

5. The biological sample analysis system according to claim 2, further comprising a detection section having at least two detector arrays for detecting fluorescence generated by the light irradiation, the at least two detector arrays being configured to respectively detect fluorescence generated by the light irradiation at the at least two positions.

6. The biological sample analyzing system according to claim 2, wherein the average fluorescence signal intensity data includes average fluorescence signal intensity data generated by irradiating one type of fluorescent dye with light.

7. The biological sample analyzing system according to claim 2, wherein the light irradiated to the at least two positions has a wavelength different from each other.

8. The biological sample analyzing system according to claim 1, wherein the average signal intensity data is average signal intensity data of scattered light generated by irradiating the particles with light.

9. The biological sample analyzing system according to claim 8, wherein the information processing section uses the average signal intensity data of the scattered light as a trigger in the analysis process.

10. The biological sample analysis system of claim 8, further comprising a detection section having at least two detectors for detecting scattered light generated by the light irradiation, the at least two detectors being configured to respectively detect scattered light generated by the light irradiation at the at least two positions.

11. The biological sample analysis system of claim 1, wherein the biological sample analysis system is configured to allow a user to select whether to perform an analytical process using the average signal intensity data or to perform an analytical process without using the average signal intensity data.

12. The biological sample analyzing system of claim 11, wherein the biological sample analyzing system is configured to display a screen prompting the user to make the selection.

13. The biological sample analyzing system according to claim 11, further comprising a display unit that displays a screen for making said selection.

14. The biological sample analysis system of claim 1, wherein the biological sample analysis system is configured to display a screen for selecting the type of light for which the average signal intensity data is generated.

15. The biological sample analysis system of claim 1, wherein the biological sample analysis system is configured to be capable of selecting at least two light irradiation positions from which signal intensity data used to calculate the average signal intensity data is derived.

16. The biological sample analysis system of claim 1, wherein the biological sample analysis system is configured to display an area that prompts a user to select a fluorescence channel to be used for calculating the average signal intensity data.

17. The biological sample analysis system of claim 1, wherein the biological sample analysis system is a flow cytometer.

18. An analytical method comprising: calculating average signal intensity data of particles from the signal intensity of light generated by irradiating light at at least two positions on the particles flowing through a flow path; and performing analytical processing using the average signal intensity data.

19. An information processing device configured to calculate average signal intensity data for particles flowing through a flow path from the signal intensity of light generated by irradiating the particles with light at each of at least two positions, and to perform analytical processing using the average signal intensity data.

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