Flow cytometer and flow cytometer signal analysis method

Abstract

To keep forward-scattered light from mixing even when capillaries are concentrated in a narrow space, the present invention has a light sheet (121) that irradiates laser light (141) at a plurality of capillaries (122), a line sensor (123) that receives forward-scattered light (142) that is scattered on the reverse side of the capillaries (122) from the side at which the laser light (141) is irradiated, first laser blockers (111) that are provided at positions that are near a light source unit that includes an alignment surface for the capillaries (122) on the side at which the laser light (141) is incident and are offset from projections of the center axes of the capillaries (122) in the direction of the light sheet (121), second laser blockers (112) that are provided between the capillaries (122) between the capillaries (122) and the line sensor (123), and third laser blockers (113) that are provided at a light reception surface of the line sensor (123) at positions that coincide with projections of the center axes of the capillaries (122) in the direction of the line sensor (123).

Description

Flow cytometer and flow cytometer signal analysis method

[0001] The present invention relates to the technology of a flow cytometer and a method for analyzing a signal from a flow cytometer.

[0002] Flow cytometry (FCM) is a technique for analyzing the expression of molecules on or within samples such as cells. In flow cytometry, samples are aligned and flowed, and laser light is used to measure each sample in turn. The intensity of scattered light and fluorescence emitted from each sample is then recorded.

[0003] There are two types of scattered light: forward scattered light (FSC), which is emitted in a direction of 1 to 10 degrees relative to the incident laser, and side scattered light (SSC), which is reflected in a direction of 90 degrees. The intensity of forward scattered light reflects information about the size of the sample. The intensity of side scattered light reflects the complexity of the sample's internal structure.

[0004] When measuring protein expression on the cell surface, the cells are labeled with fluorescently labeled antibodies for the surface antigens to be observed before flow cytometry. The fluorescence emitted by the fluorescently labeled antibodies allows users to determine the presence or absence of protein expression and the intensity of expression for each cell.

[0005] In this way, flow cytometry can obtain parameters such as the size, internal structure, and protein expression of individual samples, allowing users to classify sample groups using these parameters and understand the populations of each group.

[0006] Conventional flow cytometry uses the hydrodynamic focusing effect to align samples, etc. The hydrodynamic focusing effect is achieved by forming a flow of sheath fluid on the outside and a flow of sample solution on the inside of a flow cell, thereby aligning the samples.

[0007] On the other hand, capillary flow cytometry is a type of flow cytometry that uses a different method from the hydrodynamic focusing effect. In capillary flow cytometry, a sample solution is passed through a capillary with a diameter slightly larger than the sample. This aligns the individual samples, allowing measurements to be performed on each sample. Capillary flow cytometry has the advantage of not requiring sheath fluid and producing little waste liquid. In addition, capillary flow cytometry makes it relatively easy to multiply sample measurements by N by parallelizing the sample flow. For this reason, capillary flow cytometry is expected to be used in applications that require measuring a large number of samples in a short period of time, especially in clinical settings.

[0008] Non-Patent Document 1 describes a method for aligning cells in a capillary with high precision by using ultrasound in parallel capillary flow cytometry.

[0009] Menake E. Piyasena, “Multinode Acoustic Focusing for Parallel Flow Cytometry”, Analytical Chemistry, 84, 4(2012) 1831-1839.

[0010] In capillary flow cytometry, it is desirable to integrate capillaries in a small space in order to reduce the size of the device and the number of parts. However, integrating capillaries in a small space poses the problem of making it difficult to distinguish between forward scattered light from adjacent capillaries and the forward scattered light that is the intended measurement. In other words, signals from adjacent flow cytometers are mixed together.

[0011] As mentioned above, the forward scattered light emitted in a direction of 1° to 10° relative to the incident laser is important information that reflects the size of the sample, so it is necessary to devise a way to prevent the forward scattered light from being mixed in.

[0012] For example, in Non-Patent Document 1, in the parallel capillary flow cytometry described above, ultrasound was used to successfully align cells in the capillaries with high precision. However, forward scattered light scattered in directions between 1° and 10° was not detected, and instead, backscattered light data at 25° was acquired. In other words, the technology described in Non-Patent Document 1 cannot prevent the mixing of forward scattered light.

[0013] The present invention has been made in view of the above background, and an object of the present invention is to prevent the mixing of forward scattered light even when capillaries are integrated in a narrow space.

[0014] In order to solve the above-mentioned problems, the present invention provides a flow cytometer including a light source unit that irradiates a laser beam onto a plurality of capillaries, a light receiving unit that receives forward scattered light that is scattered in the opposite direction of the capillaries from the side where the laser beam is irradiated, and laser blockers that block light, wherein the laser blockers include a first laser blocker that is disposed at a position close to the light source unit including an alignment surface of the capillaries on the side where the laser beam is incident and at a position shifted from the position where the axial centers of the capillaries are projected in the direction of the light source unit, a second laser blocker that is disposed between the capillaries and the light receiving unit and between the capillaries, and a third laser blocker that is disposed at a position where the axial centers of the capillaries are projected in the direction of the light receiving unit on the light receiving surface of the light receiving unit. Other solutions will be described as appropriate in the embodiments.

[0015] According to the present invention, even if capillaries are integrated in a narrow space, it is possible to prevent the mixing of forward scattered light.

[0016] FIG. 1 is a schematic diagram showing the configuration of a flow cytometer according to the present embodiment; FIG. 2 is a diagram showing the position of a laser blocker; FIG. 3 is a diagram (part 1) of a flow cytometer viewed from the direction of incidence of laser light; FIG. 4 is a diagram (part 2) of a flow cytometer viewed from the direction of incidence of laser light; FIG. 5 is a diagram showing the configuration for acquiring fluorescence and side-scattered light; FIG. 6 is a diagram showing the detected intensity when each sample is measured once; FIG. 7 is an example showing the results of gating; FIG. 8 is a diagram showing the results of measuring one sample at each intersection; FIG. 9 is a diagram showing an example of gating a sample population by plotting average values; FIG. 10 is a box plot plotting the fluorescence intensity (fluorescence intensity) derived from 33 fluorescent beads in each of measurements 1 to 10; FIG. 11 is a box plot plot using the results of averaging 10 measurements for each fluorescent bead; FIG. 12 is a result showing how the variation in fluorescence intensity derived from a sample decreases with each measurement; FIG. 13 is a diagram showing the configuration of a processing device; FIG. 14 is a flowchart showing the steps of a flow cytometer signal analysis method according to the present embodiment;

[0017] Embodiments of the present invention will be described with reference to the drawings. However, the present invention should not be construed as being limited to the description of the embodiments shown below. Furthermore, the position, size, shape, range, etc. of each component shown in the drawings may not represent the actual position, size, shape, range, etc., in order to facilitate understanding of the invention. Therefore, the present invention is not necessarily limited to the position, size, shape, range, etc., disclosed in the drawings, etc.

[0018] In this embodiment, the flow cytometer 1 is an apparatus that uses the technology of capillary flow cytometry, which is one type of flow cytometry.

[0019] [First Embodiment] Fig. 1 is a schematic diagram showing the configuration of a flow cytometer 1 according to this embodiment. Fig. 2 is a diagram showing the arrangement position of a laser blocker 110.

[0020] The flow cytometer 1 includes a light sheet 121 serving as a light source, multiple capillaries 122, and a line sensor 123 serving as a light-receiving unit. The light sheet 121 emits laser light 141. FIG. 1 shows the flow cytometer 1 viewed from a direction 90 degrees relative to the incident direction of the laser light 141. The capillary 122 is shown in cross section along a plane perpendicular to the flow direction of the sample 131, which flows from the depth of the page to the front of the page. The sample 131 flows through the capillary 122 together with the liquid flowing therethrough. The liquid flowing through the capillary 122 is driven by the discharge force of a pump (not shown). The capillary 122 is made of glass such as quartz, soda lime, or borosilicate, or a resin such as polycarbonate.

[0021] The light sheet 121 irradiates a plurality of capillaries 122 with laser light 141. The line sensor 123 receives forward scattered light 142 that is scattered in the opposite direction to the side of the capillaries 122 that is irradiated with the laser light 141. The opposite side is the side of the line sensor 123, which is a light receiving unit.

[0022] The flow cytometer 1 is provided with laser blockers 110 that block light. Three types of laser blockers 110 are provided: a first laser blocker 111, a second laser blocker 112, and a third laser blocker 113. The second laser blocker 112 and the third laser blocker 113 are provided to block forward scattered light 142 originating from the adjacent capillary 122.

[0023] As shown in FIG. 2 , the first laser blocker 111 is positioned near the light sheet 121, including the alignment surface 151a of the capillary on the side where the laser light 141 is incident. The first laser blocker 111 is positioned at a position offset from the position (arrow 171) where the axial center 122a of the capillary 122 is projected toward the light sheet 121. Specifically, the first laser blocker 111 is installed on the alignment surface 151a of the capillary 122 on the side where the laser light 141 is incident. Note that in the example shown in FIG. 2 , the first laser blocker 111 is installed on the alignment surface 151a of the capillary 122 on the side where the laser light 141 is incident, but this is not limited thereto. For example, the first laser blocker 111 may be installed at a position closer to the light sheet 121 than the alignment surface 151a of the capillary 122 (above the alignment surface 151a). This is possible, for example, by making the thickness of the transparent member 151 thicker than the outer diameter of the capillary 122 .

[0024] 2, the second laser blocker 112 is installed between the capillary 122 and the line sensor 123 and between the capillaries 122. Specifically, the second laser blocker 112 is installed from the alignment surface 151a of the capillary 122 on the side where the laser light 141 is incident to the light receiving surface 123a of the line sensor 123.

[0025] As shown in FIG. 2, the third laser blocker 113 is installed on the light receiving surface 123 a of the line sensor 123 at a position (arrow 172 ) where the axial center 122 a of the capillary 122 is projected in the direction of the line sensor 123 .

[0026] The laser blocker 110 is made of resin such as polycarbonate, acrylic, or polypropylene, or metal such as glass or aluminum. It is desirable that the laser blocker 110 be anti-reflection treated. This treatment prevents the laser light 141 from being diffused and absorbed by the laser blocker 110. Care must be taken when the laser blocker 110 is not anti-reflection treated, as stray light due to diffused reflection may be detected by the line sensor 123. Examples of anti-reflection treatments include coloring the laser blocker 110 (black, for example), providing a finely textured surface, or laminating a dielectric film on the surface. The size of each laser blocker 110 in the axial direction of the capillary 122 (the size in the depth direction in FIGS. 1 and 2 ) is a size that can block the laser light 141 and forward scattered light 142.

[0027] The capillary 122 is embedded in the transparent member 151. Furthermore, the second laser blocker 112 is embedded in the transparent members 151 and 152. The transparent members 151 and 152 are highly transparent members and are made of resins such as polycarbonate, acrylic, polypropylene, or glass (they are made of highly transparent members regardless of whether they are organic or inorganic materials). In the example shown in FIG. 1, two transparent members, 151 and 152, are provided, but the transparent members 151 and 152 may be made of a single transparent member.

[0028] 1, the diameter of the capillaries 122 is 30 μm. The capillaries 122 are arranged in parallel at intervals of 30 μm. However, the diameter of the capillaries 122 and the intervals at which the capillaries 122 are arranged are not limited to 30 μm.

[0029] Furthermore, the forward scattered light 142 is light scattered by the sample 131 in a range of 1° to 10° with respect to the direction directly below the capillary 122. In this embodiment, the direction of the light sheet 121 is defined as the upward direction, and the direction of the line sensor 123 is defined as the downward direction. The scattering range of the forward scattered light 142 (1° to 10°) is an approximate value. In the configuration shown in FIG. 1, the width of the forward scattered light 142 when it reaches the line sensor 123 is approximately 30 μm.

[0030] 1, the line sensor 123 is disposed at a distance of 180 μm from the capillary 122 so that the forward scattered light 142 can reach the line sensor 123 before its intensity is excessively attenuated by the transparent member 152. However, the distance between the line sensor 123 and the capillary 122 is not limited to 180 μm. When the line sensor 123 receives the forward scattered light 142, it sends a signal to the processing device 200. Incidentally, as the distance between the capillary 122 and the line sensor 123 increases, the width of the forward scattered light 142 increases, and therefore the intensity per unit area of ​​the forward scattered light 142 that reaches the line sensor 123 decreases.

[0031] 3A and 3B are views of the flow cytometer 1 as viewed from the direction of incidence of the laser light 141. Note that FIG. 1 is a view of FIGS. 3A and 3B as viewed from the direction of the arrows A-A. Note that the laser blocker 110 is omitted from FIGS. 3A and 3B. Also, reference will be made as appropriate to FIG. 1, and descriptions of elements already described in FIG. 1 will be omitted as appropriate.

[0032] FIG. 3A shows an example in which a plurality of linear capillaries 122 are arranged independently. That is, in the flow cytometer 1 shown in FIG. 3A, each capillary 122 has an inlet and an outlet (arrows shown in FIG. 3A). FIG. 3B shows an example in which the capillary 122 is a single capillary 122 having a meandering path. FIG. 3B shows an example in which a single capillary 122 forms a continuous flow path by meandering. That is, the flow cytometer 1 shown in FIG. 3B has one inlet and one outlet (arrows shown in FIG. 3B). Note that, although the capillary 122 has five meanders (five U-shaped portions) in FIG. 3B, other numbers of meanders are also possible.

[0033] In the example shown in Fig. 3A, a light sheet 121 is provided so that laser light 141 is irradiated onto a plurality of capillaries 122. In Fig. 3B, a light sheet 121 is provided so that laser light 141 is irradiated onto a plurality of points of the capillary 122. The intersection 161 will be described later.

[0034] The laser light 141 is irradiated onto the capillaries 122 from the front side of the paper in FIGS. 3A and 3B toward the back side of the paper. Then, forward scattered light 142, side scattered light 144 (see FIG. 4), and fluorescence 143 (see FIG. 4) originating from each capillary 122 are detected. The laser light 141 may be irradiated onto each capillary 122 using a general laser light 141. However, to simplify the flow cytometer 1, a method of irradiating all capillaries 122 simultaneously using a surface such as the light sheet 121 is desirable. In this way, by using the light sheet 121 as a light source, it is possible to irradiate all of the parallel-arranged capillaries 122 with laser light 141 of the same wavelength using a single light source (light sheet 121). Note that the general laser light 141 irradiates a single irradiation point with pinpoint laser light 141.

[0035] Measurement of the sample 131 is performed when the sample 131 passes through the illuminated portion of the light sheet 121 in each capillary 122.

[0036] (Acquisition of Fluorescent Light 143 and Side Scattered Light 144) FIG. 4 is a diagram showing a configuration for acquiring the fluorescent light 143 and side scattered light 144. As shown in FIG.

[0037] In FIG. 4, the sample 131 (see FIG. 1) flows inside the capillary 122 in the direction of the white arrow.

[0038] When the laser light 141 is irradiated onto the sample 131 flowing inside the capillary 122, the sample 131 emits not only forward scattered light 142 (see FIG. 1), but also fluorescent light 143 and side scattered light 144.

[0039] The flow cytometer 1 includes a focusing lens 311, a laser notch filter 312, a diffraction grating 313, and a 2D CCD camera 314 as components for acquiring the fluorescence 143. The fluorescence 143 emitted from the sample 131 is received by the 2D CCD camera 314 via the focusing lens 311, the laser notch filter 312, and the diffraction grating 313. Upon receiving the fluorescence 143, the 2D CCD camera 314 sends a signal to the processing device 200.

[0040] The flow cytometer 1 also includes a focusing lens 321, a laser bandpass filter 322, and a line sensor 323 as components for acquiring side scattered light 144. The side scattered light 144 scattered by the sample 131 is received by the line sensor 323 via the focusing lens 321 and the laser bandpass filter 322. When the line sensor 323 receives the side scattered light 144, it sends a signal to the processing device 200.

[0041] The flow cytometer 1 acquires forward scattered light 142 using the configuration shown in FIGS. 1, 3A, and 3B, and acquires fluorescent light 143 and side scattered light 144 using the configuration shown in FIG.

[0042] That is, by combining the configuration shown in FIG. 4, it is possible to measure and analyze the forward scattered light 142 originating from the plurality of capillaries 122, as well as the fluorescent light 143 and the side scattered light 144, all at once.

[0043] According to the flow cytometer 1 shown in the first embodiment, in the flow cytometer 1 in which the capillaries 122 are arranged in parallel, three types of laser blockers 110 are arranged at the locations described with reference to FIG.

[0044] This makes it possible to acquire the forward scattered light 142 originating from each capillary 122 without being affected by the forward scattered light 142 originating from adjacent capillaries 122. In other words, it is possible to acquire the forward scattered light 142 originating from the sample 131 for each capillary 122 without being affected by the signal originating from the adjacent capillary 122. This makes it possible to reduce the distance between the capillaries 122, thereby enabling the capillaries 122 to be integrated.

[0045] Furthermore, as shown in FIG. 3B, the meandering of the capillary 122 makes it possible to measure the forward scattered light 142, the side scattered light 144, and the fluorescence 143 for each sample 131 multiple times.

[0046] 5A to 9, a method for analyzing signals acquired by the flow cytometer 1 from the line sensors 123, 323 and the 2DCCD camera 314 will be described. In the following description, FIGS. 1 to 4 will be referenced as appropriate.

[0047] In the following description, we will use the measurement results obtained by capillaries 122 arranged in parallel as a serpentine flow path as shown in Figure 3B. By using capillaries 122 arranged in parallel as a serpentine flow path, the same sample 131 can be measured multiple times. Figures 5A to 9 show examples of gating using the measurement results obtained by such a flow cytometer 1. A method for clarifying the boundaries of a sample population 401 will be described with reference to Figures 5A to 9.

[0048] In the second embodiment, a sample 131 flows through a serpentine capillary 122 as shown in Fig. 3B , and the same sample 131 is measured at each intersection 161. Then, a sample population 401 (see Fig. 6B ) is extracted based on the average value of the measurement results. In this way, the second embodiment shows that by extracting the sample population 401 based on the average value of the measurement results, the boundaries of the sample population 401 become clear.

[0049] In this way, when extracting a sample population 401 based on the average value of the measurement results, the technique of capillary flow cytometry, which has a capillary 122 through which only one sample 131 flows, is useful. In particular, a flow cytometer 1 having a serpentine capillary 122 as shown in Figure 3B is useful. Furthermore, when miniaturizing a flow cytometer 1 having a serpentine capillary 122 as shown in Figure 3B and reducing the number of parts, the three types of laser blockers 110 shown in Figure 1 are useful.

[0050] Fig. 5A is a diagram showing the detection intensity when each sample 131 (cells in the example shown in this embodiment) is measured once, and Fig. 5B is an example showing the results of gating using the results shown in Fig. 5A.

[0051] As described above, the arrangement of the capillaries 122 is as shown in Figure 3B, and the light sheet 121 is used as the light source. Also, as described above, each sample 131 is measured six times at the intersection 161 of the capillaries 122 and the light sheet 121 shown in Figure 3B. At this time, in order to avoid interference of forward scattered light 142 originating from adjacent capillaries 122, a laser blocker 110 is placed as shown in Figure 1. In Figures 5A and 5B, the results measured at each intersection 161 are shown as is.

[0052] The arrangement of the three types of laser blockers 110 (first laser blocker 111, second laser blocker 112, and third laser blocker 113) is as described in FIG.

[0053] 1, the line sensor 123 receives forward scattered light 142 scattered in a direction of 1° to 10° forward by the sample 131. Furthermore, with the configuration shown in FIG. 4, the processing device 200 acquires signals due to fluorescent light 143 and side scattered light 144 in addition to the forward scattered light 142.

[0054] 5A, the horizontal axis represents the sample 131 (cells in the example shown in this embodiment), and the vertical axis represents the detection intensity. The detection intensity is the fluorescence intensity or the scattered light intensity. Note that the intensity refers to the signal intensity output by the line sensor 123, the 2DCCD camera 314, and the line sensor 323. Furthermore, hereinafter, the scattered light refers to the forward scattered light 142 or the side scattered light 144. In other words, the scattered light intensity refers to the signal intensity of the forward scattered light 142 or the signal intensity of the side scattered light 144.

[0055] In the measurement by the flow cytometer 1, it is important to gate a sample population 401 at plot points 402 as shown in Fig. 5B. Gating means clustering the sample population 401.

[0056] 5A, if only one measurement is performed for one sample 131, variations in the detected intensity occur. Such variations are thought to be influenced by the positional deviation of the sample 131 relative to the laser light 141 during measurement of the sample 131 and differences in the orientation of the sample 131.

[0057] Figure 5B plots the results of a single measurement performed on one sample 131 using the same method as in Figure 5A. In Figure 5B, the horizontal and vertical axes represent fluorescence intensity or scattered light intensity. That is, in Figure 5B, the horizontal and vertical axes represent either fluorescence intensity, the signal intensity of forward scattered light 142, or the signal intensity of side scattered light 144.

[0058] 5B, each plot point 402 represents a sample 131 (a cell in the example shown in this embodiment). In FIG. 5B, gating is performed to group (cluster) samples 131 with similar properties into a sample population 401.

[0059] In the diagram shown in Figure 5B, some of the sample populations 401 overlap. This situation often occurs when the sample 131 is measured only once. This overlap of the sample populations 401 is thought to be due to variations in detection intensity as shown in Figure 5A.

[0060] 6A is a diagram showing the results of measuring one sample 131 at each intersection 161 (see FIG. 3B) using the flow cytometer 1 shown in FIG. 3B. In the example shown in this embodiment, one sample 131 is measured six times.

[0061] 6A shows the average value and standard deviation of the results measured for each intersection 161 on the sample 131. Note that the average value and standard deviation may be calculated by the processing device 200 after the user has removed outliers from the measurement results.

[0062] FIG. 6B is a diagram showing an example in which the average values ​​calculated by the method shown in FIG. 6A are plotted and the sample population 401 is gated.

[0063] In Fig. 6B , as in Fig. 5A , each plot point 402 represents a sample 131. However, in Fig. 6B , the same sample 131 is measured multiple times using the flow cytometer 1 shown in Fig. 3B , the results are averaged, and the average value is plotted. Then, gating of the plot points 402 is performed.

[0064] 6B , by measuring the same sample 131 multiple times and averaging the results, the sample population 401 is narrowed. This results in clear boundaries between the sample populations 401 (no overlap between the sample populations 401). In this way, by measuring the same sample 131 multiple times and averaging the results, the accuracy of gating can be improved.

[0065] Next, data verifying that the signal variation can be suppressed by actually measuring the same sample 131 multiple times is shown in FIGS.

[0066] 7 to 9 show the results of aligning 33 fluorescent beads in a single microscopic flow path filled with water, and measuring the fluorescence intensity derived from each bead using a fluorescence microscope. Note that the results of Figures 7 to 9 were obtained from an experiment in which fluorescent beads were aligned in a flow path simulating the capillary 122. In other words, fluorescent beads were not actually circulated through the capillary 122 of the flow cytometer 1.

[0067] The measurement was repeated 10 times, and between each measurement, the fluorescent beads were gently moved within the flow channel so as not to change their order. However, the fluorescent beads were moved without flowing. Note that although fluorescence 143 is used in Figures 7 to 9, a similar discussion can be made for forward scattered light 142 by replacing fluorescence 143 with forward scattered light 142.

[0068] The 33 fluorescent beads represent samples 131 belonging to the same sample population 401. Each fluorescent bead basically exhibits the same level of fluorescence intensity, but there is some variation between the individual fluorescent beads. Furthermore, moving the fluorescent beads within the flow channel represents the following: In other words, it represents the situation in the flow cytometer 1 where the sample 131 moving through the capillary 122 is measured at a slightly different position and angle relative to the laser light 141 for each measurement.

[0069] Figure 7 is a box plot plotting the intensities of fluorescence 143 (fluorescence intensity) derived from 33 fluorescent beads for each of measurements 1 to 10. Each plot point 411 shown in Figure 7 represents the fluorescence intensity of a fluorescent bead. However, since there are multiple results with the same numerical value, the number of plots shown in Figure 7 is less than 33. Note that the "x" marks in the box plot indicate the average value.

[0070] On the other hand, Fig. 8 is a box plot using the average results of 10 measurements for each fluorescent bead. In Fig. 8, each plot point 412 represents the average of 10 measurements of the fluorescence intensity for each fluorescent bead. As in Fig. 7, the number of plots in Fig. 7 is less than 10 because the same numerical results exist.

[0071] Comparing the results shown in Figures 7 and 8, it can be seen that when the fluorescence intensity was averaged based on multiple measurements (Figure 8), the variation in fluorescence intensity between fluorescent beads was smaller than when a single measurement was made (Figure 7).

[0072] FIG. 9 shows the results of an experiment similar to those shown in FIGS. 7 and 8, showing how the variation in fluorescence intensity originating from sample 131 (fluorescent beads) decreases with each measurement.

[0073] 9, the vertical axis represents the coefficient of variation (CV) as the variation in fluorescence intensity among the 33 fluorescent beads. The horizontal axis represents the cumulative number of measurements used to calculate the variation. For example, when the cumulative number of measurements is three, the average value of the fluorescence intensity from the first to third measurements for each fluorescent bead is used to calculate the variation in fluorescence intensity among the fluorescent beads.

[0074] 9, the variation in fluorescence intensity originating from sample 131 is minimized after about six measurements. As mentioned above, there are inherent differences (variations) in fluorescence intensity among the fluorescent beads, just like sample 131 (cells) in flow cytometer 1. Therefore, even if the number of measurements continues to increase, the effect of suppressing variation reaches a plateau.

[0075] 7 to 9 simulate the case where the same sample 131 is measured multiple times by the flow cytometer 1 and the average value is treated as the signal value of each sample 131. This makes it possible to reduce data variations due to positional deviation or differences in orientation of the sample 131 relative to irradiation with the laser light 141. This makes it possible to clearly separate the sample population 401 in the analysis by the flow cytometer 1.

[0076] (Processing Device 200) Fig. 10 is a diagram showing the configuration of the processing device 200. Figs. 1 and 4 will be referred to as appropriate.

[0077] The processing device 200 includes an arithmetic unit 201, a storage unit 202, an input unit 203, an output unit 204, a communication unit 205, a memory 210, and the like.

[0078] The arithmetic unit 201 is composed of a CPU (Central Processing Unit), a GPU (Graphic User Interface), etc. The storage device 202 is composed of an HDD (Hard Disk Drive), an SSD (Solid State Drive), etc. The input device 203 is composed of a keyboard, a mouse, etc. The output device 204 is composed of a display, etc. The communication device 205 receives signals from the line sensors 123 and 323 shown in FIGS. 1 and 4 and the 2D CCD camera 314. The memory 210 is composed of a volatile memory such as a RAM (Random Access Memory).

[0079] Then, the program stored in the storage device 202 is loaded into the memory 210, and the loaded program is executed by the arithmetic device 201. As a result, a calculation unit 211, a gating unit 212, and a determination unit 213 are realized.

[0080] 5A to 9, the calculation unit 211 calculates the average value of the measurement values ​​obtained as a result of N measurements. The gating unit 212 gates the measurement results based on the average value calculated by the calculation unit 211, and extracts the sample population 401. The determination unit 213 determines whether adjacent sample populations 401 are clearly separated.

[0081] 11 is a flowchart showing the procedure of the flow cytometer signal analysis method according to this embodiment, with reference to FIGS. 1 and 10 as appropriate.

[0082] First, each sample 131 is measured N times (N=6 or 10 in the examples shown in FIGS. 5A to 9) by the flow cytometer 1 (S1). Step S1 is a measurement step in which multiple measurements are performed on each of the multiple samples 131.

[0083] Next, the calculation unit 211 calculates (S2) the average values ​​of the scattered light intensity and the fluorescent intensity measured N times for each sample 131. Step S2 is a statistical processing step in which statistical processing is performed on the measurement results from the measurement step (S1). In step S2, the calculation unit 211 calculates the average values ​​of the results of the multiple measurements for each sample 131.

[0084] The gating unit 212 then plots the N-time average values ​​of each sample 131 calculated in step S2 on a plot diagram as shown in FIGS. 5B and 6B. The gating unit 212 then performs gating (clustering) on ​​the plot points 402 (see FIGS. 5B and 6B) to extract a sample group 401 (S3). Step S3 is a gating step that performs gating to extract the sample group 401 based on the results of the statistical processing step (S2). In step S3, the gating unit 212 performs gating using the k-means method, a support vector machine, or the like. In step S3, the gating unit 212 extracts the sample group 401 based on the average values.

[0085] Next, the determination unit 213 determines whether the boundaries of the sample populations 401 extracted in step S3 are clear (S4). The determination unit 213 determines whether the boundaries of the sample populations 401 are clear by determining whether the boundaries of adjacent sample populations 401 do not overlap. If the boundaries of adjacent sample populations 401 do not overlap, the determination unit 213 determines that the boundaries of the sample populations 401 are clear.

[0086] If the boundary of the sample group 401 is clear (S4→Yes), the analysis ends.

[0087] If the boundary of the sample group 401 is not clear (S4→No), outliers are removed from the N measured values ​​(S5). The removal of outliers may be performed manually by the user based on the plot diagram, or may be performed by the gating unit 212 using Mahalanobis distance, box plots, etc.

[0088] After the outliers are removed in step S5, the processing device 200 returns to step S2. The processing device 200 then performs N-time averaging and gating using the data from which the outliers have been removed. Extracting the sample population 401 based on the average value of multiple measurements can reduce the number of times the outliers are removed in step S5.

[0089] According to the second embodiment, by analyzing the results of multiple measurements, the spread of the population at the plot points 402 (see FIGS. 5B and 6B ) can be reduced compared to the case of a single measurement, and as a result, the boundary of the sample population 401 can be clearly defined during gating.

[0090] The present invention is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present invention, and the present invention is not necessarily limited to those having all of the described configurations. Furthermore, it is possible to replace part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace part of the configuration of each embodiment with other configurations.

[0091] Furthermore, the flow cytometer 1 may be an analyzer or a cell sorter.

[0092] Furthermore, the above-described configurations, functions, calculation unit 211 to determination unit 213, storage device 202, etc. may be partly or entirely implemented in hardware by, for example, designing them as integrated circuits. As shown in FIG. 10 , the above-described configurations, functions, etc. may be implemented in software by a processor such as a CPU interpreting and executing a program that implements each function. Information such as the programs, tables, and files that implement each function can be stored in a memory, a storage device such as an SSD, or a storage medium such as an IC (Integrated Circuit) card, an SD (Secure Digital) card, or a DVD (Digital Versatile Disc), in addition to being stored on a HD.

[0093] In addition, in each embodiment, the control lines and information lines shown are those that are considered necessary for explanation, and not all control lines and information lines in the product are necessarily shown. In reality, it can be considered that almost all components are interconnected.

[0094] 1 Flow cytometer 110 Laser blocker 111 First laser blocker 112 Second laser blocker 113 Third laser blocker 121 Light sheet (light source unit) 122 Capillary 122a Axial center 123 Line sensor (light receiving unit) 123a Light receiving surface 131 Sample 141 Laser light 142 Forward scattered light 151a Alignment surface 171 Arrow (position where the axial center of the capillary is projected in the direction of the light source unit) 172 Arrow (position where the axial center of the capillary is projected in the direction of the light receiving unit) 200 Processing device 401 Sample population S1 Measure each sample N times (measurement step) S2 Calculate the average value of N times (statistical processing step) S3 Extraction of sample population (gating step)

Claims

1. A flow cytometer comprising: a light source unit that irradiates laser light onto a plurality of capillaries; a light receiving unit that receives forward scattered light that scatters on the capillaries in the direction opposite to the side where the laser light is irradiated; and a laser blocker that blocks light, wherein the laser blockers include: a first laser blocker that is located close to the light source unit including the alignment surface of the capillaries on the side where the laser light is incident, and at a position shifted from the position where the axial center of the capillaries is projected in the direction of the light source unit; a second laser blocker that is installed between the capillaries and the light receiving unit and between the capillaries; and a third laser blocker that is installed on the light receiving surface of the light receiving unit at a position where the axial center of the capillaries is projected in the direction of the light receiving unit.

2. The flow cytometer described in claim 1, characterized in that the first laser blocker is installed on the alignment surface of the capillary on the side where the laser light is incident, and the second laser blocker is installed from the alignment surface of the capillary on the side where the laser light is incident to the light receiving surface of the light receiving unit.

3. The flow cytometer according to claim 1, wherein the capillary is a single capillary having a serpentine path.

4. The flow cytometer according to claim 1, wherein a plurality of linear capillaries are arranged.

5. The flow cytometer according to claim 1, wherein the light source unit is a light sheet.

6. A device comprising: a light source unit that irradiates laser light onto a plurality of capillaries; a light receiving unit that receives forward scattered light that scatters on the capillaries in the direction opposite to the side where the laser light is irradiated; a laser blocker that blocks light; and a processing device, wherein the laser blockers include: a first laser blocker that is located at a position close to the light source unit including the alignment surface of the capillaries on the side where the laser light is incident and at a position shifted from the position where the capillaries are projected in the direction of the light source unit; a second laser blocker that is located between the capillaries and the light receiving unit and between the capillaries; and a third laser blocker that is located between the capillaries and the light receiving surface of the light receiving unit, wherein the processing device executes: a measuring step that measures a plurality of samples each multiple times; a statistical processing step that performs statistical processing on the measurement results from the measuring step; and a gating step that performs gating to extract a sample population based on the results of the statistical processing step, and wherein the processing device performs a mean value of the results of the multiple measurements for each sample is calculated; and in the gating step, the sample population is extracted based on the mean value.

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