JP6948354B2 - Fluorescence image analyzer and fluorescence image analysis method - Google Patents

Description

The present invention relates to a fluorescence image analyzer and a method for analyzing a fluorescence image.

Patent Document 1 describes a method for treating cells when a flow cytometer or the like is applied to the detection of a fluorescence in situ hybridization method (FISH method). In the FISH method, first, a pretreatment for hybridizing a fluorescently labeled probe to the base sequence of a target site existing in the nucleus of a cell is performed, and the target site is fluorescently labeled. Subsequently, the fluorescence (bright spot) generated from the fluorescently labeled probe is detected. The FISH method can detect chromosomal abnormalities even in non-dividing cells using a fluorescently labeled probe that binds to a target site on the chromosome. Furthermore, the FISH method can detect cells having chromosomal abnormalities mixed with normal cells, and can analyze the proportion thereof.

Special Table 2005-515408

Currently, analysis by the FISH method is mainly performed by the human (operator) eye using a fluorescence microscope. The operator must memorize the positive pattern for determining that there is an abnormality and the negative pattern for determining that there is no abnormality for each probe used for detection. However, there are currently dozens of probes for detecting chromosomal abnormalities by the FISH method, and among the positive patterns, there are typical chromosomal aberration patterns and atypical chromosomal aberration patterns. The operator has to grasp the positive pattern and the negative pattern for all of them, and the analysis by the FISH method is complicated. Furthermore, since the determination of the bright spot depends on the operator's sense, the criterion for determining whether the bright spot pattern of the fluorescent dye of the observed cell is a positive pattern or a negative pattern changes depending on the skill level of the operator, and FISH It is difficult to maintain the accuracy of determining the presence or absence of chromosomal abnormalities by the method.

In view of the current problem of measurement analysis by the FISH method, it is an object of the present invention to improve the accuracy of determining whether or not the bright spot pattern is a positive pattern.

The first aspect of the present invention relates to a fluorescence image analyzer. The fluorescence image analyzer (1) according to this embodiment measures and analyzes a sample (10) prepared by pretreatment including a step of labeling a target site in a cell with a fluorescent dye using a labeling reagent. The apparatus includes light sources (120) to (123) that irradiate the sample (10) with light, an imaging unit (160) that images the fluorescence emitted from the sample (10) when the sample (10) is irradiated with light, and an imaging unit (160). A processing unit (11) for processing the fluorescence image captured by the unit (160) is provided. The processing unit (11) acquires a bright spot pattern determined based on the color and number of fluorescence generated from the fluorescent dye that labels the target site in the fluorescent image, and is set according to the bright spot pattern and the measurement item. Based on each of the plurality of positive patterns, at least one piece of information is generated of the number of cells determined to be abnormal cells in the sample (10) and the ratio of the number of cells.

Preferably, the fluorescence image analyzer (1) determines that the processing unit (11) is an abnormal cell in the sample (10) based on the positive pattern selected from the plurality of positive patterns by the input unit (14). At least one of the number of cells and the ratio of the number of cells is displayed on the display unit (13).

Preferably, the fluorescence image analyzer (1) determines that the processing unit (11) is an abnormal cell in the sample (10) based on the positive pattern selected from the plurality of positive patterns by the input unit (14). The fluorescent image of is displayed on the display unit (13).

Preferably, in the fluorescence image analyzer (1), the processing unit (11) is revised when the determination is corrected by the input unit (14) from the fluorescence images of the abnormal cells displayed on the display unit (13). At least one of the number of abnormal cells and the ratio of the number of abnormal cells in the subsequent sample (10) is displayed on the display unit (13).

Preferably, in the fluorescence image analyzer (1), the processing unit (11) is revised when the determination is corrected by the input unit (14) from the fluorescence images of the abnormal cells displayed on the display unit (13). The fluorescence image of the abnormal cells in the later sample (10) is displayed on the display unit (13).

Preferably, the fluorescent image analyzer (1) further includes a storage unit (12) that stores a plurality of positive patterns for each measurement item for a plurality of measurement items, and the processing unit (11) is stored in the storage unit (12). From the memorized positive patterns for each measurement item, the positive pattern corresponding to the measurement item is selected.

A second aspect of the present invention analyzes a fluorescent image obtained by imaging a sample (10) prepared by performing pretreatment including a step of labeling a target site in a cell with a fluorescent dye using a labeling reagent. Regarding the analysis method. In the analysis method according to this aspect, the measurement item of the sample (10) is received, a plurality of positive patterns set corresponding to the measurement item are read, and the color of fluorescence generated from the fluorescent dye that labels the target site in the fluorescence image. A cell determined to be an abnormal cell in the sample (10) based on the bright spot pattern determined based on the number and the bright spot pattern and each of the plurality of positive patterns set corresponding to the measurement items. Generates at least one piece of information on the number of cells and the proportion of the number of cells, and displays at least one of the number of cells and the proportion of the number of cells.

Preferably, the method for analyzing a fluorescent image displays at least one of the number of cells and the ratio of the number of cells in the sample (10) determined to be abnormal cells based on a positive pattern selected from a plurality of positive patterns. do.

Preferably, the method for analyzing a fluorescent image displays a fluorescent image of cells in a sample (10) determined to be an abnormal cell based on a positive pattern selected from a plurality of positive patterns.

Preferably, the method for analyzing the fluorescent image is at least one of the number of abnormal cells and the ratio of the number of abnormal cells in the revised sample (10) when the determination is corrected from the fluorescent images of the abnormal cells. Is displayed.

Preferably, the method for analyzing the fluorescent image displays the fluorescent image of the abnormal cell in the revised sample (10) when the determination is corrected from the fluorescent image of the abnormal cell.

Preferably, the fluorescent image analysis method obtains a positive pattern corresponding to the measurement item of the received sample (10) from the storage unit (12) of the computer that stores a plurality of positive patterns for each measurement item for the plurality of measurement items. Read.

According to the first and second aspects, it is not necessary for the operator or the like to memorize various bright spot patterns indicating abnormal cells having chromosomal abnormalities and determine whether they are abnormal cells, and the abnormal cells can be determined. It does not depend on the operator's feeling. Therefore, the accuracy of determining abnormal cells can be improved, and as a result, the accuracy of information used for determining the sample (10) can be improved. Whether or not the sample (10) contains abnormal cells, and the proportion of the abnormal cells contained in the cells in the sample (10) can be grasped, and whether the sample (10) is positive or negative can be determined. It can be judged with high accuracy.

According to the present invention, it is possible to improve the accuracy of determining whether or not the bright spot pattern of a cell is the bright spot pattern of an abnormal cell having a chromosomal abnormality.

It is the schematic which shows the structure of one Embodiment of a fluorescence image analyzer. (A) is a figure illustrating the first image to the third image and the bright field image acquired by the fluorescent image analyzer, and (b) is for explaining the extraction of the nuclear region performed by the fluorescent image analyzer. (C) and (d) are schematic views for explaining the extraction of bright spots performed by the fluorescent image analyzer. It is a flowchart explaining operation of a processing part. It is a flowchart explaining operation of a processing part. It is a flowchart explaining operation of a processing part. (A) to (d) are schematic diagrams for explaining a negative pattern and a bright spot pattern of positive patterns 1 to 3, respectively. It is a figure which shows the example of the target site where each probe which targets the BCR / ABL fusion gene hybridizes. It is a figure which shows the example of the reference pattern of the typical positive pattern of the BCR / ABL fusion gene. It is a figure which shows the example of the reference pattern of the atypical positive pattern of the BCR / ABL fusion gene. It is a figure which shows the probe example of the ALK locus. It is a figure which shows the reference pattern of the negative pattern and the positive pattern when the chromosomal abnormality related to the ALK locus is detected, and the example of the chromosomal abnormality in which the long arm (5q) of chromosome 5 is deleted. This is an example of the display screen of the display unit of the fluorescence image analyzer. This is an example of the display screen of the display unit of the fluorescence image analyzer. It is a schematic diagram which shows the structure of another example of a measuring device. It is the schematic which shows the structure of the other embodiment of the fluorescence image analyzer.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following embodiment, a nucleic acid probe (hereinafter, simply referred to as a probe) containing a target site (target sequence) existing in the nucleus of a cell and a nucleic acid sequence having a sequence complementary to the target sequence and labeled with a fluorescent dye. The present invention is applied to an apparatus and method for measuring a sample subjected to pretreatment for hybridizing with and analyzing a fluorescent image obtained for each cell for a plurality of cells in the sample.

In one example of this embodiment, chromosomal aberrations are analyzed by the Fluorescence In Situ Hybridization (FISH) method using, for example, a flow cytometer (for example, an imaging flow cytometer), a fluorescence microscope, or the like. In the following embodiments, as an example, the target sites in the nucleic acid are the BCR gene on chromosome 22 and the ABL gene on chromosome 9, and the flow cytometer is used to detect chronic myelogenous leukemia by the FISH method. Measurement and analysis of cells having a translocation (BCR / ABL fusion gene, also called Philadelphia chromosome: t (9; 22) (q34.12; q11.23)) between chromosomes 9 and 22 The mode to be used is shown. Chromosomal abnormalities detected in fluorescence image analyzers are not limited as long as they can be detected by the (FISH) method. Examples of chromosomal abnormalities include translocations, deletions, inversions, duplications, and the like. Specifically, for example, chromosomal abnormalities related to loci such as BCR / ABL fusion gene and ALK gene can be mentioned. FIGS. 8, 9 and 11 show positive patterns for each gene for determining that there is a chromosomal abnormality.

Further, in the following embodiments, the cells to be measured are not limited as long as they are nucleated cells. For example, nucleated cells in a sample collected from a subject can be mentioned, preferably nucleated cells in a blood sample. In the present specification and the like, the sample is a cell suspension to be measured, which contains cells derived from the sample containing a target site hybridized with the probe. The sample contains multiple cells. The plurality of cells, at least 10 ² or more, preferably 10 ³ or more, more preferably 10 ⁴ or more, more preferably 10 ⁵ or more, even more preferably 10 ⁶ or more.

In this embodiment, the abnormal cell means a cell having a chromosomal abnormality. Examples of abnormal cells include tumor cells such as cancer cells. Preferred are hematopoietic tumor cells such as leukemia and cancer cells such as lung cancer.

FIG. 1 shows a schematic configuration of the fluorescence image analyzer 1 of the present embodiment. The fluorescence image analyzer 1 shown in FIG. 1 includes a measuring device 100 and a processing device 200, and measures and analyzes a sample 10 prepared by pretreatment by the pretreatment device 300. The operator collects the nucleated cells, which are the cells to be measured, by centrifuging the blood sample collected from the subject using a cell separation medium such as Ficoll. In the recovery of nucleated cells, the nucleated cells may be left by hemolyzing erythrocytes or the like with a hemolytic agent instead of recovering the nucleated cells by centrifugation. The pretreatment device 300 includes a mixing container for mixing the nucleated cell suspension and the reagent obtained by centrifugation or the like, a dispensing unit for dispensing the nucleated cell suspension and the reagent into the mixing container, and mixing. Includes a heating unit for heating the container. The pretreatment apparatus 300 performs a pretreatment including a step of labeling the target site in the cell collected from the subject with a fluorescent dye and a step of staining the nucleus of the cell with a dye for nuclear staining, and prepares the sample 10. Prepare. Specifically, in the step of labeling the target site with a fluorescent dye, the target sequence is hybridized with a probe containing a nucleic acid sequence having a sequence complementary to the target sequence and labeled with the fluorescent dye.

The FISH method uses one or more fluorescent dyes to detect a target site on a chromosome. Preferably, the FISH method uses two or more fluorescent dyes to detect a target site on the first chromosome and a target site on the second chromosome (the "first" and "second" that modify the "chromosome". Is a comprehensive concept of numbers that does not mean chromosome numbers). For example, a probe that hybridizes with the BCR locus is a first fluorescent dye in which a nucleic acid having a sequence complementary to the base sequence of the BCR locus produces first fluorescence of wavelength λ21 when irradiated with light of wavelength λ11. It is labeled by. By using this probe, the BCR locus is labeled with the first fluorescent dye. In the probe that hybridizes with the ABL locus, a nucleic acid having a sequence complementary to the base sequence of the ABL locus is labeled with a second fluorescent dye that produces a second fluorescence of the wavelength λ22 when irradiated with light of the wavelength λ12. It was done. By using this probe, the ABL locus is labeled with a second fluorescent dye. The nuclei are stained with a dye for nuclear staining that produces a third fluorescence of wavelength λ23 when irradiated with light of wavelength λ13. Wavelength 11, wavelength λ12 and wavelength λ13 are so-called excitation light.

More specifically, the pretreatment apparatus 300 includes a process of fixing the cell so that the cell does not contract due to dehydration, a membrane permeation process of making a hole in the cell large enough to introduce a probe into the cell, and a heat of applying heat to the cell. It includes a denaturation process, a process of hybridizing a target site with a probe, a washing process of removing unnecessary probes from cells, and a process of staining the nucleus.

The measuring device 100 includes a flow cell 110, a light source 120 to 123, a condenser lens 130 to 133, a dichroic mirror 140 to 141, a condenser lens 150, an optical unit 151, a condenser lens 152, and an imaging unit 160. And have. The sample 10 is flowed through the flow path 111 of the flow cell 110.

The light sources 120 to 123 irradiate the sample 10 flowing through the flow cell 110 with light. The light sources 120 to 123 are composed of, for example, a semiconductor laser light source. Lights having wavelengths λ11 to λ14 are emitted from the light sources 120 to 123, respectively.

The condenser lenses 130 to 133 collect light having wavelengths λ11 to λ14 emitted from light sources 120 to 123, respectively. The dichroic mirror 140 transmits light having a wavelength of λ11 and refracts light having a wavelength of λ12. The dichroic mirror 141 transmits light having wavelengths λ11 and λ12 and refracts light having wavelength λ13. In this way, the light having wavelengths λ11 to λ14 is applied to the sample 10 flowing through the flow path 111 of the flow cell 110. The number of semiconductor laser light sources included in the measuring device 100 is not limited as long as it is 1 or more. The number of semiconductor laser light sources can be selected from, for example, 1, 2, 3, 4, 5 or 6.

When the sample 10 flowing through the flow cell 110 is irradiated with light having wavelengths λ11 to λ13, fluorescence is generated from the fluorescent dye that stains the cells. Specifically, when light having a wavelength of λ11 is applied to the first fluorescent dye that labels the BCR locus, the first fluorescent dye having a wavelength of λ21 is generated from the first fluorescent dye. When light of wavelength λ12 is applied to the second fluorescent dye that labels the ABL locus, the second fluorescent dye produces second fluorescence of wavelength λ22. When light having a wavelength of λ13 is applied to a dye for nuclear dyeing that stains the nucleus, a third fluorescence having a wavelength of λ23 is generated from the dye for nuclear dyeing. When the sample 10 flowing through the flow cell 110 is irradiated with light having a wavelength of λ14, this light passes through the cells. The transmitted light of wavelength λ14 transmitted through the cells is used to generate a bright-field image. For example, in the embodiment, the first fluorescence is the wavelength band of green light, the second fluorescence is the wavelength band of red light, and the third fluorescence is the wavelength band of blue light.

The condenser lens 150 collects the first fluorescence to the third fluorescence generated from the sample 10 flowing through the flow path 111 of the flow cell 110 and the transmitted light transmitted through the sample 10 flowing through the flow path 111 of the flow cell 110. The optical unit 151 has a configuration in which four dichroic mirrors are combined. The four dichroic mirrors of the optical unit 151 reflect the first fluorescence to the third fluorescence and the transmitted light at slightly different angles, and separate them on the light receiving surface of the imaging unit 160. The condenser lens 152 collects the first fluorescence to the third fluorescence and the transmitted light.

The imaging unit 160 is composed of a TDI (Time Delay Integration) camera. The imaging unit 160 captures the first fluorescence to the third fluorescence and the transmitted light, and processes a fluorescence image corresponding to the first fluorescence to the third fluorescence and a bright field image corresponding to the transmitted light as an imaging signal. Output to device 200. Fluorescent images corresponding to the first fluorescence to the third fluorescence are hereinafter referred to as "first image", "second image", and "third image", respectively. The "first image", "second image", and "third image" are preferably the same size for analyzing the overlap of bright spots. The "first image", "second image" and "third image" may be a color image or a grayscale image.

FIG. 2A is an example of a fluorescence image. The black dot-like portion in the first image of FIG. 2A indicates the bright spot of the first fluorescence, that is, the target site labeled with the first fluorescent dye. In the second image, dark gray dots are observed in the light gray showing the nucleus, though not as much as in the first image. This indicates the bright spot of the second fluorescence, i.e. the target site labeled with the second fluorescent dye. In the third image, the substantially circular core region is shown in black. In the bright field image, the actual state of cells can be observed. Each image of FIG. 2A is an image showing an example in which the pretreated leukocytes are placed on a slide glass and observed with a microscope, and the fluorescence image is fluorescence in the raw data (raw data). The higher the intensity, the whiter the image, and the lower the fluorescence intensity, the blacker the image. The first to third images of FIG. 2A are grayscale representations of the captured raw data with the gradations inverted. When the sample 10 flowing through the flow cell 110 is imaged by the imaging unit 160 as described above, the cells flow through the flow path 111 in a state of being separated from each other, so that the fluorescence image and the bright field image are acquired for each cell. Become.

Returning to FIG. 1, the processing device 200 includes a processing unit 11, a storage unit 12, a display unit 13, and an input unit 14 as a hardware configuration. The processing unit 11 is composed of a processor (CPU). The storage unit 12 is composed of a readable / writable memory (RAM) used for the work area of various processes of the processing unit 11, a read-only memory (ROM) for storing a computer program and data, a hard disk, and the like. The processing unit 11 and the storage unit 12 can be configured by a general-purpose computer. The hard disk may be included in the computer or may be placed as an external device of the computer. The display unit 13 is composed of a display. The input unit 14 is composed of a mouse, a keyboard, a touch panel device, and the like. The processing unit 11 transmits data to and from the storage unit 12 via the bus 15, and inputs and outputs data to and from the display unit 13, the input unit 14, and the measuring device 100 via the interface 16.

The processing unit 11 reads various computer programs stored in the ROM or the hard disk into the RAM and executes them to process the fluorescent image of the cells acquired by the measurement of the sample 10 by the measuring device 100, and the display unit 13 performs the processing. , Controls the operation of the input unit 14, and the like. Specifically, the processing unit 11 processes the fluorescence image to acquire the fluorescence bright spot pattern in the fluorescence image, and from the plurality of reference patterns corresponding to the plurality of measurement items stored in the storage unit 12, the sample 10 Select the reference pattern corresponding to the measurement item. Then, based on the fluorescence bright spot pattern in the fluorescence image and the selected reference pattern, information used for determining the sample 10 is generated.

Hereinafter, an example of a fluorescence image analysis method executed by the processing unit 11 based on a computer program that defines a processing procedure for analyzing a fluorescence image of cells will be described with reference to FIGS. 3 to 5. Although the computer program is stored in the storage unit 12 in advance, it may be installed from a computer-readable portable recording medium (not shown) such as a CD-ROM, or an external server (not shown), for example. It may be downloaded and installed from (not shown) via a network (not shown).

As shown in FIGS. 3 to 5, the processing unit 11 includes an image acquisition step S1, a bright spot pattern acquisition step S2, a bright spot pattern and reference pattern comparison step S3, and a determination result of whether the cells are abnormal cells or normal cells. Each process is performed in step S4 of displaying information regarding the sample, step S5 of generating information used for determining the sample, and step S6 of displaying information used for determining the sample.

First, in S1, the processing unit 11 reverses the gradation of the raw data captured by the imaging unit 160 and acquires the first to third images displayed in grayscale. The processing unit 11 stores the acquired first to third images in the storage unit 12.

Next, in S2, the processing unit 11 acquires the bright spot pattern of the first fluorescence in the first image based on the first fluorescence and the bright spot pattern of the second fluorescence in the second image based on the second fluorescence. ..

In this S2, as shown in FIG. 4, first, in S20, the processing unit 11 starts with the first fluorescence bright spot (first bright spot) from the first image, the second image, and the third image, respectively. A process for extracting the bright spot (second bright spot) of the second fluorescence and the nuclear region is performed. Specifically, to explain using FIGS. 2 (b) to 2 (d), the third image shown at the left end of FIG. 2 (b), the first image shown at the left end of FIG. 2 (c), and FIG. 2 ( The second image shown at the left end of d) is obtained from one cell flowing through the flow cell 110.

When a third image as shown at the left end of FIG. 2B is acquired, the processing unit 11 first comprises m horizontal (x-direction) × n vertical (y-direction) images constituting the third image. Based on the pixel value in the pixel, a graph of the pixel value and the number of pixels is created as shown in the center of FIG. 2 (b). The number of pixels on the vertical axis indicates the number of pixels. The number of pixels of the image is not particularly limited, but is, for example, 51 horizontal × 51 vertical. Further, the spatial resolution per pixel is not particularly limited. Then, the processing unit 11 sets the threshold value of the pixel value in the graph of FIG. 2B, and the range in which the pixels having the pixel value larger than the threshold value are distributed is shown by a broken line at the right end of FIG. 2B. In addition, it is extracted as a nuclear region.

When the first image as shown at the left end of FIG. 2C is acquired, the processing unit 11 has m horizontal (x direction) × n vertical (y direction) pixels constituting the first image. Based on the pixel value, a graph of the pixel value and the number of pixels is created as shown in the center of FIG. 2C. Then, in the graph of FIG. 2C, the processing unit 11 sets a pixel value threshold value as a boundary between the bright spot and the background based on, for example, the Otsu method, and pixels having a pixel value larger than the threshold value are distributed. The range to be used is extracted as a bright spot at the right end of FIG. 2 (c) as shown by a broken line. When extracting bright spots from the first image, bright spots having an extremely small range and bright spots having an extremely large range are excluded.

When the second image as shown at the left end of FIG. 2D is acquired, the processing unit 11 constitutes m horizontal (x directions) × vertical (y) images as in the case of the first image. Direction) Based on the pixel values in each of the n pixels, a graph of the pixel values and the number of pixels is created as shown in the center of FIG. 2 (d). Then, the processing unit 11 sets the threshold value of the pixel value in the graph of FIG. 2 (c), and the range in which the pixels having the pixel value larger than the threshold value are distributed is indicated by a broken line at the right end of FIG. 2 (d). As such, it is extracted as a bright spot. When extracting the bright spots from the second image, the bright spots having an extremely small range and the bright spots having an extremely large range are excluded.

Next, in S21 of FIG. 4, the processing unit 11 compares the position of the nuclear region extracted from the third image with the position of the bright spot extracted from the first image, and the processing unit 11 compares the position of the bright spot extracted from the first image with the brightness not included in the nuclear region. Exclude points. As a result, the first bright spot of the first fluorescence is extracted from the first image, and the number and position of the first bright spot in the first image are derived. Similarly, the position of the nuclear region extracted from the third image is compared with the position of the bright spot extracted from the second image, and the bright spots not included in the nuclear region are excluded. As a result, the second bright spot is extracted from the second image, and the number and position of the second bright spot in the second image are derived.

For the positions of the nuclear region and the bright spot in each image, for example, coordinate information (x, y) is predetermined for m horizontal (x direction) × n vertical (y direction) pixels constituting each image, and the nuclear region and the bright spots are located. It can be measured based on the coordinate information of a plurality of pixels included in the bright spot.

The processing unit 11 extracts the nuclear region from the third image by calculation according to the above procedure without creating the graph shown in the center of FIGS. 2 (b) to 2 (d). , Each bright spot may be extracted from the first image and the second image. Further, in the extraction of bright spots, the degree of matching between the distribution waveform of the bright spots that is regarded as normal and the region to be determined is determined, and when the degree of matching is high, the region to be judged is extracted as the bright spot. May be good. Although the processing unit 11 detected the cells by extracting the nuclear region from the third image, the cells may be detected based on the bright field image. When cells are detected based on a bright-field image, acquisition of a third image can be omitted. The bright spot in the present embodiment means a small fluorescent spot generated in a fluorescent image. More specifically, the bright spot means the center point of the bright spot (the position of the pixel having the highest pixel value (fluorescence intensity)). The bright spots can be extracted, for example, by converting the color gradations of pixels other than the pixels designated as bright spots to the same level as the background.

The "pixel value" in the present specification refers to a digital value assigned to each pixel of an image, and in particular, in an output image from a camera (so-called raw image), the brightness of the object to be imaged becomes a digital signal. Refers to the converted value.

Next, in S22, the processing unit 11 of the first image and the second image based on the arrangement example (number, position) of the first bright spot and the second bright spot extracted from the first image and the second image. The first and second bright spots that overlap each other in the composite image are extracted.

Here, first, a method for determining whether or not a cell is an abnormal cell having a chromosomal abnormality will be described.

FIG. 6A exemplifies an example of arrangement of bright spots of normal cells having no chromosomal abnormality, that is, a bright spot pattern (negative pattern), and FIGS. 6 (b) to 6 (d) show bright spot patterns of abnormal cells. (Positive pattern) is illustrated. In any of FIGS. 6A to 6D, the third image is superposed on each image.

As shown in FIG. 6A, when there are no abnormalities in the chromosomes such as translocations of the BCR locus and the ABL locus, each gene is paired in one nucleus, and each allele is independent. Exists. Therefore, in the first image, there are two first bright spots in one nuclear region. Further, in the second image, there are two second bright spots in one nuclear region. In this case, when the first image and the second image captured in the same size are superimposed and combined, in the composite image, the two first bright spots and the two second bright spots are one nuclear region. It will exist without overlapping inside. Therefore, as shown in FIG. 6A, cells having two first bright spots and two second bright spots in the nuclear region are normal cells in which no chromosomal abnormality is observed, that is, the chromosomal abnormality is negative. Is determined.

An example of a positive pattern was used as a probe targeting the BCR / ABL fusion gene [Cytocell BCR / ABL Translocation, Extra Signal (ES) Probe (Sysmex Corporation)] (hereinafter, may be simply referred to as "ES probe"). A case will be described as an example. There are multiple types of probes that target the BCR / ABL fusion gene. An example of the target site where each probe hybridizes is shown in FIG. The BCR gene is located on chromosome 22q11.22-q11.23 and the probe that hybridizes to the BCR locus is labeled with the first fluorescence (eg, green). The ABL gene is located on chromosome 9q34.11-q34.12. And the probe that hybridizes to the ABL locus is labeled with a second fluorescence (eg, red). FIG. 7A shows the binding site of the ES probe described above, and FIG. 7B shows the Cytocell BCR / ABL Translocation, Dual Fusion (DF) Probe (Sysmex Corporation, Cat No. LPH007) (hereinafter, simply referred to as “DF probe”). ) Indicates the binding site.

As shown in FIG. 6 (b), when a part of the ABL locus is moved to chromosome 9 by translocation, two first bright spots exist in the nucleus and the second bright spot exists in the nucleus in the first image. In the image, there are three second bright spots in the nucleus. In this case, when the first image and the second image are combined, one first bright spot, two second bright spots, and the first bright spot and the second bright spot overlap each other in the composite image. One fourth fluorescence (for example, yellow) bright spot (fusion bright spot) exists in one nucleus. Therefore, as shown in FIG. 6B, the cell in which each bright spot exists is determined to be an abnormal cell in which translocation has occurred in the BCR gene and the ABL gene, that is, a chromosomal abnormality is positive.

As shown in FIG. 6 (c), when a part of the BCR locus is moved to chromosome 22 and a part of the ABL gene is moved to chromosome 9 due to the translocation, the first image shows the first There are three bright spots in the nucleus, and in the second image, there are three bright spots in the nucleus. In this case, when the first image and the second image are combined, one first bright spot, one second bright spot, and the first bright spot and the second bright spot overlap each other in the composite image. Two fusion bright spots will exist in one nucleus. Therefore, as shown in FIG. 6 (c), the cell in which each bright spot exists is determined to be an abnormal cell in which a translocation has occurred at the BCR locus and the ABL locus, that is, a chromosomal abnormality is positive.

As shown in FIG. 6 (d), when the ABL locus is moved to chromosome 9 by translocation, two first bright spots exist in the nucleus in the first image, and in the second image, there are two spots. There are two second bright spots in the nucleus. In this case, when the first image and the second image are combined, one first bright spot, one second bright spot, and one first bright spot and the second bright spot overlap each other in the composite image. The fusion bright spot will exist in one nucleus. Therefore, as shown in FIG. 6 (d), the cell in which each bright spot is present is determined to be an abnormal cell in which a translocation has occurred at the BCR locus and the ABL locus, that is, a chromosomal abnormality is positive.

As described above, it is possible to determine whether or not each cell is an abnormal cell having a chromosomal abnormality based on the position and number of each bright spot in the composite image obtained by synthesizing the first image and the second image. Therefore, in S22 of FIG. 4, the processing unit 11 sets the number of fluorescent bright spot patterns of the fluorescent image as the fluorescent bright spot pattern of the first image and the second image for each cell, that is, the number of each bright spot in the composite image of the first image and the second image, that is, the first. The number of 1st bright spots that do not overlap with the 2 bright spots, the number of 2nd bright spots that do not overlap with the 1st bright spot, and the fusion bright spots at the positions where the 1st and 2nd bright spots overlap each other. And count the number of. For example, in FIG. 6D, the bright spot pattern is calculated from the number of the first bright spots of a single unit being "1", the number of the second bright spots of a single unit being "1", and the first bright spots and the second bright spots. The number of fusion bright spots can be created as "1".

The first bright spot, the second bright spot, and the fused bright spot may be shown as colors, and a bright spot pattern and a reference pattern described later may be created. For example, the first bright spot of a single substance can be represented as green (G), the second bright spot of a single substance can be represented as red (R), and the fused bright spot can be represented as yellow (F). By indicating the number of bright spots of G, R, and F, a bright spot pattern can be obtained. For example, in FIG. 6D, the bright spot pattern can be displayed as "G1R1F1".

As the fluorescence bright spot pattern of the fluorescent image, the number of the first bright spots described above is the total number of the first bright spots in the first image, and the number of the second bright spots described above is the second bright spot in the second image. It can be created as the total number of 2 bright spots. For example, in FIG. 6D, the bright spot pattern has the number of first bright spots in the first image as “2”, the number of second bright spots in the second image as “2”, and the number of bright spots in the composite image as the first bright spot. The number of fused bright spots where the dots and the second bright spot overlap each other can be created as "1" and displayed as "G2R2F1", but the meaning is the same.

In the composite image, whether or not the first bright spot of the first image and the second bright spot of the second image overlap is the ratio of the regions where the first bright spot and the second bright spot overlap each other, for example, the first. It can be determined whether or not the ratio of the pixels at the same position (coordinate information (x, y)) as each pixel included in the second bright spot among the plurality of pixels included in the bright spot is larger than the threshold value. can. Also, depending on whether the distance between the center point of the first bright spot (the position of the pixel with the highest fluorescence intensity) and the center point of the second bright spot (the position of the pixel with the highest fluorescence intensity) is smaller than the threshold value. It can be determined.

The fluorescence bright spot pattern in the fluorescent image acquired for each cell may be the number of bright spots in the composite image for each color. That is, instead of displaying each image in grayscale, the color of each pixel of the first image is displayed with a green color gradation (RGB value) based on the pixel value, and the color of each pixel of the second image is displayed. Is displayed in red color gradation (RGB value). Then, when each image is superimposed and synthesized, based on the combination of RGB values of each pixel of the composite image, if the cell is an abnormal cell, a green first bright spot and a red second bright spot are formed in the nuclear region. The bright spot and the yellow fusion bright spot exist due to the overlap of the first bright spot and the second bright spot. Therefore, it is possible to determine whether or not the cell is an abnormal cell by counting the number of bright spots for each color as the bright spot pattern.

The processing unit 11 stores the composite image of the first image and the second image created in S2 of FIG. 3 and the fluorescence bright spot pattern in the composite image in the storage unit 12 for each cell.

As described above, when the fluorescence bright spot pattern in the fluorescence image of the cell is acquired, the processing unit 11 then determines whether the cell is an abnormal cell or a normal cell based on the bright spot pattern acquired for each cell. Is determined. In the present embodiment, the storage unit 12 stores a reference pattern for determining whether a cell is an abnormal cell or a normal cell for each measurement item for a plurality of measurement items. In S3, the processing unit 11 compares the bright spot pattern acquired for each cell with the reference pattern corresponding to the measurement item of the sample 10 from the plurality of reference patterns stored in the storage unit 12 for each cell. Determine if it is an abnormal cell. The determination of abnormal cells by the processing unit 11 will be described later with reference to FIG.

The reference pattern is, for example, a bright spot pattern (positive pattern) of fluorescence in a fluorescent image of an abnormal cell having a chromosomal abnormality, as shown in FIG. 6, and a fluorescent image of a normal cell having no chromosomal abnormality. It contains at least one of the bright spot patterns (negative patterns) of fluorescence in. In the present embodiment, the reference pattern includes both the bright spot pattern of abnormal cells (positive pattern) and the bright spot pattern of normal cells (negative pattern).

In this embodiment, for example, as shown in FIGS. 8, 9 and 11, BCR / ABL fusion gene, ALK gene, and reference pattern (negative pattern and positive pattern) of chromosome 5 long arm deletion are stored as measurement items. It is stored in part 12. Further, in the present embodiment, the measurement items are further classified for each probe that hybridizes to the target site, and the reference pattern is stored for each probe in the storage unit 12. Further, in the present embodiment, the storage unit 12 has a reference pattern of typical abnormal cells (typical positive pattern) and a reference pattern of atypical abnormal cells (non-standard) for each probe. Type positive pattern) is classified and memorized.

FIG. 8 is an example of a reference pattern of a typical positive pattern (major pattern) of the BCR / ABL fusion gene. In the state where the first image and the second image are superimposed, when the ES probe is used, the negative pattern has two first bright spots, two second bright spots, and a fusion bright spot. The number of is 0. An example of a typical positive pattern using an ES probe is that the number of first bright spots is one, the number of second bright spots is two, and the number of fusion bright spots is one. When the DF probe is used, in the state where the first image and the second image are superimposed, the negative pattern has two first bright spots, two second bright spots, and a fusion bright spot. The number of is 0. An example of a typical positive pattern using a DF probe is that the number of first bright spots is one, the number of second bright spots is one, and the number of fusion bright spots is two.

FIG. 9 is an example of a reference pattern of the atypical positive pattern of the BCR / ABL fusion gene. One example of the atypical positive pattern is the minor BCR / ABL pattern, and since the cut point of the BCR gene is relatively upstream of the BCR gene, three first bright spots are detected by the ES probe. Another example of the atypical positive pattern is the deletion of a part of the binding region of the probe targeting the ABL gene on chromosome 9, which is essentially two when the DF probe is used. Only one fusion bright spot that should have been detected has been detected. In another example of the atypical positive pattern, a part of the binding region of the probe targeting the ABL gene on chromosome 9 and a part of the binding region of the probe targeting the BCR gene on chromosome 22 are both. This is an example of deletion. Depending on this, only one fusion bright spot, which should have been detected twice when the DF probe is used, is detected.

In addition to the BCR / ABL fusion gene, the chromosomal translocations that can detect fusion bright spots by the FISH method include the AML1 / ETO (MTG8) fusion gene (t (8; 21)) and the PML / RARα fusion gene (t (15;). 17)), AML1 (21q22) translocation, MLL (11q23) translocation, TEL (12p13) translocation, TEL / AML1 fusion gene (t (12; 21)), IgH (14q32) translocation, CCND1 (BCL1) / IgH fusion gene (t (11; 14)), BCL2 (18q21) translocation, IgH / MAF fusion gene (t (14; 16)), IgH / BCL2 fusion gene (t (14; 18)), c- myc / IgH fusion gene (t (8; 14)), FGFR3 / IgH fusion gene (t (4; 14)), BCL6 (3q27) translocation, c-myc (8q24) translocation, MALT1 (18q21) translocation , API2 / MALT1 fusion gene (t (11; 18) translocation), TCF3 / PBX1 fusion gene (t (1; 19) translocation), EWSR1 (22q12) translocation, PDGFRβ (5q32) translocation, etc. Can be done.

FIG. 10 shows an example of a probe at the ALK locus. In addition, FIG. 11 shows an example of a negative pattern and a reference pattern of a positive pattern when detecting a chromosomal abnormality related to the ALK locus. As shown in FIG. 10, the chromosomal abnormality of the ALK gene has a cut point near D2S405, and a part of the ALK gene is translocated to another site. Therefore, the first fluorescently labeled probe and the second fluorescently labeled probe are designed with the cutting point in between. In the negative pattern shown in FIG. 11, since the ALK gene is not cleaved, there are two fusion bright spots. On the other hand, in the positive pattern, since the ALK gene is cleaved, there is only one fusion bright spot (when only one of the alleles is cleaved), or no fusion bright spot is observed (both alleles). If is disconnected). The negative pattern and the positive pattern are the same for the ROS1 gene and the RET gene as well as the ALK gene.

Furthermore, FIG. 11 shows an example of a reference pattern of a chromosomal abnormality in which the long arm (5q) of chromosome 5 is deleted. For example, the first fluorescently labeled probe is designed to bind to the long arm of chromosome 5, and the second fluorescently labeled probe is designed to bind to the centromere of chromosome 5. In the negative pattern, the number of centromeres on chromosome 5 and the number of long arms on chromosome 5 are the same, so there are two bright spots and two bright spots, reflecting the number of homologous chromosomes. .. In the positive pattern, deletion of the long arm occurs on one or both of chromosome 5, and the number of first bright spots is only one or zero. This negative and positive pattern is the same for deletions of the short or long arms of other chromosomes. Examples of long-arm deletions of other chromosomes include long-arm deletions of chromosomes 7 and 20. In addition, as examples showing similar positive and negative patterns, 7q31 (deletion), p16 (9p21 deletion analysis), IRF-1 (5q31) deletion, D20S108 (20q12) deletion, D13S319 (13q14). ) Deletion, 4q12 deletion, ATM (11q22.3) deletion, p53 (17p13.1) deletion and the like.

Furthermore, FIG. 11 shows an example of chromosome 8 trisomy. The first fluorescently labeled probe binds, for example, to the centromere on chromosome 8. The positive pattern has three first bright spots. The negative pattern has two first bright spots. Such a bright spot pattern is the same in trisomy 12 of chromosome. Furthermore, in the chromosome 7 monosomy, for example, when a first fluorescently labeled probe that binds to the centromere of chromosome 7 is used, the positive pattern has one first bright spot. The negative pattern has two first bright spots.

Next, the details of S3 of FIG. 3 will be shown with reference to FIG. First, in S30, the processing unit 11 performs a process of selecting a reference pattern corresponding to the measurement item of the sample 10 from a plurality of reference patterns corresponding to the plurality of measurement items stored in the storage unit 12. Then, in S31, a process of comparing the bright spot pattern acquired for each cell with the negative pattern in the reference pattern selected according to the measurement item of the sample 10 is performed. As shown in FIG. 12, for example, the selection of the measurement item can be received by displaying the reception screen 30 for selecting the measurement item on the display unit 13. On the reception screen 30 of FIG. 12, a selection column for selecting measurement items for a plurality of cells to be analyzed (8 vertical (A to H) cells x 12 horizontal (1 to 12) cells = 96 cells in the illustrated example) is displayed. 31 is provided, and measurement items (for example, BCR / ABL fusion gene, ALK gene, chromosome 5 long arm deletion, etc.) can be selected for each cell by a pull-down method, for example. In FIG. 12, in the selection field 31, it is possible to select a probe that further classifies the measurement item in addition to the measurement item (for example, if the measurement item is a BCR / ABL fusion gene, a DF probe or an ES probe). be.

The processing unit 11 has a plurality of reference patterns stored in the storage unit 12 corresponding to a plurality of measurement items, and a reference pattern (negative pattern and a reference pattern) corresponding to the measurement item selected on the reception screen 30 or the probe in the measurement item. (Positive pattern) is read and compared with the bright spot pattern of the cell to be analyzed. When the bright spot pattern of the cell to be analyzed matches the negative pattern, S31 becomes YES and the process proceeds to S32, and it is determined that the cell is a normal cell. On the other hand, when the bright spot pattern of the cell to be analyzed does not match the negative pattern, S31 becomes NO and proceeds to S33, which is compared with the typical positive pattern. If the cell matches the typical positive pattern, S33 becomes YES and the process proceeds to S34, and it is determined that the cell is a typical abnormal cell. On the other hand, if it does not match the typical positive pattern, S33 becomes NO and proceeds to S35, and it is determined that the cell is an atypical abnormal cell. The processing unit 11 repeatedly performs the same comparative processing on all the cells to be analyzed, and determines whether the cells are abnormal cells or normal cells for each cell. The processing unit 11 stores the determination result according to S3 in FIG. 3 in the storage unit 12 for each cell.

Returning to FIG. 3, the processing unit 11 then causes the display unit 13 to display information on the determination result for each cell to be analyzed in S4. As information regarding the determination result, the processing unit 11 may use, for example, a fluorescent image of cells determined to be abnormal cells (composite image of the first image, the second image and the third image), and a fluorescent image of cells determined to be normal cells (first image). 1 image, a composite image of the second image and the third image) and the like are displayed on the display unit 13.

FIG. 13 is an example of the information display screen 32 of the display unit 13. On the information display screen 32 of FIG. 13, fluorescence images are displayed vertically and horizontally for each cell detected based on a predetermined analysis method among the cells to be analyzed. The information display screen 32 displays the measurement items 33 used for cell analysis, and is provided with an analysis method selection field 34 in which an analysis method can be selected. In the analysis method selection field 34, as a method for analyzing abnormal cells, it is possible to select whether to detect typical abnormal cells (ES major pattern) or atypical abnormal cells (ES minor pattern, ES delay pattern). Further, on the information display screen 32, for the cells detected based on the analysis method selected in the analysis method selection field 34, an option to display a fluorescent image of cells determined to be abnormal cells (positive), normal cells (negative). ) Is provided, for example, a display image selection field 38 is provided in which an option for displaying a fluorescent image of the cells determined to be) and an option for displaying a fluorescent image of all the analyzed cells can be selected by a pull-down method.

The processing unit 11 causes the fluorescent image of the cells selected in the display image selection field 38 to be displayed in the image display field 35 of the display screen 32 for the cells detected based on the analysis method selected in the selection field 34. In the fluorescent image of the cell displayed in the image display field 35, in addition to the Cell ID, the determination result of whether the cell is an abnormal cell (positive) or a normal cell (negative) is displayed for each fluorescent image of the cell. There is. In FIG. 13, since the analysis method for detecting typical abnormal cells (ES major pattern) is selected, the option of displaying the fluorescent image of the cells determined to be abnormal cells (positive) is selected in the display image selection field 38. Then, in the image display field 35, a fluorescent image of the cell determined to be a typical abnormal cell (ES major pattern) is displayed. Depending on the selection in the display image selection field 38, a fluorescent image of cells determined to be normal cells or a fluorescent image of all the analyzed cells can be displayed.

As a result, the operator or the like can observe the fluorescence image of the cells determined to be abnormal cells on the display unit 13. When the cell determined to be an abnormal cell is determined to be a normal cell by observation by an operator or the like, the processing unit 11 selects the fluorescent image of the abnormal cell displayed on the display unit 13. , The operator or the like selects the abnormal cell as a normal cell by the input unit 14, and corrects the determination result to the normal cell for the abnormal cell revised by the revision button 36. Then, the processing unit 11 stores the revised determination result in the storage unit 12. Similarly, when the cells determined to be normal cells are determined to be abnormal cells by the observation of an observer such as an operator, the processing unit 11 displays the fluorescence of the normal cells displayed on the display unit 13. From the image, the normal cell selected by the operator or the like as an abnormal cell by the input unit 14 and revised by the revision button 36 is corrected to the abnormal cell as the determination result. Then, the processing unit 11 stores the revised determination result in the storage unit 12. Thereby, the detection accuracy of abnormal cells and normal cells can be improved. In addition, the processing unit 11 can redisplay the fluorescent image of the revised abnormal cell or the cell determined to be a normal cell on the display unit 13.

Returning to FIG. 3, the processing unit 11 then generates information used in the determination of the sample 10 in S5 based on the determination result of whether or not the cells are abnormal cells for each cell.

For example, the processing unit 11 is based on the number of abnormal cells, the ratio of the number of abnormal cells, the number of normal cells, and the ratio of the number of normal cells based on the analysis result by the analysis method selected in the analysis method selection field 34. Performs a process to generate at least one piece of information selected from the group. The ratio of the number of abnormal cells and the ratio of the number of normal cells are the ratios to the total number of detected cells (the total value of the number of cells determined to be abnormal cells and the number of cells determined to be normal cells). It may be a ratio to the total number of cells analyzed.

Then, in S6 of FIG. 3, the processing unit 11 stores the information generated in S5 in the storage unit 12 and displays it on the display unit 13. For example, in the information display screen 32 of FIG. 13, a determination result column 37 is provided, and in the determination result column 37, the number and proportion of cells determined to be abnormal cells and the number and proportion of cells determined to be normal cells are displayed. It is displayed. Here, since the analysis method for detecting the typical abnormal cells (ES major pattern) is selected in the analysis method selection column 34, the number of typical abnormal cells (positive) whose determination result is "G1R2F1" is selected in the determination result column 37. And the ratio, the number and ratio of normal cells (negative) whose determination result is "G2R2F0" are displayed. In addition, when an analysis method for detecting atypical abnormal cells (ES minor pattern, ES dilation pattern) is selected in the analysis method selection field 34, the determination result is the number of atypical abnormal cells (positive) and the number of atypical abnormal cells (positive). The ratio and the number and ratio of normal cells (negative) as the determination result are displayed in the analysis result column 37.

As a result, the doctor or the like refers to the information displayed on the information display screen 32 to determine whether or not the sample 10 contains abnormal cells, and further, the abnormal cells contained in the nucleated cells in the sample 10. The ratio can be grasped, and whether the sample 10 is positive or negative can be determined with high accuracy.

As the information used for determining the sample 10, the processing unit 11 generates various other information such as character information such as "possibility of positiveness?" And "possibility of negativeness?" It can be displayed on 13. "Possibility of positiveness?" Is displayed when the proportion of abnormal cells is greater than a predetermined threshold and when the proportion of normal cells is smaller than a predetermined threshold. If the proportion of normal cells is greater than a predetermined threshold and the proportion of abnormal cells is less than a predetermined threshold, "possible negative?" Is displayed.

As described above, according to the present invention, it is based on the fluorescence bright spot pattern of the fluorescence image acquired for each cell to be analyzed and the reference pattern corresponding to the measurement item of the sample 10 from the reference patterns for each measurement item. The information used for the determination of the sample 10 is generated. Therefore, it is not necessary for the operator or the like to memorize various types of bright spot patterns indicating abnormal cells having chromosomal abnormalities and determine whether each cell is an abnormal cell, and the determination of the abnormal cell does not depend on the operator's sense. .. Therefore, the accuracy of determining abnormal cells can be improved, and as a result, the accuracy of information used for determining the sample 10 can be improved.

Although one embodiment of the present invention has been described above, the present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.

For example, in the fluorescence image analyzer 1 of the present embodiment described above, the measuring device 100 shown in FIG. 1 may be replaced with the measuring device 400 including the fluorescence microscope shown in FIG.

The measuring device 400 shown in FIG. 14 includes a light source 410 to 412, a mirror 420, a dichroic mirror 421 to 422, a shutter 430, a 1/4 wavelength plate 431, a beam expander 432, a condenser lens 433, and the like. It includes a dichroic mirror 434, an objective lens 435, a stage 440, a condenser lens 450, an imaging unit 451 and a controller 460-461. A slide glass 441 is installed on the stage 440. Sample 10 (shown in FIG. 1) prepared by pretreatment by the pretreatment apparatus 300 is placed on the slide glass 441.

The light sources 410 to 412 are the same as the light sources 120 to 122 shown in FIG. 1, respectively. The mirror 420 reflects the light from the light source 410. The dichroic mirror 421 transmits the light from the light source 410 and reflects the light from the light source 411. The dichroic mirror 422 transmits the light from the light sources 410 to 411 and reflects the light from the light source 412. The optical axes of the light from the light sources 410 to 412 are aligned with each other by the mirror 420 and the dichroic mirrors 421 to 422.

The shutter 430 is driven by the controller 460 and switches between a state in which the light emitted from the light sources 410 to 412 is passed through and a state in which the light emitted from the light sources 410 to 412 is blocked. As a result, the irradiation time of light on the sample 10 is adjusted. The quarter wave plate 431 converts the linearly polarized light emitted from the light sources 410 to 412 into circularly polarized light. The fluorescent dye bound to the probe reacts to light in a predetermined polarization direction. Therefore, by converting the excitation light emitted from the light sources 410 to 412 into circularly polarized light, the polarization direction of the excitation light can easily match the polarization direction in which the fluorescent dye reacts. This makes it possible to efficiently excite the fluorescence of the fluorescent dye. The beam expander 432 expands the irradiation area of light on the slide glass 441. The condensing lens 433 condenses the light so that the slide glass 441 is irradiated with the parallel light from the objective lens 435.

The dichroic mirror 434 reflects the light emitted from the light sources 410 to 412 and transmits the fluorescence generated from the sample 10. The objective lens 435 guides the light reflected by the dichroic mirror 434 to the slide glass 441. The stage 440 is driven by the controller 461. The fluorescence generated from the sample 10 passes through the objective lens 435 and passes through the dichroic mirror 434. The condenser lens 450 collects the fluorescence transmitted through the dichroic mirror 434 and guides it to the image pickup surface 452 of the image pickup unit 451. The imaging unit 451 captures an image of fluorescence irradiated on the imaging surface 452 and generates a fluorescence image. The image pickup unit 451 is composed of, for example, a CCD or the like.

The controllers 460 to 461 and the imaging unit 451 are connected to the processing unit 11 shown in FIG. 1, and the processing unit 11 controls the controllers 460 to 461 and the imaging unit 451 to obtain fluorescence imaged by the imaging unit 451. Receive the image. The fluorescent image captured by the imaging unit 451 may have cells in close contact with each other as shown in FIG. 2A, unlike the case where the flow cell 110 is used as shown in FIG. .. Therefore, the processing unit 11 performs a process of dividing the acquired fluorescence image for each cell nucleus, a process of setting a region corresponding to the nucleus of one cell in the fluorescence image, and the like.

Similarly to the present embodiment, the measuring device 400 shown in FIG. 14 can also acquire three fluorescent images (first image to third image), so that a bright spot pattern is extracted for each cell based on each fluorescent image. By comparing each cell with the reference pattern based on the extracted bright spot pattern, it is possible to determine whether each cell is an abnormal cell or a normal cell.

Further, as in the embodiment shown in FIG. 15, in the fluorescence image analyzer 1, the processing unit 11 may be connected to the preprocessing device 300 via the interface 16 so that data can be input and output. In this embodiment, the processing unit 11 can receive information on the measurement item and the reagent including the probe as the information from the pretreatment device 300. As a result, when the processing unit 11 selects the reference pattern corresponding to the measurement item of the sample 10 from the plurality of reference patterns corresponding to the plurality of measurement items stored in the storage unit 12 in S30 of FIG. The reference pattern corresponding to the measurement items transmitted from the pretreatment apparatus 300 and the information about the reagent including the probe is automatically read, and in S31, the processing unit 11 selects the selected reference pattern and the bright spot pattern acquired for each cell. May be compared with.

Further, in the fluorescence image analyzer 1 of the present embodiment described above, the reference pattern is stored in advance in the storage unit 12 in the fluorescence image analyzer 1, but is acquired from an external server (not shown) via a network. You may.

Further, in the fluorescence image analyzer 1 of the present embodiment described above, the processing unit 11 stores the fluorescence bright spot pattern in the newly acquired fluorescence image of the abnormal cell as a reference pattern in the storage unit 12 for each measurement item. You may. The new reference pattern may be acquired by the user inputting via the input unit 14, or may be acquired by the processing unit 11 from an external server (not shown) via the network.

It is also possible to provide a storage medium in which a computer program that defines a processing procedure for processing a fluorescent image of cells by the processing unit 11 of the processing device 200 described above is stored.

1 Fluorescence image analyzer 10 Sample 11 Processing unit 12 Storage unit 13 Display unit 14 Input unit 100 Measuring device 120 to 123 Light source 160 Imaging unit 200 Processing device 300 Preprocessing device 400 Measuring device

Claims (12)

A fluorescence image analyzer that measures and analyzes a sample prepared by performing pretreatment including a step of labeling a target site in a cell with a fluorescent dye using a labeling reagent.
A light source that irradiates the sample with light,
An imaging unit that captures the fluorescence emitted from the sample by being irradiated with the light, and an imaging unit.
A processing unit that processes the fluorescence image captured by the imaging unit, and
A display unit that displays the fluorescent image and
With
The processing unit
In the fluorescence image, a bright spot pattern determined based on the color and number of fluorescence generated from the fluorescent dye that labels the target site is acquired.
Based on the bright spot pattern and each of the plurality of positive patterns set corresponding to the measurement items, at least one piece of information on the number of cells determined to be abnormal cells and the ratio of the number of cells in the sample is provided. Generate and
A fluorescence image analyzer that displays the fluorescence image and at least one piece of information on the number of cells and the ratio of the number of cells on the display unit. Said processing unit, said at least one information of the number and ratio of the number of cells in the cell where it is determined that the abnormal cells in the sample based on the selected positive pattern from the plurality of positive patterns displayed by the input unit The fluorescent image analyzer according to claim 1, which is displayed on the unit. Wherein the processing unit displays a fluorescence image of the cells where it is determined that the abnormal cells in the sample based on the positive pattern selected from the plurality of positive pattern by the input unit on the display unit, to claim 1 or 2 The fluorescent image analyzer according to the description. When the determination is corrected by the input unit from the fluorescence images of the abnormal cells displayed on the display unit, the processing unit determines the number of abnormal cells and the ratio of the number of abnormal cells in the modified sample. The fluorescence image analyzer according to claim 3, wherein at least one of the above information is displayed on the display unit. The processing unit, wherein when it is determined by the input unit from the fluorescence image of the abnormal cells is displayed on the display unit is corrected, displays a fluorescence image of the abnormal cells in said sample after modification on the display unit The fluorescence image analyzer according to claim 3 or 4. A storage unit that stores multiple positive patterns for each measurement item is further provided for multiple measurement items.
The fluorescence image analyzer according to claim 1, wherein the processing unit selects a positive pattern corresponding to the measurement item from the positive patterns for each measurement item stored in the storage unit. It is an analysis method for analyzing a fluorescent image obtained by imaging a sample prepared by performing pretreatment including a step of labeling a target site in a cell with a fluorescent dye using a labeling reagent.
Accepting the measurement items of the sample, reading a plurality of positive patterns set corresponding to the measurement items,
In the fluorescence image, a bright spot pattern determined based on the color and number of fluorescence generated from the fluorescent dye that labels the target site is acquired.
Based on the bright spot pattern and each of the plurality of positive patterns set corresponding to the measurement items, at least one piece of information on the number of cells determined to be abnormal cells and the ratio of the number of cells in the sample is provided. Generate and
A method for analyzing a fluorescent image, which displays the fluorescent image and at least one piece of information on the number of cells and the ratio of the number of cells. The fluorescence according to claim 7, wherein at least one information of the number of cells and the ratio of the number of cells in the sample determined to be abnormal cells based on the positive pattern selected from the plurality of positive patterns is displayed. Image analysis method. The method for analyzing a fluorescent image according to claim 7 or 8, wherein a fluorescent image of cells in the sample determined to be abnormal cells based on a positive pattern selected from the plurality of positive patterns is displayed. The ninth aspect of claim 9, wherein when the determination is modified from the fluorescent image of the abnormal cells, at least one information of the number of abnormal cells and the ratio of the number of abnormal cells in the modified sample is displayed. Fluorescent image analysis method. The method for analyzing a fluorescent image according to claim 9 or 10, wherein when the determination is corrected from the fluorescent image of the abnormal cell, the corrected fluorescent image of the abnormal cell in the sample is displayed. The method for analyzing a fluorescent image according to claim 7, wherein a positive pattern corresponding to the received measurement item of the sample is read from a storage unit of a computer that stores a plurality of positive patterns for each measurement item.

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