JP2016128821A - Cell analyzer, and cell analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for improving analytic accuracy of an epithelial cell of a measuring object.SOLUTION: A cell analysis method includes: detecting fluorescence and scattered light from each cell contained in a measurement sample containing epithelial cells; outputting a fluorescence signal and a scattered light signal; discriminating between an epithelial cell and a leukocyte, based on a waveform width of the outputted scattered light signal and a waveform area of the outputted fluorescence signal; and sorting an abnormal cell based on a fluorescence signal acquired from the discriminated epithelial cell.SELECTED DRAWING: Figure 10

Description

  The present invention relates to a cell analysis method and a cell analysis apparatus that analyze cells and provide information related to canceration of cells.

  An analyzer that automatically analyzes a subject's cells and provides information related to canceration of the cells is known (see, for example, Patent Document 1). In Patent Document 1, a measurement sample including cells collected from a subject is flowed through a flow cell, and the measurement sample flowing through the flow cell is irradiated with light to obtain a scattered light signal for each cell. A device is disclosed that extracts characteristic parameters by analyzing the waveform of, and discriminates cancer / atypical cells from a plurality of cells using the characteristic parameters.

International Publication No. 2006/103920

White blood cells may be contained in the measurement sample, and it is desired to discriminate white blood cells from epithelial cells to increase the analysis accuracy of the epithelial cells to be measured.

In the first aspect of the present invention, fluorescence and scattered light are detected from each cell included in a measurement sample including epithelial cells, a fluorescence signal and a scattered light signal are output, and the width of the waveform of the output scattered light signal And a method for analyzing cells characterized in that epithelial cells and leukocytes are discriminated based on the area of the waveform of the outputted fluorescent signal, and abnormal cells are classified based on the fluorescent signal obtained from the discriminated epithelial cells. It is.

The second aspect of the present invention is a detector that irradiates a measurement sample including epithelial cells with light, detects fluorescence and scattered light from each cell included in the measurement sample, and outputs a fluorescence signal and a scattered light signal. And discriminating epithelial cells and leukocytes based on the width of the waveform of the scattered light signal output from the detection unit and the area of the waveform of the output fluorescence signal, and the fluorescence signal obtained from the differentiated epithelial cell And a control unit that classifies abnormal cells based on the cell analyzer.

As described above, according to the present invention, leukocytes and epithelial cells can be discriminated and the analysis accuracy of epithelial cells to be measured can be improved.

  The effects and significance of the present invention will become more apparent from the following description of embodiments. However, the embodiment described below is merely an example when the present invention is implemented, and the present invention is not limited to the following embodiment.

It is a perspective view which shows typically the structure of the external appearance of the canceration information provision apparatus which concerns on embodiment. It is a top view which shows typically the internal structure of the measuring apparatus which concerns on embodiment. It is a figure which shows the structure of the flow cytometer which concerns on embodiment. It is a figure which shows the structure of the measuring apparatus which concerns on embodiment. It is a figure which shows the structure of the data processor which concerns on embodiment. It is a flowchart which shows the analysis operation | movement of the canceration information provision apparatus which concerns on embodiment. The figure explaining the forward scattered light signal and the side fluorescence signal which concern on embodiment, the figure which shows the expanded cross section of the epithelial cell of a cervix, and the figure which shows the relationship between N / C ratio and a cell size It is. It is a figure which shows the relationship between the amount of DNA in the cell cycle which concerns on embodiment, and the number of cells, and the figure which shows the amount of DNA which changes for every cell cycle. It is a flowchart which shows the analysis process in the data processor which concerns on embodiment. It is a figure which shows the scattergram and histogram which are produced | generated by the analysis process which concerns on embodiment. It is a figure which shows the dialog displayed on the display part which concerns on embodiment. It is a figure which shows the scattergram of the test substance from which the progression degree of the cancer which concerns on embodiment differs. It is the histogram which extracted and created the cell group contained in the area | region with a high N / C ratio in the specimen from which the progression degree of the cancer which concerns on embodiment differs. It is the histogram which extracted and produced the cell group contained in the area | region with a low N / C ratio in the specimen from which the progression degree of the cancer which concerns on embodiment differs. It is a figure explaining the relationship between the determination result of the tissue examination which concerns on embodiment, and the determination result by the determination 1. FIG. It is a figure explaining the relationship between the determination result of the tissue examination which concerns on embodiment, and the determination result by the determination 2, and the figure explaining the relationship between the determination result of a tissue diagnosis, and the final determination result. It is a flowchart which shows the example of a change of the analysis process in the data processor which concerns on embodiment. It is a figure which shows the example of a change of the area | region set to the scattergram and histogram which concern on embodiment.

  Hereinafter, the canceration information providing apparatus 1 according to the present embodiment will be described with reference to the drawings.

  The canceration information providing apparatus 1 causes a measurement sample including cells (biological sample) collected from a patient (subject) to flow through the flow cell, and irradiates the measurement sample flowing through the flow cell with laser light. Then, by detecting light (forward scattered light, side scattered light, side fluorescence) from the measurement sample and analyzing the optical signal, the cells are transformed into cells that are cancerous or in the process (hereinafter these are summarized). Whether or not it is also referred to as “cancerous cells”). Specifically, when screening cervical cancer using cervical epithelial cells collected from a patient, the canceration information providing apparatus 1 is used.

  FIG. 1 is a perspective view schematically showing an external configuration of the canceration information providing apparatus 1.

  The canceration information providing apparatus 1 includes a measurement apparatus 2 that performs measurement of a biological sample collected from a patient, and a data processing apparatus 3 that is connected to the measurement apparatus 2 and that analyzes and displays (outputs) measurement results. I have. Specimen setting unit for setting a plurality of biological containers 4 (see FIG. 2) containing a liquid mixture (sample) of a preservative solution mainly composed of methanol and a biological sample collected from a patient on the front surface of the measuring device 2 2a is installed. The data processing device 3 includes an input unit 31 and a display unit 32.

  FIG. 2 is a plan view schematically showing the internal configuration of the measuring apparatus 2.

  The sample setting unit 2a sequentially transports the rack 4a in which a plurality of biological containers 4 are set to the sample suction position by the sample pipette unit 11.

  The sample pipette unit 11 transfers the sample in the biological container 4 to the first dispersion unit 12. The sample pipette unit 11 also transfers the sample in the first dispersion unit 12 to the sub-detection unit 13 and the discrimination / substitution unit 14. Further, the sample pipette unit 11 supplies the concentrated liquid concentrated in the discrimination / substitution unit 14 to the measurement sample container 5. The sample pipette unit 11 includes a sample storage unit 12a of the first dispersion unit 12, a sample take-in unit 13a of the sub-detection unit 13, a discrimination / substitution unit 14, and a measurement sample container 5 positioned in the sample delivery unit 11b. It is configured to be movable to an upper position.

  The sample pipette unit 11 has a pipette 11a for aspirating and discharging the sample, and the sample is quantified by a sample quantification unit (quantitative cylinder, motor for driving a piston in the quantification cylinder, etc.) (not shown). It is comprised so that a sample can be supplied to each part mentioned above.

  The first dispersion unit 12 performs a first dispersion process for dispersing the aggregated cells included in the sample on the sample. Specifically, the first dispersion treatment is a shearing force imparting treatment that imparts a shearing force to the aggregated cells and disperses them. The first dispersion unit 12 includes a sample storage unit 12a that can store a sample, and is configured to mechanically apply a shearing force to the aggregated cells in the sample supplied to the sample storage unit 12a.

  The sub-detection unit 13 measures the concentration of the sample before the main measurement by the main detection unit 22. The sub-detecting unit 13 employs a flow cytometer 40 (see FIG. 3A) having substantially the same configuration as a main detecting unit 22 described later.

  The discrimination / substitution unit 14 receives the sample after the first dispersion treatment by the first dispersion unit 12, and replaces the preserving solution mainly containing methanol contained in the sample with a diluent. Further, the discrimination / substitution unit 14 discriminates measurement target cells (epithelial cells of the cervix, glandular cells) contained in the sample from other cells (red blood cells, white blood cells, bacteria, etc.) and contaminants. In addition, the discrimination / substitution unit 14 concentrates the measurement target cells contained in the sample in order to obtain the number of cell measurements necessary for the measurement by the main detection unit 22. Two discrimination / replacement units 14 are provided in order to improve processing efficiency.

  The container transfer unit 15 holds the measurement sample container 5 set in the reaction unit 18 with a scissor-shaped holding unit 15a, and performs a sample delivery unit 11b, a second dispersion unit 16, a liquid removal unit 17, and a reaction. Transfer to section 18. The container transfer part 15 is configured to move the grip part 15a along a predetermined circumferential trajectory. Moreover, the container transfer part 15 is comprised so that the holding part 15a can be moved to an up-down direction. In addition, the sample delivery part 11b, the 2nd dispersion | distribution part 16, the liquid removal part 17, and the reaction part 18 are arrange | positioned on this circumferential locus. As a result, the measurement sample container 5 set in the reaction unit 18 can be held by the holding unit 15a of the container transfer unit 15 and transferred to each part on the circumferential trajectory.

  The second dispersion unit 16 performs a second dispersion process different from the first dispersion process on the sample on which the first dispersion process by the first dispersion unit 12 has been performed. Specifically, the second dispersion unit 16 performs the first dispersion process by the first dispersion unit 12 and ultrasonically applies the sample concentrated in the discrimination / substitution unit 14 (the concentration of the measurement target cell is increased). It is comprised so that a vibration may be provided. The aggregated cells remaining after the first dispersion process are dispersed into single cells by the second dispersion unit 16.

  The liquid removing unit 17 removes (drains) the liquid adhering to the outer surface of the measurement sample container 5 after the second dispersion process by the second dispersing unit 16. The second dispersion process is performed in a state where the measurement sample container 5 is immersed in a liquid. The liquid removing unit 17 is configured to remove water droplets attached to the outer surface of the measurement sample container 5 by supplying an air flow to the outer surface of the measurement sample container 5. This prevents the liquid from adhering to each part when the measurement sample container 5 is set in each part such as the reaction part 18.

  The reaction unit 18 promotes the reaction between the sample in the measurement sample container 5 and the reagent added by the first reagent addition unit 19 and the second reagent addition unit 20. The reaction unit 18 includes a circular turntable 18a configured to be rotatable by a drive unit (not shown). A plurality of holding portions 18b on which the measurement sample container 5 can be set are provided on the outer peripheral edge of the rotary table 18a. The measurement sample container 5 is set in the holding portion 18b. Further, the trajectory of the holding unit 18b due to the rotation of the rotary table 18a and the circumferential trajectory of the holding unit 15a of the container transfer unit 15 intersect at a predetermined position, and the container transfer unit 15 at this intersecting position is the measurement sample container. 5 can be set in the holding portion 18b. In addition, the reaction unit 18 warms the measurement sample container 5 set in the holding unit 18b to a predetermined temperature (about 37 degrees) to promote the reaction between the sample and the reagent.

  The 1st reagent addition part 19 and the 2nd reagent addition part 20 supply a reagent in the measurement sample container 5 set to the holding | maintenance part 18b. The first reagent addition unit 19 and the second reagent addition unit 20 are installed at positions near the peripheral edge of the turntable 18a, and move to positions P1 and P2 above the measurement sample container 5 set on the turntable 18a, respectively. It has possible supply parts 19a, 20a. Thus, when the measurement sample container 5 is transported to the positions P1 and P2 by the rotary table 18a, a predetermined amount of reagent can be added from the supply units 19a and 20a into the measurement sample container 5.

  The reagent added by the first reagent adding unit 19 is an RNase for performing RNA removal processing on cells, and the reagent added by the second reagent adding unit 20 is a staining solution for performing DNA staining processing on cells. It is. By removing the RNA, the RNA in the cell is degraded, and only the DNA of the cell nucleus can be measured. The DNA staining treatment is performed with propidium iodide (PI), which is a fluorescent staining solution containing a dye, and intracellular nuclei are selectively stained by the DNA staining treatment. Thereby, fluorescence from the nucleus can be detected.

  The sample suction unit 21 sucks the sample (measurement sample) in the measurement sample container 5 set in the holding unit 18 b and transfers the sucked measurement sample to the main detection unit 22. The sample aspirating unit 21 has a pipette 21a that is installed at a position in the vicinity of the peripheral edge of the rotary table 18a and is movable to a position P3 above the measurement sample container 5 set on the rotary table 18a. Thereby, when the measurement sample container 5 is conveyed to the position P3 by the rotary table 18a, the measurement sample in the measurement sample container 5 can be sucked. The sample suction unit 21 is connected to a flow cell 43 (see FIG. 3A) of the main detection unit 22 via a flow path (not shown), and the measurement sample sucked by the pipette 21a is used as the main detection unit 22. The flow cell 43 can be supplied.

  The main detection unit 22 includes a flow cytometer 40 for detecting light (forward scattered light, side scattered light, side fluorescence) from the measurement sample, and outputs a signal based on each light to a subsequent circuit. To do. The flow cytometer 40 will be described later with reference to FIGS. 3 (a) and 3 (b).

  The container cleaning unit 23 cleans the inside of the measurement sample container 5 after the measurement sample is supplied to the main detection unit 22 by the sample suction unit 21. The container cleaning unit 23 is configured to clean the inside of the measurement sample container 5 by discharging the cleaning liquid into the measurement sample container 5 held by the holding unit 18b of the rotary table 18a. Thereby, when the same measurement sample container 5 is used in the subsequent measurement process, contamination with other samples can be suppressed.

  FIG. 3A is a diagram illustrating a configuration of the flow cytometer 40 of the main detection unit 22.

  Laser light emitted from the semiconductor laser 41 is focused on a measurement sample flowing through the flow cell 43 by a lens system 42 including a plurality of lenses. As described above, the sample sucked by the pipette 21a of the sample suction unit 21 is supplied to the flow cell 43.

  As shown in FIG. 3B, the lens system 42 includes a collimator lens 42a and a cylinder lens system (plano-convex cylinder lens 42b + both in order from the semiconductor laser 41 side (left side of FIGS. 3A and 3B)). Concave cylinder lens 42c) and a condenser lens system (condenser lens 42d + condenser lens 42e).

  The condensing lens 44 condenses the forward scattered light of the cells in the measurement sample on the scattered light detector composed of the photodiode 45. The side condensing lens 46 condenses the side scattered light and the side fluorescence of the measurement target cell and the nucleus in the cell and guides it to the dichroic mirror 47. The dichroic mirror 47 reflects side scattered light to a photomultiplier (photomultiplier tube) 48 and transmits side fluorescence to a photomultiplier (photomultiplier tube) 49. Thus, the side scattered light is collected on the photomultiplier 48 and the side fluorescence is collected on the photomultiplier 49. These lights reflect the characteristics of cells and nuclei in the measurement sample.

  The photodiode 45 and the photomultipliers 48 and 49 convert the received optical signal into an electrical signal, respectively, and forward scattered light signal (FSC), side scattered light signal (SSC), and side fluorescent signal ( SFL) is output. These output signals are amplified by a preamplifier (not shown) and output to the signal processing unit 24 (see FIG. 4) of the measuring apparatus 2.

  FIG. 4 is a diagram showing the configuration of the measuring device 2.

  The measuring apparatus 2 includes the main detection unit 22 and the sub-detection unit 13 shown in FIG. 2 and the preparation device unit 29 including each unit for automatically performing component adjustment on the sample as described above. The measuring apparatus 2 includes a signal processing unit 24, a measurement control unit 25, an I / O interface 26, a signal processing unit 27, and a preparation control unit 28.

  As described above, the main detection unit 22 includes the flow cytometer 40 illustrated in FIG. 3A, and from the measurement sample, a forward scattered light signal (FSC), a side scattered light signal (SSC), A side fluorescent signal (SFL) is output. The signal processing unit 24 includes a signal processing circuit that performs necessary signal processing on the output signal from the main detection unit 22, processes the signals FSC, SSC, and SFL output from the main detection unit 22, and performs measurement control. To the unit 25.

  The measurement control unit 25 includes a microprocessor 251 and a storage unit 252. The microprocessor 251 is connected to the data processing device 3 and the microprocessor 281 of the preparation control unit 28 via the I / O interface 26. As a result, the microprocessor 251 can transmit and receive various data between the data processing device 3 and the microprocessor 281 of the preparation control unit 28. The storage unit 252 includes a ROM for storing a control program such as the main detection unit 22 and data, a RAM, and the like.

  Each signal FSC, SSC, SFL processed by the signal processing unit 24 of the measuring device 2 is transmitted from the I / O interface 26 to the data processing device 3 by the microprocessor 251.

  Since the sub-detecting unit 13 employs the flow cytometer 40 having substantially the same configuration as that of the main detecting unit 22, the description of the configuration is omitted. Since the sub-detection unit 13 measures the concentration of the sample before the main measurement by the main detection unit 22, it is sufficient that the sub-detection unit 13 can output a signal for counting the number of cells. That is, it is sufficient for the sub-detecting unit 13 to acquire a forward scattered light signal (FSC). The signal processing unit 27 includes a signal processing circuit that performs necessary signal processing on the output signal from the sub-detection unit 13, processes the forward scattered signal FSC output from the sub-detection unit 13, and sends the signal to the preparation control unit 28. Output.

  The preparation control unit 28 includes a microprocessor 281, a storage unit 282, a sensor driver 283, and a drive unit driver 284. The microprocessor 281 is connected to the microprocessor 251 of the measurement control unit 25 via the I / O interface 26. Thereby, the microprocessor 281 can transmit and receive various data to and from the microprocessor 251 of the measurement control unit 25.

  The storage unit 282 includes a ROM that stores a control program for controlling the sub-detection unit 13, the preparation device unit 29, and the like, and a RAM. The adjustment device unit 29 includes the sample setting unit 2a, the sample pipette unit 11, the first dispersion unit 12, the discrimination / substitution unit 14, the container transfer unit 15, the second dispersion unit 16, and the like shown in FIG. The liquid removal unit 17, the reaction unit 18, the first reagent addition unit 19, the second reagent addition unit 20, the sample suction unit 21, and the container cleaning unit 23 are included.

  Further, the microprocessor 281 is connected to the sensors and the drive motor of each part of the preparation device unit 29 via the sensor driver 283 or the drive unit driver 284. Thereby, the microprocessor 281 executes the control program based on the detection signal from the sensor and controls the operation of the drive motor.

  FIG. 5 is a diagram illustrating a configuration of the data processing device 3.

  The data processing device 3 includes a personal computer, and includes a main body 30, an input unit 31, and a display unit 32. The main body 30 includes a CPU 301, a ROM 302, a RAM 303, a hard disk 304, a reading device 305, an input / output interface 306, an image output interface 307, and a communication interface 308.

  The CPU 301 executes a computer program stored in the ROM 302 and a computer program loaded in the RAM 303. The RAM 303 is used for reading out computer programs recorded in the ROM 302 and the hard disk 304. The RAM 303 is also used as a work area for the CPU 301 when executing these computer programs.

  The hard disk 304 is installed with various computer programs to be executed by the CPU 301 such as an operating system and application programs, and data used for executing the computer programs. Specifically, the hard disk 304 is installed with a program that analyzes the measurement result transmitted from the measurement device 2 and displays the result on the display unit 32 based on the generated analysis result.

  The CPU 301 executes a program installed in the hard disk 304 to acquire characteristic parameters such as forward scattered light intensity and side fluorescent intensity based on the signals FSC, SSC, and SFL, and based on these characteristic parameters. To create frequency distribution data for cell and nucleus analysis. Then, the CPU 301 discriminates the particles in the measurement sample based on the frequency distribution data, and determines whether or not the measurement target cell (epithelial cell) is abnormal, specifically a cancerous cell (atypical cell). ). Such determination by the CPU 301 will be described later with reference to FIG.

  The reading device 305 is configured by a CD drive, a DVD drive, or the like, and can read a computer program and data recorded on a recording medium. An input unit 31 such as a keyboard is connected to the input / output interface 306, and instructions and data are input to the data processing device 3 when the operator uses the input unit 31. The image output interface 307 is connected to the display unit 32 configured by a display or the like, and outputs a video signal corresponding to the image data to the display unit 32.

  The display unit 32 displays an image based on the input video signal. Various program screens are displayed on the display unit 32. In addition, the communication interface 308 enables data transmission / reception with respect to the measurement apparatus 2.

  FIG. 6 is a flowchart showing the analysis operation of the canceration information providing apparatus 1.

  Operation control of the main detection unit 22 and the signal processing unit 24 of the measurement apparatus 2 is executed by the microprocessor 251 of the measurement control unit 25. Further, the operation control of the sub-detection unit 13, the signal processing unit 27, and the preparation device unit 29 of the measurement apparatus 2 is executed by the microprocessor 281 of the preparation control unit 28. Further, the control of the data processing device 3 is executed by the CPU 301.

  Upon analysis by the canceration information providing apparatus 1, a biological container 4 containing a biological sample and a preservation solution containing methanol as a main component is set in the specimen setting unit 2a (see FIG. 2) by the operator, and canceration information is obtained. Analysis by the providing device 1 is started.

  When the measurement is started, the first dispersion unit 12 performs a dispersion process (first dispersion process) on the aggregated cells in the sample (S11). Specifically, the sample in the biological container 4 set in the sample setting unit 2a is aspirated by the sample pipette unit 11 and supplied into the sample storage unit 12a. The sample supplied to the sample storage unit 12a is then dispersed by the first dispersion unit 12.

  When the first dispersion process is completed, the sample pipette unit 11 supplies the dispersed sample to the sample take-in unit 13a of the sub-detection unit 13, and the flow cell of the sub-detection unit 13 is the same as the flow cell 43 of FIG. Then, a predetermined amount of the dispersed sample is flowed. In the sub-detecting unit 13, the number of cells contained in the sample is detected (pre-measurement) by the flow cytometry method (S12). The concentration of this sample is calculated from the number of cells obtained by the pre-measurement and the volume of the sample supplied to the sub-detecting unit 13.

  Subsequently, based on the calculated concentration, the microprocessor 281 determines the suction amount of the sample for preparing the measurement sample used for the main measurement (S13). That is, based on the sample concentration (number of cells per unit volume) used for the pre-measurement and the number of significant cells necessary for cancer cell detection in this measurement, The liquid amount of the sample necessary for performing the measurement is calculated. In the present embodiment, for example, it is assumed that the number of single epithelial cells (number of significant cells) supplied to the flow cell 43 of the main detection unit 22 is about 20,000. In this case, the sample supplied to the discrimination / replacement unit 14 needs to include about 100,000 cells. Therefore, the sample in S13 is set so that about 100,000 cells are supplied to the discrimination / replacement unit 14. The amount of liquid is calculated.

  In addition, the number of cells obtained by the pre-measurement includes a single cell and an aggregated cell of measurement target cells (epithelial cells), and also includes leukocytes and the like in addition to the measurement target cells. For this reason, the number of cells obtained does not accurately indicate the number of cells to be measured. However, based on the number of cells obtained by the pre-measurement, it becomes possible to secure the number of significant cells required for the main measurement to some extent.

  Next, discrimination / substitution processing is executed for the sample of the calculated liquid amount (S14). That is, the sample pipette unit 11 is driven by the preparation control unit 28, and the sample after the first dispersion process is aspirated from the sample storage unit 12a of the first dispersion unit 12 by the calculated liquid amount. The aspirated sample is supplied to the discrimination / substitution unit 14 to start discrimination / substitution processing.

  Next, a dispersion process (second dispersion process) of aggregated cells in the sample is performed by the second dispersion unit 16 (S15). Specifically, the container transfer unit 15 grasps and takes out the measurement sample container 5 set in the holding unit 18b of the reaction unit 18, and positions it on the sample delivery unit 11b. Then, the sample aspirated by the sample pipette unit 11 from the discrimination / substitution unit 14 is supplied to the measurement sample container 5 positioned in the sample delivery unit 11b. Thereafter, the measurement sample container 5 is transferred to the second dispersion unit 16 by the container transfer unit 15 and the second dispersion process is executed.

  When the measurement sample container 5 containing the sample after the second dispersion treatment is set in the holding unit 18b of the reaction unit 18, a reagent (RNase) is added by the first reagent addition unit 19 and heated by the reaction unit 18, The RNA removal process of the measurement object cell in the measurement sample container 5 is performed (S16). After the RNA removal process, a reagent (staining solution) is added by the second reagent adding unit 20, heated by the reaction unit 18, and the DNA staining process of the measurement target cells in the measurement sample container 5 is performed (S17).

  Next, the measurement sample that has been subjected to the DNA staining process is sucked by the sample suction unit 21. The sucked measurement sample is sent to the flow cell 43 (see FIG. 3A) of the main detection unit 22, and the main measurement is performed on the cells in the measurement sample (S18).

  After the actual measurement, the obtained measurement data is transmitted from the measurement control unit 25 of the measurement device 2 to the data processing device 3 (S19). Specifically, the forward scattered light signal (FSC), the side scattered light signal (SSC), and the side fluorescent signal (SFL) obtained for each cell in the measurement sample are transmitted to the data processing device 3. The The CPU 301 of the data processing device 3 always determines whether or not measurement data has been received from the measurement device 2. When the measurement data is received from the measurement device 2, the CPU 301 of the data processing device 3 performs an analysis process based on the measurement data (S20). Details of the analysis processing in S20 will be described later with reference to FIG.

  Next, a procedure for acquiring canceration information in the present embodiment will be described.

  Fig.7 (a) is a figure explaining the forward scattered light signal (FSC) and side fluorescence signal (SFL) which are obtained in this measurement (S18 of FIG. 6).

  FIG. 7A shows a schematic diagram of a cell including a cell nucleus, a waveform of a forward scattered light signal obtained from the cell, and a waveform of a side fluorescence signal. The vertical axis represents the light intensity. The waveform width of the forward scattered light intensity represents a numerical value indicating the cell width (cell size C), and the waveform width of the lateral fluorescence intensity is a numerical value indicating the cell nucleus width (cell nucleus size N). ). Further, as indicated by the oblique lines, the area of the region determined by the waveform of the side fluorescence intensity and the predetermined baseline represents the amount of DNA in the cell.

  FIG. 7B is a diagram schematically showing an enlarged cross section of the epithelial cells of the cervix.

  In the cervix, in order from the basement membrane side, a layer formed by basal cells (basal layer), a layer formed by parabasal cells (parabasal layer), and a layer formed by middle layer cells (middle layer) And a layer (surface layer) formed by surface cells is formed. The basal cells near the basement membrane are differentiated into parabasal cells, parabasal cells are differentiated into intermediate cells, and intermediate cells are differentiated into surface cells.

  Among these epithelial cells, cells related to canceration are basal cells in cervical epithelial cells. In the process leading to cancer, basal cells acquire dysplasia and become atypical cells. Atypical cells acquire proliferative ability and occupy from the basal layer side to the surface layer side. For this reason, in the initial stage leading to cancer, in the epithelial cells of the cervix, many cancerous cells are present in cells in the basal layer, the parabasal layer, and the middle layer. Conversely, at the initial stage leading to cancer, the cells existing on the surface layer of the epithelial cells of the cervix have very few cancerous cells.

  In epithelial cells, it is known that the size of the cell nucleus gradually increases as the size of the cell gradually decreases from the surface layer toward the basement membrane layer. Therefore, the ratio of the cell nucleus size (N) to the cell size (C) (hereinafter referred to as “N / C ratio”) also increases sequentially from the surface layer side toward the basement membrane side layer. For this reason, the N / C ratio and the cell size C have a relationship as shown in FIG. Therefore, parabasal cells and basal cells can be extracted by extracting cells having a large N / C ratio.

  FIG. 8A shows the relationship between the amount of DNA and the number of cells in the cell cycle.

  According to a certain cycle (cell cycle), the cell returns to the starting point through two events such as DNA replication, chromosome distribution, nuclear division, and cytokinesis. Depending on the stage of the cell cycle, the G1 phase (time for preparation and inspection to enter S phase), S phase (DNA synthesis phase), and G2 phase (time to prepare and check for M phase) ) And M phase (mitotic phase), and when G0 phase (resting phase) in which cell growth is halted is added to this 4 phase, cells are On stage.

  When a cell grows according to the cell cycle, the chromosome of the nucleus in the cell also increases. Therefore, by measuring the amount of DNA in the cell, it can be estimated in which state the cell is in the cell cycle. In the case of normal cells, as shown in FIG. 8 (b), the DNA amount in the G0 / G1 phase is a constant value (2C), the DNA amount gradually increases in the subsequent S phase, and then in the G2 phase. When it enters, it becomes a constant value (4C), and this value is maintained even in the M period. Here, C represents the genomic DNA content per haploid. That is, 2C represents a DNA amount that is twice the genomic DNA content per haploid, and 4C represents a DNA amount that is four times the genomic DNA content per haploid. The DNA amount of normal cells in the G0 or G1 phase of the cell cycle is 2C. When a histogram of DNA amount is created for normal cells, a histogram as shown in FIG. 8A is obtained. The peak with the highest peak corresponds to the cell in the G0 / G1 phase with the least amount of DNA, and the peak with the next highest peak corresponds to the cell in the G2 / M phase with the highest amount of DNA. Corresponds to cells in S phase.

  In the case of normal cells, the number of cells in the S phase and G2 / M phase is extremely small compared to the number of cells in the G0 / G1 phase. However, in the case of cancerous cells, the number of cells in the S phase and G2 / M phase is greater than that of normal cells. In addition, in the case of cancerous cells, the number of chromosomes in the cells increases, so the amount of DNA also increases.

  Therefore, in the present embodiment, two determination methods (determinations 1 and 2) focusing on the N / C ratio and the DNA amount are used for determining canceration. In “analysis processing by the data processing device 3” (S20 in FIG. 6), the determination of canceration is performed based on the determinations 1 and 2.

  In the determination 1, basal cells, parabasal cells, and middle layer cells, which are considered to be easily cancerated, are extracted by extracting cells having a large N / C ratio. Subsequently, by extracting cells having a large amount of DNA from the cell group thus extracted, cells having a high possibility of being cancerous cells are effectively extracted (second counting step). In determination 1, when the number of cells obtained by the second counting step is large, it is determined that the possibility of canceration is high.

  In determination 2, by extracting cells having a small N / C ratio, middle layer cells and surface layer cells that are difficult to progress to canceration are extracted. Subsequently, by extracting cells having a small amount of DNA from the cell group thus extracted, cells having a low possibility of being cancerous cells are effectively extracted (first counting step). In general, when the canceration of a tissue progresses, the number of cells obtained by the second counting step increases, and the number of cells obtained by the first counting step decreases. For this reason, the ratio of the number of both cells differs greatly depending on whether the tissue is normal or cancerous. In the determination 2, the tissue canceration is determined based on this ratio. In addition, when the ratio of the two cell numbers having the opposite increase / decrease tendency is used as described above, a highly reliable determination result can be obtained even if the number of measurement target cells included in the measurement sample is relatively small.

  FIG. 9 is a flowchart showing analysis processing in the data processing device 3.

  When the CPU 301 of the data processing device 3 receives the measurement data from the measurement device 2, it creates a scattergram as shown in FIG. In FIG. 10A, the horizontal axis represents the cell size (the width of the forward scattered light signal waveform), and the vertical axis represents the DNA amount (the sum of the side fluorescence signal waveforms).

  Subsequently, the CPU 301 separates leukocytes and epithelial cells (S101). Specifically, the CPU 301 sets a region A1 in which the lower left region corresponding to white blood cells is missing in the scattergram of FIG. 10A, and extracts cells included in the region A1.

  Next, the CPU 301 creates a scattergram shown in FIG. 10B from the cell group extracted in S101. In FIG. 10B, the horizontal axis represents (the sum of the differences of the side fluorescence signals / the peak value of the side fluorescence signals), and the vertical axis (the sum of the differences of the forward scattered light signals / the peak value of the forward scattered light signals). Represents.

  Subsequently, the CPU 301 separates single epithelial cells and aggregated epithelial cells (S102). Specifically, the CPU 301 sets a region A2 corresponding to a single epithelial cell in the scattergram of FIG. 10B, and extracts cells included in this region A2. In addition, the removal of the aggregated cells is performed in order to prevent a decrease in analysis accuracy due to an abnormal value of the measured DNA amount due to aggregation of a plurality of cells even though the amount of DNA is normal as a single cell. .

  Next, the CPU 301 creates a scattergram shown in FIG. 10C from the cell group extracted in S102. In the histogram of FIG. 10C, the horizontal axis represents a value (N / C ratio) obtained by dividing the cell nucleus size N by the cell size C, and the vertical axis represents the cell size. .

  Subsequently, the CPU 301 extracts a cell group of V11 ≦ N / C ratio ≦ V12 in the histogram of FIG. 10C (S103). Specifically, the CPU 301 sets an area A4 in the scattergram of FIG. 10C, and extracts a cell group included in the area A4. It should be noted that the value at the left end and the value at the right end of the area A4 are set to V11 and V12, respectively. V11 is a threshold value for dividing the middle layer cell and the parabasal cell. V11 is appropriately set from the viewpoint of sensitivity and specificity. In the present embodiment, V11 is set in the range of 0.2 to 0.4. V12 is a threshold value that separates basal cells and cells of unknown significance. V12 is appropriately set from the viewpoint of sensitivity and specificity. In the present embodiment, V12 is set in the range of 0.6-1.

  Next, the CPU 301 determines whether or not the number of cells extracted in S103 is greater than or equal to a threshold value S0 (S104). If the number of cells is not sufficient, the canceration determination accuracy may be reduced. Therefore, when the number of cells is less than the threshold value S0 (S104: NO), the CPU 301 displays that this sample is an inappropriate sample (S116). Specifically, as illustrated in FIG. 11A, the CPU 301 displays a dialog D <b> 1 in which “unsuitable sample” is displayed on the display unit 32. In the dialog D1, messages such as “NG” and “analysis impossible” may be displayed. And CPU301 complete | finishes a process, without outputting canceration information. The threshold value S0 is a threshold value for determining whether or not the specimen is unsuitable for collection. The threshold value S0 is appropriately set from the viewpoint of sensitivity and specificity. In the present embodiment, the threshold value S0 is set in the range of 50 to 1000.

  If the number of cells extracted in S103 is equal to or greater than the threshold S0 (S104: YES), the CPU 301 resets the value of flag 1 stored in the hard disk 304 (S105). The flag 1 is for indicating a determination result by the above-described “determination 1”. Subsequently, the CPU 301 extracts a cell group of V11 ≦ N / C ratio ≦ V12 in the scattergram of FIG. 10C as in S103, and creates a histogram (DNA ployy) shown in FIG. S106). In the histogram of FIG. 10 (d), the horizontal axis represents the amount of DNA, and the vertical axis represents the number of cells.

  Next, the CPU 301 has, in the histogram created in S106, the number of cells whose DNA amount is equal to or higher than the S phase of normal cells, that is, the amount of DNA exceeding the DNA amount of normal cells whose cell cycle is in the G0 phase or G1 phase. It is determined whether the number of cells is equal to or greater than a threshold value S1 (S107). Specifically, the CPU 301 sets a region A5 in the histogram shown in FIG. 10D, and determines whether the number of cells included in the region A5 is equal to or greater than the threshold value S1. The leftmost value V21 of the region A5 is set to be the upper limit value of the range of DNA amount detected as the DNA amount of normal cells whose cell cycle is in the G0 / G1 phase in the canceration information providing apparatus 1, and the region A5 Is set to include all cells in the right direction. The cell number threshold S1 used for the determination of the number of cells is determined corresponding to the fact that there are about 20,000 single epithelial cells supplied to the flow cell 43 of the main detection unit 22. The threshold value S1 is a threshold value for fractionating cancer samples and negative samples. Since the threshold value S1 varies depending on the number of cells measured, it is appropriately set from the viewpoint of sensitivity and specificity. In the present embodiment, the threshold value S1 is set in the range of 2000 to 4000.

  If the number of cells included in the region A5 is equal to or greater than the threshold S1 (S107: YES), the CPU 301 sets “Cancer” in the flag 1 (S108). When the number of cells included in the region A5 is less than the threshold value S1 (S107: NO), S108 is skipped. Note that the processing of S105 to S108 corresponds to “determination 1” described above.

  Next, the CPU 301 resets the value of the flag 2 stored in the hard disk 304 (S109). The flag 2 is for indicating a determination result by the above-described “determination 2”. Subsequently, the CPU 301 extracts a cell group (cell group included in the region A3) with V13 ≦ N / C ratio <V11 in the scattergram of FIG. 10 (c), and displays a histogram (DNA ployid) shown in FIG. 10 (e). ) Is created (S110). V13 is a threshold value for allowing the surface layer cell or the middle layer cell to be included in the range of V13 ≦ N / C ratio <V11. V13 is appropriately set from the viewpoint of sensitivity and specificity. In the present embodiment, V13 is set in a range that is smaller than V11 and not less than 0. In the histogram of FIG. 10 (e), the horizontal axis represents the amount of DNA, and the vertical axis represents the number of cells.

  Next, the CPU 301 obtains the number of cells whose DNA amount is 2C of normal cells in the histogram created in S110, that is, the number of cells having the DNA amount of normal cells whose cell cycle is in the G0 phase or G1 phase. Specifically, the CPU 301 sets a region A6 in the histogram shown in FIG. 10E, and obtains the number of cells included in this region A6. The value V32 at the right end of the area A6 is set to be the same as the value V21 at the left end of the area A5.

  V32 (V21) set here is set as a value that divides the amount of DNA (2C) of normal cells in the G0 phase or G1 phase and the amount of DNA of normal cells in the S phase. Specifically, a value V30 indicating the amount of DNA of normal cells whose cell cycle is in the G0 phase or G1 phase is set, and V31 and V32 (V21) include V30 in the range of V31 and V32 (V21). , V31 and V32 are set to have a width A.

  Subsequently, the CPU 301 determines the number of cells whose DNA amount is equal to or higher than the S phase of normal cells in the histogram created in S106, that is, a cell having a DNA amount exceeding the DNA amount of normal cells whose cell cycle is in the G0 phase or G1 phase. A value obtained by dividing the number by the number of cells having a DNA amount of 2C in the histogram created in S110, that is, the number of cells having a DNA amount of normal cells having a cell cycle in the G0 phase or G1 phase (hereinafter referred to as “cancer ratio”). "). Then, the CPU 301 determines whether or not the canceration ratio is greater than or equal to a predetermined threshold S2 (S111). When the canceration ratio is equal to or greater than the threshold value S2 (S111: YES), the CPU 301 sets “Cancer” in the flag 2 (S112). When the canceration ratio is less than the threshold value S2 (S111: NO), S112 is skipped. Note that the processing of S109 to S112 corresponds to “determination 2” described above. The threshold value S2 is a threshold value for fractionating cancer samples and negative samples. The threshold S2 is appropriately set from the viewpoint of sensitivity and specificity. In the present embodiment, the threshold value S2 is set in the range of 0.5 to 1.5.

  Next, if any of the flags 1 and 2 is “Cancer” (S113: YES), that is, if it is determined as “Cancer” in the determination 1 or the determination 2, the CPU 301 re-executes as information on canceration. A display indicating that inspection is necessary is performed (S114). Specifically, as illustrated in FIG. 11B, the CPU 301 displays a dialog D <b> 2 indicating “re-examination required” on the display unit 32. In addition, in order to show that the suspicion of canceration is high in the dialog D2, as shown in FIG. 11C, a display of “Cancer?” May be further added.

  On the other hand, if both the flags 1 and 2 are not “Cancer” (S113: NO), that is, if it is not determined as “Cancer” in both the determinations 1 and 2, the CPU 301 displays an indication that re-examination is unnecessary. It performs (S115). Specifically, as illustrated in FIG. 11D, the CPU 301 displays a dialog D <b> 3 indicating “No reexamination is necessary” on the display unit 32.

  In addition, since there are few biological samples contained in the biological container 4, when the number of significant cells required for a cancer cell detection cannot be ensured, there exists a possibility that an appropriate result may not be obtained in the determination 1. In such a case, in S113, it may be determined based on only the value of the flag 2 whether or not it is “Cancer”.

  12A to 12E are scattergrams of five specimens having different degrees of cancer progression. 12A to 12E correspond to “Cancer”, “CIN3”, “CIN2”, “CIN1”, and “Normal” indicating the degree of progression of cancer. As the cancer progresses from “Normal” to “Cancer”, the number of cells contained in the region A4 with a high N / C ratio increases as compared to the cells contained in the region A3 with a low N / C ratio. I understand.

  FIGS. 13A to 13E are histograms created by extracting cell groups included in the region A4 with a high N / C ratio in FIGS. 12A to 12E, respectively. Here, 6785, 875, 4042, 589, and 121 cells are included in the region A5 corresponding to the DNA amount in the S phase or higher in FIGS. 13A to 13E, respectively. At this time, if the value of the threshold value S0 is set to 100 and the value of the threshold value S1 is set between 875 and 4042, the sample in FIGS. 13A and 13C is determined as “Cancer” in the determination 1 in FIG. The specimens in FIGS. 13B, 13D, and 13E are not determined as “Cancer”. Therefore, in this case, it is determined whether or not the samples of FIGS. 13A, 13B, 13D, and 13E are properly cancerous in the determination 1, but the sample of FIG. In the determination 1, it is not determined whether the cancer is appropriately cancerous.

  14A to 14E are histograms created by extracting cell groups included in the region A4 having a low N / C ratio in FIGS. 12A to 12E, respectively. Here, 738 cells, 1878 cells, 8270 cells, 28787 cells, and 19485 cells are included in the region A6 corresponding to the 2C DNA amount in FIGS. 14A to 14E, respectively. Accordingly, the canceration ratios obtained in S111 of FIG. 9 are 9.19, 0.47, 0.49, 0.02, and 0.01 in the specimens of FIGS. 14 (a) to (e), respectively. Become. At this time, if the value of the threshold value S0 is set to 100 and the value of the threshold value S2 is set between 0.49 and 9.19, in the determination 2 of FIG. 9, the specimen of FIG. 14A is determined to be “Cancer”. The samples in FIGS. 14B to 14E are not determined as “Cancer”. Therefore, in this case, in the determination 2, it is determined whether or not all of the specimens in FIGS. 14A to 14E are properly cancerous.

  FIGS. 15A to 15C are diagrams for explaining the relationship between the determination result of the histological diagnosis and the determination result by the determination 1 of FIG. Here, determination is performed on 1035 samples.

  In FIG. 15A, the left area A11 is a box-and-whisker diagram showing data variation and the right area A12 is a histogram. In the areas A11 and A12, the five values on the horizontal axis correspond to “Normal”, “CIN1”, “CIN2”, “CIN3”, and “Cancer” as a result of the histological diagnosis from the left. The vertical axis in FIG. 15A represents the number of cells that are equal to or greater than the amount of DNA of normal cells in S phase counted in S107 in FIG.

  In a region A11 in FIG. 15A, one dot corresponds to one sample (specimen). Each sample diagnosed as “Normal” in the histological examination is plotted at the position of the corresponding number of cells (vertical axis) in the leftmost column. For example, for a sample diagnosed as “Normal”, if the number of cells that are greater than or equal to the amount of DNA of normal cells in S phase counted in S107 in FIG. In the vertical axis direction of the column (“Normal” column), a plot is made at a position corresponding to this cell number. Similarly, each sample diagnosed as “CIN1”, “CIN2”, “CIN3”, “Cancer” is plotted at a position corresponding to the number of cells in the vertical axis direction on the column of the corresponding canceration stage. . Region A12 in FIG. 15 (a) is a bar graph showing at what rate the samples included in each canceration stage are distributed on the vertical axis. In FIG. 15A, the position of the threshold value S1 of the number of cells set in S107 of FIG. 9 is indicated by a dotted line. That is, the sample plotted at a position where the number of cells is larger than the dotted line is determined as “Cancer” in the determination 1 of FIG.

  Referring to FIG. 15 (a), among the samples determined as “Normal” by histological examination, the samples determined as “Cancer” in determination 1, that is, the amount of DNA more than that of normal cells in S phase It can be seen that there is one specimen that is positive because the number of cells is equal to or greater than the threshold value S1. It can be seen that all the specimens determined as “CIN1” by the histological diagnosis were appropriately determined as “Normal” in the determination 1. It can be seen that there are two samples each determined as “Cancer” in the determination 1 among the samples determined as “CIN2” and “CIN3” by the histological examination. It can be seen that all of the samples determined as “Cancer” by the histological diagnosis were appropriately determined as “Cancer” in the determination 1.

  FIG. 15B is a table summarizing the contents of FIG. In the histological diagnosis item, positive indicates that the sample is cancer, and negative indicates a sample of CIN3 or lower. In the item of determination 1, positive indicates a sample determined as YES in S107 of FIG. 9, and negative indicates a sample determined as NO in S107 of FIG.

  Referring to FIG. 15B, out of 11 samples determined to be positive by determination 1, there are 6 samples that were positive in histology. That is, five specimens are false positives (differing from histological diagnosis) by determination 1. In addition, all 1024 specimens determined to be negative by determination 1 are diagnosed as negative in the histological diagnosis.

  FIG. 15C shows the determination result of FIG. 15B as a percentage.

  Since all six samples that were positive in the histological diagnosis were determined to be positive by determination 1, the sensitivity according to determination 1 is 6/6 = 100.0%. Moreover, since 1024 samples were determined to be negative among 1029 samples that were negative in the histological diagnosis by the determination 1, the specificity according to the determination 1 is 1024/1029 = 99.5%.

  FIGS. 16A to 16C are diagrams illustrating the relationship between the determination result of the histological diagnosis and the determination result based on the determination 2 of FIG. 9, as in FIGS. 15A to 15C. In addition, the vertical axis | shaft of Fig.16 (a) represents the canceration ratio (ratio calculated | required by S111) calculated by the determination 2 of FIG. In FIG. 16A, the position of the canceration ratio threshold S2 set in S111 of FIG. 9 is indicated by a dotted line. That is, the sample plotted at a position where the canceration ratio is higher than the dotted line is determined as “Cancer” in the determination 2 of FIG.

  Referring to FIG. 16A, among samples determined as “Normal” by histological examination, samples determined as “Cancer” in determination 2, that is, positive because the canceration ratio is greater than or equal to threshold S2. It can be seen that there are 8 samples. It can be seen that all the specimens determined as “CIN1” and “CIN2” by the histological diagnosis were appropriately determined as “Normal” in the determination 2. It can be seen that among the samples determined as “CIN3” by the histological diagnosis, there is one sample determined as “Cancer” in the determination 2. Among the samples determined as “Cancer” by histological examination, it was not determined as “Cancer” in Determination 2, that is, the place that should be determined as “Cancer” originally was not “Cancer”. It can be seen that there is one specimen.

  FIG. 16B is a table summarizing the contents of FIG. 16A in the same manner as FIG. 15B.

  Referring to FIG. 16B, out of the 14 samples determined to be positive by determination 2, there are 5 samples that were positive in the histological diagnosis. That is, nine samples are false positives (differing from histological diagnosis) by the determination 2. Of the 1021 samples determined to be negative by determination 2, 1020 samples were negative in the histological diagnosis. That is, one specimen is false negative (differing from the histological diagnosis) by determination 2.

  FIG.16 (c) displays the determination result of FIG.16 (b) in percent.

  According to the determination 2, five of the six samples that were positive in the histological diagnosis were determined to be positive. Therefore, the sensitivity according to the determination 2 is 5/6 = 83.3%. In addition, according to the determination 2, 1020 samples out of 1029 samples that are negative in the histological diagnosis are determined to be negative. Therefore, the specificity according to the determination 2 is 1020/1029 = 99.1%.

  Here, as shown in the present embodiment, when the determination of “Cancer” is performed by combining the determinations 1 and 2 in S113 of FIG. 9, the determination result of the histological diagnosis and the determination result of S113 of FIG. The relationship is as shown in FIGS. 16 (d) and 16 (e).

  Referring to FIG. 16D, of the 18 specimens determined to be positive in S113, 6 specimens were positive in the histological examination. That is, 12 specimens are false positives (different from the histological diagnosis) as determined in S113. In addition, all the 1017 specimens determined to be negative by the determination in S113 are actually negative.

  With reference to FIG.16 (e), since all six samples which were positive in the histological diagnosis were determined to be positive by determination of S113, the sensitivity by S113 will be 6/6 = 100.0%. Further, as a result of the determination in S113, 1017 samples out of 1029 samples that were negative in the histological diagnosis were determined to be negative, so the specificity according to S113 is 1017/1029 = 98.8%.

  As mentioned above, according to this Embodiment, it can be determined by the determination 1 of FIG. 9 whether it is cancer. According to the determination 1, as described with reference to FIGS. 15A to 15C, five of 1029 negative samples were false positives, but were determined to be “Cancer” by histology. All the six specimens can be properly determined as “Cancer”.

  Moreover, in the determination 1, the cell group whose DNA amount is V21 or more was extracted from the cell group having the N / C ratio of V11 to V12. Thus, since the N / C ratio of the cells to be extracted is limited to a high range, the determination accuracy by determination 1 can be improved.

  In the determination 1, concentration detection (pre-measurement) is performed in the sub-detection unit 13, and a necessary sample is supplied to the main detection unit 22 to such an extent that the number of significant cells necessary for cancer cell detection is ensured. . Thus, in S107 of FIG. 9, it is possible to accurately determine whether or not the cancer is by comparing the number of cells equal to or greater than the amount of DNA of normal cells in S phase with a predetermined number (S1). It becomes.

  Moreover, according to this Embodiment, it can be determined whether it is cancer by the determination 2 of FIG. According to the determination 2, as described with reference to FIGS. 16A to 16C, 9 samples out of 1029 negative samples become false positive, and one sample out of 6 positive samples is false negative. However, most of the specimens that are determined to be Cancer by histological diagnosis can be appropriately determined to be “Cancer”.

  Further, according to the present embodiment, if any of the determinations 1 and 2 is “Cancer” in S113 of FIG. 9, it is determined as “Cancer” as a final result, and this is notified to the operator. . Thereby, even if a false negative sample is included in the result of any one of the determinations 1 and 2, if it is determined as “Cancer” in the other determination, the result is determined as “Cancer”. The sample to be determined can be reliably determined as “Cancer”.

  In the present embodiment, determinations 1 and 2 are arranged in parallel, and the result of determinations 1 and 2 is determined in S113 in FIG. 9 to finally determine whether or not it is “Cancer”. The final determination may be performed using only one of the determinations 1 and 2.

  FIG. 17A is a diagram illustrating a case where only determination 1 is used, and FIG. 17B is a diagram illustrating a case where only determination 2 is used. In FIGS. 17A and 17B, the same number is assigned to the same part as the process of FIG. 9, and only the vicinity of the determinations 1 and 2 is shown.

  In FIG. 17A, after the same determination 1 as in FIG. 9 is performed, the CPU 301 of the data processing device 3 determines whether or not the flag 1 is “Cancer” (S121). In FIG. 17B, after the same determination 2 as in FIG. 9 is performed, the CPU 301 determines whether or not the flag 2 is “Cancer” (S122). When final determination is performed as shown in FIGS. 17A and 17B, the determination results of FIGS. 15A to 15C and the determination results of FIGS. 16A to 16C are respectively obtained. Final decision. Further, when the determination is performed as shown in FIGS. 17A and 17B, the same effects as those of the determinations 1 and 2 described above are obtained.

  In the present embodiment, leukocytes and epithelial cells (single epithelial cells and aggregated epithelial cells) are separated in S101 of FIG. Thereby, the determination precision of canceration of the epithelial cell used as a measuring object can be improved. In the present embodiment, single epithelial cells and aggregated epithelial cells are separated in S102 of FIG. Thereby, the determination precision of canceration of the epithelial cell used as a measuring object can be improved.

  In the present embodiment, if NO is determined in S104 of FIG. 9, “inappropriate sample” is displayed and the process ends. As a result, even when the number of extracted cells is small and proper determination cannot be performed, it is possible to prevent information with poor reliability from being output by prohibiting output of information related to canceration of cells. And appropriate diagnosis can be carried out smoothly. In this case, it is desirable that information notifying that the number of cells to be measured is insufficient is output. In this way, measures such as re-collecting epithelial cells can be smoothly advanced.

  Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, and the embodiments of the present invention can be variously modified in addition to the above.

  For example, in the above embodiment, cervical epithelial cells are analyzed, but other epithelial cells such as oral cells, bladder, pharynx, and organ epithelial cells are analyzed, and these cells are analyzed. Determination of canceration may be performed.

  In the above embodiment, the sub-detection unit 13 detects the number of cells (pre-measurement). However, when the variation in the concentration of the measurement target cell is small for each biological sample and the influence on the analysis result is small. The sub-detecting unit 13 may be omitted.

  In the above embodiment, the N / C ratio is a numerical value (cell nucleus size N) indicating the width of the cell nucleus obtained based on the width of the waveform of the lateral fluorescence intensity and the width of the waveform of the forward scattered light intensity. Was calculated as a ratio of numerical values (cell size C) indicating the cell width obtained based on However, the present invention is not limited to this, and the N / C ratio may be calculated as a ratio of the cell nucleus area to the cell area. In the above embodiment, a numerical value (cell size C) indicating the cell width is obtained based on the waveform width of the forward scattered light intensity. Thereby, even when a cell having a long shape in a predetermined direction flows through the flow cell, the size of the cell can be accurately expressed.

  FIG. 18A is a diagram showing a state where the upper limit of the cell group extraction range in S106 of FIG. 9 is eliminated, that is, a state where the right boundary of the region A4 of FIG. 10C is expanded to the right. is there. FIG. 18B is a diagram showing a state in which both the upper limit and the lower limit of the cell group extraction range (region A4) in S106 of FIG. 9 are eliminated.

  As shown in FIGS. 18A and 18B, the region A4 is set, and the determination of canceration is performed on 1035 specimens (samples) as described above. Similarly to the above embodiment, it was 83.3%, and the specificities were 94.6% and 93.3%, respectively, which were only slightly decreased from the above embodiment. Therefore, even if the range of cells to be extracted is expanded in this way, it is possible to appropriately determine “Cancer” as in the above embodiment. From this result, when the region A4 is set as shown in FIGS. 18A and 18B, it can be inferred that the sensitivity and specificity are similarly maintained for the determination 1.

  In the above embodiment, the value at the left end of the region A5 in which the amount of DNA equal to or greater than the amount of DNA of normal cells in the S phase shown in FIG. 10 (d) is set to V21, and the value at the right end is the S phase. However, the minimum and maximum values of the setting range are not fixed as described above, so that sensitivity and specificity are improved. As appropriate, it may be set other than the above values. In addition, the values at the left end and the right end of the region A6 for setting the 2C DNA amount shown in FIG. 10E are set to V31 and V32, respectively. The minimum and maximum values of the setting range are the above V31 and V32. Instead of being fixed, it may be set appropriately other than the above values so as to improve sensitivity and specificity.

  FIG. 18C is a diagram showing a state in which the range of 2C cells extracted in S111 in FIG. 9 is changed, that is, a state in which the right end of the region A6 in FIG. V31 and V32 in the region A6 in FIG. 18C include V30 in the range of V31 and V32, and the width of the range of V31 and V32 is smaller than the width A of the range of V31 and V32 in FIG. B is set. FIG. 18 (d) shows a state where the range of cells exceeding the amount of DNA of normal cells in S phase extracted in S107 of FIG. 9, ie, the left end of region A5 in FIG. 10 (d) is widened. It is a figure which shows the state made. The value V21 at the left end of the region A5 in FIG. 18D is greater than V30 and greater than V32 when V30 is included in the range of V31 and V32 and the width of the range of V31 and V32 is set to A. It is set to be a small value.

  As shown in FIGS. 18C and 18D, the regions A5 and A6 are set, and the determination of canceration is performed on 1035 specimens (samples) as described above. Similar to the above embodiment, it was 83.3%, and the specificities were 98.7% and 98.5%, respectively, which were substantially the same as the above embodiment. Therefore, even if the range of cells to be extracted is changed in this way, it is possible to appropriately determine “Cancer” as in the above embodiment.

  In the above embodiment, after S105 in FIG. 9, first, a cell group of V11 ≦ N / C ratio ≦ V12 is extracted, and among the extracted cell group, a normal cell having a DNA amount in S phase has. A group of cells greater than or equal to the amount of DNA was counted, and this count was used for the number of cells in S107 or the molecule of canceration ratio in S111. However, instead of this, first, a cell group having a DNA amount equal to or greater than the DNA amount of normal cells in the S phase is extracted, and among the extracted cell groups, a cell group having V11 ≦ N / C ratio ≦ V12 is counted. This count value may be used for the number of cells in S107 or the molecule of canceration ratio in S111.

  In the above embodiment, after S109 in FIG. 9, first, a cell group with V13 ≦ N / C ratio <V11 is extracted, and among the extracted cell groups, a cell group with a DNA amount of 2C is counted, This count value was set as the denominator of the canceration ratio. However, instead of this, first, a cell group with a DNA amount of 2C is extracted, and among the extracted cell groups, a cell group with V13 ≦ N / C ratio <V11 is counted, and this counted value is the canceration in S111. It may be set as the denominator of the ratio.

  In addition, the embodiment of the present invention can be variously modified as appropriate within the scope of the technical idea shown in the claims.

DESCRIPTION OF SYMBOLS 1 ... Canceration information provision apparatus 40 ... Flow cytometer (detection part)
43 ... Flow cell 301 ... CPU (control part)

Claims (14)

  Fluorescence and scattered light is detected from each cell contained in the measurement sample including epithelial cells, and a fluorescence signal and scattered light signal are output.
  Discriminate epithelial cells from white blood cells based on the width of the waveform of the output scattered light signal and the area of the waveform of the output fluorescence signal,
  Classify abnormal cells based on fluorescence signals obtained from the differentiated epithelial cells,
A cell analysis method characterized by the above.   The step of discriminating between the epithelial cells and the white blood cells discriminates, as epithelial cells, cells whose width of the waveform of the scattered light signal is larger than the threshold or cells whose area of the waveform of the fluorescence signal is larger than the threshold. The cell analysis method described.   The cell analysis method according to claim 1, wherein the scattered light is forward scattered light.   Further comprising a step of discriminating an aggregated cell obtained by aggregating a plurality of epithelial cells from a single epithelial cell based on a scattered light signal and a fluorescence signal obtained from the differentiated epithelial cells;
  The cell analysis method according to any one of claims 1 to 3, wherein in the step of classifying the abnormal cells, the abnormal cells are classified based on a fluorescence signal obtained from the single epithelial cells thus discriminated.   In the step of discriminating between the aggregated cells and the single epithelial cells, based on at least one of the difference integrated value and peak value of the scattered light signal waveform, and the difference integrated value and peak value of the fluorescent signal waveform The cell analysis method according to claim 4, wherein the aggregated cells and the single epithelial cells are discriminated.   For a single epithelial cell that has been discriminated, first data indicating the size of the cell nucleus and second data indicating the size of the cell are obtained, and the first data and the second data satisfy a predetermined condition. Further comprising the step of extracting groups,
  The cell analysis method according to claim 5, wherein in the step of classifying the abnormal cells, the abnormal cells are classified from the cell group based on a fluorescence signal obtained from each cell included in the cell group.   The cell analysis method according to claim 6, wherein the predetermined condition is a condition for extracting a cell selected from epidermal cells, middle layer cells, parabasal cells, and basal cells.   The cell analysis method according to claim 1, wherein the epithelial cells are cervical epithelial cells.   A detection unit that irradiates light to a measurement sample including epithelial cells, detects fluorescence and scattered light from each cell included in the measurement sample, and outputs a fluorescence signal and a scattered light signal;
  Based on the width of the waveform of the scattered light signal output from the detection unit and the area of the waveform of the output fluorescence signal, the epithelial cells are discriminated from the white blood cells, and based on the fluorescence signals obtained from the differentiated epithelial cells. A control unit for classifying abnormal cells;
A cell analyzer comprising:   The cell analyzer according to claim 9, wherein the control unit discriminates a cell having a waveform width of a scattered light signal larger than a threshold value or a cell having an area of a fluorescence signal waveform larger than a threshold value as an epithelial cell.   The cell analyzer according to claim 9 or 10, wherein the scattered light is forward scattered light.   The control unit discriminates an aggregated cell obtained by aggregating a plurality of epithelial cells from a single epithelial cell based on a scattered light signal and a fluorescence signal obtained from the discriminated epithelial cell, and discriminates a single epithelium. The cell analyzer according to any one of claims 9 to 11, wherein abnormal cells are classified based on a fluorescence signal obtained from the cells.   The control unit obtains first data indicating the size of the cell nucleus and second data indicating the size of the cell for the discriminated single epithelial cell, and the first data and the second data are in a predetermined condition. The cell analysis apparatus according to claim 12, wherein a cell group that matches is extracted, and abnormal cells are classified from the cell group based on fluorescence signals obtained from the respective cells included in the cell group.   The cell analyzer according to claim 13, wherein the predetermined condition is a condition for extracting a cell selected from epidermal cells, middle layer cells, parabasal cells and basal cells.

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