WO2011099565A1 - Method for detecting cancer cells in blood, and program used therefor - Google Patents

Method for detecting cancer cells in blood, and program used therefor Download PDF

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Publication number
WO2011099565A1
WO2011099565A1 PCT/JP2011/052892 JP2011052892W WO2011099565A1 WO 2011099565 A1 WO2011099565 A1 WO 2011099565A1 JP 2011052892 W JP2011052892 W JP 2011052892W WO 2011099565 A1 WO2011099565 A1 WO 2011099565A1
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cell
fluorescence
cells
oncolytic virus
blood
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PCT/JP2011/052892
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French (fr)
Japanese (ja)
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佐藤　淳
眞砂　明典
幸夫 辻野
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シスメックス株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage

Definitions

  • the present invention relates to a method for detecting cancer cells from a blood sample collected from a subject and a program used therefor.
  • Cancer metastasis is considered to occur when cancer cells in the primary lesion spread throughout the body through blood vessels and lymphatic vessels, and some of them engraft in other parts of the organ. Cancer cells circulating in the blood are called CTC (CirculatingirTumor Cell). It has been reported that the number of CTCs in the blood correlates with cancer metastasis and prognosis. Therefore, measurement of the number of CTCs is considered useful as a method for predicting the prognosis and therapeutic effect of metastatic cancer such as metastatic breast cancer.
  • Cancer cells are known to have increased telomerase activity that is hardly detected in most normal cells. Therefore, a virus (Oncolytic Virus) carrying a replication cassette containing a telomerase promoter and a labeled cassette containing a gene for a labeled protein (eg, green fluorescent protein (GFP)) is grown in cancer cells.
  • a technique for specifically labeling cancer cells is known (Patent Document 1).
  • An oncolytic virus having a GFP gene is commercially available as Telomescan (registered trademark) (OBP-401). Telomescan (registered trademark) can specifically grow in cancer cells and produce GFP, whereby the cancer cells can specifically emit fluorescence.
  • telomescan registered trademark
  • OBP-401 telomescan (registered trademark)
  • GFP fluorescence emission of GFP
  • Non-Patent Document 1 a cancer cell in blood, that is, CTC is specifically identified. Can be detected.
  • oncolytic virus infects not only cancer cells but also some normal cells such as leukocytes (some monocytes and lymphocytes) and proliferates. . Therefore, when CTC is detected using an oncolytic virus that expresses a fluorescent protein, the fluorescence intensity may be detected in normal cells other than cancer cells. As a result, cancer cells may not be detected accurately.
  • the present invention uses an oncolytic virus that expresses a fluorescent protein to detect cancer cells in blood that can accurately determine whether cells contained in a blood sample collected from a subject are cancer cells. It is an object to provide a method and a program used therefor.
  • the present inventors have found that it is possible to accurately determine whether a cell is a cancer cell by using an oncolytic virus that expresses a fluorescent protein and using the fluorescence information and cell size information of the cell. Completed the invention.
  • the present invention includes a step of preparing a measurement sample by treating cells contained in a blood sample collected from a subject with an oncolytic virus that expresses a fluorescent protein, and the measurement sample includes Blood, including a step of acquiring fluorescence information and cell size information of a cell, and a step of determining whether or not the cell is a cancer cell based on the fluorescence information and cell size information acquired in the acquisition step A method for detecting a middle cancer cell is provided.
  • the present invention provides a computer based on the information acquired by the acquisition means, the acquisition means for acquiring fluorescence information and cell size information of cells that have been infected and proliferated by an oncolytic virus in a blood sample, Provided is a blood cancer cell detection program for functioning as determination means for determining whether or not a cell is a cancer cell, and display means for displaying the determination result of the determination means.
  • the present invention it is possible to accurately determine whether a cell contained in a blood sample collected from a subject is a cancer cell using an oncolytic virus that expresses a fluorescent protein. It can be detected with high accuracy.
  • FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and a volume on the Y axis.
  • FIG. 2 is a scattergram in Example 1 with a peak value on the X axis and a volume on the Y axis.
  • FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and an area on the Y axis.
  • FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and a diameter on the Y axis.
  • FIG. 2 is a scattergram in Example 1 with an integral value on the X axis and a perimeter on the Y axis.
  • Example 1 it is a three-dimensional frequency distribution diagram which took the area on the X-axis, the peak value on the Y-axis, and the integrated value on the Z-axis.
  • the method for detecting cancer cells in blood is as follows. (1) A step of preparing a measurement sample (preparation step) by treating cells contained in a blood sample collected from a subject with an oncolytic virus expressing a fluorescent protein; (2) a step of acquiring fluorescence information and cell size information of cells contained in the measurement sample (acquisition step); (3) A step of determining whether or not the cell is a cancer cell based on the fluorescence information and the cell size information acquired in the acquisition step (determination step) including.
  • the “blood sample” may be either blood collected from a subject or processed blood obtained by processing blood.
  • the blood sample is preferably a blood sample obtained by removing serum from whole blood, particularly peripheral blood, in that the determination efficiency is further improved.
  • Subjects include patients who are trying to determine whether they have cancer cells, such as patients suspected of suffering from cancer or patients suffering from cancer.
  • a known method can be used as a method for removing serum from whole blood.
  • a method of centrifuging whole blood to which an anticoagulant for example, ethylenediaminetetraacetic acid, sodium citrate, heparin, etc.
  • Centrifugation is preferably performed at 500 rpm to 3500 rpm for 3 to 30 minutes.
  • the fluorescent protein a known protein that is usually used in the biochemical field can be used.
  • fluorescent proteins such as green fluorescent protein (GFP) and variants thereof (for example, Enhanced-humanized GFP (EGFP), red-shiftGFP (rsGFP)), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), etc. are used. be able to.
  • the fluorescent protein is preferably a green fluorescent protein.
  • the “oncolytic virus” is a restricted-proliferating virus that cannot propagate in normal cells but can specifically propagate in cancer cells.
  • the virus in which the promoter which shows a promoter activity specifically in a cancer cell was integrated is mentioned.
  • Examples of promoters that exhibit specific promoter activity in cancer cells include human telomerase promoter, human prostate cancer specific antigen (PSA) promoter, human alpha protein (AFP) promoter, fetus Sex cancer antigen (CEA) promoter and the like.
  • PSA human prostate cancer specific antigen
  • AFP human alpha protein
  • CEA fetus Sex cancer antigen
  • the human telomerase promoter is preferred in that it can exhibit promoter activity in many types of cancer cells.
  • the hTERT promoter which is a gene encoding human telomerase reverse transcriptase, is more preferred.
  • SEQ ID NO: 1 is the nucleic acid sequence of the hTERT promoter.
  • the hTERT promoter the entire 455 bp nucleic acid sequence shown in SEQ ID NO: 1 may be used, but the 181 bp region 5 ′ upstream of SEQ ID NO: 1 is considered to be an important core region for downstream gene expression. Therefore, a nucleic acid sequence containing at least this core region may be used.
  • the above-mentioned oncolytic virus expresses a fluorescent protein. That is, the oncolytic virus has a gene encoding a fluorescent protein.
  • the gene for the fluorescent protein can be placed under the control of the cancer cell-specific promoter.
  • the above-mentioned fluorescent protein gene can be placed under the control of a promoter capable of exhibiting promoter activity in an oncolytic virus.
  • promoters that can control the expression of the fluorescent protein gene include cytomegalovirus (CMV) promoter, SV40 late promoter, MMTV LTR promoter, RSV LTR promoter, SR ⁇ promoter, and the like.
  • CMV cytomegalovirus
  • SV40 late promoter SV40 late promoter
  • MMTV LTR promoter MMTV LTR promoter
  • RSV LTR promoter SR ⁇ promoter
  • the fluorescent protein gene is preferably placed under the control of a CMV promoter or hTERT promoter.
  • the nucleic acid sequences of these promoters are known.
  • An expression cassette comprising at least a gene encoding the above fluorescent protein and a promoter for expressing it (a cancer cell-specific promoter or a promoter capable of exhibiting promoter activity in an oncolytic virus) is a common genetic engineering It can be obtained by a technique. For example, a gene encoding a fluorescent protein and a promoter are amplified by PCR or the like based on a known sequence, each gene obtained is ligated to an appropriate plasmid, and a necessary portion is excised, thereby expressing an expression cassette. Can be obtained. See, for example, International Publication No. 2006/36004.
  • the above-mentioned oncolytic virus may have a replication cassette in which a gene required for virus growth is linked downstream of the above cancer cell-specific promoter.
  • genes necessary for virus growth include early genes (EIA, EIB, etc.), IRES that are protein synthesis initiation signals specific to the Picornaviridae family, and the like. The nucleic acid sequences of these genes are known.
  • the above replication cassette can be prepared by a normal genetic engineering technique.
  • the above cancer cell-specific promoter and, if necessary, a gene necessary for virus growth are amplified by polymerase chain reaction (PCR) or the like based on a known sequence, and each of the obtained genes is converted into an appropriate plasmid.
  • the replication cassette can be obtained by linking to and cutting out the necessary part. See, for example, International Publication No. 2006/36004.
  • virus examples include those derived from adenovirus, herpes simplex virus, Sendai virus, and reovirus.
  • human adenovirus is preferred.
  • Telomescan registered trademark
  • E1A gene E1A gene
  • IRES gene and E1B gene E1B gene
  • an expression cassette containing the CMV promoter and GFP gene OBP-401 (manufactured by Oncoris Bioformer) is preferred.
  • This embodiment is a step of preparing a measurement sample by treating cells contained in a blood sample with an oncolytic virus that expresses a fluorescent protein (hereinafter referred to as “preparation step”). Included).
  • the oncolytic virus expressing the fluorescent protein is infected with cancer cells in the blood sample.
  • fluorescent protein can be expressed in cancer cells.
  • the treatment of the cells contained in the blood sample with the oncolytic virus may be performed by a known method, and is not particularly limited.
  • an oncolytic virus can be infected with cancer cells in a blood sample by incubating the blood sample with an oncolytic virus in the presence of a medium commonly used for animal cell culture. Examples of the medium used for incubating the blood sample and oncolytic virus include RPMI-1640, Dulbecco's modified Eagle medium (DMEM), minimal essential medium (MEM), and the like.
  • the oncolytic virus When incubating a blood sample with an oncolytic virus, it is preferable to incubate the oncolytic virus with the blood sample in an amount of 6 ⁇ 10 4 to 6 ⁇ 10 8 PFU (Plaque Forming Unit) per ml of blood sample. .
  • the time and temperature at which the above blood sample is incubated with the oncolytic virus may be any conditions as long as the oncolytic virus can grow on cancer cells in the blood sample, and can be appropriately adjusted depending on the type of oncolytic virus.
  • the incubation can be performed at a temperature of 25 to 40 ° C. for 1 to 36 hours, more preferably at 30 to 37 ° C. for 12 to 24 hours.
  • the preparation step of the present embodiment may further include mixing a blood sample treated with an oncolytic virus with a nonionic surfactant and a fixing agent.
  • Leukocytes monoocytes, lymphocytes, etc.
  • telomerase enzymes that are specifically expressed in cancer cells such as telomerase. Therefore, when cells contained in a blood sample are treated with an oncolytic virus that expresses a fluorescent protein, the oncolytic virus may grow on normal white blood cells. As a result, fluorescent protein-derived fluorescence may occur from normal white blood cells. Therefore, in the acquisition process to be described later, fluorescence information that becomes noise increases.
  • the order of mixing the blood sample treated with the oncolytic virus with the nonionic surfactant and the immobilizing agent is not particularly limited.
  • the immobilizing agent may be mixed after mixing a nonionic surfactant and a blood sample treated with oncolytic virus.
  • a nonionic surfactant may be mixed after mixing the immobilizing agent and the blood sample treated with the oncolytic virus.
  • a blood sample treated with a nonionic surfactant, an immobilizing agent and an oncolytic virus may be mixed simultaneously.
  • the order of mixing the nonionic surfactant after mixing the immobilizing agent and the blood sample treated with the oncolytic virus is more preferable.
  • the nonionic surfactant may be any one of a polyoxyethylene surfactant and a fatty acid ester surfactant of a polyoxy compound, but a polyoxyethylene nonionic surfactant is more preferable.
  • Polyoxyethylene nonionic surfactants include higher alcohol / ethylene oxide adducts, alkylphenol / ethylene oxide adducts, higher fatty acids / ethylene oxide adducts, higher aliphatic amines / ethylene oxide adducts, higher fatty acid amide / ethylene oxide adducts.
  • the nonionic surfactant in the present embodiment is preferably a higher alcohol / ethylene oxide adduct.
  • the alkyl group of the higher alcohol of the higher alcohol / ethylene oxide adduct may be linear or branched, and preferably has 12 to 20 carbon atoms, more preferably 15 to 20 carbon atoms.
  • the higher alcohol / ethylene oxide adduct preferably has an ethylene oxide polymerization number of 15 to 40, more preferably 20 to 30.
  • polyoxyethylene octyldodecyl ether in which the alkyl group of the higher alcohol is branched, the number of carbon atoms is 18, and the polymerization number of ethylene oxide is 25 is more preferable.
  • the concentration of the nonionic surfactant when mixed with the blood sample and the fixing agent can be appropriately selected depending on the kind of the nonionic surfactant, but is preferably 0.005 to 0.5% by weight, more preferably 0.045 to 0.055% by weight.
  • aldehyde compounds such as paraformaldehyde, glutaraldehyde, and formaldehyde are preferable. Of these, paraformaldehyde is more preferable.
  • the concentration of the fixing agent when mixed with the blood sample and the nonionic surfactant can be appropriately selected depending on the type of the fixing agent, but is preferably 0.005 to 0.5% by weight, and preferably 0.045 to 0.8%. 055% by weight is more preferred.
  • the above mixing is preferably performed at pH 6 to 9 and osmotic pressure 200 to 400 m0sm.
  • This embodiment includes a step of acquiring cell fluorescence information and cell size information contained in the measurement sample obtained in the preparation step (hereinafter also referred to as “acquisition step”).
  • Fluorescence information in the present embodiment is information obtained from fluorescence emitted by a fluorescent protein.
  • the fluorescence intensity detected from the fluorescence emitted by the fluorescent protein the peak value of the fluorescence intensity, the minimum value of the fluorescence intensity, the average value of the fluorescence intensity, the calculated numerical value derived from the fluorescence intensity such as the integrated value of the fluorescence intensity, etc. It is done.
  • fluorescence information in the present embodiment a peak value of fluorescence intensity and an integral value of fluorescence intensity are preferable.
  • Cell fluorescence information can be obtained by a known method. More specifically, the fluorescence information of a cell can be acquired using a fluorescence microscope, a flow cytometer, or the like.
  • a measurement sample obtained in the above-described preparation step is smeared on a slide glass to prepare a smear. At this time, it is preferable to create a slide so that cells do not overlap.
  • the fluorescence in the cells present in the smear prepared as described above is observed with a fluorescence microscope equipped with a CCD camera.
  • the excitation light irradiated by the fluorescence microscope can be appropriately selected depending on the type of fluorescent protein used.
  • fluorescent protein for example, when GFP is used as the fluorescent protein, green light (G excitation / 546 nm) can be used as excitation light.
  • the fluorescence in the above-described cells is imaged using a CCD camera of a fluorescence microscope.
  • the above-described fluorescence information of the cell is acquired by performing image processing on an image obtained by imaging.
  • the image processing for acquiring the fluorescence information can be appropriately performed according to the type of the fluorescence information. More specifically, the fluorescence information can be acquired by processing an image obtained by imaging using a computer in which a computer program for image processing is installed.
  • Computer programs for image processing are well known, and storage media (image processing software) in which such computer programs are stored are generally commercially available. Examples of the image processing software include ImagePro (registered trademark) (manufactured by Roper Industries), WinROOF (registered trademark) (Mitani Corporation), and MetaMorph (registered trademark) (Molecular Devices).
  • the brightness (maximum brightness) of the pixel having the highest brightness among the pixels having the brightness values within the range selected in the above image is set to the fluorescence intensity. Get as the peak value of.
  • the integrated value of the fluorescence intensity a value obtained by integrating the luminance of each pixel having the luminance value within the range selected in the above-described image is acquired as the integrated value of the fluorescence intensity.
  • the flow cytometer which can be used is a flow cytometer which can detect fluorescence.
  • a conventionally known flow cytometer including a flow cell, a light source, a detection unit (fluorescence detection unit) that detects fluorescence, an analysis unit that analyzes data output from the fluorescence detection unit, and the like can be used.
  • ⁇ A measurement sample passing through the flow cell of the flow cytometer is irradiated with light from a light source.
  • the light source of the flow cytometer only needs to have a wavelength suitable for excitation of the fluorescent protein to be used.
  • the light source include a semiconductor laser, an argon laser, and a He—Ne laser.
  • the fluorescence detection unit may include a light receiving element that is generally used in a flow cytometer and can detect fluorescence. Specific examples of the fluorescence detection unit include a photomultiplier tube and an avalanche photodiode.
  • the wavelength of fluorescence detected by the fluorescence detection unit can be appropriately selected depending on the type of fluorescent protein used. For example, when GFP is used as the fluorescent protein, fluorescence at 480 to 580 nm may be detected by the fluorescence detection unit.
  • Fluorescence detected by the fluorescence detection unit is output to the analysis unit as an electrical signal.
  • the analysis unit performs analysis using the electrical signal output from the fluorescence detection unit, and acquires fluorescence information.
  • the analysis unit can analyze the waveform of the fluorescence intensity over time by obtaining the fluorescence intensity when an electrical signal is output from the fluorescence detection unit.
  • An example of the analysis result of the waveform of the fluorescence intensity over time by this analysis unit is shown in FIG. In FIG. 1, the vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the detection time. In this case, for example, the maximum height of the fluorescence intensity waveform can be acquired as the peak value of the fluorescence intensity. Further, the area value of the waveform of the fluorescence intensity can be acquired as an integrated value of the fluorescence intensity.
  • the “cell size information” in the present embodiment is not particularly limited as long as it is information reflecting the cell size.
  • Examples of the cell size information include a cell diameter, a cell area, a cell volume, a cell perimeter, and a roundness.
  • cell diameter, cell area, cell volume, and cell perimeter are preferred.
  • the cell size information can be obtained by a known method. More specifically, cell size information can be acquired using a fluorescence microscope, a flow cytometer, or the like.
  • the area occupied by the cell in the above-mentioned image is measured to calculate an average value and converted to a diameter when the cell is assumed to be spherical.
  • the area consisting of pixels having a luminance value within the range selected in the above-mentioned image is measured.
  • the volume of a cell the three-dimensional image of a cell is constructed
  • the length of the outer periphery (outline) of a cell is measured.
  • examples of the flow cytometer that can be used include conventionally known flow cytometers that can acquire cell size information by electrical or optical measurement. . More specifically, an electric resistance type flow cytometer that can acquire cell size information by electric resistance, an optical flow cytometer that can acquire cell size information by scattered light, and the like can be mentioned. A flow cytometer that can perform both electrical and optical measurements can also be used.
  • An electric resistance type flow cytometer for acquiring cell size information is a measuring unit comprising an orifice for introducing the above-described measurement sample and a pair of electrodes electrically connected by an electrolyte through the orifice. And an analysis unit. The interelectrode electrical resistance generated when the measurement sample passes through the orifice of the flow cytometer is measured by the measurement unit of the flow cytometer.
  • the inter-electrode electrical resistance measured by the measurement unit is output as an electrical signal to the analysis unit of the flow cytometer.
  • the analysis unit performs analysis based on the electrical signal output from the measurement unit, and acquires cell size information.
  • the analysis unit can analyze the waveform of the electrical resistance over time by obtaining the electrical resistance when the electrical signal is output from the measurement unit.
  • An example of the analysis result of the electrical resistance waveform over time by the analysis unit is shown in FIG. In FIG. 2, the vertical axis represents electric resistance, and the horizontal axis represents detection time.
  • the magnitude of electrical resistance strongly correlates with cell volume.
  • the maximum height of the waveform of electric resistance can be acquired as the volume of the cell.
  • the cell diameter, the cell area, and the cell perimeter can be obtained from the obtained cell volume.
  • An optical flow cytometer for acquiring cell size information is a detection unit that detects scattered light in addition to or in addition to the fluorescence detection unit of the flow cytometer used in the above-described fluorescence detection.
  • the scattered light detection unit may include a light receiving element that is generally used in a flow cytometer and can detect scattered light. Specific examples of the scattered light detection unit include a photodiode and an avalanche photodiode. Scattered light generated when the measurement sample passes through the flow cell of the flow cytometer is measured by the scattered light detector. Here, forward scattered light is preferable as the scattered light.
  • Scattered light measured by the scattered light detection unit is output as an electrical signal to the analysis unit of the flow cytometer.
  • the analysis unit performs analysis based on the electrical signal output from the scattered light detection unit, and acquires cell size information.
  • the analysis unit can analyze the waveform of the forward scattered light intensity over time by obtaining the intensity of the forward scattered light when the electrical signal is output from the scattered light detection unit.
  • An example of the analysis result of the waveform of the forward scattered light intensity over time by this analysis unit is shown in FIG.
  • the vertical axis represents the forward scattered light intensity
  • the horizontal axis represents the detection time.
  • the forward scattered light intensity strongly correlates with the cell surface area
  • the waveform width of the forward scattered light intensity strongly correlates with the cell length.
  • the maximum height of the waveform of the forward scattered light intensity can be acquired as the cell surface area.
  • the maximum width of the waveform of the forward scattered light intensity can be acquired as the cell diameter.
  • the cell volume, cell area, and cell perimeter can also be obtained from the obtained cell surface area and cell diameter.
  • This embodiment includes a step of determining whether a cell is a cancer cell based on the fluorescence information and cell size information acquired in the acquisition step (hereinafter also referred to as “determination step”). Including.
  • the fluorescence information and the cell size information obtained in the acquisition step are used to separate cancer cells in the measurement sample from other cells, debris, and the like in the measurement sample. More specifically, a cell in a measurement sample having predetermined fluorescence information and predetermined cell size information is determined as a cancer cell.
  • the threshold value is determined from the accumulation of data related to fluorescence information and cell size information of cancer cells in blood, and data related to fluorescence information and cell size information of cells other than cancer cells contained in blood.
  • a plurality of data including a value related to fluorescence information and a value related to cell size information is acquired from a plurality of measurement samples including only cancer cells based on the procedure of the acquisition step described above.
  • a plurality of data including a value related to fluorescence information and a value related to cell size information is obtained from a measurement sample prepared from a blood sample not containing cancer cells. Based on the acquired data, it is possible to determine whether or not the cell is a cancer cell from cells and debris contained in a measurement sample prepared from a blood sample. Threshold values of a value relating to fluorescence information and a value relating to cell size information, respectively. Can be set.
  • FIG. 4 shows a schematic diagram of a scattergram based on fluorescence information and cell size information obtained in the acquisition step.
  • the X axis is fluorescence information
  • the Y axis is cell information.
  • cancer cells mainly appear in the area A in FIG.
  • oncolytic viruses may grow on leukocytes. Therefore, white blood cells may show values related to fluorescence information similar to cancer cells.
  • the inventors' investigation has revealed that the value relating to the cell size information from leukocytes where the oncolytic virus grows is smaller than that of cancer cells. Therefore, white blood cells mainly appear in the region B in FIG.
  • oncolytic viruses do not grow in cells other than cancer cells and leukocytes and debris. Therefore, the values relating to fluorescence information of cells other than cancer cells and leukocytes and debris are extremely small.
  • cells other than cancer cells and leukocytes, debris, and the like mainly appear in the region C of FIG. That is, the cells in which the fluorescence information and the cell size information obtained in the acquisition step described above appear in the area A in FIG. 4 can be determined as cancer cells.
  • the computer 100 mainly includes a main body 110, a display unit 120, and an input unit 130.
  • a CPU 110a, a ROM 110b, a RAM 110c, a hard disk 110d, a reading device 110e, an input / output interface 110f, and an image output interface 110h are connected to each other via a bus 110i so that data communication is possible.
  • the CPU 110a can execute computer programs stored in the ROM 110b and computer programs loaded in the RAM 110c.
  • the ROM 110b is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and is recorded with a computer program executed by the CPU 110a and data used therefor.
  • the RAM 110c is configured by SRAM, DRAM, or the like. The RAM 110c is used for reading computer programs recorded in the ROM 110B and the hard disk 110d. Further, when these computer programs are executed, they are used as a work area of the CPU 110a.
  • the hard disk 110d is installed with various computer programs to be executed by the CPU 110a such as operating system and application system program, and data used for executing the computer program.
  • An application program 140a described later is also installed in the hard disk 110d.
  • the reading device 110e is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data recorded on the portable storage medium 140.
  • the portable storage medium 140 stores an application program 140a related to the acquisition process and the determination process in a computer.
  • the CPU 110a reads the application program 140a from the portable storage medium 140, and the application program 140a is stored in the hard disk 110d. It is also possible to install.
  • an operating system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by Microsoft Corporation in the United States is installed in the hard disk 110d.
  • Windows registered trademark
  • the application program 140a related to the acquisition process and the determination process described above operates on the operating system.
  • the input / output interface 110f includes, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter, an A / D converter, and the like. ing.
  • An input device 130 including a keyboard and a mouse is connected to the input / output interface 110f, and the user can input data to the computer main body 110 by using the input device 130.
  • the input / output interface 110f is connected to a fluorescence microscope 200 equipped with a CCD camera. Thereby, the computer main body 110 can acquire the imaged image data from the fluorescence microscope 200.
  • the image output interface 110h is connected to a display unit 120 constituted by an LCD, a CRT, or the like, and outputs a video signal corresponding to the image data given from the CPU 110a to the display unit 120.
  • the display unit 120 outputs image data according to the input video signal. Further, the display unit 120 outputs a determination result given from a CPU 110a described later.
  • FIG. 6 is an example of a flowchart showing processing for determining whether or not the cancer cell is executed by the CPU 110a.
  • image data obtained by imaging cells included in the measurement sample from the fluorescence microscope 200 is acquired by the CPU 110a via the input / output interface 110f and stored in the hard disk 110d (step S11).
  • the CPU 110a reads the application program 140a stored in the hard disk 110d, performs image processing on the image data, and executes processing for calculating the integral value of fluorescence intensity and the volume of cells as parameters (step S12). .
  • the CPU 110a determines whether or not the cell is a cancer cell based on the integrated value of the fluorescence intensity, which is the calculated parameter, and the cell volume (step S13).
  • the hard disk 110d stores the coordinates of the parameter area where the cancer cells appear in the area A of FIG. 7 with respect to the integrated value of the fluorescence intensity and the volume of the cell.
  • the CPU 110a determines whether or not the cell is a cancer cell from the integrated value of the fluorescence intensity, which is the calculated parameter, the volume of the cell, and the parameter area. Specifically, when each parameter regarding a cell enters the parameter area, it is determined that the cell is a cancer cell. If the cell is detached, it is determined that the cell is not a cancer cell. Then, the CPU 110a stores the scattergram shown in FIG. 7 as a determination result in the RAM 110c and outputs it to the display unit 120 via the image output interface 110f (step S14).
  • the scattergram is used as the determination result described above, but the determination result is not limited to this.
  • the determination result include the count number of cells determined to be cancer cells, image data of cells determined to be cancer cells, and a value (positive or negative) indicating the presence or absence of cancer cells in the measurement sample.
  • the determination result output to the display unit is not limited to one. At least one selected from the determination results can be output to the display unit.
  • the parameters of the fluorescence information and the cell size information are not limited thereto. Is not to be done.
  • the parameters of the fluorescence information include a peak value of fluorescence intensity, a minimum value of fluorescence intensity, and an average value of fluorescence intensity.
  • the cell size information parameters include cell area, cell perimeter, cell diameter, and cell roundness.
  • the parameter region of the integrated value of the fluorescence intensity and the cell area is used as the parameter region where cancer cells appear in the determination step performed by the computer. It is not limited to.
  • the parameter area for example, the peak value of the fluorescence intensity shown in the area A of FIG. 8 and the parameter area for the cell volume, the integrated value of the fluorescence intensity shown in the area A of FIG. 9 and the parameter area for the cell area, FIG.
  • the number of parameters used in the parameter area is not limited to two.
  • At least one selected from the parameters of the fluorescence information and at least one selected from the parameters of the cell size information can be used as parameters.
  • three parameters including a peak value of fluorescence intensity, an integrated value of fluorescence intensity, and a cell area can also be used as parameters used in the parameter region.
  • the example in which the fluorescence microscope 200 equipped with the CCD camera is connected to the computer 100 via the input / output interface 110f is shown, but these aspects are not limited thereto.
  • a flow cytometer 300 may be connected instead of the fluorescence microscope 200.
  • the computer main body 110 acquires data relating to fluorescence and data relating to electrical resistance and / or scattered light from the flow cytometer 300.
  • CPU110a calculates the parameter of fluorescence information from the acquired data regarding fluorescence.
  • parameters of cell size information are calculated from data relating to electrical resistance and / or scattered light.
  • the computer 100 and the flow cytometer 300 may be separate or integrated.
  • the measurement sample used in this example was prepared as follows.
  • a mononuclear cell that is one of white blood cells (WBC) was prepared as follows. 1. Into a 12-15 mL centrifuge tube, put 5.0 mL of Polymorphprep TM (Daiichi Kagaku), a reagent for separating mononuclear cells, and collect a vacuum blood collection tube (Benoject II vacuum blood collection tube, 3.8% citric acid) The sample was collected from 4 healthy individuals using 4.5 ml of sodium (manufactured by Terumo), and 5.0 mL of blood within 2 hours after the collection was overlaid. 2.
  • MB468 which is one of the breast cancer cells, was cultured in a culture solution (DMEM-F12 (Sigma) containing 10% FBS (Hyclone)) so as to be 90% confluent in a 125 cm 2 flask.
  • the obtained cultured cells were suspended in 1000 ⁇ l of DMEM-F12 so as to have about 3 ⁇ 10 5 cells to prepare MB468 cell samples.
  • RPMI-1640 was added to the obtained sample to make up to 10 ml.
  • Oncolytic virus Telomescan (registered trademark) (OBP-401) was added to a final concentration of 6 ⁇ 10 6 PFU / ml and cultured at 37 ° C. for 24 hours while rotating. The culture solution was centrifuged at 1500 rpm for 5 minutes with a weak brake, and the supernatant was removed to obtain cells on which oncolytic virus had grown.
  • PFA paraformaldehyde
  • Emulgen 2025G has the following structural formula:
  • n 25.
  • Imaging 7 types of beads (7 Peaks Beads (Cyto-Cal Multifluor Fluorescence Intensity Calibrator, Thermo Scientific, FC3M)) with known fluorescence intensities under exposure times of 20 ms, 50 ms, 100 ms, 200 ms and 400 ms A calibration curve for each exposure time was prepared.
  • the maximum fluorescence intensity (cell peak value (MEFL)) from the treated sample and the integration of the fluorescence intensity Values (MEFL), fluorescence distribution area (cell area (pixel 2 )), and fluorescence distribution perimeter (cell perimeter (pixel)) were digitized.
  • the cell volume (pixel 3 ) was calculated using the fluorescence distribution area assuming that the cell was a true sphere.
  • the cell diameter (pixel) was similarly calculated using the fluorescence distribution area.

Abstract

Provided is a method for detecting cancer cells in blood, which involves: processing, by means of an oncolytic virus which expresses fluorescent protein, the cells contained in a blood sample collected from a subject; obtaining fluorescence data of the cells and information regarding the size of the cells; and determining whether or not the cells are cancer cells on the basis of the obtained fluorescence data and information regarding the size of the cells. Also provided is a program used for said method.

Description

血液中癌細胞の検出方法及びそれに用いるプログラムMethod for detecting cancer cells in blood and program used therefor
　本発明は、被験者から採取された血液試料から癌細胞を検出するための方法及びそれに用いるプログラムに関する。 The present invention relates to a method for detecting cancer cells from a blood sample collected from a subject and a program used therefor.
　癌の転移は、原発巣の癌細胞が血管やリンパ管を通って全身に広がり、その一部が他部位の臓器などに生着して増殖することにより起こると考えられている。血液中を循環する癌細胞は、CTC(Circulating Tumor Cell)と呼ばれている。血液中のCTC数と癌の転移や予後とが相関することが報告されている。そのため、CTC数の測定が、転移性癌、例えば転移性乳癌の予後や治療効果を予測する方法として有用であると考えられている。 Cancer metastasis is considered to occur when cancer cells in the primary lesion spread throughout the body through blood vessels and lymphatic vessels, and some of them engraft in other parts of the organ. Cancer cells circulating in the blood are called CTC (CirculatingirTumor Cell). It has been reported that the number of CTCs in the blood correlates with cancer metastasis and prognosis. Therefore, measurement of the number of CTCs is considered useful as a method for predicting the prognosis and therapeutic effect of metastatic cancer such as metastatic breast cancer.
　癌細胞では、正常な細胞の大部分ではほとんど検出されないテロメラーゼの活性が亢進していることが知られている。そこで、テロメラーゼプロモーターを含む複製カセットと、標識タンパク質(例えば緑色蛍光タンパク質(GFP))の遺伝子を含む標識カセットとを保持するウイルス（腫瘍溶解性ウイルス(Oncolytic Virus)）を、癌細胞で増殖させて、癌細胞を特異的に標識する技術が知られている(特許文献１)。GFP遺伝子を有する腫瘍溶解性ウイルスは、テロメスキャン(登録商標)(OBP-401)として市販されている。テロメスキャン(登録商標)は、癌細胞で特異的に増殖し、GFPを産生することにより、癌細胞を特異的に蛍光発光させることができる。 Cancer cells are known to have increased telomerase activity that is hardly detected in most normal cells. Therefore, a virus (Oncolytic Virus) carrying a replication cassette containing a telomerase promoter and a labeled cassette containing a gene for a labeled protein (eg, green fluorescent protein (GFP)) is grown in cancer cells. A technique for specifically labeling cancer cells is known (Patent Document 1). An oncolytic virus having a GFP gene is commercially available as Telomescan (registered trademark) (OBP-401). Telomescan (registered trademark) can specifically grow in cancer cells and produce GFP, whereby the cancer cells can specifically emit fluorescence.
　このテロメスキャン(登録商標)(OBP-401)を利用したCTC検出方法として、テロメスキャン(登録商標)(OBP-401)を血液中の癌細胞に感染させ増殖させて、GFPの蛍光発光を利用することにより血液中の癌細胞を検出する方法が知られている(非特許文献１)。 As a CTC detection method using this telomescan (registered trademark) (OBP-401), telomescan (registered trademark) (OBP-401) is infected with cancer cells in blood and allowed to grow, and the fluorescence emission of GFP is used. Thus, a method for detecting cancer cells in blood is known (Non-patent Document 1).
国際公開公報２００６／３６００４号パンフレットInternational Publication No. 2006/36004 Pamphlet
　非特許文献１における、テロメスキャン(登録商標)(OBP-401)のような蛍光タンパク質を発現する腫瘍溶解性ウイルスを利用したCTC検出方法を用いれば、血液中の癌細胞、すなわちCTCを特異的に検出できると考えられる。 If the CTC detection method using an oncolytic virus that expresses a fluorescent protein such as Telomescan (registered trademark) (OBP-401) in Non-Patent Document 1, a cancer cell in blood, that is, CTC is specifically identified. Can be detected.
　しかしながら、発明者らが検討したところ、腫瘍溶解性ウイルスは、癌細胞だけではなく一部の正常細胞、例えば白血球(一部の単球及びリンパ球)にも感染し、増殖することがわかった。そのため、蛍光タンパク質を発現する腫瘍溶解性ウイルスを用いてCTCの検出を行うと、癌細胞以外の正常細胞においても蛍光強度が検出されることがある。結果として、正確に癌細胞を検出できない場合がある。 However, when the inventors examined, it was found that oncolytic virus infects not only cancer cells but also some normal cells such as leukocytes (some monocytes and lymphocytes) and proliferates. . Therefore, when CTC is detected using an oncolytic virus that expresses a fluorescent protein, the fluorescence intensity may be detected in normal cells other than cancer cells. As a result, cancer cells may not be detected accurately.
　したがって、本発明は、蛍光タンパク質を発現する腫瘍溶解性ウイルスを用いて、被験者から採取された血液試料に含まれる細胞が癌細胞か否かを正確に判定することのできる血液中癌細胞の検出方法及びそれに用いるプログラムを提供することを目的とする。 Therefore, the present invention uses an oncolytic virus that expresses a fluorescent protein to detect cancer cells in blood that can accurately determine whether cells contained in a blood sample collected from a subject are cancer cells. It is an object to provide a method and a program used therefor.
　本発明者らは、蛍光タンパク質を発現する腫瘍溶解性ウイルスを用い、細胞の蛍光情報と細胞の大きさ情報を利用することにより、細胞が癌細胞か否かを正確に判定できることを見出し、本発明を完成した。 The present inventors have found that it is possible to accurately determine whether a cell is a cancer cell by using an oncolytic virus that expresses a fluorescent protein and using the fluorescence information and cell size information of the cell. Completed the invention.
　すなわち、本発明は、被験者から採取された血液試料に含まれる細胞を、蛍光タンパク質を発現する腫瘍溶解性ウイルスで処理することにより、測定用試料を調製する工程と、前記測定用試料に含まれる細胞の蛍光情報及び細胞の大きさ情報を取得する工程と、取得工程で取得された蛍光情報及び細胞の大きさ情報に基づいて、細胞が癌細胞か否かを判定する工程とを含む、血液中癌細胞の検出方法を提供する。
　また、本発明は、コンピュータを、血液試料中の腫瘍溶解性ウイルスが感染し増殖した細胞の蛍光情報及び細胞の大きさ情報を取得する取得手段、前記取得手段で取得された情報に基づいて、細胞が癌細胞か否かを判定する判定手段、前記判定手段の判定結果を表示する表示手段、として機能させるための血液中癌細胞の検出プログラムを提供する。 That is, the present invention includes a step of preparing a measurement sample by treating cells contained in a blood sample collected from a subject with an oncolytic virus that expresses a fluorescent protein, and the measurement sample includes Blood, including a step of acquiring fluorescence information and cell size information of a cell, and a step of determining whether or not the cell is a cancer cell based on the fluorescence information and cell size information acquired in the acquisition step A method for detecting a middle cancer cell is provided.
Further, the present invention provides a computer based on the information acquired by the acquisition means, the acquisition means for acquiring fluorescence information and cell size information of cells that have been infected and proliferated by an oncolytic virus in a blood sample, Provided is a blood cancer cell detection program for functioning as determination means for determining whether or not a cell is a cancer cell, and display means for displaying the determination result of the determination means.
　本発明によれば、蛍光タンパク質を発現する腫瘍溶解性ウイルスを用いて、被験者から採取された血液試料に含まれる細胞が癌細胞か否かを正確に判定することができ、これによって癌細胞を精度良く検出することができる。 According to the present invention, it is possible to accurately determine whether a cell contained in a blood sample collected from a subject is a cancer cell using an oncolytic virus that expresses a fluorescent protein. It can be detected with high accuracy.
経時的な蛍光強度の波形の分析結果の一例を示す模式図である。It is a schematic diagram which shows an example of the analysis result of the waveform of the fluorescence intensity with time. 経時的な電気抵抗の波形の分析結果の一例を示す模式図である。It is a schematic diagram which shows an example of the analysis result of the waveform of an electrical resistance with time. 経時的な散乱光強度の波形の分析結果の一例を示す模式図である。It is a schematic diagram which shows an example of the analysis result of the waveform of scattered light intensity with time. 判定工程におけるスキャッタグラムの一例を示す模式図である。It is a schematic diagram which shows an example of the scattergram in a determination process. 取得工程及び判定工程を実施するコンピュータの一例を示す模式図である。It is a schematic diagram which shows an example of the computer which implements an acquisition process and a determination process. コンピュータによって実行される癌細胞か否かを判定する処理を示すフローチャートの一例である。It is an example of the flowchart which shows the process which determines whether it is a cancer cell performed by the computer. 実施例１における、Ｘ軸に積分値、Ｙ軸に体積を取ったスキャッタグラムである。FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and a volume on the Y axis. FIG. 実施例１における、Ｘ軸にピーク値、Ｙ軸に体積を取ったスキャッタグラムである。2 is a scattergram in Example 1 with a peak value on the X axis and a volume on the Y axis. 実施例１における、Ｘ軸に積分値、Ｙ軸に面積を取ったスキャッタグラムである。FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and an area on the Y axis. 実施例１における、Ｘ軸に積分値、Ｙ軸に直径を取ったスキャッタグラムである。FIG. 3 is a scattergram in Example 1 with an integral value on the X axis and a diameter on the Y axis. 実施例１における、Ｘ軸に積分値、Ｙ軸に周囲長を取ったスキャッタグラムである。FIG. 2 is a scattergram in Example 1 with an integral value on the X axis and a perimeter on the Y axis. 実施例１における、Ｘ軸に面積、Ｙ軸にピーク値、Ｚ軸に積分値を取った三次元頻度分布図である。In Example 1, it is a three-dimensional frequency distribution diagram which took the area on the X-axis, the peak value on the Y-axis, and the integrated value on the Z-axis.
　本実施形態の血液中癌細胞の検出方法は、
（１）被験者から採取された血液試料に含まれる細胞を、蛍光タンパク質を発現する腫瘍溶解性ウイルスで処理することにより、測定用試料を調製する工程（調製工程）；
（２）前記測定用試料に含まれる細胞の蛍光情報及び細胞の大きさ情報を取得する工程（取得工程）；
（３）取得工程で取得された蛍光情報及び細胞の大きさ情報に基づいて、細胞が癌細胞か否かを判定する工程（判定工程）
を含む。 The method for detecting cancer cells in blood according to the present embodiment is as follows.
(1) A step of preparing a measurement sample (preparation step) by treating cells contained in a blood sample collected from a subject with an oncolytic virus expressing a fluorescent protein;
(2) a step of acquiring fluorescence information and cell size information of cells contained in the measurement sample (acquisition step);
(3) A step of determining whether or not the cell is a cancer cell based on the fluorescence information and the cell size information acquired in the acquisition step (determination step)
including.
　本実施形態において、「血液試料」は、被験者から採取された血液、及び血液を処理して得られた処理血液のいずれかであってもよい。判定効率がより向上される点で、血液試料は、全血、特に末梢血から血清を除去したものが好ましい。被験者は、通常、癌に羅患したことが疑われる患者、癌に羅患している患者などの癌細胞を有するか否かを判定しようとする患者が挙げられる。 In this embodiment, the “blood sample” may be either blood collected from a subject or processed blood obtained by processing blood. The blood sample is preferably a blood sample obtained by removing serum from whole blood, particularly peripheral blood, in that the determination efficiency is further improved. Subjects include patients who are trying to determine whether they have cancer cells, such as patients suspected of suffering from cancer or patients suffering from cancer.
　全血から血清を除去する方法としては、公知の方法を用いることができる。例えば、抗凝固剤（例えばエチレンジアミン四酢酸、クエン酸ナトリウム、へパリンなど）を加えた全血を、遠心分離する方法が挙げられる。遠心分離は、５００ｒｐｍ～３５００ｒｐｍで、３～３０分間行うことが好ましい。 As a method for removing serum from whole blood, a known method can be used. For example, a method of centrifuging whole blood to which an anticoagulant (for example, ethylenediaminetetraacetic acid, sodium citrate, heparin, etc.) is added can be mentioned. Centrifugation is preferably performed at 500 rpm to 3500 rpm for 3 to 30 minutes.
　本実施形態において、蛍光タンパク質としては、生化学分野で通常用いられる公知のタンパク質を用いることができる。例えば、緑色蛍光タンパク質（ＧＦＰ）及びその変異体（例えばＥｎｈａｎｃｅｄ－ｈｕｍａｎｉｚｅｄ　ＧＦＰ（ＥＧＦＰ）、ｒｅｄ－ｓｈｉｆｔＧＦＰ（ｒｓＧＦＰ））、黄色蛍光タンパク質（ＹＦＰ）、青色蛍光タンパク質（ＢＦＰ）などの蛍光タンパク質などを用いることができる。本実施形態では、蛍光タンパク質は、緑色蛍光タンパク質が好ましい。 In the present embodiment, as the fluorescent protein, a known protein that is usually used in the biochemical field can be used. For example, fluorescent proteins such as green fluorescent protein (GFP) and variants thereof (for example, Enhanced-humanized GFP (EGFP), red-shiftGFP (rsGFP)), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), etc. are used. be able to. In the present embodiment, the fluorescent protein is preferably a green fluorescent protein.
　本実施形態において、「腫瘍溶解性ウイルス」とは、正常細胞内では増殖できず、癌細胞内で特異的に増殖可能な制限増殖型ウイルスである。腫瘍溶解性ウイルスとしては、特に限定されないが、癌細胞において特異的にプロモーター活性を示すプロモーターが組み込まれたウイルスが挙げられる。癌細胞において特異的にプロモーター活性を示すプロモーター（以下、「癌細胞特異的プロモーター」ともいう）としては、ヒトテロメラーゼプロモーター、ヒト前立腺癌特異抗原(ＰＳＡ)プロモーター、ヒトアルファプロテイン（ＡＦＰ）プロモーター、胎児性癌抗原（ＣＥＡ）プロモーターなどが挙げられる。多種類の癌細胞でプロモーター活性を示すことができる点で、ヒトテロメラーゼプロモーターが好ましい。なかでも、ヒトテロメラーゼ逆転写酵素をコードする遺伝子であるｈＴＥＲＴのプロモーターがより好ましい。 In the present embodiment, the “oncolytic virus” is a restricted-proliferating virus that cannot propagate in normal cells but can specifically propagate in cancer cells. Although it does not specifically limit as oncolytic virus, The virus in which the promoter which shows a promoter activity specifically in a cancer cell was integrated is mentioned. Examples of promoters that exhibit specific promoter activity in cancer cells (hereinafter also referred to as “cancer cell-specific promoters”) include human telomerase promoter, human prostate cancer specific antigen (PSA) promoter, human alpha protein (AFP) promoter, fetus Sex cancer antigen (CEA) promoter and the like. The human telomerase promoter is preferred in that it can exhibit promoter activity in many types of cancer cells. Of these, the hTERT promoter, which is a gene encoding human telomerase reverse transcriptase, is more preferred.
　配列番号１は、ｈＴＥＲＴプロモーターの核酸配列である。ｈＴＥＲＴプロモーターとしては、配列番号１に示す４５５ｂｐの核酸配列の全体を用いてもよいが、配列番号１の５’上流の１８１ｂｐの領域が、下流の遺伝子発現に重要なコア領域であると考えられているので、このコア領域を少なくとも含む核酸配列を用いればよい。 SEQ ID NO: 1 is the nucleic acid sequence of the hTERT promoter. As the hTERT promoter, the entire 455 bp nucleic acid sequence shown in SEQ ID NO: 1 may be used, but the 181 bp region 5 ′ upstream of SEQ ID NO: 1 is considered to be an important core region for downstream gene expression. Therefore, a nucleic acid sequence containing at least this core region may be used.
　上記の腫瘍溶解性ウイルスは、蛍光タンパク質を発現する。すなわち、上記の腫瘍溶解性ウイルスは、蛍光タンパク質をコードする遺伝子を有する。 The above-mentioned oncolytic virus expresses a fluorescent protein. That is, the oncolytic virus has a gene encoding a fluorescent protein.
　上記の蛍光タンパク質の遺伝子は、上記の癌細胞特異的プロモーターの制御下に置くことができる。
　また、上記の蛍光タンパク質の遺伝子は、腫瘍溶解性ウイルス内でプロモーター活性を示し得るプロモーターの制御下におくこともできる。蛍光タンパク質遺伝子の発現を制御し得るプロモーターとしては、サイトメガロウイルス（ＣＭＶ）プロモーター、ＳＶ４０後期プロモーター、ＭＭＴＶ　ＬＴＲプロモーター、ＲＳＶ　ＬＴＲプロモーター、ＳＲαプロモーターなどが挙げられる。
　本実施例では、蛍光タンパク質の遺伝子は、ＣＭＶプロモーター又はｈＴＥＲＴプロモーターの制御下におくことが好ましい。
　これらのプロモーターの核酸配列は、公知である。 The gene for the fluorescent protein can be placed under the control of the cancer cell-specific promoter.
In addition, the above-mentioned fluorescent protein gene can be placed under the control of a promoter capable of exhibiting promoter activity in an oncolytic virus. Examples of promoters that can control the expression of the fluorescent protein gene include cytomegalovirus (CMV) promoter, SV40 late promoter, MMTV LTR promoter, RSV LTR promoter, SRα promoter, and the like.
In this example, the fluorescent protein gene is preferably placed under the control of a CMV promoter or hTERT promoter.
The nucleic acid sequences of these promoters are known.
　上記の蛍光タンパク質をコードする遺伝子と、それを発現するためのプロモーター（癌細胞特異的プロモーター又は腫瘍溶解性ウイルス内でプロモーター活性を示し得るプロモーター）とを少なくとも含む発現カセットは、通常の遺伝子工学的手法により得ることができる。例えば、蛍光タンパク質をコードする遺伝子、及びプロモーターを、公知の配列に基づいて、ＰＣＲなどにより増幅させ、得られた各遺伝子を、適切なプラスミドに連結し、必要な部分を切り出すことにより、発現カセットを得ることができる。例えば、国際公開２００６／３６００４号を参照されたい。 An expression cassette comprising at least a gene encoding the above fluorescent protein and a promoter for expressing it (a cancer cell-specific promoter or a promoter capable of exhibiting promoter activity in an oncolytic virus) is a common genetic engineering It can be obtained by a technique. For example, a gene encoding a fluorescent protein and a promoter are amplified by PCR or the like based on a known sequence, each gene obtained is ligated to an appropriate plasmid, and a necessary portion is excised, thereby expressing an expression cassette. Can be obtained. See, for example, International Publication No. 2006/36004.
　上記の腫瘍溶解性ウイルスは、上記の癌細胞特異的プロモーターの下流に、ウイルスの増殖に必要な遺伝子を連結した複製カセットを有するものであってもよい。ウイルスの増殖に必要な遺伝子としては、初期遺伝子（ＥＩＡ、ＥＩＢなど）、ピコルナウイルス科に特異的なタンパク質合成開始シグナルであるＩＲＥＳなどが挙げられる。これらの遺伝子の核酸配列は公知である。 The above-mentioned oncolytic virus may have a replication cassette in which a gene required for virus growth is linked downstream of the above cancer cell-specific promoter. Examples of genes necessary for virus growth include early genes (EIA, EIB, etc.), IRES that are protein synthesis initiation signals specific to the Picornaviridae family, and the like. The nucleic acid sequences of these genes are known.
　上記の複製カセットは、通常の遺伝子工学的手法により作製できる。例えば、上記の癌細胞特異的プロモーター、及び所望によりウイルスの増殖に必要な遺伝子を、公知の配列に基づいて、ポリメラーゼ連鎖反応（ＰＣＲ）などにより増幅させ、得られた各遺伝子を、適切なプラスミドに連結し、必要な部分を切り出すことにより、複製カセットを得ることができる。例えば、国際公開２００６／３６００４号を参照されたい。 The above replication cassette can be prepared by a normal genetic engineering technique. For example, the above cancer cell-specific promoter and, if necessary, a gene necessary for virus growth are amplified by polymerase chain reaction (PCR) or the like based on a known sequence, and each of the obtained genes is converted into an appropriate plasmid. The replication cassette can be obtained by linking to and cutting out the necessary part. See, for example, International Publication No. 2006/36004.
　上記のウイルスとしては、アデノウイルス、単純ヘルペスウイルス、センダイウイルス、レオウイルスに由来するものが挙げられる。本実施形態では、ヒトアデノウイルスが好ましい。 Examples of the virus include those derived from adenovirus, herpes simplex virus, Sendai virus, and reovirus. In this embodiment, human adenovirus is preferred.
　上記の腫瘍溶解性ウイルスは、市販のものを用いることもできる。本明細書における腫瘍溶解性ウイルスとしては、ｈＴＥＲＴプロモーター、Ｅ１Ａ遺伝子、ＩＲＥＳ遺伝子及びＥ１Ｂ遺伝子を含む複製カセットと、ＣＭＶプロモーター及びＧＦＰ遺伝子を含む発現カセットとを有するアデノウイルスであるテロメスキャン（登録商標）（ＯＢＰ－４０１）（オンコリスバイオフォーマ社製）が好ましい。 Commercially available oncolytic viruses can be used. As the oncolytic virus in the present specification, Telomescan (registered trademark), which is an adenovirus having a replication cassette containing the hTERT promoter, E1A gene, IRES gene and E1B gene, and an expression cassette containing the CMV promoter and GFP gene. (OBP-401) (manufactured by Oncoris Bioformer) is preferred.
（１）調製工程
　本実施形態は、血液試料に含まれる細胞を、蛍光タンパク質を発現する腫瘍溶解性ウイルスで処理することにより、測定用試料を調製する工程（以下、「調製工程」ということがある）を含む。血液試料に含まれる細胞を、腫瘍溶解性ウイルスで処理することで、蛍光タンパク質を発現する腫瘍溶解性ウイルスを、血液試料中の癌細胞に感染させる。これにより、蛍光タンパク質を癌細胞内で発現させることができる。腫瘍溶解性ウイルスによる、血液試料に含まれる細胞の処理は、公知の手法により行ってよく、特に制限されない。例えば、動物細胞培養に通常使用される培地の存在下で、血液試料と腫瘍溶解性ウイルスとをインキュベートすることにより、腫瘍溶解性ウイルスを、血液試料中の癌細胞に感染させることができる。血液試料と腫瘍溶解性ウイルスのインキュベートに用いる培地としては、例えば、ＲＰＭＩ－１６４０、ダルベッコ改変イーグル培地（ＤＭＥＭ）、最少必須培地（ＭＥＭ）などが挙げられる。 (1) Preparation Step This embodiment is a step of preparing a measurement sample by treating cells contained in a blood sample with an oncolytic virus that expresses a fluorescent protein (hereinafter referred to as “preparation step”). Included). By treating cells contained in the blood sample with an oncolytic virus, the oncolytic virus expressing the fluorescent protein is infected with cancer cells in the blood sample. Thereby, fluorescent protein can be expressed in cancer cells. The treatment of the cells contained in the blood sample with the oncolytic virus may be performed by a known method, and is not particularly limited. For example, an oncolytic virus can be infected with cancer cells in a blood sample by incubating the blood sample with an oncolytic virus in the presence of a medium commonly used for animal cell culture. Examples of the medium used for incubating the blood sample and oncolytic virus include RPMI-1640, Dulbecco's modified Eagle medium (DMEM), minimal essential medium (MEM), and the like.
　血液試料と腫瘍溶解性ウイルスとをインキュベートする場合、腫瘍溶解性ウイルスを、血液試料１ｍｌあたり６×１０4～６×１０8ＰＦＵ（Ｐｌａｑｕｅ　Ｆｏｒｍｉｎｇ　Ｕｎｉｔ）の量で、血液試料とインキュベートすることが好ましい。上記の血液試料を、腫瘍溶解性ウイルスとインキュベーションする時間及び温度は、腫瘍溶解性ウイルスが血液試料中の癌細胞で増殖できる条件であればよく、腫瘍溶解性ウイルスの種類により適宜調節できる。本実施形態において、インキュベーションは、２５～４０℃の温度で、１～３６時間、より好ましくは３０～３７℃にて１２～２４時間行うことができる。 When incubating a blood sample with an oncolytic virus, it is preferable to incubate the oncolytic virus with the blood sample in an amount of 6 × 10 4 to 6 × 10 8 PFU (Plaque Forming Unit) per ml of blood sample. . The time and temperature at which the above blood sample is incubated with the oncolytic virus may be any conditions as long as the oncolytic virus can grow on cancer cells in the blood sample, and can be appropriately adjusted depending on the type of oncolytic virus. In this embodiment, the incubation can be performed at a temperature of 25 to 40 ° C. for 1 to 36 hours, more preferably at 30 to 37 ° C. for 12 to 24 hours.
　本実施形態の調製工程は、腫瘍溶解性ウイルスで処理した血液試料を、非イオン性界面活性剤及び固定化剤と混合することをさらに含み得る。血液試料中に存在する白血球（単球、リンパ球など）は、テロメラーゼのような癌細胞で特異的に発現する酵素を稀に発現する。そのため、血液試料に含まれる細胞を、蛍光タンパク質を発現する腫瘍溶解性ウイルスで処理すると、正常な白血球で腫瘍溶解性ウイルスが増殖する場合がある。結果として、正常な白血球から、蛍光タンパク質由来の蛍光が生じることがある。従って、後述する取得工程において、ノイズとなる蛍光情報が増えてしまう。ここで、腫瘍溶解性ウイルスで処理した血液試料を、非イオン性界面活性剤及び固定化剤と混合すると、正常な白血球などに損傷を与え、かつ、赤血球を溶血することができる。これにより、取得工程におけるノイズを減らすことができる。結果として、後述する判定工程において、細胞が癌細胞か否かをより正確に判定することが可能となる。 The preparation step of the present embodiment may further include mixing a blood sample treated with an oncolytic virus with a nonionic surfactant and a fixing agent. Leukocytes (monocytes, lymphocytes, etc.) present in blood samples rarely express enzymes that are specifically expressed in cancer cells such as telomerase. Therefore, when cells contained in a blood sample are treated with an oncolytic virus that expresses a fluorescent protein, the oncolytic virus may grow on normal white blood cells. As a result, fluorescent protein-derived fluorescence may occur from normal white blood cells. Therefore, in the acquisition process to be described later, fluorescence information that becomes noise increases. Here, when a blood sample treated with an oncolytic virus is mixed with a nonionic surfactant and a fixing agent, normal white blood cells and the like are damaged, and red blood cells can be hemolyzed. Thereby, the noise in an acquisition process can be reduced. As a result, it becomes possible to more accurately determine whether or not the cell is a cancer cell in the determination step described later.
　ここで、腫瘍溶解性ウイルスで処理した血液試料と、非イオン性界面活性剤及び固定化剤との混合の順序は、特に制限されない。例えば、非イオン性界面活性剤と腫瘍溶解性ウイルスで処理した血液試料とを混合した後に、固定化剤を混合してもよい。また、固定化剤と腫瘍溶解性ウイルスで処理した血液試料とを混合した後に、非イオン性界面活性剤を混合してもよい。さらに、非イオン性界面活性剤、固定化剤及び腫瘍溶解性ウイルスで処理した血液試料を同時に混合してもよい。本実施形態において、固定化剤と腫瘍溶解性ウイルスで処理した血液試料とを混合した後に、非イオン性界面活性剤を混合する順序がより好ましい。 Here, the order of mixing the blood sample treated with the oncolytic virus with the nonionic surfactant and the immobilizing agent is not particularly limited. For example, the immobilizing agent may be mixed after mixing a nonionic surfactant and a blood sample treated with oncolytic virus. Further, after mixing the immobilizing agent and the blood sample treated with the oncolytic virus, a nonionic surfactant may be mixed. Furthermore, a blood sample treated with a nonionic surfactant, an immobilizing agent and an oncolytic virus may be mixed simultaneously. In the present embodiment, the order of mixing the nonionic surfactant after mixing the immobilizing agent and the blood sample treated with the oncolytic virus is more preferable.
　上記の非イオン性界面活性剤は、ポリオキシエチレン系界面活性剤、及びポリオキシ化合物の脂肪酸エステル系界面活性剤のいずれであってもよいが、ポリオキシエチレン系の非イオン性界面活性剤がより好ましい。ポリオキシエチレン系の非イオン性界面活性剤としては、高級アルコール・エチレンオキシド付加物、アルキルフェノール・エチレンオキシド付加物、高級脂肪酸・エチレンオキシド付加物、高級脂肪族アミン・エチレンオキシド付加物、高級脂肪酸アミド・エチレンオキシド付加物などが挙げられる。本実施形態における非イオン性界面活性剤としては、高級アルコール・エチレンオキシド付加物が好ましい。 The nonionic surfactant may be any one of a polyoxyethylene surfactant and a fatty acid ester surfactant of a polyoxy compound, but a polyoxyethylene nonionic surfactant is more preferable. preferable. Polyoxyethylene nonionic surfactants include higher alcohol / ethylene oxide adducts, alkylphenol / ethylene oxide adducts, higher fatty acids / ethylene oxide adducts, higher aliphatic amines / ethylene oxide adducts, higher fatty acid amide / ethylene oxide adducts. Etc. The nonionic surfactant in the present embodiment is preferably a higher alcohol / ethylene oxide adduct.
　高級アルコール・エチレンオキシド付加物の高級アルコールのアルキル基は、直鎖状又は分枝鎖状のいずれであってもよく、炭素数が１２～２０が好ましく、より好ましくは１５～２０である。高級アルコール・エチレンオキシド付加物は、エチレンオキシドの重合数が１５～４０のものが好ましく、より好ましくは２０～３０である。本実施形態における非イオン性界面活性剤としては、高級アルコールのアルキル基が分岐鎖であり、炭素数が１８であり、エチレンオキシドの重合数が２５であるポリオキシエチレンオクチルドデシルエーテルがより好ましい。 The alkyl group of the higher alcohol of the higher alcohol / ethylene oxide adduct may be linear or branched, and preferably has 12 to 20 carbon atoms, more preferably 15 to 20 carbon atoms. The higher alcohol / ethylene oxide adduct preferably has an ethylene oxide polymerization number of 15 to 40, more preferably 20 to 30. As the nonionic surfactant in the present embodiment, polyoxyethylene octyldodecyl ether in which the alkyl group of the higher alcohol is branched, the number of carbon atoms is 18, and the polymerization number of ethylene oxide is 25 is more preferable.
　血液試料及び固定化剤と混合したときの非イオン性界面活性剤の濃度は、非イオン性界面活性剤の種類により適宜選択できるが、０．００５～０．５重量％が好ましく、より好ましくは０．０４５～０．０５５重量％である。 The concentration of the nonionic surfactant when mixed with the blood sample and the fixing agent can be appropriately selected depending on the kind of the nonionic surfactant, but is preferably 0.005 to 0.5% by weight, more preferably 0.045 to 0.055% by weight.
　上記の固定化剤としては、組織の固定のために通常用いられるタンパク質を架橋させる物質を用いることができ、パラホルムアルデヒド、グルタルアルデヒド、ホルムアルデヒドなどのアルデヒド化合物が好ましい。なかでも、パラホルムアルデヒドがより好ましい。 As the above-mentioned fixing agent, a substance that crosslinks proteins usually used for tissue fixation can be used, and aldehyde compounds such as paraformaldehyde, glutaraldehyde, and formaldehyde are preferable. Of these, paraformaldehyde is more preferable.
　血液試料及び非イオン性界面活性剤と混合したときの固定化剤の濃度は、固定化剤の種類により適宜選択できるが、０．００５～０．５重量％が好ましく、０．０４５～０．０５５重量％がより好ましい。 The concentration of the fixing agent when mixed with the blood sample and the nonionic surfactant can be appropriately selected depending on the type of the fixing agent, but is preferably 0.005 to 0.5% by weight, and preferably 0.045 to 0.8%. 055% by weight is more preferred.
また、上記の混合は、ｐＨ６～９、浸透圧２００～４００m0smで行うことが好ましい。 The above mixing is preferably performed at pH 6 to 9 and osmotic pressure 200 to 400 m0sm.
（２）取得工程
　本実施形態は、調製工程で得られた測定用試料に含まれる細胞の蛍光情報及び細胞の大きさ情報を取得する工程（以下、「取得工程」ともいう）を含む。 (2) Acquisition Step This embodiment includes a step of acquiring cell fluorescence information and cell size information contained in the measurement sample obtained in the preparation step (hereinafter also referred to as “acquisition step”).
　本実施形態における「蛍光情報」とは、蛍光タンパク質の発する蛍光から得られる情報である。例えば、蛍光タンパク質が発する蛍光から検出した蛍光強度、蛍光強度のピーク値、蛍光強度の最小値、蛍光強度の平均値、蛍光強度の積分値などの蛍光強度から導かれる算出された数値などが挙げられる。本実施形態における蛍光情報としては、蛍光強度のピーク値、蛍光強度の積分値が好ましい。 “Fluorescence information” in the present embodiment is information obtained from fluorescence emitted by a fluorescent protein. For example, the fluorescence intensity detected from the fluorescence emitted by the fluorescent protein, the peak value of the fluorescence intensity, the minimum value of the fluorescence intensity, the average value of the fluorescence intensity, the calculated numerical value derived from the fluorescence intensity such as the integrated value of the fluorescence intensity, etc. It is done. As fluorescence information in the present embodiment, a peak value of fluorescence intensity and an integral value of fluorescence intensity are preferable.
　細胞の蛍光情報は、公知の手法で取得できる。より具体的には、蛍光顕微鏡、フローサイトメータなどを用いて、細胞の蛍光情報を取得することができる。 Cell fluorescence information can be obtained by a known method. More specifically, the fluorescence information of a cell can be acquired using a fluorescence microscope, a flow cytometer, or the like.
　例えば、蛍光顕微鏡を用いて、細胞の蛍光情報を取得する場合、まず上述の調製工程で得られた測定用試料をスライドグラスに塗抹して、塗抹標本を作製する。この際、細胞が重層しないようスライドを作成することが好ましい。 For example, when acquiring fluorescence information of cells using a fluorescence microscope, first, a measurement sample obtained in the above-described preparation step is smeared on a slide glass to prepare a smear. At this time, it is preferable to create a slide so that cells do not overlap.
　次に、上述で作製した塗抹標本に存在する、細胞における蛍光を、ＣＣＤカメラを備えた蛍光顕微鏡で観察する。ここで、蛍光顕微鏡が照射する励起光は、用いる蛍光タンパク質の種類により適宜選択することができる。例えば、蛍光タンパク質として、ＧＦＰを用いる場合、緑色光（Ｇ励起・５４６ｎｍ）を励起光として用いることができる。 Next, the fluorescence in the cells present in the smear prepared as described above is observed with a fluorescence microscope equipped with a CCD camera. Here, the excitation light irradiated by the fluorescence microscope can be appropriately selected depending on the type of fluorescent protein used. For example, when GFP is used as the fluorescent protein, green light (G excitation / 546 nm) can be used as excitation light.
　次に、蛍光顕微鏡のＣＣＤカメラを用いて、上述の細胞における蛍光を撮像する。 Next, the fluorescence in the above-described cells is imaged using a CCD camera of a fluorescence microscope.
　次に、撮像により得られた画像を画像処理することで、上述の細胞の蛍光情報を取得する。ここで、蛍光情報を取得するための画像処理は、蛍光情報の種類に応じ、適宜行うことができる。より具体的には、蛍光情報は、撮像により得られた画像を、画像処理用のコンピュータプログラムがインストールされたコンピュータを用いて処理することにより取得できる。画像処理用のコンピュータプログラムは公知であり、このようなコンピュータプログラムが記憶された記憶媒体（画像処理ソフト）も一般に市販されている。画像処理ソフトとしては、例えば、ＩｍａｇｅＰｒｏ（登録商標）（ローパーインダストリーズ社製）、ＷｉｎＲＯＯＦ（登録商標）（三谷商事）、及びＭｅｔａＭｏｒｐｈ（登録商標）（Ｍｏｌｅｃｕｌａｒ　Ｄｅｃｉｃｅｓ）などが挙げられる。 Next, the above-described fluorescence information of the cell is acquired by performing image processing on an image obtained by imaging. Here, the image processing for acquiring the fluorescence information can be appropriately performed according to the type of the fluorescence information. More specifically, the fluorescence information can be acquired by processing an image obtained by imaging using a computer in which a computer program for image processing is installed. Computer programs for image processing are well known, and storage media (image processing software) in which such computer programs are stored are generally commercially available. Examples of the image processing software include ImagePro (registered trademark) (manufactured by Roper Industries), WinROOF (registered trademark) (Mitani Corporation), and MetaMorph (registered trademark) (Molecular Devices).
　例えば、ＩｍａｇｅＰｒｏを用いて、蛍光強度のピーク値を取得する場合、上述の画像において選択された範囲内の輝度値を持つピクセルのうち、最も大きい輝度を有するピクセルの輝度（最大輝度）を蛍光強度のピーク値として取得する。また、蛍光強度の積分値を取得する場合、上述の画像において選択された範囲内の輝度値を持つ各ピクセルの輝度を積算した値を蛍光強度の積分値として取得する。 For example, when acquiring the peak value of the fluorescence intensity using ImagePro, the brightness (maximum brightness) of the pixel having the highest brightness among the pixels having the brightness values within the range selected in the above image is set to the fluorescence intensity. Get as the peak value of. Further, when acquiring the integrated value of the fluorescence intensity, a value obtained by integrating the luminance of each pixel having the luminance value within the range selected in the above-described image is acquired as the integrated value of the fluorescence intensity.
　また、細胞の蛍光情報をフローサイトメータで取得する場合、用い得るフローサイトメータは、蛍光の検出が可能なフローサイトメータである。
　例えば、フローセル、光源、蛍光を検出する検出部（蛍光検出部）、蛍光検出部から出力されるデータを分析する分析部などを備える従来公知のフローサイトメータを用いることができる。 Moreover, when acquiring the fluorescence information of a cell with a flow cytometer, the flow cytometer which can be used is a flow cytometer which can detect fluorescence.
For example, a conventionally known flow cytometer including a flow cell, a light source, a detection unit (fluorescence detection unit) that detects fluorescence, an analysis unit that analyzes data output from the fluorescence detection unit, and the like can be used.
　上記のフローサイトメータのフローセルを通過する測定用試料に、光源からの光を照射する。ここで、フローサイトメータの光源は、用いる蛍光タンパク質の励起に好適な波長を有するものであればよい。例えば、蛍光タンパク質としてGFPを用いる場合、２９５～４９５ｎｍの光を励起光として用いることができる。具体的な光源としては、例えば、半導体レーザ、アルゴンレーザ、Ｈｅ－Ｎｅレーザなどが挙げられる。 ¡A measurement sample passing through the flow cell of the flow cytometer is irradiated with light from a light source. Here, the light source of the flow cytometer only needs to have a wavelength suitable for excitation of the fluorescent protein to be used. For example, when GFP is used as the fluorescent protein, light of 295 to 495 nm can be used as excitation light. Specific examples of the light source include a semiconductor laser, an argon laser, and a He—Ne laser.
　上記の光源からの光を照射された測定用試料からの蛍光は、蛍光検出部によって検出される。蛍光検出部は、フローサイトメータにおいて一般的に用いられる、蛍光の検出が可能な受光素子を有し得る。蛍光検出部の具体例としては、光電子増倍管やアバランシェフォトダイオードなどが挙げられる。蛍光検出部によって検出する蛍光の波長は、用いる蛍光タンパク質の種類により適宜選択することができる。例えば、蛍光タンパク質として、GFPを用いる場合、４８０～５８０ｎｍの蛍光を、蛍光検出部で検出すればよい。 Fluorescence from the measurement sample irradiated with light from the light source is detected by the fluorescence detection unit. The fluorescence detection unit may include a light receiving element that is generally used in a flow cytometer and can detect fluorescence. Specific examples of the fluorescence detection unit include a photomultiplier tube and an avalanche photodiode. The wavelength of fluorescence detected by the fluorescence detection unit can be appropriately selected depending on the type of fluorescent protein used. For example, when GFP is used as the fluorescent protein, fluorescence at 480 to 580 nm may be detected by the fluorescence detection unit.
　蛍光検出部により検出された蛍光は、電気信号として分析部に出力される。分析部は、蛍光検出部から出力された電気信号を用いて分析を行い、蛍光情報を取得する。例えば、分析部は、蛍光検出部から電気信号が出力されたときの蛍光強度を求めることで、経時的な蛍光強度の波形の分析ができる。この分析部による、経時的な蛍光強度の波形の分析結果の一例を、図１に示す。図１において、縦軸は蛍光強度を示し、横軸は検出時間を示す。この場合、例えば蛍光強度の波形の最大の高さを、蛍光強度のピーク値として取得することができる。また、蛍光強度の波形の面積値を、蛍光強度の積分値として取得することもできる。 Fluorescence detected by the fluorescence detection unit is output to the analysis unit as an electrical signal. The analysis unit performs analysis using the electrical signal output from the fluorescence detection unit, and acquires fluorescence information. For example, the analysis unit can analyze the waveform of the fluorescence intensity over time by obtaining the fluorescence intensity when an electrical signal is output from the fluorescence detection unit. An example of the analysis result of the waveform of the fluorescence intensity over time by this analysis unit is shown in FIG. In FIG. 1, the vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the detection time. In this case, for example, the maximum height of the fluorescence intensity waveform can be acquired as the peak value of the fluorescence intensity. Further, the area value of the waveform of the fluorescence intensity can be acquired as an integrated value of the fluorescence intensity.
　本実施形態における「細胞の大きさ情報」とは、細胞の大きさを反映する情報であれば、特に制限されない。細胞の大きさ情報は、例えば、細胞の直径、細胞の面積、細胞の体積、細胞の周囲長、真円度などが挙げられる。本実施形態おける細胞の大きさ情報としては、細胞の直径、細胞の面積、細胞の体積、細胞の周囲長が好ましい。 The “cell size information” in the present embodiment is not particularly limited as long as it is information reflecting the cell size. Examples of the cell size information include a cell diameter, a cell area, a cell volume, a cell perimeter, and a roundness. As cell size information in the present embodiment, cell diameter, cell area, cell volume, and cell perimeter are preferred.
　細胞の大きさ情報は、公知の手法で取得できる。より具体的には、蛍光顕微鏡、フローサイトメータなどを用いて、細胞の大きさ情報を取得することができる。 The cell size information can be obtained by a known method. More specifically, cell size information can be acquired using a fluorescence microscope, a flow cytometer, or the like.
　例えば、蛍光顕微鏡を用いて、細胞の大きさ情報を取得する場合、上述した蛍光情報を取得する場合と同様に、撮像により得られた画像を市販の画像処理ソフトで画像処理することにより、細胞の大きさ情報を取得することができる。 For example, when cell size information is acquired using a fluorescence microscope, an image obtained by imaging is image-processed with commercially available image processing software in the same manner as in the case of acquiring fluorescence information described above. Size information can be acquired.
　例えば、ＩｍａｇｅＰｒｏを用いて、細胞の直径を取得する場合、上述の画像において細胞の占める面積を測定して平均値を算出し、細胞が球状と仮定したときの直径に換算する。また、細胞の面積を取得する場合は、上述の画像において選択された範囲内の輝度値を持つピクセルからなる面積を測定する。また、細胞の体積を取得する場合は、上述の画像において細胞の3次元画像を構築し、体積を測定する。また、細胞の周囲長を取得する場合は、細胞の外周（アウトライン）の長さを測定する。 For example, when acquiring the diameter of a cell using ImagePro, the area occupied by the cell in the above-mentioned image is measured to calculate an average value and converted to a diameter when the cell is assumed to be spherical. When acquiring the area of a cell, the area consisting of pixels having a luminance value within the range selected in the above-mentioned image is measured. Moreover, when acquiring the volume of a cell, the three-dimensional image of a cell is constructed | assembled in the above-mentioned image, and a volume is measured. Moreover, when acquiring the perimeter of a cell, the length of the outer periphery (outline) of a cell is measured.
　また、細胞の大きさ情報をフローサイトメータで取得する場合、用い得るフローサイトメータとしては、例えば、電気的又は光学的測定により細胞の大きさ情報を取得できる従来公知のフローサイトメータが挙げられる。より具体的には、電気抵抗により細胞の大きさ情報が取得できる電気抵抗式フローサイトメータ、又は散乱光により細胞の大きさ情報が取得できる光学式フローサイトメータなどが挙げられる。また、電気的及び光学的測定の両方を行うことができるフローサイトメータを用いることもできる。 Moreover, when acquiring cell size information with a flow cytometer, examples of the flow cytometer that can be used include conventionally known flow cytometers that can acquire cell size information by electrical or optical measurement. . More specifically, an electric resistance type flow cytometer that can acquire cell size information by electric resistance, an optical flow cytometer that can acquire cell size information by scattered light, and the like can be mentioned. A flow cytometer that can perform both electrical and optical measurements can also be used.
　細胞の大きさ情報を取得するための電気抵抗式フローサイトメータは、上記の測定用試料を導入するオリフィスと、オリフィスを介して電解液によって電気的に接続された一対の電極を備えた測定部と、分析部を備える。上記のフローサイトメータのオリフィスに測定用試料が通過する際に生じる電極間電気抵抗は、フローサイトメータの測定部により測定される。 An electric resistance type flow cytometer for acquiring cell size information is a measuring unit comprising an orifice for introducing the above-described measurement sample and a pair of electrodes electrically connected by an electrolyte through the orifice. And an analysis unit. The interelectrode electrical resistance generated when the measurement sample passes through the orifice of the flow cytometer is measured by the measurement unit of the flow cytometer.
　測定部により測定された電極間電気抵抗は、電気信号としてフローサイトメータの分析部に出力される。分析部は、測定部から出力された電気信号に基づいて分析を行い、細胞の大きさ情報を取得する。例えば、分析部は、測定部から電気信号が出力されたときの電気抵抗を求めることで、経時的な電気抵抗の波形の分析ができる。この分析部による、経時的な電気抵抗の波形の分析結果の一例を、図２に示す。図２において、縦軸は電気抵抗を示し、横軸は検出時間を示す。一般に、電気抵抗の大きさは細胞の体積に強く相関することが知られている。この場合、例えば電気抵抗の波形の最大の高さを、細胞の体積として取得することができる。また、得られた細胞の体積から、細胞の直径、細胞の面積及び細胞の周囲長を取得することもできる。 The inter-electrode electrical resistance measured by the measurement unit is output as an electrical signal to the analysis unit of the flow cytometer. The analysis unit performs analysis based on the electrical signal output from the measurement unit, and acquires cell size information. For example, the analysis unit can analyze the waveform of the electrical resistance over time by obtaining the electrical resistance when the electrical signal is output from the measurement unit. An example of the analysis result of the electrical resistance waveform over time by the analysis unit is shown in FIG. In FIG. 2, the vertical axis represents electric resistance, and the horizontal axis represents detection time. In general, it is known that the magnitude of electrical resistance strongly correlates with cell volume. In this case, for example, the maximum height of the waveform of electric resistance can be acquired as the volume of the cell. In addition, the cell diameter, the cell area, and the cell perimeter can be obtained from the obtained cell volume.
　また、細胞の大きさ情報を取得するための光学式フローサイトメータは、上述の蛍光検出に用いたフローサイトメータの蛍光検出部の代わり又は蛍光検出部に加えて、散乱光を検出する検出部（散乱光検出部）を備える。散乱光検出部は、フローサイトメータにおいて一般的に用いられる、散乱光の検出が可能な受光素子を有し得る。散乱光検出部の具体例としては、例えば、フォトダイオードやアバランシェフフォトダイオードなどが挙げられる。フローサイトメータのフローセルに測定用試料が通過する際に生じる散乱光は、この散乱光検出部により測定される。ここで、散乱光としては、前方散乱光が好ましい。 An optical flow cytometer for acquiring cell size information is a detection unit that detects scattered light in addition to or in addition to the fluorescence detection unit of the flow cytometer used in the above-described fluorescence detection. (Scattered light detector). The scattered light detection unit may include a light receiving element that is generally used in a flow cytometer and can detect scattered light. Specific examples of the scattered light detection unit include a photodiode and an avalanche photodiode. Scattered light generated when the measurement sample passes through the flow cell of the flow cytometer is measured by the scattered light detector. Here, forward scattered light is preferable as the scattered light.
　散乱光検出部により測定された散乱光は、電気信号としてフローサイトメータの分析部に出力される。分析部は、散乱光検出部から出力された電気信号に基づいて分析を行い、細胞の大きさ情報を取得する。例えば、分析部は、散乱光検出部から電気信号が出力されたときの前方散乱光の強度を求めることで、経時的な前方散乱光強度の波形の分析ができる。この分析部による、経時的な前方散乱光強度の波形の分析結果の一例を、図３に示す。図３において、縦軸は前方散乱光強度を示し、横軸は検出時間を示す。一般に、前方散乱光強度は細胞の表面積に強く相関し、前方散乱光強度の波形の幅は細胞の長さに強く相関することが知られている。この場合、例えば、前方散乱光強度の波形の最大の高さを細胞の表面積として取得することができる。また、前方散乱光強度の波形の最大の幅を細胞の直径として取得することができる。また、取得された細胞の表面積や細胞の直径から、細胞の体積、細胞の面積及び細胞の周囲長を取得することもできる。 Scattered light measured by the scattered light detection unit is output as an electrical signal to the analysis unit of the flow cytometer. The analysis unit performs analysis based on the electrical signal output from the scattered light detection unit, and acquires cell size information. For example, the analysis unit can analyze the waveform of the forward scattered light intensity over time by obtaining the intensity of the forward scattered light when the electrical signal is output from the scattered light detection unit. An example of the analysis result of the waveform of the forward scattered light intensity over time by this analysis unit is shown in FIG. In FIG. 3, the vertical axis represents the forward scattered light intensity, and the horizontal axis represents the detection time. In general, it is known that the forward scattered light intensity strongly correlates with the cell surface area, and the waveform width of the forward scattered light intensity strongly correlates with the cell length. In this case, for example, the maximum height of the waveform of the forward scattered light intensity can be acquired as the cell surface area. Further, the maximum width of the waveform of the forward scattered light intensity can be acquired as the cell diameter. The cell volume, cell area, and cell perimeter can also be obtained from the obtained cell surface area and cell diameter.
（３）判定工程
　本実施形態は、取得工程で取得された蛍光情報及び細胞の大きさ情報に基づいて、細胞が癌細胞か否かを判定する工程（以下、「判定工程」ともいう）を含む。 (3) Determination Step This embodiment includes a step of determining whether a cell is a cancer cell based on the fluorescence information and cell size information acquired in the acquisition step (hereinafter also referred to as “determination step”). Including.
　判定工程では、取得工程で得られた蛍光情報及び細胞の大きさ情報を用い、測定用試料中の癌細胞を、測定用試料中の他の細胞及びデブリス等から分別する。より具体的には、所定の蛍光情報及び所定の細胞の大きさ情報を有する測定用試料中の細胞を癌細胞と判定する。 In the determination step, the fluorescence information and the cell size information obtained in the acquisition step are used to separate cancer cells in the measurement sample from other cells, debris, and the like in the measurement sample. More specifically, a cell in a measurement sample having predetermined fluorescence information and predetermined cell size information is determined as a cancer cell.
　例えば、細胞の蛍光情報に関する値が、所定の閾値より大きく、かつ細胞の大きさ情報に関する値が、所定の閾値より大きい場合、この細胞を癌細胞と判定することができる。ここで、閾値は、血液中の癌細胞の蛍光情報及び細胞の大きさ情報に関するデータや、血液中に含まれる癌細胞以外の細胞の蛍光情報と細胞の大きさ情報に関するデータの蓄積から、経験的に導き出すことができる。例えば、癌細胞のみを含む複数の測定試料から、上述の取得工程の手順に基づいて、蛍光情報に関する値及び細胞の大きさ情報に関する値を含む、複数のデータを取得する。さらに、癌細胞を含まない血液試料から調製された測定用試料から、同様に蛍光情報に関する値及び細胞の大きさ情報に関する値を含む、複数のデータを取得する。これら取得されたデータに基づき、血液試料から調製された測定用試料に含まれる細胞及びデブリス等から、癌細胞か否かを判定できる、蛍光情報に関する値及び細胞の大きさ情報に関する値それぞれの閾値を設定することができる。 For example, when a value related to fluorescence information of a cell is larger than a predetermined threshold value and a value related to cell size information is larger than a predetermined threshold value, this cell can be determined as a cancer cell. Here, the threshold value is determined from the accumulation of data related to fluorescence information and cell size information of cancer cells in blood, and data related to fluorescence information and cell size information of cells other than cancer cells contained in blood. Can be derived. For example, a plurality of data including a value related to fluorescence information and a value related to cell size information is acquired from a plurality of measurement samples including only cancer cells based on the procedure of the acquisition step described above. Furthermore, a plurality of data including a value related to fluorescence information and a value related to cell size information is obtained from a measurement sample prepared from a blood sample not containing cancer cells. Based on the acquired data, it is possible to determine whether or not the cell is a cancer cell from cells and debris contained in a measurement sample prepared from a blood sample. Threshold values of a value relating to fluorescence information and a value relating to cell size information, respectively. Can be set.
　また、例えば、蛍光情報及び細胞の大きさ情報に基づくスキャッタグラムにおいて、所定の領域に出現する細胞を癌細胞と判定することもできる。図４に、取得工程で得られる、蛍光情報及び細胞の大きさ情報に基づく、スキャッタグラムの模式図を示す。図４のスキャッタグラムは、Ｘ軸が蛍光情報、Ｙ軸が細胞情報である。上述のように、調製工程において、癌細胞で腫瘍溶解性ウイルスが増殖する。そのため、腫瘍溶解性ウイルスの増殖に伴い、腫瘍溶解性ウイルスにコードされた蛍光タンパク質が、癌細胞内に多く蓄積する。結果として、癌細胞からの蛍光情報に関する値は、大きい。また、癌細胞は、血液試料に含まれる細胞において、細胞の大きさが大きい。そのため、癌細胞からの細胞の大きさに関する値は、大きい。よって、癌細胞は図４のＡの領域に主に出現する。また、上述のように、白血球で腫瘍溶解性ウイルスが増殖する場合がある。そのため、白血球は、癌細胞と同様の蛍光情報に関する値を示す場合がある。しかし、発明者らの検討により、腫瘍溶解性ウイルスが増殖するような白血球からの細胞の大きさ情報に関する値は、癌細胞よりも小さいことがわかった。よって、白血球は図４のＢの領域に主に出現する。また、癌細胞及び白血球以外の細胞及びデブリス等では、腫瘍溶解性ウイルスが増殖しない。従って、癌細胞及び白血球以外の細胞及びデブリス等の蛍光情報に関する値は、極めて小さい。よって、癌細胞及び白血球以外の細胞及びデブリス等は、図４のＣの領域に主に出現する。すなわち、上述した取得工程で得られた蛍光情報及び細胞の大きさ情報が、図４のＡの領域に出現する細胞を癌細胞と判定ができる。 Also, for example, in a scattergram based on fluorescence information and cell size information, a cell appearing in a predetermined region can be determined as a cancer cell. FIG. 4 shows a schematic diagram of a scattergram based on fluorescence information and cell size information obtained in the acquisition step. In the scattergram of FIG. 4, the X axis is fluorescence information and the Y axis is cell information. As described above, oncolytic virus grows in cancer cells in the preparation process. Therefore, as the oncolytic virus grows, a large amount of fluorescent protein encoded by the oncolytic virus accumulates in the cancer cells. As a result, the value for fluorescence information from cancer cells is large. Cancer cells have a large cell size among cells contained in a blood sample. Therefore, the value related to the size of cells from cancer cells is large. Therefore, cancer cells mainly appear in the area A in FIG. Moreover, as described above, oncolytic viruses may grow on leukocytes. Therefore, white blood cells may show values related to fluorescence information similar to cancer cells. However, the inventors' investigation has revealed that the value relating to the cell size information from leukocytes where the oncolytic virus grows is smaller than that of cancer cells. Therefore, white blood cells mainly appear in the region B in FIG. In addition, oncolytic viruses do not grow in cells other than cancer cells and leukocytes and debris. Therefore, the values relating to fluorescence information of cells other than cancer cells and leukocytes and debris are extremely small. Therefore, cells other than cancer cells and leukocytes, debris, and the like mainly appear in the region C of FIG. That is, the cells in which the fluorescence information and the cell size information obtained in the acquisition step described above appear in the area A in FIG. 4 can be determined as cancer cells.
　なお、上記の取得工程及び判定工程は、例えば、コンピュータにおいて実施することができ、その具体的なコンピュータの一例を、図５に示す。
　コンピュータ１００は、本体１１０と表示部１２０と、入力部１３０とから主として構成されている。本体１１０は、ＣＰＵ１１０ａと、ＲＯＭ１１０ｂと、ＲＡＭ１１０ｃと、ハードディスク１１０ｄと、読出装置１１０ｅと、入出力インターフェイス１１０ｆ、及び画像出力インターフェイス１１０ｈは、お互いにバス１１０ｉによってデータ通信可能に接続されている。 In addition, said acquisition process and determination process can be implemented in a computer, for example, An example of the specific computer is shown in FIG.
The computer 100 mainly includes a main body 110, a display unit 120, and an input unit 130. In the main body 110, a CPU 110a, a ROM 110b, a RAM 110c, a hard disk 110d, a reading device 110e, an input / output interface 110f, and an image output interface 110h are connected to each other via a bus 110i so that data communication is possible.
　ＣＰＵ１１０ａは、ＲＯＭ１１０ｂに記憶されているコンピュータプログラム及びＲＡＭ１１０ｃにロードされたコンピュータプログラムを実行することが可能である。
　ＲＯＭ１１０ｂは、マスクＲＯＭ、ＰＲＯＭ、ＥＰＲＯＭ、ＥＥＰＲＯＭ等によって構成されており、ＣＰＵ１１０ａに実行されるコンピュータプログラム及びこれに用いるデータが記録されている。
　ＲＡＭ１１０ｃは、ＳＲＡＭ又はＤＲＡＭ等によって構成されている。ＲＡＭ１１０ｃは、ＲＯＭ１１０Ｂ及びハードディスク１１０ｄに記録されているコンピュータプログラムの読み出しに用いられる。また、これらのコンピュータプログラムを実行するときに、ＣＰＵ１１０ａの作業領域として利用される。 The CPU 110a can execute computer programs stored in the ROM 110b and computer programs loaded in the RAM 110c.
The ROM 110b is configured by a mask ROM, PROM, EPROM, EEPROM, or the like, and is recorded with a computer program executed by the CPU 110a and data used therefor.
The RAM 110c is configured by SRAM, DRAM, or the like. The RAM 110c is used for reading computer programs recorded in the ROM 110B and the hard disk 110d. Further, when these computer programs are executed, they are used as a work area of the CPU 110a.
　ハードディスク１１０ｄは、オペレーティングシステム及びアプリケーションシステムプログラム等、ＣＰＵ１１０ａに実行させるための種々のコンピュータプログラム及びコンピュータプログラムの実行に用いるデータがインストールされている。後述するアプリケーションプログラム１４０ａも、このハードディスク１１０ｄにインストールされている。 The hard disk 110d is installed with various computer programs to be executed by the CPU 110a such as operating system and application system program, and data used for executing the computer program. An application program 140a described later is also installed in the hard disk 110d.
　読出装置１１０ｅは、フレキシブルディスクドライブ、ＣＤ－ＲＯＭドライブ、又はＤＶＤ－ＲＯＭドライブ等によって構成されており、可搬型記憶媒体１４０に記録されたコンピュータプログラム又はデータを読み出すことができる。また、可搬型記憶媒体１４０には、コンピュータに取得工程及び判定工程に係るアプリケーションプログラム１４０ａが格納されており、ＣＰＵ１１０ａが可搬型記憶媒体１４０から当該アプリケーションプログラム１４０ａを読み出し、アプリケーションプログラム１４０ａをハードディスク１１０ｄにインストールすることも可能である。 The reading device 110e is configured by a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data recorded on the portable storage medium 140. The portable storage medium 140 stores an application program 140a related to the acquisition process and the determination process in a computer. The CPU 110a reads the application program 140a from the portable storage medium 140, and the application program 140a is stored in the hard disk 110d. It is also possible to install.
　また、ハードディスク１１０ｄには、例えば米国マイクロソフト社が製造販売するＷｉｎｄｏｗｓ（登録商標）などのグラフィカルユーザインターフェイス環境を提供するオペレーションシステムがインストールされている。以下の説明においては、上述した取得工程及び判定工程に係るアプリケーションプログラム１４０ａは、当該オペレーティングシステム上で動作するものとしている。 In addition, an operating system that provides a graphical user interface environment such as Windows (registered trademark) manufactured and sold by Microsoft Corporation in the United States is installed in the hard disk 110d. In the following description, it is assumed that the application program 140a related to the acquisition process and the determination process described above operates on the operating system.
　入出力インターフェイス１１０ｆは、例えばＵＳＢ、ＩＥＥＥ１３９４、ＲＳ－２３２Ｃ等のシリアルインターフェイス、ＳＣＳＩ、ＩＤＥ、ＩＥＥＥ１２８４等のパラレルインターフェイス、及びＤ／Ａ変換器、Ａ／Ｄ変換器等からなるアナログインターフェイス等から構成されている。入出力インターフェイス１１０ｆには、キーボード及びマウスからなる入力デバイス１３０が接続されており、ユーザが当該入力デバイス１３０を使用することにより、コンピュータ本体１１０にデータを入力することが可能である。 The input / output interface 110f includes, for example, a serial interface such as USB, IEEE1394, RS-232C, a parallel interface such as SCSI, IDE, IEEE1284, and an analog interface including a D / A converter, an A / D converter, and the like. ing. An input device 130 including a keyboard and a mouse is connected to the input / output interface 110f, and the user can input data to the computer main body 110 by using the input device 130.
　また、入出力インターフェイス１１０ｆには、ＣＣＤカメラを搭載した蛍光顕微鏡２００が接続されている。これにより、コンピュータ本体１１０は、蛍光顕微鏡２００から、撮像された画像データを取得することが可能である。 The input / output interface 110f is connected to a fluorescence microscope 200 equipped with a CCD camera. Thereby, the computer main body 110 can acquire the imaged image data from the fluorescence microscope 200.
　画像出力インターフェイス１１０ｈは、ＬＣＤ又はＣＲＴ等で構成された表示部１２０に接続されており、ＣＰＵ１１０ａから与えられた画像データに応じた映像信号を表示部１２０に出力するようになっている。表示部１２０は、入力された映像信号にしたがって、画像データを出力する。また、表示部１２０は、後述するＣＰＵ１１０ａから与えられた判定結果を出力する。 The image output interface 110h is connected to a display unit 120 constituted by an LCD, a CRT, or the like, and outputs a video signal corresponding to the image data given from the CPU 110a to the display unit 120. The display unit 120 outputs image data according to the input video signal. Further, the display unit 120 outputs a determination result given from a CPU 110a described later.
　図６は、ＣＰＵ１１０ａによって実行される癌細胞か否かを判定する処理を示すフローチャートの一例である。
まず、蛍光顕微鏡２００から測定用試料に含まれる細胞を撮像した画像データを、ＣＰＵ１１０ａが入出力インターフェイス１１０ｆを介して取得し、ハードディスク１１０ｄに記憶させる（ステップＳ１１）。 FIG. 6 is an example of a flowchart showing processing for determining whether or not the cancer cell is executed by the CPU 110a.
First, image data obtained by imaging cells included in the measurement sample from the fluorescence microscope 200 is acquired by the CPU 110a via the input / output interface 110f and stored in the hard disk 110d (step S11).
　ＣＰＵ１１０ａは、ハードディスク１１０ｄに格納された上記プリケーションプログラム１４０ａを読み出して、画像データに対して画像処理を行い、パラメータとして蛍光強度の積分値及び細胞の体積を算出する処理を実行する（ステップＳ１２）。 The CPU 110a reads the application program 140a stored in the hard disk 110d, performs image processing on the image data, and executes processing for calculating the integral value of fluorescence intensity and the volume of cells as parameters (step S12). .
　次に、ＣＰＵ１１０ａは、算出されたパラメータである蛍光強度の積分値及び細胞の体積に基づいて、細胞が癌細胞か否かを判定する（ステップＳ１３）。
　ハードディスク１１０ｄには、蛍光強度の積分値及び細胞の体積について、図７の領域Ａに示される癌細胞が出現するパラメータ領域の座標が記憶されている。 Next, the CPU 110a determines whether or not the cell is a cancer cell based on the integrated value of the fluorescence intensity, which is the calculated parameter, and the cell volume (step S13).
The hard disk 110d stores the coordinates of the parameter area where the cancer cells appear in the area A of FIG. 7 with respect to the integrated value of the fluorescence intensity and the volume of the cell.
　ＣＰＵ１１０ａは、算出されたパラメータである蛍光強度の積分値及び細胞の体積と当該パラメータ領域から、細胞が癌細胞か否かを判定する。具体的には、細胞に関してそれぞれのパラメータが当該パラメータ領域に入る場合は、該細胞が癌細胞であると判定する。また外れる場合は、該細胞が癌細胞でないと判定する。
　そして、ＣＰＵ１１０ａは、判定結果として図７に示されるスキャッタグラムを、ＲＡＭ１１０ｃに格納するとともに画像出力インターフェイス１１０ｆを介して表示部１２０に出力する（ステップＳ１４）。 The CPU 110a determines whether or not the cell is a cancer cell from the integrated value of the fluorescence intensity, which is the calculated parameter, the volume of the cell, and the parameter area. Specifically, when each parameter regarding a cell enters the parameter area, it is determined that the cell is a cancer cell. If the cell is detached, it is determined that the cell is not a cancer cell.
Then, the CPU 110a stores the scattergram shown in FIG. 7 as a determination result in the RAM 110c and outputs it to the display unit 120 via the image output interface 110f (step S14).
　なお、本実施形態においては、上述した判定結果として、スキャッタグラムを用いる例を示したが、判定結果はこれに限定されるものではない。判定結果としては、例えば、癌細胞と判定された細胞のカウント数、癌細胞と判定された細胞の画像データ、測定用試料中の癌細胞の有無を示す値（陽性又は陰性）などが挙げられる。また、表示部に出力される判定結果は一つに限定されるものではない。上記の判定結果より選択される少なくとも一つを表示部に出力することができる。 In the present embodiment, the scattergram is used as the determination result described above, but the determination result is not limited to this. Examples of the determination result include the count number of cells determined to be cancer cells, image data of cells determined to be cancer cells, and a value (positive or negative) indicating the presence or absence of cancer cells in the measurement sample. . Further, the determination result output to the display unit is not limited to one. At least one selected from the determination results can be output to the display unit.
　なお、本実施形態においては、コンピュータにより実施する取得工程において、蛍光強度の積分値及び細胞の体積を算出する例を示したが、蛍光情報及び細胞の大きさ情報のパラメータとしては、これに限定されるものではない。例えば、蛍光情報のパラメータとしては、蛍光強度のピーク値、蛍光強度の最小値及び蛍光強度の平均値が挙げられる。また、細胞の大きさ情報のパラメータとしては細胞の面積、細胞の周囲長、細胞の直径及び細胞の真円度が挙げられる。 In the present embodiment, an example in which the integrated value of the fluorescence intensity and the volume of the cell are calculated in the acquisition step performed by the computer is shown. However, the parameters of the fluorescence information and the cell size information are not limited thereto. Is not to be done. For example, the parameters of the fluorescence information include a peak value of fluorescence intensity, a minimum value of fluorescence intensity, and an average value of fluorescence intensity. In addition, the cell size information parameters include cell area, cell perimeter, cell diameter, and cell roundness.
　また、本実施形態においては、コンピュータにより実施する判定工程において、癌細胞が出現するパラメータ領域として、蛍光強度の積分値と細胞の面積とのパラメータ領域を用いる例を示したが、パラメータ領域はこれに限定されるものではない。パラメータ領域としては、例えば、図８の領域Ａに示される蛍光強度のピーク値及び細胞の体積に対するパラメータ領域、図９の領域Ａに示される蛍光強度の積分値及び細胞の面積に対するパラメータ領域、図１０の領域Ａに示される蛍光強度の積分値及び細胞の直径に対するパラメータ領域、又は図１１の領域Ａに示される蛍光強度の積分値及び細胞の周囲長に対するパラメータ領域などが挙げられる。また、パラメータ領域に用いるパラメータの数は二つに限定されるものではない。上記の蛍光情報のパラメータより選択される少なくとも一つと、上記の細胞の大きさ情報のパラメータより選択される少なくとも一つを、パラメータとして用いることができる。例えば、パラメータ領域に用いるパラメータとして、蛍光強度のピーク値、蛍光強度の積分値及び細胞の面積からなる３つのパラメータを用いることもできる。 In the present embodiment, an example is shown in which the parameter region of the integrated value of the fluorescence intensity and the cell area is used as the parameter region where cancer cells appear in the determination step performed by the computer. It is not limited to. As the parameter area, for example, the peak value of the fluorescence intensity shown in the area A of FIG. 8 and the parameter area for the cell volume, the integrated value of the fluorescence intensity shown in the area A of FIG. 9 and the parameter area for the cell area, FIG. For example, the integrated value of the fluorescence intensity shown in the area A of 10 and the parameter area for the cell diameter, or the integrated value of the fluorescence intensity shown in the area A of FIG. Further, the number of parameters used in the parameter area is not limited to two. At least one selected from the parameters of the fluorescence information and at least one selected from the parameters of the cell size information can be used as parameters. For example, three parameters including a peak value of fluorescence intensity, an integrated value of fluorescence intensity, and a cell area can also be used as parameters used in the parameter region.
　また、本実施形態においては、コンピュータ１００と蛍光顕微鏡２００が別体に備えられる例を示したが、これらの態様はこれに限定されるものではなく、例えば一体型でもよい。 Further, in the present embodiment, an example in which the computer 100 and the fluorescence microscope 200 are provided separately is shown, but these aspects are not limited to this, and may be an integrated type, for example.
　また、本実施形態においては、入出力インターフェイス１１０ｆを介して、ＣＣＤカメラを搭載した蛍光顕微鏡２００が、コンピュータ１００に接続される例を示したが、これらの態様はこれに限定されるものではない。例えば、蛍光顕微鏡２００の代わりに、フローサイトメータ３００が接続されていてもよい。この場合、コンピュータ本体１１０は、フローサイトメータ３００から、蛍光に関するデータと、電気抵抗及び／又は散乱光に関するデータとを取得する。ＣＰＵ１１０ａは、取得された蛍光に関するデータから、蛍光情報のパラメータを算出する。また、電気抵抗及び／又は散乱光に関するデータから、細胞の大きさ情報のパラメータを算出する。 Further, in the present embodiment, the example in which the fluorescence microscope 200 equipped with the CCD camera is connected to the computer 100 via the input / output interface 110f is shown, but these aspects are not limited thereto. . For example, a flow cytometer 300 may be connected instead of the fluorescence microscope 200. In this case, the computer main body 110 acquires data relating to fluorescence and data relating to electrical resistance and / or scattered light from the flow cytometer 300. CPU110a calculates the parameter of fluorescence information from the acquired data regarding fluorescence. In addition, parameters of cell size information are calculated from data relating to electrical resistance and / or scattered light.
　また、コンピュータ１００とフローサイトメータ３００は別体型でも、一体型でもよい。 Further, the computer 100 and the flow cytometer 300 may be separate or integrated.
＜蛍光情報と細胞の体積による癌細胞か否かの判定＞
　細胞画像から得られた蛍光情報と細胞の大きさ情報により、細胞が癌細胞か否かを判定できることを調べるため、Ｙ軸は細胞の大きさ情報として体積を共通のパラメータとしてプロットし、Ｘ軸は蛍光情報としてピーク値又は積分値をプロットした二次元スキャッタグラムを作製した。 <Determining whether a cancer cell is based on fluorescence information and cell volume>
In order to investigate whether or not a cell is a cancer cell based on fluorescence information and cell size information obtained from a cell image, the Y axis plots volume as a common parameter as cell size information, and the X axis Produced a two-dimensional scattergram in which peak values or integral values were plotted as fluorescence information.
　本実施例で使用する測定用試料は、以下の通りに調製した。
＜単核球試料の調製＞
　白血球（WBC）の一つである単核球を以下の通り調製した。
１．12～15mLの遠心チューブに、単核球を分離するための試薬であるPolymorphprep(商標)(第一化学薬品)5.0mLを入れ、真空採血管(ベノジェクトII真空採血管、3.8％クエン酸ナトリウム 4.5ML含有、テルモ社製)を用いて４人の健常人から採取された、採取後２時間以内の血液5.0ｍLを重層した。
２．スウィングローターを用いて、室温で500×g、30分間遠心分離した。
３．パスツールピペットを用いて単核球浮遊液を採取した。
４．単核球浮遊液に同量のリン酸緩衝液(PBS)(-)を加え、混和した。
５．室温で400×g、10分間遠心分離した。
６．上清を除去し、単核球をPBS(-)に懸濁し、５．と同様に遠心分離して上清を除去し、単核球試料を調製した。 The measurement sample used in this example was prepared as follows.
<Preparation of mononuclear cell sample>
A mononuclear cell that is one of white blood cells (WBC) was prepared as follows.
1. Into a 12-15 mL centrifuge tube, put 5.0 mL of Polymorphprep ™ (Daiichi Kagaku), a reagent for separating mononuclear cells, and collect a vacuum blood collection tube (Benoject II vacuum blood collection tube, 3.8% citric acid) The sample was collected from 4 healthy individuals using 4.5 ml of sodium (manufactured by Terumo), and 5.0 mL of blood within 2 hours after the collection was overlaid.
2. Centrifugation was performed at 500 × g for 30 minutes at room temperature using a swing rotor.
3. Mononuclear cell suspension was collected using a Pasteur pipette.
4). The same amount of phosphate buffer (PBS) (−) was added to the mononuclear cell suspension and mixed.
5. Centrifuge at 400 × g for 10 minutes at room temperature.
6). 4. Remove supernatant and suspend mononuclear cells in PBS (-). In the same manner as above, the supernatant was removed by centrifugation, and a mononuclear cell sample was prepared.
＜癌細胞試料の調製＞
　乳癌細胞の一つであるMB468を、１２５cm2のフラスコにおいて９０%コンフルエントとなるように、培養液(１０％FBS(Hyclone社製)を含むDMEM-F12(Sigma社製))中で培養した。得られた培養細胞を、DMEM-F12 1000μl中に約3×105個となるように懸濁して、MB468細胞試料を調製した。 <Preparation of cancer cell sample>
MB468, which is one of the breast cancer cells, was cultured in a culture solution (DMEM-F12 (Sigma) containing 10% FBS (Hyclone)) so as to be 90% confluent in a 125 cm 2 flask. The obtained cultured cells were suspended in 1000 μl of DMEM-F12 so as to have about 3 × 10 5 cells to prepare MB468 cell samples.
　次に、上記の単核球試料及び癌細胞試料について、それぞれ以下に示す工程を行った。 Next, the following steps were performed on the above mononuclear cell sample and cancer cell sample, respectively.
＜ウイルス増殖＞
　得られた試料に、RPMI-1640を加え、10mlにメスアップした。腫瘍溶解性ウイルス(テロメスキャン(商標登録)(OBP-401))を、最終濃度6×106PFU/mlとなるように加え、37℃にてローテーションしながら24時間培養した。培養液を、1500rpmで5分間、弱ブレーキで遠心分離し、上清を除去して、腫瘍溶解性ウイルスを増殖させた細胞を得た。 <Virus propagation>
RPMI-1640 was added to the obtained sample to make up to 10 ml. Oncolytic virus (Telomescan (registered trademark) (OBP-401)) was added to a final concentration of 6 × 10 6 PFU / ml and cultured at 37 ° C. for 24 hours while rotating. The culture solution was centrifuged at 1500 rpm for 5 minutes with a weak brake, and the supernatant was removed to obtain cells on which oncolytic virus had grown.
＜固定化剤及び非イオン性界面活性剤での処理＞
　得られたウイルスが増殖した沈殿細胞に、該細胞と同体積の４％パラホルムアルデヒド(PFA)を加え、室温にて20分間静置した。
次いで、沈殿細胞及びPFAの合計体積の２倍の体積の非イオン性界面活性剤(エマルゲン2025G、花王社製)のPBS(-)溶液(0.05重量％)を加えて懸濁し、攪拌しながら40℃にて5分間インキュベートした。
　エマルゲン2025Gは、以下の構造式を有する。 <Treatment with immobilizing agent and nonionic surfactant>
4% paraformaldehyde (PFA) having the same volume as the cells was added to the precipitated cells on which the obtained virus had grown, and the mixture was allowed to stand at room temperature for 20 minutes.
Next, a PBS (−) solution (0.05% by weight) of a nonionic surfactant (Emulgen 2025G, manufactured by Kao Corporation) in a volume twice the total volume of the precipitated cells and PFA was added and suspended, and the mixture was stirred while stirring. Incubated for 5 minutes at ° C.
Emulgen 2025G has the following structural formula:
Figure JPOXMLDOC01-appb-C000001
 （式中、ｎは２５である。）
Figure JPOXMLDOC01-appb-C000001
 (In the formula, n is 25.)
＜スライド作製及び顕微鏡観察＞
　1500rpmで５分間、弱ブレーキで遠心分離し、上清を除去し、沈殿物を1ml  PBSに懸濁した。1500rpmで５分間、上清を除去し、サイトスピンを用いて(1000rpm、４分)、スライドを作製した。マウンティングメディウム(Fluorescent Mounting Medium, Dako, S3032)を載せ、カバーガラスを載せて、倒立型リサーチ顕微鏡(Power IX71、オリンパス社製)を用いて、露光時間20ms、50ms、100ms、200ms及び400msの条件下で、蛍光強度を測定した。
　なお、上記と同様の条件下で、蛍光強度が既知の異なる蛍光強度を有する7種のビーズ(7 Peaks Beads (Cyto-Cal Multifluor Fluorescence Intensity Calibrator, Thermo Scientific, FC3M))を撮像して、それぞれの露光時間における検量線を作成した。得られた検量線に基づいて、上記の処理試料からの蛍光強度を数値化した。 <Slide preparation and microscope observation>
Centrifugation was performed with a weak brake at 1500 rpm for 5 minutes, the supernatant was removed, and the precipitate was suspended in 1 ml PBS. The supernatant was removed at 1500 rpm for 5 minutes, and a slide was prepared using cytospin (1000 rpm, 4 minutes). Mount mounting medium (Fluorescent Mounting Medium, Dako, S3032), cover glass, and using inverted research microscope (Power IX71, Olympus), exposure time of 20ms, 50ms, 100ms, 200ms and 400ms Then, the fluorescence intensity was measured.
Under the same conditions as described above, seven types of beads having different fluorescence intensities with known fluorescence intensity (7 Peaks Beads (Cyto-Cal Multifluor Fluorescence Intensity Calibrator, Thermo Scientific, FC3M)) were imaged. A calibration curve for the exposure time was prepared. Based on the obtained calibration curve, the fluorescence intensity from the treated sample was quantified.
＜蛍光情報及び細胞の大きさ情報の取得＞
　露光時間20ms、50ms、100ms、200ms及び400msの条件下で蛍光強度が既知の異なる蛍光強度を有する7種のビーズ(7 Peaks Beads (Cyto-Cal Multifluor Fluorescence Intensity Calibrator, Thermo Scientific, FC3M))を撮像して、それぞれの露光時間における検量線を作成した。次にImage-Pro Plus Ver6.1（Media Cybernetics, Inc.）を用いて、得られた検量線に基づいて、処理試料からの最大蛍光強度（細胞のピーク値(MEFL)）、蛍光強度の積分値(MEFL)、蛍光分布面積（細胞の面積(pixel2)）、蛍光分布周囲長(細胞の周囲長(pixel))を数値化した。細胞の体積(pixel3)は細胞が真球であると仮定し、蛍光分布面積を用いて算出した。細胞の直径(pixel)も同様に蛍光分布面積を用いて算出した。 <Acquisition of fluorescence information and cell size information>
Imaging 7 types of beads (7 Peaks Beads (Cyto-Cal Multifluor Fluorescence Intensity Calibrator, Thermo Scientific, FC3M)) with known fluorescence intensities under exposure times of 20 ms, 50 ms, 100 ms, 200 ms and 400 ms A calibration curve for each exposure time was prepared. Next, using Image-Pro Plus Ver6.1 (Media Cybernetics, Inc.), based on the calibration curve obtained, the maximum fluorescence intensity (cell peak value (MEFL)) from the treated sample and the integration of the fluorescence intensity Values (MEFL), fluorescence distribution area (cell area (pixel 2 )), and fluorescence distribution perimeter (cell perimeter (pixel)) were digitized. The cell volume (pixel 3 ) was calculated using the fluorescence distribution area assuming that the cell was a true sphere. The cell diameter (pixel) was similarly calculated using the fluorescence distribution area.
＜スキャッタグラム、及び三次元頻度分布図の作成＞
　次に、Image-Pro Plus Ver6.1（Media Cybernetics, Inc.）を用いて算出したパラメータである蛍光強度のピーク値、蛍光強度の積分値、細胞の面積、細胞の周囲長、細胞の体積、細胞の直径をプロットした二次元スキャッタグラムを作成した。本実施例では、蛍光強度の積分値－細胞の体積、蛍光強度のピーク値－細胞の体積、蛍光強度の積分値－細胞の面積、蛍光強度の積分値－細胞の直径、及び蛍光強度の積分値－細胞の周囲長をプロットしたスキャッタグラムを作成した。また、グラフ作成フリーソフト（GNUPLOT）を用いて、上述したパラメータをプロットした三次元頻度分布図を作成した。本実施例では、細胞の面積－蛍光強度のピーク値－蛍光強度の積分値をプロットした三次元頻度分布図を作成した。 <Creation of scattergram and 3D frequency distribution map>
Next, the peak value of fluorescence intensity, which is a parameter calculated using Image-Pro Plus Ver6.1 (Media Cybernetics, Inc.), the integrated value of fluorescence intensity, the cell area, the cell perimeter, the cell volume, A two-dimensional scattergram plotting the cell diameter was generated. In this example, the integrated value of fluorescence intensity—cell volume, peak value of fluorescence intensity—cell volume, integrated value of fluorescence intensity—cell area, integrated value of fluorescence intensity—cell diameter, and integration of fluorescence intensity. Values—A scattergram plotting the perimeter of the cells was generated. In addition, using the graph creation free software (GNUPLOT), a three-dimensional frequency distribution diagram in which the above parameters were plotted was created. In the present example, a three-dimensional frequency distribution diagram in which the cell area—the peak value of the fluorescence intensity—the integrated value of the fluorescence intensity was plotted was created.
　上記の工程により得られた結果を、スキャッタグラムを図７～図１２に示す。 Scattergrams of the results obtained by the above steps are shown in FIGS.
　図７及び８の結果から、蛍光情報のパラメータの一つ(積分値及びピーク値)と細胞の大きさ情報の一つとしての体積を用いることにより、白血球と癌細胞を分別し、細胞が癌細胞か否かを判定できることがわかる。さらに図９～１１の結果から、蛍光情報と細胞の大きさ情報のパラメータの一つ（直径、面積及び周囲長）を用いることにより、白血球と癌細胞を分別し、細胞が癌細胞か否かを判定できることがわかる。この結果から、蛍光情報及び細胞の大きさ情報により、細胞が癌細胞か否かを判定できることが示された。また、図１２の結果から、蛍光情報及び細胞の大きさ情報から複数のパラメータを選択しても、白血球と癌細胞を分別し、細胞が癌細胞か否かを判定できることがわかる。 From the results of FIGS. 7 and 8, by using one of the parameters of fluorescence information (integrated value and peak value) and the volume as one of cell size information, leukocytes and cancer cells are separated, and the cells are cancerous. It turns out that it can be determined whether it is a cell. Further, from the results shown in FIGS. 9 to 11, by using one of the parameters of the fluorescence information and the cell size information (diameter, area and perimeter), the leukocytes and the cancer cells are separated, and whether or not the cells are cancer cells. Can be determined. From this result, it was shown that whether or not a cell is a cancer cell can be determined from fluorescence information and cell size information. In addition, it can be seen from the results of FIG. 12 that even if a plurality of parameters are selected from the fluorescence information and the cell size information, it is possible to distinguish white blood cells from cancer cells and determine whether the cells are cancer cells.
　本出願は、２０１０年２月１０日に出願された日本国特許出願　特願２０１０－２７９２８号に関し、この特許請求の範囲、明細書、図面及び要約書の全ては本明細書中に参照として組み込まれる。 This application is related to Japanese Patent Application No. 2010-27928 filed on Feb. 10, 2010. All of the claims, description, drawings and abstract are incorporated herein by reference. It is.

Claims (14)

  1. 　被験者から採取された血液試料に含まれる細胞を、蛍光タンパク質を発現する腫瘍溶解性ウイルスで処理することにより、測定用試料を調製する工程と、
    　前記測定用試料に含まれる細胞の蛍光情報及び細胞の大きさ情報を取得する工程と、
    　取得工程で取得された蛍光情報及び細胞の大きさ情報に基づいて、細胞が癌細胞か否かを判定する工程と、
    を含む、血液中癌細胞の検出方法。     Preparing a measurement sample by treating cells contained in a blood sample collected from a subject with an oncolytic virus expressing a fluorescent protein;
    Obtaining fluorescence information and cell size information of cells contained in the measurement sample;
    Determining whether the cell is a cancer cell based on the fluorescence information and cell size information acquired in the acquisition step; and
    A method for detecting cancer cells in blood, comprising:
  2. 　蛍光情報が、蛍光強度のピーク値及び積分値の少なくとも一つである請求項１に記載の方法。 2. The method according to claim 1, wherein the fluorescence information is at least one of a peak value and an integral value of the fluorescence intensity.
  3. 　細胞の大きさ情報が、細胞の直径、細胞の面積、細胞の体積、及び細胞の周円長の少なくとも一つである請求項１又は２に記載の方法。 3. The method according to claim 1 or 2, wherein the cell size information is at least one of a cell diameter, a cell area, a cell volume, and a cell circumferential length.
  4. 　調製工程が、前記腫瘍溶解性ウイルスで処理した血液試料を、非イオン性界面活性剤及び固定化剤と混合することをさらに含む、請求項１～３のいずれか１項に記載の方法。 The method according to any one of claims 1 to 3, wherein the preparing step further comprises mixing a blood sample treated with the oncolytic virus with a nonionic surfactant and a fixing agent.
  5. 　非イオン性界面活性剤が、ポリオキシエチレン系の非イオン性界面活性剤である請求項４に記載の方法。 The method according to claim 4, wherein the nonionic surfactant is a polyoxyethylene-based nonionic surfactant.
  6. 　固定化剤が、タンパク質を架橋させる物質であり、パラホルムアルデヒド、グルタルアルデヒド及びホルムアルデヒドから選択される請求項４又は５に記載の方法。 The method according to claim 4 or 5, wherein the immobilizing agent is a substance that crosslinks proteins, and is selected from paraformaldehyde, glutaraldehyde, and formaldehyde.
  7. 　血液試料が、末梢血から血清を除去したものである請求項１～６のいずれか１項に記載の方法。 The method according to any one of claims 1 to 6, wherein the blood sample is obtained by removing serum from peripheral blood.
  8. 　蛍光タンパク質が、緑色蛍光タンパク質である請求項１～７のいずれか１項に記載の方法。 The method according to any one of claims 1 to 7, wherein the fluorescent protein is a green fluorescent protein.
  9. 　前記腫瘍溶解性ウイルスが、テロメアーゼプロモーターが組み込まれた腫瘍溶解性ウイルスである請求項１～８のいずれか１項に記載の方法。 The method according to any one of claims 1 to 8, wherein the oncolytic virus is an oncolytic virus in which a telomerase promoter is incorporated.
  10. 　前記腫瘍溶解性ウイルスが、ヒトアデノウイルス由来である請求項１～９のいずれか１項に記載の方法。 The method according to any one of claims 1 to 9, wherein the oncolytic virus is derived from a human adenovirus.
  11. 　コンピュータを、
    血液試料中の腫瘍溶解性ウイルスが感染し増殖した細胞の蛍光情報及び細胞の大きさ情報を取得する取得手段、
    　前記取得手段で取得された情報に基づいて、細胞が癌細胞か否かを判定する判定手段、
    　前記判定手段の判定結果を表示する表示手段、
    として機能させるための血液中癌細胞の検出プログラム。     Computer
    An acquisition means for acquiring fluorescence information and cell size information of cells that have been infected and proliferated by an oncolytic virus in a blood sample;
    Determination means for determining whether the cell is a cancer cell based on the information acquired by the acquisition means;
    Display means for displaying the determination result of the determination means;
    For detecting cancer cells in blood to function as
  12. 　前記取得手段が、血液試料中の腫瘍溶解性ウイルスが感染し増殖した細胞を撮像することにより得られた細胞画像から、蛍光タンパク質由来の蛍光情報及び細胞の大きさ情報を取得する、請求項１１に記載のプログラム。 The said acquisition means acquires the fluorescence information derived from fluorescent protein and the size information of a cell from the cell image obtained by imaging the cell which the oncolytic virus in the blood sample infected and proliferated. The program described in.
  13. 前記取得手段で取得される蛍光情報が、蛍光強度のピーク値及び積分値の少なくとも一つである請求項１１又は１２に記載のプログラム。 The program according to claim 11 or 12, wherein the fluorescence information acquired by the acquisition means is at least one of a peak value and an integrated value of fluorescence intensity.
  14. 　前記取得手段で取得される細胞の大きさ情報が、細胞の直径、細胞の面積、細胞の体積、及び細胞の周円長の少なくとも一つである請求項１１～１３のいずれか１項に記載のプログラム。 The cell size information acquired by the acquisition means is at least one of a cell diameter, a cell area, a cell volume, and a circumferential length of the cell. Program.
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