JP6639906B2 - Biological sample detection method - Google Patents

Description

  The present invention relates to a method for detecting target cells in a biological sample, particularly preferably a method for efficiently separating living cells from a sample containing cells such as a body fluid, a dispersed tissue specimen or a culture, and capturing the living cells in a holding hole. And a method for detecting target cells with high sensitivity using cell detection parameters.

  Researches are underway to separate target cells from biological fluids such as body fluids and cell cultures, or from dispersed specimens in which tissues and cells are suspended and dispersed, and to apply them to clinical diagnosis and therapy. For example, a very small amount of cancer cells present in blood, that is, circulating tumor cell (CTC) is a tumor cell that has infiltrated into a blood vessel from a primary tumor of a cancer patient, and CTC flowing in the blood vessel is a blood vessel. It is thought that cancer metastasis occurs by attaching to the inner wall and invading extravascular organs. Therefore, a method of detecting CTC has attracted attention as one of the methods for early detection of malignant tumors involved in metastasis (Non-Patent Document 1).

  For example, after enriching CTC with magnetic microparticles modified with an anti-epithelial cell adhesion molecule (EpCAM) antibody in whole blood, and labeling CTC and normal cells (leukocytes) by immunostaining, an automatic fluorescence detector is used. A method of counting CTCs using the method has been proposed (CellSearch method, see Non-Patent Document 3).

  Also, taking advantage of the fact that cancer cells in the blood are larger in size than red blood cells, platelets, and white blood cells, which are blood cells, the blood is filtered to discharge smaller blood cells, and the blood is filtered onto the filter surface. There has been proposed a detection method using a technique of capturing cancer cells therein (ISET method, for example, see Non-Patent Document 4 and Patent Document 1).

  CTC is a rare cell in which only about a few to several tens of cells are present in 1 mL of blood (about 5 billion blood cells) of a cancer patient (Non-patent Document 2). In addition, it has been reported that during the metastasis process, molecules that are targets for the separation and detection of CTC, such as epithelial cell adhesion molecule (EpCAM), which is a cell surface protein, and cytokeratin, which is an intracellular skeletal protein, have disappeared. Therefore, it is estimated that the cells are rich in diversity. Furthermore, many CTCs are killed by apoptosis (cell death) or attack by immune cells, and only a few surviving CTCs can establish distant metastasis. I was

JP 2011-163830 A International Publication No. 2014/192919

Pantel K, et al. 2008. Detection, clinical relevance and specific biological biological properties of disseminating tumor cells. Nat Rev3. Cristofanilli M, et. al. 2004. Circulating tumour cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 351 (8): 781.13 (3): 920. Riethdorf S, et. al. 2007. Detection of circulating tumour cells in peripheral blood of patients with metastatic breast cancer: a validation study of the cellular search. Clin Cancer Res 13 (3): 920. Rostagno P, Moll JL, Bisconte JC, Caldani C.I. 1997. Detection of rare circulating breaking cancer cells by filtration cytometry and identification by DNA content: sensitivity in an experimental element. Anticancer Res 17 (4A): 2481.

  For the detection of CTC, a technique is needed that can separate and detect CTC without depending on the detection target (detection marker) possessed by CTC, and selectively and highly sensitively detects CTC that survives in blood and establishes metastasis. (Non-Patent Document 3).

  However, the above-described CellSearch method cannot separate CTCs that do not express EpCAM, and cannot selectively detect only strictly living cells. On the other hand, the ISET method employs a CTC separation method utilizing a difference in cell size independent of a CTC-carrying marker, but cannot distinguish between live and dead cells, and removes live CTCs involved in metastasis. There is a problem that qualitative evaluation for detection is difficult. Therefore, an object of the present invention is to provide a method for detecting living target cells with high sensitivity.

  The present invention, which has been completed in view of the above object, has a method for detecting a biological sample, comprising: concentrating living cells from a biological sample, developing the living cells on a substrate, and detecting the expanded living target cells. About.

  In the present invention, a live target cell can be detected with high sensitivity by performing a step of developing a concentrated solution and a step of detecting a target cell after performing a concentration step of concentrating living cells. In the enrichment step, living target cells can be efficiently enriched by separating live cells and dead cells based on the difference in specific gravity. In addition, the developing step captures approximately one cell in each holding hole provided with live cells on the substrate by dielectrophoretic force, thereby detecting target cells with high sensitivity using cell detection parameters. Can be. Therefore, living cells can be more selectively separated and detected as compared with a method of separating cells based on cell surface antigens or cell size. In addition, even if there is no target cell labeling marker, the target cells can be detected by labeling and eliminating unnecessary cells. Such effects enable early detection of malignant tumors and diagnosis of metastasis by detecting live cancer cells.

It is a figure for explaining the separation concentration structure object of the present invention. It is a figure for explaining the structure concerning a 1st embodiment used for the present invention. It is a figure for explaining the structure concerning a 2nd embodiment used for the present invention. It is a figure for explaining a structure and a biological sample detecting device used for the present invention. It is a figure for explaining the separation method of the present invention. It is a figure for explaining the separation method of the present invention. It is a figure which shows the dielectrophoretic frequency and the cancer cell detection rate in Example 4 of this invention. It is a figure in Example 6 of this invention which shows the detection result of the CTC count of a breast cancer patient sample. It is a figure in Example 7 of this invention which shows the detection flow of abnormal cells.

Hereinafter, the present invention will be described in detail.
The present invention relates to a method for detecting living target cells in a biological sample. More specifically, the detection method of the present invention comprises the following steps:
A concentration step of concentrating living cells from a biological sample to obtain a concentrated solution;
A developing step of developing the concentrated solution obtained in the concentration step on a substrate,
An optical detection step of optically detecting a living target cell developed on the substrate,
including.

  In the present invention, a biological sample refers to any sample as long as it is a sample containing cells, and includes a biological sample obtained from a living body and a culture sample obtained by cell culture, tissue culture, or the like. Biological samples include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, amniotic fluid, lymph fluid, cell aggregates, tumors, samples derived from organs or tissues such as lymph nodes or arteries. . Culture samples include cell cultures, tissue cultures, or cultures thereof. These biological samples can be subjected to treatments such as dilution, mixing, dispersion, suspension, etc., depending on the type of the sample, to prepare liquid samples in advance.

  An object of the present invention is to detect living target cells (hereinafter, also referred to as target cells) in a biological sample. The target cells detected in the present invention can be any cells depending on experiments, such as red blood cells, white blood cells, ES cells, stem cells represented by iPS cells, endothelial cells, bacteria, microorganisms, etc. From the viewpoint of performing early diagnosis and metastasis diagnosis of cancer, it is preferable to mainly target cancer cells. Among the cancer cells targeted by the present invention, it is preferable to target circulating tumor cancer cells (CTC) that metastasize remotely through a circulatory system such as blood or lymph. Examples of such cancer cells include cells derived from gastric cancer, colon cancer, esophageal cancer, liver cancer, lung cancer, pancreatic cancer, bladder cancer, uterine cancer (epithelial tumor), and blood cell cancer (lymphoma, leukemia). .

Concentration Step The concentration step is a step of concentrating living cells from a biological sample to obtain a concentrated solution. In general, since the specific gravity is different between a living cell and a dead cell, a living cell can be separated from a dead cell and concentrated by utilizing a difference in specific gravity of a biological sample. As such a concentration step, viable cells can be separated and concentrated by specifically using a density gradient solution. Such a concentration step is not intended to completely separate living cells, and is sufficient as long as the proportion of living cells increases. The ratio of dead cells after the enrichment step is less than 20%, preferably less than 15%, more preferably less than 10%, even more preferably less than 5%, although it may vary depending on the contamination ratio of dead cells in the biological sample. It is preferable that

  The enrichment step is a step of separating and enriching living cells, but can also enrich target cells at the same time. In a biological sample, for example, in a blood sample, various cells including red blood cells, white blood cells, and platelets are present, but in order to detect target cells in a later step, target cells in all cells It is desirable to increase the ratio of. The concentration step can be performed by overlaying a biological sample on a density gradient solution and performing centrifugation. Live target cells can be recovered from the interface with the density gradient solution, thereby enriching the live target cells. Those skilled in the art can appropriately select the centrifugal speed and the density gradient that can achieve the concentration.

  As an instrument for performing the concentration step, for example, the cell separation / concentration structure shown in FIG. 1 can be used. The separation / concentration structure 1 is composed of two cylindrical members 2 and 3. The cylindrical member 2 constituting the upper part of the separation / concentration structure has an opening, and the cylindrical member 3 has one end closed to form a bottom part 5. Each of the tubular members 2 and 3 is provided with a communication opening 6 at an end opposite to the opening or the bottom, and when the two members are connected, the internal spaces of both tubular members communicate with each other, so that one separation and concentration as a whole is achieved. Form a structure.

  The density gradient solution is injected from the bottom (closed end 5 of the cylindrical member 3) to the vicinity of the separation part in the separation and concentration structure 1. More specifically, when the separation / concentration structure is allowed to stand, the liquid level height of the density gradient solution is higher than the communication port end of the upper cylindrical member 2 (on the side of the cylindrical member 2), that is, When the lower cylindrical member (cylindrical member 3) is separated, the components that have passed through the density gradient solution and moved to the closed end 5 side of the cylindrical member 3 by centrifugation are removed together with the majority of the density gradient solution. The target component (cell) maintained on the density gradient solution is injected into the cylindrical member 3 so that the target component (cell) can be separated while maintaining the cylindrical member 2, preferably about 1 mm. Thereafter, the biological sample solution is overlaid on the density gradient solution, the opening is closed with the cap 4, and centrifugation is performed. Generally, the centrifugation operation may be performed at a low speed of about 1000 to 2000 × g. However, in consideration of the density of the target cells and the density of the density gradient solution to be used, the conditions maintained on the density gradient solution are considered. Select For example, if the target cell is a living tumor cell and the above-mentioned centrifugation is performed, the density of the density gradient solution is set in the range of 1.060 to 1.095 g / mL according to the type of the tumor cell. can do. The density of the density gradient solution is preferably 1.075 g / ml or more, and more preferably 1.080 g / ml or more, from the viewpoint of increasing the concentration rate of the target cells. From the viewpoint of increasing the concentration rate of the target cells, the concentration is preferably 1.100 g / ml or less, more preferably 1.096 or less, and even more preferably 1.093 or less. More specifically, the density of the density gradient solution is preferably in the range of 1.082 to 1.091 g / mL. The physiological osmotic pressure can be set in the range of 200 to 450 mOsm / kg. From the viewpoint of reducing the contamination rate of dead cells, 280 mOsm / kg or more is preferable, and 300 mOsm / kg or more is still more preferable. From the viewpoint of increasing the enrichment rate of the target cells, it is preferably at least 300 mOsm / kg, more preferably at least 350 mOsm / kg, even more preferably at least 380 mOsm / kg. More specifically, the osmotic pressure of the density gradient solution is more preferably from 300 to 400 mOsm / kg. The pH of the solution can be arbitrarily selected within a range where the cells are not damaged, and for example, adjustment to a range of 6.8 to 7.8 can be exemplified.

  Due to the centrifugation operation, components having a density higher than the density of the density gradient solution (eg, dead cells) move through the gradient layer of the density gradient solution into the lower cylindrical member (cylindrical member 3). . On the other hand, target cells having a lower density than the density gradient solution (for example, living tumor cells) are maintained on the density gradient solution in the upper cylindrical member (cylindrical member 2). Therefore, if the connected tubular members are separated so as to be in the state shown in FIG. 1 while maintaining the hermetic sealing of the opening, the upper tubular member (tubular member 2) contains the target cell. Images can be collected. This fraction can be easily collected without special skill if, for example, the cap 4 is removed and the sealed state is opened to allow the fraction to be dropped downward. On the other hand, the fraction moved into the lower tubular member (the tubular member 3) can be discarded together with the tubular member, for example.

  For the measurement of the collected cells, a method of microscopic observation of cells applied to a slide or captured in a well, a flow cytometry method, or the like can be used. In particular, the technique of capturing and measuring cells one by one in a plurality of holding holes provided on the substrate by dielectrophoretic force used in the present invention is more preferable because the cells can be observed and analyzed with high sensitivity and high accuracy.

Developing Step The developing step is performed by developing the concentrated solution obtained through the concentration step on a substrate. By developing the concentrate, cells contained in the concentrate can be distributed on the substrate at intervals suitable for detection. It is preferable to develop in a non-aggregated state, and it is preferable to sufficiently suspend the concentrated solution before developing. The developing step can use any method as long as the cells can be distributed on the substrate at intervals suitable for detection, and may simply apply the concentrated solution on the substrate, but if necessary, Further processing may be performed. As an example, cells can be developed by applying a vibration or an induced electrophoretic force after applying the concentrate on the substrate. In order to spread the cells uniformly, it is preferable that a holding hole is formed on the substrate.By arranging approximately one cell in each holding hole, detection of target cells in the subsequent detection step is easy. become. Counting the number of cells in the concentrate, depending on the desired cell development density, may be diluted so that an appropriate cell number is developed, and measuring and developing the concentrate to be developed You can also.

  For example, the biological sample detecting structure shown in FIG. 2 can be used as an instrument used in the developing step of the present invention. The present structure is a structure 8 having a plurality of holding holes (through holes) 7 for holding cells in order to detect light emitted by a substance indicating the presence of cells in a subsequent detection step, and is a flat plate-like structure. On the substrate 9. Preferably, the substrate and the upper cover substrate 10 are made of a light-transmitting material, and the electrodes provided on the holding hole side of the substrate and on the surface of the upper cover substrate are transparent electrodes such as ITO. This makes it possible to observe the light emitted from the holding hole from above or below the substrate. The holding hole is made of the insulator film 11, but may be provided with the light shielding film 12. By providing the light-shielding film, it is possible to reduce light noise such as background noise caused by autofluorescence of the insulator film itself and crosstalk noise caused by light leaking from the adjacent holding holes. Only the light emitted by the observation target substance in the inside can be detected with high sensitivity and high accuracy.

  In addition, the structure includes a storage unit 13 that stores the suspension containing the cells on the holding hole, and the storage hole is provided so as to communicate with the storage unit. In addition, the accommodation section has an inlet 14 for introducing the cell suspension and an outlet 15 for discharging the cell suspension.

  As a method of capturing cells in the holding holes, dielectrophoretic force is used. With this dielectrophoretic force, living cells can be captured in many holding holes in a very short time of about several seconds. In order for the dielectrophoretic force to act on the cells, an AC electric field may be applied so that the lines of electric force concentrate on the holding holes while the container and the holding holes are filled with the suspension. As a configuration for applying such an AC electric field, as shown in FIG. 3, in addition to the structure of FIG. 2, the configuration is arranged on the surface of the substrate on the holding hole side at positions corresponding to the holding holes different from each other. Electrodes 16 and 17 forming a pair of electrodes (comb-shaped electrodes 18) are provided, and the holding hole extends from the upper surface of the holding hole to the comb-shaped electrode on the substrate. Can be. In any case, by exposing the electrode to the bottom of the holding hole and applying an AC voltage having a predetermined waveform between the two electrodes, the cells in the suspension are moved in the holding hole by dielectrophoretic force. It is possible to capture to. In addition, by arranging the holding holes in an array, the electric field generated by the voltage applied between the electrodes is almost equally generated in all the holding holes, and the cells are guided and trapped in all the holding holes in the same way can do.

  FIG. 4 is a diagram showing a biological sample detection device used in the present invention. As an example of the biological sample detecting device used in the present invention, the substrate 9, the upper cover substrate 10, the AC power source 19 for applying an AC voltage for generating the dielectrophoretic force 24 to the electrodes, and the AC power source shown in FIG. And a detector 21 for detecting light 20 emitted by a substance indicating the presence of the cells 23 captured in the holding hole of the structure after the application of a voltage from. As an example of the detection unit, a fluorescence microscope can be exemplified.

  An AC power supply is connected to the pair of electrodes of the structure via a conductive line 22. The AC power supply only needs to be able to apply a voltage between the electrodes sufficient to move the cells to the holding hole and generate an electric field necessary for the dielectrophoretic force to be captured. Specifically, a power supply which can apply an AC voltage having a peak voltage of about 1 V to about 20 V and a waveform of a sine wave, a rectangular wave, a triangular wave, a trapezoidal wave, or the like having a frequency of about 10 kHz to 10 MHz can be exemplified. In particular, it is preferable to use a rectangular wave having a frequency of 100 kHz to 1 MHz as a frequency and a waveform capable of moving live cells and capturing only one cell in one holding hole, and a rectangular wave of 3 MHz to 10 MHz. Particularly preferred. As the AC voltage having such a waveform, the rectangular wave instantly reaches the set peak voltage as compared with the case where the waveform is a sine wave, a triangular wave, or a trapezoidal wave, so that the cell is quickly moved toward the holding hole. This makes it possible to reduce the probability that two or more cells enter the holding hole so as to overlap (the probability that one holding hole can capture only one cell increases). The cells can be electrically considered as capacitors, but as long as the peak voltage of the square wave does not change, it is difficult for current to flow to the cells captured in the holding hole, and it is difficult for electric lines of force to be generated. Dielectrophoretic force is less likely to be generated in the holding hole that has captured cells. Therefore, once a cell is trapped in the holding hole, the probability of another cell being trapped in the same holding hole is reduced, and instead, another line of electric force is generated to generate a dielectrophoretic force. Cells are sequentially captured in empty holding holes where no cells are captured.

  In the biological sample detection device used in the present invention, it is preferable to employ a power supply that generates an AC voltage having no DC component. When an AC voltage having a DC component is applied, cells move due to a biased force in a specific direction due to an electrostatic force (electrophoretic force) generated by the DC component, which makes it difficult to capture cells by dielectrophoretic force. is there. Also, when an AC voltage having a DC component is applied, ions contained in the suspension containing the cells generate an electric reaction on the electrode surface and generate heat, and the cells cause a thermal motion, which makes it impossible to control the movement due to the dielectrophoretic force. However, it is difficult to move to the holding hole and capture. The AC voltage having a DC component refers to a voltage having a frequency duty ratio other than 50%, a voltage having an offset, a voltage having an extremely long cycle (for example, 1 second or more), and the like.

  In the present invention, a living cell may be detected after a voltage is applied to the biological sample captured in the holding hole at least once. There is no particular limitation on the method of applying the voltage, and there is no particular limitation as long as the voltage can be applied to the biological sample. For example, application of a voltage between electrodes provided in a structure used in the present invention can be exemplified. As the voltage, a DC voltage or an AC voltage, or both a DC voltage and an AC voltage may be applied simultaneously or alternately. Conditions such as the magnitude of the voltage and the application time can be set as appropriate, and there is no particular limitation as long as most cells, including cells with high viability, do not die. For example, in the case of a DC voltage, a voltage of several volts to several tens of volts and an application time of several nanoseconds to several milliseconds can be exemplified. For example, when a voltage of about 50 V is applied for several minutes or more, most cells, including cells having high viability, will be killed. preferable. For example, when the structure of the present invention is used, 30 microseconds at 50 V can be exemplified.

  The AC voltage is not particularly limited as long as the voltage repeats positive and negative values of the voltage in several nanoseconds to several hundred milliseconds. For example, a power supply that can apply an AC voltage having a peak voltage of about 1 V to about 20 V and a waveform of a sine wave, a rectangular wave, a triangular wave, a trapezoidal wave, or the like having a frequency of about 100 kHz to 3 MHz can be exemplified. When the voltage is about 1 V to 20 V, the application time of the AC voltage is preferably 30 minutes or less, and more preferably 15 minutes or less.

  The number of repetitions of applying the DC voltage and / or the AC voltage is not particularly limited as long as it is one or more times. However, if the number of repetitions is large, most cells, including cells having high viability, die. For example, when the structure of the present invention is used, the number of repetitions of applying a DC voltage of 30 V for 30 microseconds at 50 V or the number of repetitions of applying an AC voltage of 20 V and 1 MHz can be about three times.

  By applying such a voltage (a DC voltage and / or an AC voltage), cells having a damaged cell membrane and having low survival activity can be killed, and living cells having higher survival activity can be selected. An AC power supply for generating dielectrophoresis may be used as a power supply for applying a voltage to the biological sample.

Detection step The detection step is a step of detecting target cells spread on the substrate. The biological sample detection method of the present invention may be any method as long as it can detect a target cell among the cells contained in the concentrated solution containing the living cells concentrated by the separation and concentration structure. The detection is preferably performed optically, and the target cell is obtained by imaging under a microscope or a fluorescence microscope, analyzing an image taken in a bright field, and / or an image taken using a labeling substance. Can be detected. For example, a target cell can be detected by acquiring and analyzing information on cell morphology from an image captured in a bright field. Examples of such information include a cell diameter, a cell area, a cell volume, a cell perimeter, and roundness. When a labeling substance is used, the target cells may be directly labeled, or cells other than the target cells may be labeled. When labeling cells other than target cells, target cells can be detected by comparing the captured image with another image, for example, a bright-field captured image.

  The labeling substance in the present invention is a substance that can be directly or indirectly bound to a substance (hereinafter, referred to as a “specific substance”) that is specifically present on the surface or inside of a cell to be labeled, and is identifiable. Say. When labeling a target cell, the term "specifically present in the cell to be labeled" means that the cell is present in the cell to be labeled and does not exist in the cell other than the cell to be labeled, or is labeled more than the cell other than the cell to be labeled. It means that it is abundant in cells.

  Examples of the specific substance include an antigen, a receptor, a sugar chain, an enzyme, a nucleic acid and the like. Examples of antigens include antigens on cancer cells, major histocompatibility antigens (MHC, and HLA in the case of humans). Antigens on cancer cells include EpCAM, cytokeratin (CK) and the like. Receptors include hormone receptors, Fc receptors, viral receptors and the like. In addition, proteins such as DNA and RNA and tumor markers may be used.

  More specifically, for example, if the cells are various cancer cells, a marker versatile (common) for various cancer cells can be a specific substance. If the target cells are breast cancer or lung cancer, HER2, CK, and EGFR, which are antigens specifically expressed in these cancers, can be specific substances.

  The specific substance can be observed (detected) by, for example, labeling with a substance that specifically binds to the specific substance. For example, an antigen binds to an antibody against the antigen, a receptor binds to a ligand, and a sugar chain binds to lectin. That is, a substance that specifically binds to a specific substance and a signal substance that emits an optically detectable signal can be combined to form a labeling substance. Examples of the optically detectable signal include fluorescence, phosphorescence, and luminescence, and the signal substance may itself emit these lights, or the signal substance may emit phosphorescence. It may be a substance that catalyzes the reaction.

  The labeling substance for detecting the specific substance of the present invention is not particularly limited as long as the cell to be labeled can be detected, and refers to any substance that can indicate the presence of any component constituting the cell. As a representative labeling substance, a signal substance itself that can be detected based on the property of fluorescence, phosphorescence, or luminescence, or these signal substances can specifically bind to a specific substance such as an antibody, a ligand, or a lectin described above. Examples thereof include a substance bonded to a substance. As a signal substance that emits fluorescence, there is a fluorescent dye, and the fluorescent dyes FITC (fluorescein isocyanate), PE (phycoerythrin), rhodamine, and the like can be used in combination with a substance capable of binding to a specific substance. Fluorescent substances such as DAPI (4 ′, 6-diamidino-2-phenylindole) and PI (propidium iodide) can directly stain nuclei. Examples of the substance that catalyzes the luminescence reaction include peroxidase, β-galactosidase, alkaline phosphatase, and luciferase.

  Further, the use of a labeling substance in which a quencher that inhibits fluorescence, phosphorescence, or luminescence and a substance that specifically binds to a specific substance can be exemplified. In this case, the cells are stained with a substance that emits fluorescence, phosphorescence, or luminescence, and when the labeling substance is further reacted, the fluorescence, phosphorescence, or luminescence from the holding hole in which the cells having the specific substance are captured is reduced. Since the decrease is caused by the quencher, this decrease may be detected.

  The labeling substance is, as described above, a substance that binds or reacts with a specific substance and a substance that specifically binds to a specific substance and a substance that emits an optically detectable signal. There may be. When a substance that emits an optically detectable signal is bound to a substance that specifically binds to a specific substance, the two may be directly bound to each other by a known chemical method or the like. It may be indirectly bound to a binding substance via a binding substance. For example, a substance that specifically binds to a specific substance is bound to biotin, and a substance that generates a signal is bound to avidin or streptavidin. In this case, avidin or streptavidin bound to a substance that generates a signal binds to biotin bound to a substance specifically bound to a specific substance, and as a result, (specific substance)-(specifically to a specific substance) The specific substance is indirectly labeled by forming a complex of (binding substance)-(biotin)-(avidin or streptavidin)-(signal generating substance). Such indirect cases are also included in the specific substance of the present invention.

  The labeling substance may be a substance that labels target cells, a substance that labels cells other than the target cells, or a substance that labels both of them. By using a plurality of labeling substances, the detection sensitivity of target cells is increased. Since the cell suspension concentrated in the concentration step contains a large number of white blood cells, red blood cells, and platelets in addition to the target cells, it is useful to use a labeling substance that distinguishes these cells from the target cells. It is. For example, when DAPI or PI that stains nuclei is used as a labeling substance, it becomes possible to distinguish erythrocytes without nuclei from other cells. In order to distinguish leukocytes from other cells, a labeling substance for leukocyte markers such as CD2, CD3, CD16, CD19, CD36, CD45, CD56, and CD66b can be used. It is preferable to use a labeling substance for CD45 which is considered to be used. Furthermore, since vascular endothelial cells other than leukocytes may exist in blood in very small numbers as nucleated cells, the use of a labeling substance for CD105, CD146, or the like enables more accurate detection of target cells.

  In the detection step of the present invention, the target cell can be detected by observing the cell size and cell nucleus size of the target cell from the optically detected detection image. In this detection method, target cells can be detected by observing one or a plurality of images taken by an optical detection method corresponding to one or a plurality of labeling substances.

  In particular, CTC detection includes, for example, labeling with an epithelial marker-conjugated antibody such as an anti-CK antibody labeled with FITC or PE, followed by detection with a fluorescence microscope or the like. A typical CTC detection method will be described with reference to FIG. Fluorescently labeled antibodies that recognize epithelial markers, such as CK, and leukocyte markers, such as CD45, and nuclear markers that stain nuclei, such as DAPI positive / CK Positive / CD45 negative is detected as CTC. Furthermore, for DAPI-positive / CK-negative / CD45-negative cells, cells larger than leukocytes, that is, cells having a size of 10 μm to 30 μm can also be detected as CTC. Furthermore, with respect to DAPI positive / CK negative / CD45 negative cells, the cell nucleus size is referred to, and those having a DAPI luminance region larger than that of leukocytes can be detected as CTC.

  Nucleated cells are identified by fluorescence observation at the DAPI wavelength (UV excitation and blue emission), and are represented by CK-positive and CD45-negative tumor cells, DAPI-positive, CK-negative, CD45-positive leukocytes, dust, etc. It allows the distinction from noise signals (DAPI positive, CK positive and CD45 positive or DAPI negative, CK positive). In addition, DAPI-positive, CK-negative and CD45-negative cells are compared with normal cells such as erythrocytes and leukocytes in bright-field observation to compare cell morphology and size, or by performing Papanicolaou staining or Giemsa staining to detect intracellular nuclei. CTC can be identified by morphological characteristics such as cytoplasm and cytoplasm.

  It is sufficient that the biological sample can be captured and detected by the holding hole on the substrate. Further, the biological sample detection device of the present invention is a device capable of observing a biological sample by including the substrate and optical detection means for irradiating the observation region of the substrate with light and detecting an optical signal generated. Light sources include halogen lamps, mercury lamps, and metal lamps, lasers, and LEDs. The light from the light source can be observed by optical means, such as optical filters, mirrors, and lenses as necessary. The light may be guided to the region.

  As optical signal information, an image (bright field) composed of light intensity distribution by transmitted light and reflected light in a wide wavelength range, the fluorescence intensity detected from the fluorescence emitted by the fluorescent substance, the peak value of the fluorescence intensity, the fluorescence intensity Numerical values calculated from the fluorescence intensity such as the minimum value, the average value of the fluorescence intensity, and the integrated value of the fluorescence intensity can be exemplified. The bright field information is not particularly limited as long as information on cell morphology can be obtained. For example, the diameter of the cell, the area of the cell, the volume of the cell, the perimeter of the cell, the roundness and the like can be exemplified.

  The biological sample detection device of the present invention may include means for specifying the position of the substrate to be observed. By integrating the position information of the biological sample captured in the holding hole on the substrate and the information regarding the optical signal derived from the biological sample, it is possible to accurately specify in which holding hole the target cell is present.

  As a method for detecting a biological sample using no fluorescent label or using a minimal fluorescent label, it can be applied to the detection of target cells by analyzing the shape and pattern of cells in a bright-field image. As used herein, the term “bright field” refers to an image that is specifically labeled with a fluorescent label and is not based on the intensity of light that emits fluorescence, and is an image that is obtained based on polarization, phase difference, absorbance based on wavelength, and reflectance.

  Moreover, you may detect a target cell further according to a magnitude | size. It is known that many cancer cells are larger in size than red blood cells and white blood cells (Non-patent literature: Rostagno P. et al., Anticancer Res., 17 (4A), 2481-2485 (1997)), By extracting the cells with larger cells than red blood cells and white blood cells, it becomes possible to accurately detect mesenchymal cells derived from tumor tissue that has undergone epithelial-mesenchymal metastasis. The size of the red blood cell is a disk shape having a diameter of 7 to 8 μm and a thickness of about 2 μm, and the white blood cell is a spherical shape having a diameter of about 6 to 15 μm, although the size varies depending on the type. On the other hand, since cancer cells, particularly CTC, have a size of about 10 to 30 μm, they can be detected as target cells having a size of preferably 10 μm or more, more preferably 15 μm or more.

In the present invention, a living cell may be detected after heating the biological sample captured in the holding hole. There is no particular limitation on the method of heating the biological sample, and there is no particular limitation as long as the temperature of the biological sample can be increased. For example, the structure itself for detecting cells may be heated by a heater or the like, or the temperature around the structure may be increased by a heater or the like. Furthermore, individual biological samples may be heated by irradiating a biological sample with a laser or applying electromagnetic waves. For example, as electromagnetic waves, radiation such as gamma rays (frequency 3 × 10 ¹⁸ Hz), X-rays (frequency 3 × 10 ¹⁶ Hz), ultraviolet rays (frequency 3 × 10 ¹⁵ Hz), and visible rays (frequency 3 3 × 10 ¹³ Hz), light such as infrared rays (frequency 3 × 10 ¹² Hz), microwaves (frequency 3 × 10 ¹¹ Hz), millimeter waves (frequency 3 × 10 ¹⁰ Hz), centimeter waves (frequency 3 × 10 ⁹ Hz) Hz), ultra high frequency (frequency 3 × 10 ⁸ Hz), ultra high frequency (frequency 3 × 10 ⁷ Hz), short wave (frequency 3 × 10 ⁶ Hz), medium frequency (frequency 3 × 10 ⁵ Hz), long wave (frequency 3 × 10 ⁴ Hz), a very long wave (frequency 3 × 10 ³ Hz), and a very low frequency (frequency 50 to 60 Hz).

  The measurement of the heating temperature is not particularly limited as long as the temperature in the holding hole, the temperature of the structure, or the temperature around the structure can be measured by a known means such as a general thermometer or a thermocouple. The heating temperature and time can be appropriately set, and there is no particular limitation as long as most cells, including cells with high viability, do not die. For example, as a heating temperature, if the temperature generally exceeds 40 ° C., most cells including cells having high viability will be killed. Therefore, the heating temperature may be in the range of 25 ° C. to 40 ° C., and more preferably 30 ° C. to 40 ° C. 35 ° C. is preferred. When the heating time is about 30 ° C. to 35 ° C., about 1 to 3 hours can be exemplified. By heating the biological sample in this manner, cells with weak viable activity whose cell membrane is being damaged can be killed, and living cells having higher viability can be selected.

Cancer Diagnostic Method The method for detecting a target cell in a biological sample of the present invention can be used in a method for diagnosing cancer, a method for diagnosing cancer metastasis, and a method for predicting the prognosis of cancer by detecting CTC as a target cell. it can. When CTC can be detected in a biological sample of a subject such as a health checkup, the subject can be diagnosed as having cancer. When the subject is thus diagnosed as having cancer, the subject can undergo further diagnosis to identify the site of the cancer, or be treated by administration of an anticancer drug, radiation therapy, surgery, or the like. . If the subject already has cancer or has been treated for cancer, and if CTC can be detected by the detection method of the present invention, it can be diagnosed that there is a risk of developing metastatic cancer. In that case, the administration of the anticancer drug can be continued after the diagnosis to further treat the metastatic cancer.

  All documents mentioned herein are incorporated by reference in their entirety. Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the examples.

Example 1 Concentration Step of Living Cancer Cells Using the separation-concentration structure of the embodiment shown in FIG. 1, living cancer cells were separated and concentrated. Specifically, the cylindrical member 2 is a cylindrical polypropylene member having an inner diameter of 18 mm, a length of 70 mm, and a capacity of 15 mL. In addition, the inclination angle of the tapered portion of the tubular member 2 is 30 °, and the communication opening with the tubular member 2 is Φ2 mm. The tubular member 3 is a polypropylene member having an inner diameter of 10 mm, a length of 54 mm, and a capacity of 2 mL.

As schematically shown in FIG. 5, 2 mL of various density gradient solutions 25 shown in Table 1 were injected into the lower cylindrical member 3 of the separation / concentration structure (the white portion at the bottom in the figure indicates the density). Part filled with gradient solution). Specifically, the liquid was injected such that the liquid surface height of the density gradient solution was higher than the communication opening 6 of the upper cylindrical member 2 by about 1 mm (therefore, the liquid surface was located inside the upper cylindrical member). Subsequently, a mixture 26 of 3 mL of a blood sample, 3 mL of physiological saline, and 75 μL of a binder (trademark: RosetteSep, StemCell Technologies Inc) was overlaid on the density gradient solution (in the figure, a mixture in which black portions were overlaid). Liquid part). The blood sample is a suspension in which about 30 human breast cancer cells (SKBR3), in which cell death was induced by treatment with hydrogen peroxide in blood of a healthy individual obtained with informed consent, were suspended as model dead cells. . The cancer cells to be added are labeled in advance with a fluorescent staining reagent (PI, manufactured by Dojindo Laboratories Inc.). The cancer cells were prepared by stationary culture so that the cell density became about 2 × 10 ⁵ cells / cm ² , then detaching the cells from the dish with 0.25% trypsin / 1 mM EDTA, and limiting dilution. It is.

  After injecting the sample, the opening of the separation / concentration structure was closed with a cap 4 (made of polypropylene), and centrifuged at 2,000 × g for 10 minutes at room temperature. Due to the centrifugation operation, the cells were maintained at the interface 27 between the density gradient solution and the sample as shown on the left of FIG. After separating the tubular members 2 and 3 constituting the separation / concentration structure at the separating portion without removing the cap, the cap is removed and the seal is opened as shown in the right of FIG. A part of the density gradient solution and the cells maintained thereon are drained from the communication opening 6 of 2 and collected by a 50 mL tube placed below, and the inner wall of the upper cylindrical member is washed, and the cells adhered to the wall are washed. Was also collected at the same time.

  An erythrocyte lysate containing ammonium chloride as a main component was added to the recovered cell suspension to make up to 30 mL, and centrifuged at 300 × g for 10 minutes at room temperature. After centrifugation, the liquid at the top of the pellet was removed with a pipette, and the cells in the pellet were resuspended in 30 mL of a 300 mM mannitol solution and centrifuged at 300 xg for 5 minutes at room temperature. This centrifugation operation is for removing cell debris and platelets and enriching the target cells. The number of normal leukocytes mixed with cancer cells is 300,000 to 500,000, and the density of the target cells (cancer cells) is increased by binding non-target cells (red blood cells and white blood cells) to each other with a binder. By performing separation using the separation-concentration structure of the present invention after increasing the difference, target cells can be separated with high recovery and selectivity.

  For the measurement of the collected cells, a method of microscopic observation of cells applied to a slide or captured in a well, a flow cytometry method, or the like can be used. In the present Example 1, a method was employed in which the separated cells were captured by a dielectrophoretic force in a holding hole (approximately 300,000) having a pore diameter of Φ30 μm and a depth of 40 μm provided on the substrate and measuring.

  Using the apparatus shown in FIG. 4, the percentage of dead cancer cells mixed into the recovered cell suspension was measured. The separated cell suspension was supplied to the holding section of the structure, and the cells captured in the holding holes were observed with the detection section 21 (fluorescence microscope). The right column of Table 1 shows the osmotic pressure of the density gradient solution and the mixing ratio of dead cells. When the osmotic pressure was in the range of 200 to 400 mOsm / kg, the number of dead cells mixed into the upper collected fraction was less than 10%, and living cells could be selectively collected. In particular, under conditions of an osmotic pressure of 300 to 400 mOsm / kg, contamination of dead cells was less than 5%, and living cancer cells could be more selectively recovered.

Example 2 Examination of Specific Gravity in Concentration Step Using the same separation / concentration structure as in Example 1, about 30 live cells labeled in advance with a fluorescent staining reagent (trade name: CalceinAM, manufactured by Dojin Chemical Laboratory Co., Ltd.) A sample in which human breast cancer cells (SKBR3) were mixed with blood from a healthy subject, a mixture of physiological saline and a binder were overlaid on the density gradient solution, and centrifugation and collection operations were performed. The centrifugation in the density difference separation was performed at 2000 × g for 10 minutes, and the centrifugation in the recovery was performed at 300 × g for 10 minutes at room temperature. In Example 2, 2 mL of the density gradient solution shown in Table 2 was injected into the cylindrical member 3 below the separation / concentration structure.
Using the device shown in FIG. 4, the number of recovered cancer cells was measured in the same manner as in Example 1. Table 2 shows the results of the density of the density gradient solution and the recovery rate of cancer cells. As the density of the density gradient solution increased, the recovery rate of cancer cells improved, but at a density of 1.091 g / mL and an osmotic pressure of 300 mOsm / kg, red blood cells did not sufficiently sediment and were mixed into the upper structure, resulting in poor separation. became. On the other hand, when the density is 1.091 g / mL and the osmotic pressure is in the range of 380 to 400 mOsm / kg, erythrocytes (part of leukocytes) and cancer cells can be well separated, and the recovery rate is 88.2 to 95.3. %Met.

Example 3: Examination of centrifugation conditions in the concentration step In the same manner as in Example 2, the density of a mixture of a sample obtained by mixing about 30 human breast cancer cells (SKBR3) with healthy human blood, saline, and a binder was determined. The solution was overlaid on the gradient solution, and centrifugation and recovery operations were performed. In addition, centrifugation conditions in the density difference separation were 2000 × g, 5 to 20 minutes, and were performed at room temperature. In Example 3, 2 mL of a density of 1.091 g / mL and an osmotic pressure of 380 mOsm / kg were injected into the cylindrical member 3 below the separation / concentration structure.

Using the apparatus shown in FIG. 4, it was confirmed that the separated cells were the target breast cancer cells as in Example 1. Table 3 shows the results of the density of the density gradient solution and the recovery rate of cancer cells. The centrifugation conditions in the density difference separation reached a maximum of 95.8% in 2000 × g for 10 to 15 minutes.

Example 4: Development step and detection step In the same manner as in Example 2, a mixed solution of a sample obtained by mixing about 30 human breast cancer cells (SKBR3) with healthy human blood, physiological saline, and a binder was used as a density gradient solution. Was concentrated, and a concentration step was performed by performing centrifugal separation and recovery operations to obtain a concentrated solution. In addition, centrifugation conditions in the density difference separation were 2000 × g for 10 minutes at room temperature. In Example 4, 2 mL of a density of 1.091 g / mL and an osmotic pressure of 380 mOsm / kg were injected into the cylindrical member 3 below the separation / concentration structure. After centrifugation, the cells were collected and washed in the same manner as in Example 1 to obtain a concentrated liquid in which live cancer cells were concentrated.
The cell suspension is introduced into the accommodating portion of the structure shown in FIG. 2, an AC voltage (voltage 20 Vpp, frequency 10 kHz to 10 MHz, rectangular wave) is applied between the substrates, and the cells are captured in the holding holes by dielectrophoretic force. did.

  While applying the AC voltage, a 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine was introduced into the accommodation part in which one cell was captured in one holding hole, and allowed to stand for 3 minutes. The application of the AC voltage was stopped, and the solution in the container was removed by suction. Since the cells are electrostatically bound to the holding portion by the action of poly-L-lysine, the cells are not detached from the holding portion by washing even if there is no dielectrophoretic force.

  Subsequently, an aqueous solution containing 50% ethanol and 1% formaldehyde (hereinafter sometimes referred to as a cell membrane permeating reagent) was introduced into the storage section, and allowed to stand for 10 minutes to allow the cell membrane to permeate. Thereafter, the solution in the storage section was removed by suction, PBS was introduced, and the cell membrane permeating reagent remaining in the storage section was washed.

  Next, 800 μL of a cell staining solution (a cell staining solution obtained by mixing a FITC-labeled anti-CK antibody (Miltenyi Biotec), a PE-labeled anti-CD45 antibody (Beckman-Coulter), and DAPI (0.5 μg / mL)) was sent to the storage section. After labeling the cells (25 ° C., 30 minutes), the cells were washed with an aqueous mannitol solution to detect cancer cells. Since both cancer cells and normal white blood cells have nuclei, they were stained with DAPI, and those having no nuclei, such as red blood cells and dead cell debris, were eliminated by detection software. Since the cancer cells expressed CK but did not express CD45, they were labeled only with the FITC-labeled anti-CK antibody. On the other hand, normal white blood cells express CD45 but do not express CK, and thus were labeled with a PE-labeled anti-CD45 antibody.

  In order to observe all cells captured in the plurality of holding holes, an image of the entire holding unit was taken. For this, a fluorescence microscope (IX71; Olympus) equipped with a computer-controlled motorized stage and an electron multiplying-cooled CCD camera (EMCCD; FLOVEL, ADT-100) was used. LabVIEW (National Instruments) was used for image acquisition and analysis software. The number of normal white blood cells captured in the holding hole after the treatment was about 300,000, but this amount was not hindered in detecting cancer cells. Further, since the cancer cells were captured in the holding holes, the total number of cells could be counted by scanning the holding portions. As shown in FIG. 7, the detection rate of cancer cells in the blood was about 90% at a frequency of 100 kHz to 3 MHz.

Example 5: Separation of living cells in the developing step
After fixing human breast cancer cells (SKBR3) labeled with a fluorescent labeling reagent (trade name: CFSE, manufactured by Dojindo Laboratories) from a 4% aqueous solution of formaldehyde, a PI-labeled cell suspension is shown in FIG. Introduced into the housing part of the body, and by applying an AC voltage (voltage 20 Vpp, frequency 100 kHz to 10 MHz, rectangular wave) between the substrates, PI unlabeled cells (live cell model) and PI labeled cells (dead cells) by dielectrophoretic force Model) were compared. As a result, the PI non-labeled cell (live cell model) maintained a capture rate of 98% or more in the holding hole at a frequency of 100 kHz to 10 MHz. On the other hand, the PI-labeled cells (dead cell model) had a capture rate of 99% at 100 kHz, but decreased as the frequency increased to 92% at 1 MHz, 59% at 3 MHz, and 6% at 10 MHz. Therefore, live cancer cells can be selectively captured at frequencies of 3 MHz to 10 MHz.

Example 6: Breast cancer clinical measurement data at the National Cancer Center, counting of CK positive and negative cells, correlation with prognosis In the same manner as in Example 1, a mixture of stage IV breast cancer patient blood, saline, and a binder The solution was overlaid on the density gradient solution, and centrifugation and recovery operations were performed to concentrate live cancer cells. In addition, centrifugation conditions in the density difference separation were 2000 × g for 10 minutes at room temperature. In Example 5, 2 mL of a density of 1.091 g / mL and an osmotic pressure of 380 mOsm / kg were injected into the cylindrical member 3 below the separation / concentration structure. After centrifugation, cells were collected and washed in the same manner as in Example 1 to obtain a cell suspension in which cancer cells were concentrated.

  The cell suspension was introduced into the container of the structure shown in FIG. 2, an AC voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) was applied between the substrates, and the cells were captured in the holding holes by dielectrophoretic force.

  While applying the AC voltage, a 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine was introduced into the accommodation part in which one cell was captured in one holding hole, and allowed to stand for 3 minutes. The application of the AC voltage was stopped, and the solution in the container was removed by suction. Subsequently, a cell membrane permeating reagent was introduced into the container, and allowed to stand for 10 minutes to permeate the cell membrane. Thereafter, the solution in the storage section was removed by suction, PBS was introduced, and the cell membrane permeating reagent remaining in the storage section was washed.

  Next, 800 μL of a cell staining solution (a cell staining solution obtained by mixing a FITC-labeled anti-CK antibody (Miltenyi Biotec), a PE-labeled anti-CD45 antibody (Beckman-Coulter), and DAPI (0.5 μg / mL)) was sent to the storage section. After cell labeling (25 ° C., 30 minutes), the cells were washed with an aqueous mannitol solution, and CTC was detected. Since both CTC and normal white blood cells have nuclei, they were stained with DAPI, and those that did not have nuclei such as red blood cells and dead cell debris were eliminated by detection software. In addition, although CTC does not express CD45, there are cells that express CK and cells that do not express CK. Therefore, DAPI positive, CD45 negative, CK positive and negative cells were extracted by image processing. Normal white blood cells express CD45 but do not express CK, and thus were identified as CTC by detecting DAPI positive, CK negative, and CD45 positive cells.

  In order to observe all cells captured in the plurality of holding holes, an image of the entire holding unit was taken. For this, a fluorescence microscope (IX71; Olympus) equipped with a computer-controlled motorized stage and an electron multiplying-cooled CCD camera (EMCCD; FLOVEL, ADT-100) was used. LabVIEW (National Instruments) was used for image acquisition and analysis software. Although the number of normal leukocytes captured in the holding hole after the treatment was about 100,000 to 500,000, it was an amount that would not hinder the detection of CTC. Further, since CTC was captured in the holding hole, the total number of cells could be counted by scanning the holding portion. FIG. 8 shows the CTC count of the breast cancer patient sample (15 cases).

  The number of detected CTCs was compared with that of a prior art CellSearch system (Janssen Diagnostics). As a result, CTC was not detectable in 5 cases out of a total of 15 cases with the CellSearch system (positive rate: 66.7%, diagnostic threshold:> 2 cells / 7.5 mL of blood), but all cases were detected by the method of the present invention. (Positive rate 100%, diagnostic threshold:> 1/3 mL blood).

Example 7 Method for Counting CK Positive and Negative Cells In the same manner as in Example 1, a mixture of blood of a stage IV breast cancer patient, physiological saline, and a binder was overlaid on a density gradient solution, followed by centrifugation and collection operations. Was carried out. In addition, centrifugation conditions in the density difference separation were 2000 × g for 10 minutes at room temperature. In Example 6, 2 mL of a density of 1.091 g / mL and an osmotic pressure of 380 mOsm / kg were injected into the cylindrical member 3 below the separation / concentration structure. After centrifugation, cells were collected and washed in the same manner as in Example 1 to obtain a cell suspension in which cancer cells were concentrated.
The cell suspension in the housing part of the structure shown in FIG. 2 was introduced, an AC voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) was applied between the substrates, and the cells were captured in the holding holes by dielectrophoretic force.

While applying the AC voltage, a 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine was introduced into the accommodation part in which one cell was captured in one holding hole, and allowed to stand for 3 minutes. The application of the AC voltage was stopped, and the solution in the container was removed by suction. Subsequently, a cell membrane permeating reagent was introduced into the container, and allowed to stand for 10 minutes to permeate the cell membrane. Thereafter, the solution in the storage section was removed by suction, PBS was introduced, and the cell membrane permeating reagent remaining in the storage section was washed.
Next, 800 μL of a cell staining solution (a cell staining solution obtained by mixing a FITC-labeled anti-CK antibody, a PE-labeled anti-CD45 antibody, and DAPI (0.5 μg / mL)) was sent to the storage section, and cell labeling was performed ( (25 ° C., 30 minutes), and washed with an aqueous mannitol solution to detect CTC. Since both CTC and normal white blood cells have nuclei, they were stained with DAPI, and those having no nuclei, such as red blood cells and dead cell debris, were eliminated by detection software. In addition, although CTC does not express CD45, there are CTC expressing CK (CK positive CTC) and CTC not expressing (CK negative CTC), so that DAPI positive, CD45 negative, CK positive and negative Cells were extracted by image processing. Normal white blood cells express CD45 but do not express CK, and thus were identified as CTC by detecting DAPI positive, CK negative, and CD45 positive cells.

  In addition, in order to observe all the cells captured in the plurality of holding holes, the entire holding unit was imaged. For this, a fluorescence microscope (IX71; Olympus) equipped with a computer-controlled motorized stage and an electron multiplying-cooled CCD camera (EMCCD; FLOVEL, ADT-100) was used. LabVIEW (National Instruments) was used for image acquisition and analysis software.

Further, the number of normal white blood cells captured in the holding hole after the treatment was about 100,000 to 500,000, but this amount did not hinder the detection of CTC. Although the number of CTCs was counted by the above method, DAPI-positive, CD45-negative, and CK-negative cells (determined as CK-negative CTCs that are not leukocytes and do not express CK) exist in some cells. Make sure you do.
FIG. 9 shows a series of CTC detection flows including abnormal cells that are not white blood cells.

1 Separation and concentration structure 2 Cylindrical member (upper side)
3 tubular member (lower side)
4 Cap 5 Bottom 6 Communication opening end (separation part)
7 Holding hole (through hole)
Reference Signs List 8 Structure 9 Substrate 10 Top cover substrate 11 Insulating film 12 Light shielding film 13 Housing 14 Inlet 15 Outlet 16 Electrode (+)
17 electrode (-)
DESCRIPTION OF SYMBOLS 18 Comb electrode 19 AC power supply 20 Light 21 Detection part 22 Conductive wire 23 Cell 24 Dielectrophoretic force 25 Density gradient solution 26 Mixed solution 27 Interface

Claims (6)

A method for detecting live tumor cells in blood ,
Live tumor cells from a blood sample,
A density gradient solution having a specific gravity of 1.091 to 1.093 g / mL and an osmotic pressure in a range of 380 to 400 mOsm / kg, or
Density gradient solution having a specific gravity of 1.077 to 1.084 g / mL and an osmotic pressure of 300 mOsm / kg
A first step of obtaining a concentrate using
A second step of developing the concentrated solution obtained in the first step on a substrate;
A third step of detecting target cells developed on the substrate;
Detection method of tumor cells in the blood you comprising: a. The method according to claim 1, wherein the density gradient solution has a specific gravity of 1.091 / mL and an osmotic pressure in a range of 380 to 400 mOsm / kg. The biological sample detection method according to claim 1 or 2 , wherein the first step is a step of enriching using a specific gravity difference between a living cell and a dead cell in the biological sample. In the first step, one end is closed to form a bottom, and the other end is composed of an open tubular structure and a cap for closing the opening. The structure is composed of two or more tubular members, The method for detecting a biological sample according to claim 3 , wherein the living cells are concentrated from the biological sample using a structure that can be separated in the part. A plurality of holding holes are provided on the substrate,
The biological sample detection method according to any one of claims 1 to 4 , wherein the biological sample cells contained in the concentrate are developed on a substrate by dielectrophoretic force. The biological sample detection method according to any one of claims 1 to 5 , wherein the target cell is a circulating tumor cancer cell (CTC).