WO2012091056A1 - Procédé de test d'une cellule cible dans le sang, dispositif de recherche d'une cellule cible, et biopuce - Google Patents

Procédé de test d'une cellule cible dans le sang, dispositif de recherche d'une cellule cible, et biopuce Download PDF

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WO2012091056A1
WO2012091056A1 PCT/JP2011/080306 JP2011080306W WO2012091056A1 WO 2012091056 A1 WO2012091056 A1 WO 2012091056A1 JP 2011080306 W JP2011080306 W JP 2011080306W WO 2012091056 A1 WO2012091056 A1 WO 2012091056A1
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target
target cell
image data
blood
dictionary
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PCT/JP2011/080306
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English (en)
Japanese (ja)
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森田　敏樹
貴幸 菊池
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エスシーワールド株式会社
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Priority to JP2012526214A priority Critical patent/JP5140780B2/ja
Publication of WO2012091056A1 publication Critical patent/WO2012091056A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0346—Capillary cells; Microcells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/38—Pediatrics
    • G01N2800/385—Congenital anomalies

Definitions

  • the present invention relates to a method, a target cell search device, and a biochip for examining a specific target cell such as a fetus-derived cell using blood collected for prenatal diagnosis.
  • amniotic fluid test which is known as prenatal diagnosis of the fetus, not only has a large invasion to the mother's body, but also can damage the uterus and fetus, leading to miscarriage and stillbirth.
  • fetal cell for example, nucleated red blood cells migrates in 1 ml of maternal blood. Therefore, in order to examine the clinical usefulness as a non-invasive prenatal diagnosis, a large-scale clinical study on fetal prenatal diagnosis using maternal blood in the United States and the like from 1995 to about five years (NIFTY study).
  • This clinical study uses flow cytometry to measure fluorescence intensity, target cell size, etc. at high speed with a laser light source while the target cells are specifically fluorescently labeled using an antigen-antibody method and then placed in a liquid flow.
  • Fluorescent activated cell sorting Fluorescence-activated cell ⁇ sorting
  • magnetic microbeads are specifically bound to their target cells via antibodies that react with cell surface antigens and antigens. 2744 cases were also carried out by techniques such as nuclear magnetic cell separation (MACS: Magnetic-activated cell sorting) to collect cells.
  • FACS Fluorescence-activated cell ⁇ sorting
  • MCS Nuclear magnetic cell separation
  • FISH method Fluorescence in situ hybridization
  • Patent Document 1 describes a slide glass with target cells placed on a sample stage using an optical microscope and a CCD camera provided with an XY stage.
  • a method of effectively searching for a target cell by performing image processing based on color, shape, positional relationship, area ratio, and the like divided into a nucleus region, a cell membrane region, and a cytoplasm region is disclosed.
  • specimens are spread on a glass slide, such as fetal nucleated erythrocytes, which are about 1 in 1 ml of maternal blood, that is, only about 1 in about 10 8 cells in the blood.
  • Patent Document 2 a microscope equipped with an objective lens and an imaging device, a sample stage having an XY movement mechanism, on which a slide glass on which a target object-containing sample is placed can be moved to a position where it can be observed with a microscope, and a sample are searched for.
  • An image processing unit having a nozzle that collects the target object, an analysis unit that analyzes image data of the target object and the nozzle acquired by the imaging apparatus, and a storage unit that stores the data and the position thereof, and focusing and
  • an automatic search and recovery apparatus for a target object including a sample plate in a XY plane and a control unit for controlling the relative position of the nozzle.
  • the process of acquiring images is repeated while moving the Z position of the microscope, the contrast intensity of the obtained images is calculated, and the image with the highest contrast intensity is focused. It is said.
  • the calculation of contrast intensity in a plurality of images takes time because of a large amount of calculation, and as a result, it takes time to search for a target object.
  • the present invention was made to solve the above-mentioned problems, and fetal-derived target cells that are present in a very small amount in the collected maternal blood in order to quickly and accurately perform prenatal diagnosis of the fetus in a non-invasive and safe manner Concentrate appropriately, or concentrate abnormal target cells present in the blood of infants and adults as appropriate, and search and identify the position on the flat plate or in the flow path of the biochip with high accuracy in a short time. Then, the primary purpose is to provide a simple and efficient method for testing target cells in blood, which reliably recovers the target cells and accurately analyzes the chromosomes, DNA and RNA in the cell nucleus. To do.
  • the method for examining a target cell in blood which was made to achieve the above object, Optically label cells in blood containing reference cells collected as samples for dictionary creation, irradiate light corresponding to the optical label, color image, and extract the reference cells from the captured color image data A plurality of selected cell regions are cut out with a predetermined image size as target dictionary image data, and the extracted target dictionary image data is binarized based on a predetermined color corresponding to the reference cell.
  • a target dictionary creating step in which the orthonormal basis obtained from the eigenvector is obtained by performing principal component analysis on the plurality of target binary image data obtained and processed in advance, A plurality of cells other than the reference cells are selected from the color image data, and each selected cell region is cut out as the non-target dictionary image data at the predetermined image size, and each cut out non-target dictionary is used.
  • the image data is binarized based on the predetermined color, and a plurality of non-target binarized image data obtained are subjected to principal component analysis, and orthonormal bases obtained from the eigenvectors are pre-determined into a non-target dictionary.
  • Non-target dictionary creation process Collecting a blood sample for testing or concentrating the blood to obtain a blood sample containing target cells to be tested;
  • the blood sample is irradiated with focusing light at an irradiation angle that can be totally reflected by the flat plate or the biochip, and the blood sample is based on the reflection position of the focusing light on the flat plate or the biochip.
  • the inspection image data of the predetermined image size including the connected image region indicated by each label after the secondary candidate search step is sequentially cut out, and the inspection image data and each of the target dictionary and the non-target dictionary
  • the method for examining target cells in blood according to claim 2 is the method according to claim 1, wherein the target cells are fetal nucleated red blood cells.
  • the method for examining a target cell in blood according to claim 3 is the method according to any one of claims 1 to 2, wherein the concentration is performed by a specific gravity centrifugation during the specimen acquisition step, and the specific gravity is determined therefrom.
  • the above-mentioned acquisition is performed separately.
  • the method for examining target cells in blood according to claim 4 is the method according to any one of claims 1 to 3, wherein the blood sample is formed into a film on a flat plate and fixed during the sample labeling step. From there, it is characterized in that, in the target cell recovery step, the target cells are sucked out, scraped out, or scooped out while being sorted and recovered.
  • the method for examining target cells in blood according to claim 5 is the method according to any one of claims 1 to 4, wherein the blood sample is injected into the flow path of the biochip during the sample labeling step. However, the position is specified in the flow path during the target cell recovery step.
  • the method for examining target cells in blood according to claim 6 is the method according to claim 5, wherein the sorted target cells are collected from the flow path during the target cell recovery step. It is characterized by guiding to a road route.
  • the method for examining a target cell in blood according to claim 7 is the method according to any one of claims 1 to 6, wherein the chromosome, DNA and / or RNA is detected during the target cell examining step.
  • the centromere of the chromosome to be detected or a single-stranded complementary sequence that binds to the gene sequence to be detected and a diagnostic fluorescent probe in which a fluorescent dye is combined are detected by a hybridization reaction and analyzed.
  • the method for inspecting a target cell in blood according to claim 8 is the method according to claim 7, wherein the labeling material of the diagnostic probe uses fluorescence or magnetism.
  • the method for examining a target cell in blood is the method according to any one of claims 1 to 8, wherein the target cell and / or a tissue derived therefrom is suspended during the target cell examination step.
  • a sample comprising a suspension is injected into a biochip reaction field comprising a channel, compartment, groove, capillary, fiber and / or bead, and the sample is labeled with a reagent for reacting with the chromosome, DNA and / or RNA of the cell; After reacting in the reaction field, the label of the labeling reagent is detected.
  • the target cell search device Optically label cells in blood containing reference cells collected as samples for dictionary creation, irradiate light corresponding to the optical label, color image, and extract the reference cells from the captured color image data A plurality of selected cell regions are cut out with a predetermined image size as target dictionary image data, and the extracted target dictionary image data is binarized based on a predetermined color corresponding to the reference cell.
  • a target dictionary storage unit in which orthonormal bases obtained from the eigenvectors obtained by performing principal component analysis of the obtained binary image data for the target for processing are stored in advance as a target dictionary; A plurality of cells other than the reference cells are selected from the color image data, and each selected cell region is cut out as the non-target dictionary image data at the predetermined image size, and each cut out non-target dictionary is used.
  • the image data is binarized based on the predetermined color, and a plurality of obtained non-target binarized image data is subjected to principal component analysis, and an orthonormal basis obtained from the eigenvector is used as a non-target dictionary.
  • a pre-stored non-target dictionary storage unit A blood chip in which blood is collected for testing or concentrated, and a blood sample on which target cells to be tested are optically labeled is placed on a flat plate or a biochip having a flow channel into which the blood sample is injected XYZ stage that can be held and can be moved and controlled in the XYZ axial directions, An imaging camera capable of enlarging the blood sample of the planar plate held by the XYZ stage or the biochip and capturing a color image thereof; An imaging light source that irradiates the planar plate or the biochip with optical label expression light corresponding to the optical label; A light source for focus control that irradiates light for focus control at an irradiation angle that can be totally reflected by the planar plate or the biochip; A reflected light detector for detecting a reflection position of the focus control light on the planar plate or the biochip; Focus control means for moving the XYZ stage in the Z-axis direction based on the reflection position detected by
  • a target cell search device is the target cell search device according to the tenth aspect, wherein the tertiary candidate search means shifts a position at which the inspection image data is cut out one pixel at a time with a plurality of predetermined image sizes.
  • the evaluation processing is performed for each of the inspection image data that is cut out once and shifted and shifted.
  • the target cell search device is the one according to any one of claims 10 to 11, wherein the target cell specifying means is the test image data selected by the tertiary candidate search means.
  • the connected image area whose size and / or roundness is within a predetermined specified value is determined to be the target cell, and the position of the target cell is specified.
  • a target cell search device is the device according to any one of the tenth to twelfth aspects, wherein the focus control light source is also used as the imaging light source.
  • the target cell search device is the device according to any one of claims 10 to 13, wherein the image data for examination specified as the target cell by the target cell specifying means, A principal component analysis is performed together with the plurality of target binarized image data used to create the target dictionary, and an orthonormal basis obtained from the eigenvector is updated to the target dictionary storage unit as the target dictionary. And a target dictionary learning means for storing the information.
  • a target cell search device is the target cell search device according to any one of claims 10 to 14, wherein the examination image data not selected by the tertiary candidate search means or the target cell specifying means In the principal component analysis, the inspection image data that has not been determined to be the target cell in the plurality of non-target binary image data used for the creation of the non-target dictionary,
  • the apparatus further includes non-target dictionary learning means for updating and storing the orthonormal basis obtained from the eigenvector as a non-target dictionary in the non-target dictionary storage unit.
  • the target cell search device is the device according to any one of claims 10 to 15, further comprising a display, wherein the target cell specifying means specifies the similarity of the target cell, It is possible to display on the display at least one selected from an image of the inspection image data, an image of the color image data cut out in the same range as the inspection image data, and an image of the entire color image data. It is characterized by.
  • the target cell search device is the device described in any one of claims 10 to 16, and each of the planar plates after specifying the target cells and the planar plates before specification are accommodated. And a transport mechanism for transporting the flat plate between the stocker and the XYZ stage.
  • the biochip according to claim 18 is a biochip having a flow path into which a blood sample of blood collected for testing and blood obtained by concentrating it are injected.
  • the flow path includes a target cell passage filter having a gap through which optically labeled target cells to be examined in the blood sample can pass and cells larger than the target cells cannot pass, and the target cell passage An optical observation window disposed downstream of the filter and optically observing the target cell from the outside of the biochip, and recovery of the target cell branched from the flow path downstream of the optical observation window And a valve for guiding the target cell to the branch for recovery, which is arranged in a branch portion of the branch for recovery.
  • the biochip according to claim 19 is the biochip according to claim 18, wherein a plurality of the target cell passage filters are arranged in the flow path.
  • a biochip according to claim 20 is the biochip according to any of claims 18 to 19, wherein the valve is a thermally controllable thermal control valve, an electrically controllable electrical control valve, or The magnetic control valve is magnetically controllable.
  • the biochip according to claim 21 is the biochip according to any one of claims 18 to 20, wherein the flow path in the optical observation window section is formed along the flow path without overlapping the target cells. It is formed with the flow path width which can pass in a row
  • a biochip according to claim 22 is the biochip according to any one of claims 18 to 21, wherein the target cell is optically labeled in the flow path upstream of the optical observation window.
  • the chemical solution injection path for injecting the liquid from the outside is merged.
  • the method for examining a target cell in blood of the present invention it is non-invasive and safe for a mother and a fetus, and does not induce miscarriage or stillbirth because of the examination, and prenatal diagnosis of a fetus Can be done quickly and accurately.
  • fetal-derived target cells which are present in a very small amount in the collected maternal blood, are appropriately concentrated by centrifugation or the like to reduce the volume per sample and improve efficiency.
  • fetal-derived target cells that are appropriately concentrated from the maternal blood and abnormal target cells that are appropriately concentrated from blood of infants or adults can be accurately obtained in a short time on a flat plate or in the flow path of a biochip. Since it is possible to instantly search and optically determine the position and specify it, and then reliably capture and collect the few target cells, it is highly reliable.
  • specific fluorescence can be obtained by amplifying genes such as chromosomes, DNA and RNA in the cell nucleus by polymerase chain reaction (PCR) or the like, or by performing the FISH method on the nuclear cells without amplification. Since it can detect optically with a probe or magnetically with a magnetic probe, perform chromosome diagnosis / DNA diagnosis, accurately analyze and perform highly reliable genetic diagnosis, fetal diagnosis can be performed easily and efficiently. Contributes to advanced medical diagnosis before birth and precise and advanced medical diagnosis based on blood component tests of infants and adults.
  • PCR polymerase chain reaction
  • a target dictionary is created by principally analyzing an image of a cell serving as a reference for the target cell, and images of other cells
  • a non-target dictionary is created by principal component analysis, and the vector distance between the blood image collected for examination and the dictionary and the examination image is calculated, and the similarity to the dictionary is evaluated. Since target cells can be searched with a large amount of calculation, high-speed and highly efficient search is possible.
  • the color image itself is not searched by image processing, but the color image is binarized with a predetermined color corresponding to the target cell, and the binarized image is processed and searched.
  • the amount of calculation is smaller than that of image processing, and a higher-speed search is possible. Also, by focusing the light for focus control on a flat plate etc. and performing focusing based on the reflection position, the amount of calculation is far less than when calculating the contrast intensity of the image, and focusing can be performed at high speed. Therefore, high-speed search becomes possible. In addition, when the imaging light is also used as the focus control light, a simple configuration can be obtained.
  • the target cell search device of the present invention can process a blood sample quickly and search for a target cell at high speed and with high efficiency, so that the target cell is separated, reliably captured and recovered,
  • This is a simple and high-performance target cell search apparatus suitable for a non-invasive diagnostic system in which genes such as chromosomes, DNA and RNA are accurately analyzed to perform highly reliable genetic diagnosis.
  • biochip of the present invention is suitable for searching for target cells from blood samples such as blood and its concentrated components / separated components, and the searched target cells can be immediately separated and collected.
  • FIG. 5 is a schematic diagram illustrating an overview of a focusing operation of the imaging unit in FIG. 4. It is a flowchart explaining the tertiary candidate search process in FIG. 1 in detail. It is a side view which shows the outline
  • Schematic plan view (a) of a biochip to which the present invention is applied an enlarged plan view (b) of a target cell passage filter 72, an enlarged plan view (c) of an optical observation window portion 73, and a flow in the optical observation window portion 73
  • FIG. 1 is a schematic plan view of a biochip for chromosome testing and genetic testing to which the present invention is applied. It is a cell observation image after staining (a) and a fluorescence observation image after FISH (b) on a biochip for chromosome inspection and genetic inspection to which the present invention is applied.
  • FIG. 5 shows DNA microarray fluorescence observation images for genetic testing on a biochip for chromosome testing and genetic testing to which the present invention is applied, showing normal cell DNA microarray results (a) and 21 trisomy cell DNA microarray results (b), respectively.
  • It is a color image example (a) cut out as target dictionary image data, and a color image example (b) cut out as non-target dictionary image data.
  • the color image (a) which imaged the concentrated blood sample (a), the binarized image (b) after the binarization process, and the binarized image (c) after the contraction and expansion process.
  • 1 is a target cell search device
  • 2 is an imaging unit
  • 3 is a processing unit
  • 4 is a display
  • 5 is a stocker
  • 6 is a transport mechanism
  • 8 is a flat plate
  • 9 is a target dictionary creation means
  • 10 is a non-target dictionary creation means
  • 11 is an XYZ stage
  • 12 is a laser light source
  • 13 is a focusing camera
  • 14 is an imaging light source
  • 15 is an imaging camera
  • 16 is a magnifying lens
  • 21 is an image acquisition means
  • 22 is a focus control means
  • 23 is an imaging position control means.
  • , 24 is a transport control unit
  • 31 is a target dictionary storage unit
  • 32 is a non-target dictionary storage unit
  • 41 is a search storage unit
  • 42 is a binarization processing unit
  • 43 is a contraction / expansion processing unit
  • 44 is a primary candidate search Means 45, secondary candidate search means 46, tertiary candidate search means 47, target cell specifying means 47, target dictionary learning means 51, non-target dictionary learning means 52, observation image 55, observation position 56, 61 is a sample container
  • 2 is a control valve
  • 63 is a pressure pump
  • 64 is a three-way opening / closing control valve
  • 65 is a collection container
  • 66 is a disposal container
  • 67 is one output pipe
  • 68 is the other output pipe
  • 70 and 75 are biochips.
  • a flow path 72 a target cell passage filter, 73 an optical observation window, 77 a discard path, 78 a recovery branch path, 79 an open / close control valve, 80 a chemical solution injection path, 82 a gap
  • 85 is a control electrode
  • 86 is a base plate
  • 87 is a cover plate
  • 91 is a target cell
  • 92 is a non-target cell
  • 101 is a liquid flow path
  • 102 is an open / close control valve
  • 103 a FISH chamber
  • 104 is a cell immobilization filter , 105 PCR chamber, the DNA microarray chamber 106, L1 ⁇ L2 width, Le laser beam for focus control, Le' reflected light, L T is an optical indicator expressing light, theta is the angle
  • S11 is a sample acquisition step
  • S12 is a sample labeling step
  • S13 is an optical label expression step
  • the method for examining target cells in blood to which the present invention is applied performs the target dictionary creation step S1 and the non-target dictionary creation step S2 of FIG. 1 in advance. Thereafter, the collected blood sampled is subjected to the sample acquisition step S11 and the sample labeling step S12 in the figure to optically label the target cells in the concentrated blood sample, and the optical label expression step S13 and the focus control step S14. , Color image acquisition step S15, binarization processing step S16, contraction and expansion step S17, primary candidate search step S18, secondary candidate search step S19, tertiary candidate search step S20, and target cell specifying step S21 The target cell in the blood is examined by performing the target cell recovery step S23 and the target cell test step S24.
  • the region that can be imaged at one time is narrower than that of the flat plate. Therefore, the imaging region of the flat plate is moved to perform processing from the optical labeling step S13 to the target cell specifying step S21. Is repeated to search for target cells for the entire flat plate (step S22). If the concentrated blood sample on the flat plate can be imaged at one time, it is not necessary to repeat step S22.
  • the target cells in the blood to be examined are fetal nucleated red blood cells (NRBC)
  • NRBC fetal nucleated red blood cells
  • the target cells are not limited to this, and not only undifferentiated NRBC but also non-nucleated red blood cells.
  • various blood cells may be used, and specifically, white blood cells, platelets, and the like may be examined.
  • dysmorphic red blood cells such as red blood cells of patients infected with malaria, sickle red blood cells (sickle cell disease), large red blood cells (such as vitamin B12 deficiency), small red blood cells / hypochromic ( Iron deficiency), spherical red blood cells (such as genetic spherocytosis), elliptical red blood cells (such as genetic elliptical red blood cells), oral red blood cells (such as hemolytic anemia), teardrop red blood cells (such as myelofibrosis), target red blood cells (such as myelofibrosis)
  • Non-nucleated red blood cells such as thalassemia
  • erythrocytes such as congenital abeta-lipoproteinemia
  • crescent red blood cells such as disseminated intravascular coagulation
  • the target dictionary creation step S1 cells in maternal blood containing the reference NRBC collected as a dictionary creation sample are optically labeled, and light corresponding to the optical label is irradiated for color imaging to obtain color image data. obtain.
  • color image data can be obtained in the same manner as in a sample acquisition step S11 to a color image acquisition step S15 described later.
  • the operator sorts a plurality of NRBCs from the captured color image data while viewing the images, and cuts out each sorted cell region as target dictionary image data with a predetermined image size.
  • This predetermined image size is the size of an image that allows just one NRBC to be cut out (included), and is appropriately adjusted depending on the imaging magnification.
  • the predetermined image size is 52 pixels ⁇ 52 pixels.
  • Each extracted target dictionary image data is binarized based on a predetermined color corresponding to the reference NRBC. Such binarization processing is performed in the same manner as the binarization processing step S16 described later.
  • a plurality of obtained binarized target image data is subjected to principal component analysis, and orthonormal bases obtained from the eigenvectors are set as target dictionaries in advance.
  • Fig. 2 shows a flowchart of principal component analysis.
  • a plurality of (n) NRBC target binarized image data D (p, q) are vectorized.
  • p represents the X coordinate of the pixel
  • q represents the Y coordinate of the pixel.
  • the binarized image data for target D (p, q) is a matrix of P rows and Q columns, so this is a matrix of one column of P ⁇ Q dimensions.
  • Dv (m) is an integer value of 1 to P ⁇ Q, and is 1 to 2704 in the case of 52 pixels ⁇ 52 pixels.
  • Dv (1) D (1,1)
  • Dv (2) D (1,2)
  • step S32 an average vector of dictionary vectors Dv1, Dv2,... Dvn is calculated by the following equation (1).
  • step S33 a covariance matrix Cp with the average vector is calculated by the following equation (2).
  • step S34 the covariance matrix Cp is subjected to eigenvalue decomposition.
  • step S35 eigenvectors for a plurality of (for example, 40) eigenvalues from the higher order of eigenvalues are obtained.
  • the number of eigenvalues is preferably a number that can represent 90% or more.
  • a matrix in which these eigenvectors are arranged is an orthonormal basis, and this is the target dictionary B.
  • the target dictionary creating means 9 creates the target dictionary B as described above and stores it in the target dictionary storage unit 31.
  • non-target dictionary creation step S2 a plurality of cells other than the reference NRBC are selected from the color image data used in the target dictionary creation step S1 or the color image data obtained in the same manner while viewing the image.
  • the sorted cell regions are cut out as non-target dictionary image data with a predetermined image size.
  • Each of the extracted non-target dictionary image data is binarized based on a predetermined color, and a plurality of obtained non-target binary image data are subjected to principal component analysis and orthonormal obtained from the eigenvectors.
  • the base be a non-target dictionary C in advance.
  • erythroblasts that are not NRBC as well as leukocytes such as neutrophils as cells other than NRBC.
  • the image clipping method, binarization processing, principal component analysis method, and the like in the non-target dictionary creation step S2 can be performed in the same manner as the target dictionary creation step S1 except that the target cells are different.
  • the detailed explanation is omitted.
  • the non-target dictionary creating means 10 creates the non-target dictionary C as described above and stores it in the non-target dictionary storage unit 32.
  • the target dictionary B and the non-target dictionary C may be created by the target cell search device 1, for example, the manufacturer of the target cell search device 1 creates in advance, and it is data separately from the device. May be sold at.
  • the maternal blood collected for the test is concentrated to a desired component by centrifugation or the like to acquire a concentrated blood sample containing target cells derived from the fetus to be tested.
  • a specific gravity adjusting liquid that is a suspension of immiscible suspensions of two types having specific gravity larger than that of red blood cells or white blood cells, specifically, a Percoll liquid that is a fine suspension of polyvinylpyrrolidone-coated silica gel (GE Healthcare Japan) Maternal blood is put into a centrifuge tube together with the product made by Co., Ltd. and centrifuged.
  • maternal blood collected from heparin is injected into a centrifuge tube containing two layers of Percoll solution with different specific gravities, and is centrifuged at a high speed with a centrifuge and centrifuged from the difference in specific gravities.
  • erythrocyte and leukocyte-containing layers containing NRBC are collected from the maternal blood divided into a plurality of layers with a pipette to obtain a concentrated blood sample.
  • Concentrated blood samples may be collected at high speed in parallel from different maternal blood by simultaneously centrifuging using a plurality of centrifuge tubes.
  • a concentrated blood sample may be collected by various known methods such as an external filtration method and a blood adsorption method.
  • the concentrated blood sample is formed into a film on the flat plate 8 (see FIG. 3) and fixed, and then the entire target cell is optically labeled.
  • the flat plate 8 is a sample stage suitable for optical microscope observation such as a slide glass.
  • optical labels include fluorescent and staining labels such as Giemsa staining, hematoxylin staining, and light staining; antibodies such as anti-CD71 antibody, anti-CD36 antibody, and anti-glycophorin A antibody are used with a green fluorescent dye.
  • Fluorescent labeling using a labeled fluorescent dye such as a certain fluorescein isothiocyanate (FITC) labeled monoclonal antibody, an orange fluorescent dye, phycoerythrin (PE) labeled monoclonal antibody, a Texas red labeled monoclonal antibody, an allophycocyanin labeled monoclonal antibody Can be mentioned.
  • fluorescently labeling NRBC the concentrated blood sample obtained in the sample acquisition step S11 is dropped on the flat plate 8, spread uniformly and dried, and then applied to a fluorescent staining solution such as Giemsa staining solution and dried.
  • the planar plate 8 optically labeled by staining the whole cell in this manner is repeatedly subjected to the optical labeling step S13 to the target cell specifying step S21 to specify the position of the target cell by image processing.
  • the configuration of the target cell search device 1 of the present invention suitable for performing these steps S13 to S21 will be described, and then the steps S13 to S21 will be described.
  • the target cell search device 1 includes an imaging unit 2, a processing unit 3, a display 4, a stocker 5, and a transport mechanism 6 as an example.
  • the imaging unit 2 operates under the control of the processing unit 3 to capture a color image obtained by enlarging the concentrated blood sample, and includes an XYZ stage 11, an imaging camera 15, an imaging light source 14, a laser light source 12, and a focal point.
  • the camera 13 is provided.
  • a side view of the imaging unit 2 is shown in FIG.
  • the XYZ stage 11 can position and hold the flat plate 8 and can control movement in the XYZ axial directions.
  • the XYZ stage 11 is a flat end, and the flat plate 8 is placed on the top (upper side in the figure), and the flat plate 8 is positioned and fixed by a positioning mechanism (not shown).
  • the XYZ stage 11 has an XYZ moving mechanism (not shown), is connected to the imaging position control means 23 (see FIG. 3) of the processing unit 3, and is in the X-axis direction (left-right direction in the figure) and Y-axis direction.
  • the movement in the direction (front and back in the figure) is controlled, connected to the focus control means 22 (see FIG. 3) of the processing unit 3, and the movement in the Z-axis direction (vertical direction in the figure) is controlled.
  • a known one can be adopted.
  • the imaging camera 15 is a color CCD camera as an example, and has a magnifying lens 16 such as an optical microscope in the imaging direction.
  • This imaging camera 15 is arranged at a position facing the flat plate 8 held by the XYZ stage 11, and expands the target cells contained in the concentrated blood sample on the flat plate 8 at a magnification that can be identified, The color image is taken.
  • Imaging light source 14 is for irradiating the light for imaging the plane plate 8, corresponding to the optical labeling of concentrated blood sample, irradiating the optical indicator expressing light L T of the wavelength of expressing optical label.
  • the optical label is a fluorescent label
  • an optical indicator expressing light L T is the light that can excite the fluorescent substance wavelength.
  • a light emitting diode that oscillates at a specific wavelength such as ultraviolet or near ultraviolet corresponding to a fluorescent material is used as the imaging light source 14.
  • optical label is a dyed label visible
  • optical indicator expressing light L T is visible light.
  • a white light emitting diode or a halogen lamp is used as the imaging light source 14.
  • the imaging light source 14 may be configured to be disposed on the same side as the imaging camera 15 with respect to the flat plate 8 as shown in FIG. 4A (configuration of epi-illumination), or as shown in FIG. Alternatively, the flat plate 8 may be arranged on the opposite side of the imaging camera 15 (transmission illumination configuration).
  • the imaging camera 15 images the reflected light of the concentrated blood sample
  • the transmitted illumination is the XYZ stage 11, and holds the edge portions of the flat plate 8 so as not to interfere with the irradiation of the optical indicator expressing light L T.
  • the target cell search apparatus 1 has both epi-illumination and transmitted illumination as the imaging light source 14 and can select either one. Which illumination is used to select an image is selected as appropriate based on the color of the optical marker, the sharpness, and the like.
  • the laser light source 12 is a focus control light source that emits focus control light.
  • the laser light source 12 is disposed at a position where the flat plate 8 on the XYZ stage 11 is irradiated with the laser light Le at an angle ⁇ . This angle ⁇ is an angle at which the laser beam Le is totally reflected by the flat plate 8.
  • the focus camera 13 corresponds to the reflected light detector in the present invention, and is, for example, a monochrome CCD camera. As shown in the figure, the focusing camera 13 tilts the imaging direction at an angle ⁇ with respect to the plane plate 8 so that the reflected light Le ′ of the laser beam Le reflected by the plane plate 8 can be detected. Are disposed at positions facing the laser light source 12.
  • the processing unit 3 of the target cell search device 1 controls the operation of the target cell search device 1 in an integrated manner, and although not shown, a central processing unit (not shown) that performs various calculations and processing ( CPU), ROM, RAM, hard disk, one or a plurality of computers having an input / output interface, etc., and an operation program thereof.
  • CPU calculations and processing
  • ROM read-only memory
  • RAM random access memory
  • hard disk hard disk
  • an operation program thereof an operation program thereof.
  • the processing unit 3 includes a target dictionary creation unit 9, a non-target dictionary creation unit 10, an image acquisition unit 21, a focus control unit 22, an imaging position control unit 23, a transport control unit 24, a target dictionary storage unit 31, and a non-target dictionary storage unit.
  • 32, search storage unit 41, binarization processing means 42, contraction / expansion processing means 43, primary candidate search means 44, secondary candidate search means 45, tertiary candidate search means 46, target cell specifying means 47, target dictionary A learning unit 51 and a non-target dictionary learning unit 52 are included.
  • Each storage unit 31, 32, 41 is a hard disk as an example.
  • the stocker 5 accommodates a plurality (for example, 80) of flat plates 8 before and after the search for target cells, and can control the loading and unloading of each flat plate 8.
  • the transport mechanism 6 transports the flat plate 8 between the stocker 5 and the imaging unit 2.
  • the operator accommodates the plurality of flat plates 8 labeled in the sample labeling step S12 in the stocker 5.
  • the transport control unit 24 controls the stocker 5 and the transport mechanism 6 to set one flat plate 8 from the stocker 5 on the XYZ stage 11.
  • the imaging position control means 23 controls the XYZ stage 11 so that a predetermined imaging position of the flat plate 8 enters the imaging area of the imaging camera 15.
  • the image acquisition unit 21 to irradiate the optical indicator expressing light L T to the imaging light source 14.
  • the fluorescent label of the concentrated blood sample is excited and the cell nucleus develops fluorescence.
  • the focus control means 22 irradiates the laser light source 12 with the laser light Le (see FIG. 4).
  • the focus control means 22 acquires a captured image from the focus camera 13, moves the XYZ stage 11 in the Z-axis direction based on the reflection position of the reflected light Le ′ of the laser light Le reflected by the flat plate 8, By controlling the distance between the flat plate 8 and the magnifying lens 16, the imaging camera 15 (magnifying lens 16) is focused on the concentrated blood sample.
  • FIG. 5 shows an observation image 55 of the focusing camera 13.
  • the focus camera 13 observes the reflection position of the reflected light Le ′ on the flat plate 8.
  • the distance between the magnifying lens 16 and the plane plate 8 (see FIG. 4) is the distance at which the focal point of the magnifying lens 16 is exactly aligned with the plane plate 8, and the reflected light Le ′ is the observation image 55 as shown in FIG.
  • the imaging area of the focusing camera 13 is adjusted in advance so that the image is observed in alignment with the prescribed position 56 at the center of the screen. In this way, when the flat plate 8 is close to the magnifying lens 14, the reflection position of the reflected light Le ′ is shifted as shown in FIG. Observed by shifting to the left side.
  • the focus control unit 22 performs focusing by moving the XYZ stage 11 in the Z-axis direction so that the reflected light Le ′ is observed just overlapping the specified position 56.
  • the imaging camera 15 performs imaging while gradually moving the XYZ stage 11 in the Z-axis direction. Since the amount of calculation is much smaller than that in the case of calculating the contrast of the image and controlling to find the position where the contrast becomes the highest, focusing can be performed at high speed.
  • the color image acquisition unit 21 causes the imaging camera 15 to capture the image and acquire the color image data X (i, j), along with the X and Y information (position information) on the plane plate 8, for search.
  • the data is stored in the storage unit 41. Since the area of the plane plate 8 that can be imaged at once by the imaging camera 15 is usually a narrow area, the captured color image data X (i, j) is a part of the plane plate 8 image data.
  • i represents the position in the X-axis direction in the image data
  • j represents the position in the Y-axis direction. For example, if the color image data X (i, j) has a size of 2000 pixels ⁇ 1000 pixels, i is 1 to 1.
  • An integer value of 2000, j is an integer value of 1 to 1000. Since the flat plate 8 is positioned on the XYZ stage 11, the position i, j in the obtained color image data X (i, j) is specified, and the flat plate of the color image data X (i, j) obtained is captured. By referring to the position at 8, the position on the plane plate 8 can be specified.
  • the binarization processing means 42 reads the color image data X (i, j) stored in the search storage unit 41, and based on a predetermined color corresponding to the reference cell.
  • the binarization process is performed and the search storage unit 41 stores the binarization process.
  • the predetermined color is a color of a cell nucleus that is fluorescently labeled with the target NRBC, and is obtained in advance by averaging the colors of a plurality of NRBCs as a reference obtained from the sample.
  • the red (R) component, the green (G) component, and the blue (B) component of the predetermined color are used for the calculation, and the maximum value of the gradation of each color (for example, in the case of 8-bit gradation) It is normalized so that the maximum value is 1 by dividing by 255).
  • the normalized R component of the predetermined color is Xr
  • the G component is Xg
  • the B component is Xb.
  • Xr, Xg, and Xb are stored in advance in the search storage unit 41.
  • the binarization processing unit 42 normalizes the RGB component of each pixel of the color image data X (i, j) with the maximum value of the gradation of each color, and then performs the following (3 ) Formula is calculated.
  • the normalized R component of the pixel is r
  • the G component is g
  • the B component is b.
  • a pixel in which C> 0.99, that is, a pixel having a color approximate to a predetermined color is set to 1, for example, and the other pixels are set to 0, for example, to binarize all the pixels.
  • the binarization processing unit 42 stores the binarized binarized image data IR (i, j) in the search storage unit 41 separately from the color image data X (i, j).
  • the data amount is smaller than that of the color image, so that the search can be performed at high speed in the following steps.
  • the contraction / expansion processing unit 43 reads the binarized image data IR (i, j) from the search storage unit 41, and if there is 0 among the eight pixels surrounding the pixel of interest. After performing the contraction process for setting the target pixel to 0 for all the pixels, if there is 1 in 8 pixels surrounding the target pixel, the expansion process for setting the target pixel to 1 is performed for all the pixels.
  • the binarized pixel data IR (i, j) is replaced and stored in the search storage unit 41. Noise is removed by the contraction and expansion processing.
  • the contraction / expansion process may be performed a plurality of times as necessary.
  • the primary candidate search means 44 applies to a connected pixel area in which pixels 1 are adjacent to each other in the binarized image data IR (i, j). Then, a labeling process for assigning a label is performed so that each connected pixel region can be identified, and the assigned label is stored in the search storage unit 41 in association with the connected pixel region.
  • the labeling process can be performed by a known method.
  • the secondary candidate search means 45 reads the label stored in the search storage unit 41, and re-applies the same label to the connected pixel regions of different labels that are close within a predetermined image size. It is reassigned and stored in the search storage unit 41. That is, when the inspection image data is cut out in the next tertiary candidate search step S20, the same label is re-assigned to the connected pixel regions of different labels that fall within a predetermined image size and can be cut out simultaneously.
  • This predetermined image size is the same image size as the target dictionary image data and the non-target dictionary image data when the target dictionary B and the non-target dictionary C are created. For example, when the predetermined image size to be cut out is 52 pixels ⁇ 52 pixels, when different connected pixel regions are included in the 52 pixels ⁇ 52 pixels, the same label is given again.
  • the secondary candidate search means 45 averages the X coordinate and Y coordinate of the connected pixel area of the same label, obtains the barycentric coordinates Lx and Ly of the connected pixel area, and associates them with the label for search.
  • the data is stored in the storage unit 41.
  • the tertiary candidate search means 46 sequentially cuts out inspection image data having a predetermined image size including the connected image region indicated by each label after the secondary candidate search step, and the inspection image data and Then, the vector distance between each of the target dictionary B and the non-target dictionary C is calculated, and both vector distances are sequentially evaluated to obtain test image data that is similar to the target dictionary within the predetermined evaluation value than the non-target dictionary. Select.
  • This tertiary candidate search step S20 will be described in detail with reference to the flowchart of FIG.
  • the tertiary candidate search means 46 reads the barycentric coordinates Lx and Ly of one label from the search storage unit 41, and determines the cut-out position of the inspection image data having a predetermined image size.
  • the target cell is distorted, the center of gravity is biased. Therefore, when the inspection image data is cut out around the center of gravity coordinates Lx and Ly, the target cell is biased out and protrudes from the inspection image data. May end up. For this reason, not only the inspection image data is cut out and evaluated with the center-of-gravity coordinates Lx and Ly as the center coordinates, but the inspection image data is cut out by shifting the center coordinates to be cut out from the center-of-gravity coordinates Lx and Ly one pixel at a time.
  • the maximum range for shifting the pixels is preferably about 1/8 to 1/20 in the vertical and horizontal directions of a predetermined image size.
  • the evaluation is performed by shifting from the center-of-gravity coordinates Lx, Ly to ⁇ 4 pixels. In this case, the evaluation process is performed by cutting out a total of 81 times for one label. If the search is performed by shifting one pixel at a time, the target cell can be searched with high accuracy. In addition, when there is no distortion in the shape of the target cell, or when the target cell can be cut out from the inspection image data so as not to protrude, the process of shifting by one pixel may not be performed.
  • the tertiary candidate search means 46 sequentially selects one center coordinate to be cut out within the range of ⁇ 4 pixels from the barycentric coordinates Lx and Ly. specify.
  • the tertiary candidate search means 46 binarizes the inspection image data K (p, q) having a predetermined image size (52 pixels ⁇ 52 pixels) centered on the coordinates specified in step S41.
  • the cut-out image data IR (i, j) is cut out and read from the search storage unit 41.
  • p represents a pixel in the X-axis direction
  • q represents a pixel in the Y-axis direction.
  • the predetermined image size is 52 pixels ⁇ 52 pixels
  • p and q are integer values of 1 to 52.
  • the tertiary candidate search means 46 vectorizes the inspection image data K (p, q). That is, it is expressed by a P ⁇ Q-dimensional inspection vector A, that is, a one-column matrix A (m). This vectorization is performed in the same manner as the vectorization performed in the target dictionary creation step S1.
  • the inspection vector A (m) is such that A (1) is K (1,1), A (2) is K (1,2),... A (m) is K (P, Q).
  • the inspection image data K (p, q) is 52 pixels ⁇ 52 pixels, it is a matrix of one column in which 2704 pixel values are arranged in order.
  • step S43 the tertiary candidate search means 46 calculates the addition average value h of each element (each pixel) of the inspection vector A (m) by the following equation (4).
  • step S44 the tertiary candidate search means 46 performs norm calculation of the inspection vector A (m).
  • the norm Vr of the inspection vector A (m) is calculated by the following equation (5).
  • step S45 the tertiary candidate search means 46 normalizes the inspection vector A (m) with the calculated addition average value h and norm Vr. Specifically, the inspection normalization vector Av (m) is calculated by the following equation (6).
  • step S46 the tertiary candidate search means 46 calculates the distance Gb between the test normalization vector Av and the target dictionary B, and calculates the distance Gc between the test normalization vector Av and the non-target dictionary C.
  • the distance Gb between the normalization vector Av for inspection and the target dictionary B calculates the inner product A ⁇ B of both vectors. Further, the distance Gc between the test normalization vector Av and the non-target dictionary C calculates the inner product A ⁇ C between the vectors.
  • step S ⁇ b> 47 the tertiary candidate search unit 46 calculates the similarity with the evaluation function S represented by Expression (7), and stores it in the search storage unit 41.
  • This evaluation function S takes a negative value when the test normalization vector Av (test vector A) is closer to the target dictionary B than the non-target dictionary C, and the value approaches -1 as the target dictionary B approaches. .
  • step S48 the tertiary candidate search unit 46 determines that the inspection vector A is a non-target when the value S calculated in step S46 is negative, that is, in a range smaller than the predetermined evaluation value (value 0). It is selected as being similar to the target dictionary B rather than the dictionary C, and a flag or the like is stored in the search storage unit 41 so that the selection can be understood.
  • step S49 the tertiary candidate search means 46 returns to step S41 when the evaluation has not been completed for the range of ⁇ 4 pixels of the barycentric coordinates Lx, Ly, and cuts out the inspection image data shifted by one pixel and performs S42.
  • the tertiary candidate search means 46 updates the center position where the value of the evaluation function S is closest to ⁇ 1 as the center of gravity coordinates Lx, Ly among the center positions shifted by one pixel at a time, and the degree of similarity at that time is updated.
  • the search storage unit 41 is overwritten and stored in correspondence with the label.
  • step S50 the tertiary candidate search means 46 determines whether or not the similarity has been calculated for all labels, and if all the calculations have not been completed yet, repeats steps S41 to S49 for another label. When all the labels are finished, the tertiary candidate search step S20 is finished.
  • the target cell specifying means 47 determines that the connected pixel region of the test image data selected in the tertiary candidate search step S20 is a target cell, specifies its position, and outputs it. .
  • a predetermined number for example, three
  • S is close to -1
  • the target cell specifying means 47 has the size and / or roundness of the connected image region of the test image data selected in the tertiary candidate search step S20 within predetermined specified values. You may perform the process which determines that a connection image area
  • the target cell specifying unit 47 determines the width in the X-axis direction and the width in the Y-axis direction as the size of the connected pixel region, and both widths are within predetermined predetermined values for size determination ( For example, when the number of pixels of a cell is 52 pixels ⁇ 52 pixels, a cell within a range of 20 to 40 pixels) is determined as a target cell.
  • the predetermined predetermined value for size determination is obtained by replacing the size range that can be taken by the cell nucleus of the NRBC in the inspection image data with the number of pixels.
  • the target cell specifying means 47 calculates the roundness of the connected pixel region by the following equation (8), and this roundness is within a predetermined prescribed value for roundness discrimination (for example, 0.5 to 0.5). That are within the range of 1.5) are determined to be target cells.
  • R L ⁇ ⁇ ⁇ 2 L is the perimeter of the connected pixel region.
  • the target cell specifying means 47 specifies that the size and / or roundness is within a predetermined specified value as the cell nucleus of the target cell, and searches for a flag or the like so that it can be identified.
  • the data is stored in the storage unit 41. It is preferable to determine the target cell from both the size and the roundness.
  • the target cell specifying means 47 determines all the labels selected in the tertiary candidate search step S20, and then specifies the positions Lx, Ly and the position on the flat plate 8 specified as NRBC as a cell recovery device (FIG. (Not shown).
  • the imaging position control unit 11 moves the XYZ stage 11 in the X-axis direction and the Y-axis direction so that the imaging regions overlap each other or slightly, and the target cell specifying step from the optical label expression step S13 described above. By repeating the steps up to S21, the search for one flat plate 8 is completed.
  • the XYZ stage 11 is moved in the XY directions for each imaging region to repeatedly perform imaging, and after acquiring the entire color image data of one flat plate 8, binarization processing is performed.
  • Step 16 to target cell identification step S21 may be performed.
  • the plane plate 8 for which the search has been completed is accommodated in the stocker 5 by the transport mechanism 6, the other plane plate 8 before the search is set from the stocker 5 to the XYZ stage 11, and the search is similarly started. In this way, the target cell search device 1 automatically searches all the flat plates 8 accommodated in the stocker 5.
  • the target cell search device 1 includes a position where a target cell is specified, a degree of similarity thereof, an image of test image data, an image of color image data cut out in the same range as the test image data, and an image of the entire color image data. It is preferable that at least one selected from can be displayed on the display 4. When an image of inspection image data or an image of color image data is displayed on the display 4, it is preferable to display an instruction image such as a red circle or an arrow at the specified NRBC position.
  • the target cell search device 1 uses the test image data K (p, q) determined by the target cell specifying unit S21 to be an NRBC cell nucleus and a plurality of binarizations for targets used for creating the target dictionary.
  • a target dictionary learning means 51 (FIG. 5) that performs principal component analysis together with the image data D (p, q) and updates and stores the orthonormal basis obtained from the eigenvector as the target dictionary B in the target dictionary storage unit 31. 3) may be further provided. By learning and updating the target dictionary B in this way, the target dictionary B becomes more accurate, so that the NRBC search accuracy can be improved.
  • the target cell search device 1 has the test image data K (p, q) that the tertiary candidate search means 46 did not select, or the test image data K that has not been determined that the target cell specifying means is NRBC.
  • (P, q) and a plurality of non-dictionary image data used to create the non-target dictionary C are subjected to principal component analysis, and the orthonormal basis obtained from the eigenvector is set as the non-target dictionary C.
  • the target cell is recovered based on the position of the target cell output from the target cell search device 1.
  • the flat plate 8 is set on the XY stage of an inverted microscope, and the XY stage is moved so as to observe the positions Lx and Ly where the target cells are specified.
  • the operator moves the position roughly with a motor so that the tip of the glass capillary for cell recovery having a slightly larger lumen than the target cell is located at the position Lx, Ly of the target cell.
  • Target cells can be collected by various known methods.
  • an enzyme-containing solution such as physiological saline, Ringer's solution, phosphate buffered saline (PBS), or protein kinase K is dropped on a desired target cell with a glass capillary for cell recovery, and left for a while to slide glass.
  • the target cells fixed to the cell are easily removed, the target cells are selectively sucked out by using the same glass capillary or another cell recovery glass capillary under negative pressure to obtain the target cells.
  • the target cells may be scraped or scooped with a spatula.
  • the chromosomes, DNA and / or RNA in the cell nuclei of the collected target cells are analyzed.
  • a highly sensitive analysis method is required.
  • the obtained cells are labeled with a fluorescence initusitu hybridization (FISH method: Fluorescence in situ hybridization) for labeling chromosomes and RNA in the cell nucleus, DNA, etc.
  • FISH method Fluorescence in situ hybridization
  • PCR Polymerase chain reaction
  • PEP method primer extension preamplification
  • CGH method comparative genomic hybridization
  • DOP-PCR method Degenerated Oligonucleotide Primer PCR
  • the detection biochip has a reaction field composed of, for example, a flow path, a compartment, a groove, a capillary, a fiber, and / or a bead, and a path through which a labeling reagent can be injected as necessary, an extra reagent, a by-product, It has a route through which unnecessary chemicals can be discharged, and these analysis methods can be carried out in minute amounts in the reaction field.
  • chromosome abnormality For example, by examining the obtained cells by FISH method using a monoclonal antibody against CD71 and glycophorin A, it is possible to examine a chromosome abnormality, XY chromosome number abnormality, 21 ⁇ 18 or 13 trisomy abnormality.
  • sex determination, individual identification, and Rh (D) type can be examined by PCR, and a plurality of gene abnormalities can be diagnosed by PEP.
  • a chromosome, DNA and / or RNA is used to detect a diagnostic fluorescent probe in which a chromosome-specific gene and a fluorescent dye are bound, for example, a fluorescently labeled probe that specifically binds to a centromere.
  • a hybridization reaction may be detected and analyzed.
  • the CEP series DNA CEP is a registered trademark
  • CEPbDNA ⁇ FISH probe CEP Chromosome Enumeration DNA FISH Probes
  • CEP probe kit manufactured by Abbott The labeling material for the diagnostic probe may use fluorescence or magnetism.
  • the optically labeled concentrated blood sample may be injected into the flow channel 71 of the biochip 70 and the target cell search device 1 may search for the NRBC.
  • the target cell search device 1 may search for the NRBC.
  • the biochip 70 is held on the XYZ stage 11 of the target cell search apparatus 1.
  • This biochip 70 has one flow path 71.
  • a sample container 61 containing a concentrated blood sample is connected to the input side of the flow channel 71 of the biochip 70 via a conduit, and the sample container 61 is connected via a control valve 62 that can be controlled to open and close.
  • the pressure pump 63 is connected.
  • a three-way opening / closing control valve 64 is connected to the output side of the flow path 71
  • a collection container 65 is connected to one output pipe 67 (recovery flow path) of the three-way opening / closing control valve 64, and the other output pipe. 68 is connected to a disposal container 66.
  • the three-way opening / closing control valve 64 is an opening / closing control valve having one input port and two output ports and capable of opening / closing each output port.
  • the three-way opening / closing control valve 64 controls opening / closing of the output pipe 67 side and the output pipe 68 side.
  • a separate opening / closing control valve may be provided for each of the output pipe 67 and the output pipe 68.
  • the biochip 70 is made of a transparent material such as a pressure resistant resin, specifically a polyester resin such as polyethylene terephthalate (PET) or an acrylic resin so that the NRBC flowing through the channel 71 from the outside can be optically observed. It is made of plastic resin such as cyclic polyolefin and silicone resin.
  • the channel has a channel width of 10 ⁇ m to 20 ⁇ m, preferably 10 ⁇ m to 15 ⁇ m.
  • the output pipe 68 side of the control valve 62 and the three-way opening / closing control valve 64 is opened, the output pipe 67 side is closed, each valve is closed after the concentrated blood sample flows into the flow path 71, and the optical labeling expression step S13 already described.
  • the target cell specifying step S21 is performed.
  • the control valve 62 and the three-way opening / closing control valve 64 are controlled to guide the NRBC to flow into the recovery container 65, and control is performed so that other than the NRBC flows into the disposal container 66. By repeating this process, NRBC is recovered (target cell recovery step S22).
  • step S21 is repeated and NRBC is specified, the three-way opening / closing control valve 64 is guided to control the three-way opening / closing control valve 64 so that the NRBC flows into the recovery container 65, and the three-way opening / closing control valve is flowed so as to flow into the disposal container 66 other than NRBC. 64 is controlled (target cell recovery step S22).
  • the flow path 71 is narrower than the flat plate 8, the size of the image data to be image-processed is small, so that the image processing can be performed at high speed and the NRBC can be specified at high speed. Therefore, the NRBC can be specified before passing the three-way opening / closing control valve 64, and the NRBC can be recovered.
  • biochip 70 eliminates the need to fix the concentrated blood sample on the flat plate 8 and to remove and collect the NRBC from the flat plate 8, thereby improving work efficiency and reducing the NRBC. It can be collected quickly. Therefore, inspection efficiency can be improved.
  • the biochip 70 in FIG. 7 may be replaced with the biochip 75 shown in FIG. 8 to search and collect target cells.
  • the three-way opening / closing control valve 64 shown in FIG. 7 is unnecessary, and the collection container 65 and the disposal container 66 are connected to the biochip 75.
  • the biochip 75 shown in FIG. 8 is formed of, for example, a silicone resin (for example, polydimethylsiloxane: PDMS), a plastic resin such as PET, acrylic resin, or cyclic polyolefin, or glass, and blood collected for testing. And a flow path 76 into which the concentrated blood sample is injected.
  • the channel 76 is provided with a target cell passage filter 72, an optical observation window 73, a recovery branch 78, and an open / close control valve 79.
  • the positional relationship between the flow path 76 and the target cell passage filter 72 is schematically shown.
  • the target cell passage filter 72 is a cell (non-target cell) 92 larger than the target cell 91 and capable of passing the target cell 91 to be examined in the concentrated blood sample as shown in FIG. Has a gap 82 that cannot pass through.
  • the target cell 91 is a red blood cell such as NRBC
  • the non-target cell 92 is a white blood cell larger than the red blood cell.
  • the non-target cells 92 are discharged to the outside of the biochip 75 through the disposal path 77 branched from the flow path 76 immediately upstream of the gap 82.
  • a storage tank for non-target cells 92 is formed in the biochip 75, and the non-target cells 92 removed by the target cell passage filter 72 are not discarded outside and stored in the storage tank. It is good also as a structure to accommodate.
  • a plurality of such target cell passage filters 72 may be arranged along the flow path 76. By arranging the plurality of target cell passage filters 72, the separation accuracy between the target cells 91 and the non-target cells 92 is improved, so that the search for the target cells 91 is further accelerated.
  • An optical observation window 73 is disposed downstream of the target cell passage filter 72.
  • the enlarged plan view is shown in FIG. 2C, and the enlarged side sectional view is shown in FIG.
  • the optical observation window 73 is formed of a transparent material so that optical observation of the target cell 91 flowing through the flow path 76 from the outside of the biochip 75 is possible.
  • the position of the XYZ stage 11 is controlled so that the imaging camera 15 of the target cell search device 1 images the optical observation window 73.
  • the flow path 76 of the optical observation window 73 is formed with a flow path width that allows the NRBC to pass in one row along the flow path 76 without overlapping.
  • the cross section that can pass through the disks in a single row and aligned in the direction of the surface is rectangular so that the flat surface can be observed from the optical observation window 73. It is preferable to form the flow path 76 with a hole.
  • the width of the flow path 76 parallel to the optical observation window 73 is formed with a width L1 (for example, 10 ⁇ m to 15 ⁇ m) such that a disk with a disk-like NRBC shape (diameter approximately 10 ⁇ m) recessed in the center passes.
  • the width of the flow path 76 perpendicular to the observation window 73 is formed with a width L2 (for example, 3 ⁇ m to 4 ⁇ m) that allows a disk (thickness of about 3 ⁇ m) to pass through.
  • the flat surface can be reliably observed without overlapping the NRBCs, so that it is possible to accurately distinguish between normal enucleated erythrocytes and NRBCs and further improve the NRBC search efficiency. be able to.
  • the flow path 76 is formed with such a narrow flow path width from the place past the target cell passage filter 72.
  • a recovery branch 78 (recovery flow path) for collecting NRBC from the flow path 76 is branched downstream from the optical observation window 73.
  • An opening / closing control valve 79 capable of branch control is disposed at a branch portion of the recovery branch path 78.
  • This open / close control valve 79 has the same function as the open / close control valve 64 already described, and can open and close the recovery branch 78 and the flow path 76 respectively.
  • the open / close control valve 79 can perform open / close control by inputting a control signal to the control electrode 85.
  • the on-off control valve 79 is a thermally controllable thermal control valve, an electrically controllable electrical control valve, a magnetically controllable magnetic control valve, or a pneumatic control valve that can be controlled by air pressure. It is preferable.
  • the open / close control valve 79 is a thermal control valve, for example, the valve is formed of a bimetallic material in which two metal plates having different thermal expansion coefficients are stacked, a shape memory alloy, or a thermoresponsive polymer such as poly-N-isopropylamide.
  • the valve is activated by applying heat. For example, the heat control valve is irradiated with laser light to generate heat, and the heat control valve is operated with this heat.
  • the open / close control valve 79 is an electric control valve, for example, the valve is formed of an electromagnetic control valve or a piezoelectric element that operates by energizing an electromagnetic coil. A metal terminal electrically connected to the electric control valve is formed on the surface of the biochip 75, and a control electric signal is applied to this terminal to operate the electric control valve.
  • the open / close control valve 79 is a magnetic control valve, for example, the valve is formed of a magnetostrictive element. The magnetic control valve is operated by applying a magnetic field by bringing a magnet close to the magnetic control valve.
  • the open / close control valve 79 is a pneumatic control valve
  • an air valve made of an elastic material such as PDMS is formed in the biochip, and the pneumatic control valve is operated by pressurization / decompression of air.
  • a heat control valve or a magnetic control valve since the valve can be operated without contact with the biochip 75, it is not necessary to consider contact failure, and it is preferable because it can be operated with high reliability.
  • the recovery container 65 of FIG. 7 is connected to the recovery branch 78, and the waste container 66 of FIG.
  • the target cell search device 1 has a control mechanism for operating the open / close control valve 79 (for example, a laser light source and its control means in the case of a thermal control valve).
  • the open / close control valve 79 is provided in the biochip 75 and the target cell search device 1 performs this control, whereby the specified NRBC can be reliably recovered. Further, there is no need to provide the three-way opening / closing control valve 64 outside, and when testing a plurality of specimens, there is no need to replace the three-way opening / closing control valve 64 for each specimen, and NRBC is quickly collected from a large number of specimens. can do.
  • a chemical solution injection path 80 for injecting an optical labeling chemical solution for optically labeling NRBC from the outside may be joined to the flow channel 76 upstream of the optical observation window 73.
  • NRBC can be optically labeled in the flow channel 76 by injecting a concentrated blood sample that is not optically labeled into the flow channel 76 and injecting an optically labeled chemical solution from the chemical solution injection path 80 (specimen labeling step).
  • S12 An example of the cell image after staining is shown in FIG.
  • FIG. 10 (a) is a typical image of the target cell NRBC.
  • FIG. 10B is a typical image of normal red blood cells (RBC), which is an example of a non-target cell
  • FIG. 10C is a typical image of white blood cells (WBC), which is an example of another non-target cell. It is a typical image.
  • a three-way opening / closing control valve may be arranged at a branching point of the recovery branch 78 and the flow path 76.
  • a direction switching valve that can control whether it flows may be provided.
  • a manual valve that can be opened and closed may be arranged.
  • the NRBC is specified, for example, the NRBC is tracked and imaged by the imaging camera 15, and the operator manually operates the valve while viewing the image to collect the NRBC in the collection container 65 (target Cell recovery step S22).
  • the biochip 75 is formed by bonding together two base plate materials 86 and a cover plate material 87 as shown in a side view in FIG.
  • the base plate 86 has a groove that becomes a flow path 76, a target cell passage filter 72, a disposal path 77, and a collection branch path 78, and a groove into which the opening / closing control valve 79 is fitted, and is formed by integral molding.
  • This base plate material 86 is made of transparent resin or glass. When a hard resin is used, it is manufactured by nanoimprinting, transfer molding, cutting using a mold or Si mold, or injection molding using a mold. can do.
  • a soft resin such as PDMS or elastomer
  • it can be manufactured by nanoimprinting or transfer molding using a mold or Si mold, or injection molding using a mold.
  • glass When glass is used, it can be manufactured by dry etching, wet etching, or the like.
  • electrical wiring wiring such as Cr and Au is manufactured by vacuum deposition or sputtering.
  • the cover plate 87 After the opening / closing control valve 79 is incorporated into the base plate 86, the cover plate 87, which is a transparent resin flat plate, is bonded by bonding, thermal bonding, or physical adsorption, thereby completing the biochip 75.
  • a frame-shaped instruction display indicating the position may be attached to the optical observation window 73 by printing, engraving, or the like.
  • the target cell collected in the biochip 75 (S23 in FIG. 1) is subjected to a process such as the target cell inspection step S24.
  • the focus control laser light source 12 may also be used as the imaging light source 14. More specifically, without placed the imaging light source 14 in FIGS. 4 (a) and 7, the laser light source 12 to irradiate the optical indicator expressing light L T as light Le for focusing. If it does in this way, it will become a simple structure. Since the irradiation range (irradiation area) of the laser beam Le is narrow, this configuration can be particularly preferably employed when imaging the channel 71 in FIG. 7 and the channel 76 in FIG. 8 where the imaging range is narrow.
  • NRBC fetal nucleated red blood cells
  • Example 1 The test method for fetal cells is as follows: [1] Specimen acquisition step for acquiring a concentrated blood sample by concentration of NRBC by density gradient centrifugation of maternal blood; [2] Blood is formed by smearing the concentrated blood sample on a flat plate.
  • Specimen labeling step of preparing a smear, optically labeling with Giemsa staining [3] Optical labeling step of expressing an optically labeled sample and capturing the color image, focus control step, color image acquisition step, [4 A binarization process for binarizing the color image to remove noise, a contraction / expansion process, [5] a primary candidate search process for searching and specifying the position of the NRBC, a secondary candidate search process, a tertiary candidate search process, Target cell identification step, [6] Target cell recovery step of capturing and recovering NRBC on the plate based on the position information, [7] Gene information in the nucleus of the recovered target cell
  • the target cells inspection step of analyzing the biological technique is chromosomal tests are performed.
  • Specimen acquisition step A stock solution of polyvinylpyrrolidone-coated silica gel fine powder (manufactured by GE Healthcare Japan, Inc .: Percoll) is diluted with physiological saline (0.15M NaCl aqueous solution: 0.9% NaCl aqueous solution) to obtain a specific gravity. Percoll dilutions of 1.085 g / ml and 1.075 g / ml were prepared.
  • This centrifuge tube was centrifuged for 30 minutes.
  • the erythrocyte layer, the Percoll diluent layer having a specific gravity of 1.085 g / ml, the NRBC and leukocyte-containing layer, the specific gravity of 1.075 g / ml Percoll diluent, the lymphocyte layer, and the plasma layer were separated.
  • the plasma layer was removed with a disposable pipette. Subsequently, the lymphocyte layer and the specific gravity 1.075 g / ml Percoll dilution layer were also removed.
  • the target layer in which NRBC and leukocytes are present appears on the surface layer, and was carefully collected with a pipette.
  • the collected recovery solution of the target layer is placed in a 25 ml centrifuge tube, and then physiological saline or phosphate buffered saline (PBS) pH 7.2 is added thereto, followed by centrifugation for 10 minutes, and the supernatant is removed three times. Washing was performed by repeating.
  • PBS phosphate buffered saline
  • the suspension was uniformly smeared on the slide glass using a cover glass to form a membrane. If necessary, cold air was applied to the smear to dry the smear.
  • the slide glass together with the dried smear film was immersed in methanol in a pad for 10 minutes to immobilize the smear film. The smear film was dried while applying cold air as necessary.
  • the slide glass together with the dried smear was immersed in a 2% Giemsa solution diluted solution in another pad for 10 minutes and stained.
  • the slide glass together with the stained smear was washed twice with ultrapure water in another pad.
  • the smear film was dried while applying cold air as needed to prepare a specimen-labeled blood smear sample.
  • the target cell search device described in the embodiment was prototyped. Target dictionaries and non-target dictionaries were created in advance.
  • One of the target dictionary image data used for the creation of the target dictionary is enlarged and shown in FIG.
  • FIG. 15B is an enlarged view of one of the non-target dictionary image data used for creating the non-target dictionary. These images are 52 pixels ⁇ 52 pixels in size.
  • Optical labeling step, focus control step, color image acquisition step The slide glass prepared in the specimen labeling step was set in the stocker of the prototype target cell search device. When the target cell search device 1 is operated, the target cell search device 1 transports the slide glass to the XYZ stage, irradiates the blood smear sample with the imaging light source, and automatically focuses and color images. . An example of the captured color image is shown in FIG. This color image has a size of 2448 pixels ⁇ 2050 pixels.
  • FIG. 16B shows a binarized image obtained by binarizing the color image of FIG. 16A
  • FIG. 16C shows a binarized image obtained by contracting and expanding the color image.
  • the position of the NRBC specified in the target cell specifying step described later is indicated by an arrow.
  • the target cell search device performs a primary search step to a target cell specification step, and FIG.
  • One target cell was automatically identified from the image of.
  • the size of the connected image region of the test image data selected in the tertiary candidate search step is in the range of 20 to 40 pixels, and the roundness is in the range of 0.5 to 1.5.
  • the connected image area inside was determined to be the nucleus of the target cell.
  • the identified NRBC image was visually confirmed by the operator to be NRBC.
  • Target cell recovery step A blood smear sample containing NRBC found by a target cell search apparatus was set on the stage of an inverted microscope equipped with a micromanipulator. The stage was moved to the position specified in the target cell identification step, and manually adjusted to the position where NRBC was present while looking at the measure attached to the microscope.
  • a 100 ⁇ g / ml proteinase K solution (manufactured by Wako Pure Chemical Industries, Ltd., 164-14004 dissolved in TE buffer) is sucked into a glass capillary for cell recovery, and the proteinase is recovered from the recovery capillary.
  • the solution K was discharged toward the target NRBC and allowed to stand at room temperature for 5 minutes.
  • the target NRBC was peeled off with a cell peeling glass capillary, and the NRBC was sucked with a cell collecting glass capillary, captured and collected.
  • the recovered NRBC was put in a 600 ⁇ l microtube, washed twice with PBS, and finally adjusted to a volume of about 2 ⁇ l to obtain a recovered NRBC suspension.
  • the NRBC collected by the micromanipulator can be directly transferred onto a slide glass for chromosome examination and fixed to Carnoy instead of being collected in a microtube.
  • Target cell test process chromosome test: FISH method
  • a 2 ⁇ SSC standard citric acid-added physiological saline
  • NP-40 Nonidet-40
  • 70% ethanol aqueous solution, 85% ethanol aqueous solution, and 100% ethanol were prepared.
  • a 70% formamide / 2 ⁇ SSC solution was prepared as a denaturing solution.
  • a 50% formamide / 2 ⁇ SSC solution was prepared as a formamide washing solution.
  • hybridization buffer manufactured by Abbott, attached to the FISH probe
  • FISH probe Abbott 32-190002, 32--190001, 32-192018, 32
  • 1 ⁇ l and 2 ⁇ l of ultrapure water were prepared in a microtube.
  • the mixing ratio varies depending on the number of FISH probes used. The above example is an example in the case of using one type of FISH probe. This was centrifuged for several seconds after stirring, and then heated with a block heater at 73 ° C. for 5 minutes to denature the probe. This probe solution was kept at 45 ° C. until just before use.
  • the Carnoy's solution containing this NRBC was dropped onto the center of the frontier (FRC-05, Matsunami Glass Industry Co., Ltd.), which is a cell-immobilized coated slide glass, and placed in a humidified tapper and left at 67 ° C. for 10 minutes. Then, it dried with cold air and obtained the NRBC fixed slide.
  • FRC-05 Matsunami Glass Industry Co., Ltd.
  • the aging solution (2 ⁇ SSC / 0.1% NP-40) was heated to 37 ° C., and the NRBC-fixed slide was immersed therein for 30 minutes.
  • the NRBC-immobilized slide was washed and dehydrated in a separate staining pad in the order of 70% ethanol aqueous solution, 85% ethanol aqueous solution, and 100% ethanol in order, followed by washing and dehydration. And dried.
  • the FISH success rate is improved by performing Lithium® Dodecyl® Sulfate (LIS) solution treatment and pepsin solution treatment as necessary.
  • LIS Lithium® Dodecyl® Sulfate
  • NRBC-immobilized slides were immersed in a LIS solution (10-100 mM LIS / 0.1 M Tris (tris hydroxymethylaminomethane), pH 7.4) for 30 minutes in a staining pad.
  • the NRBC-immobilized slide is then washed three times in a separate staining pad for 1 minute immersion in 2 ⁇ SSC solution at room temperature. It was then immersed in a pepsin solution (10-100 ⁇ g / ml pepsin / 0.01 M HCl, pH 2) heated to 37 ° C. in a staining pad for 10 minutes.
  • room temperature formalin refix solution 1% formaldehyde / 50 mM MgCl 2
  • room temperature enzyme stop solution 50 mM MgCl 2 / PBS solution, pH 7.4 in another staining pad. / PBS solution, pH 7.4) for 10 minutes.
  • the sample was immersed in PBS at room temperature for 5 minutes, and washed and dehydrated in the order of 70% ethanol aqueous solution, 85% ethanol aqueous solution, and 100% ethanol as an ethanol cleaning solution, followed by drying with cold air.
  • the denaturing solution (70% formamide / 2 ⁇ SSC solution) was heated to 73 ° C., and the NRBC-immobilized slide was immersed therein for 5 minutes.
  • the NRBC-immobilized slide was washed and dehydrated in a separate staining pad in the order of 70% ethanol aqueous solution, 85% ethanol aqueous solution, and 100% ethanol in order, followed by washing and dehydration. And dried.
  • a hybridization mask seal (manufactured by SC World Co., Ltd.) is attached to the cell immobilization position on the NRBC-immobilized slide, heated to 45 ° C. with a heater, and 2 ⁇ l of the probe solution stored at 45 ° C. is then added.
  • the solution was dropped on a well formed like a bank with a hybridization mask seal on an NRBC-immobilized slide, and a sealing seal was affixed to prevent bubbles from entering.
  • the NRBC-immobilized slide to which this probe solution was dropped was placed in a humidified tapper in an incubator adjusted to 37 ° C., and hybridization was performed for 4 to 18 hours.
  • the cover glass or hybridization mask seal was removed, and the NRBC-immobilized slide after hybridization was washed with 2 ⁇ SSC solution at room temperature.
  • the NRBC-immobilized slide was washed by soaking in a 2 ⁇ SSC / 0.3% NP-40 solution at room temperature for 5 minutes in a staining pad, and 2 ⁇ SSC / 0.3% at 73 ° C. in another staining pad. Wash by soaking in NP-40 solution for 2 minutes, then soak in 2 ⁇ SSC for 1 minute at room temperature in another staining pad.
  • 6-diamidino-2-phenylindole manufactured by Dojindo Laboratories Co., Ltd .; 340-07971
  • DAPI solution containing fluorescent anti-fading agent manufactured by Invitrogen; SlowFade (registered trademark) Gold Antifade Reagent with DAPI S36938 etc.
  • the cover glass was covered, and the cover glass was sealed with non-fluorescent and colorless nail polish to prevent the cover glass from slipping and the DAPI solution from drying.
  • FIG. 11 shows examples of images of normal cells and chromosome 21 trisomy cells.
  • FIG. 11 (a) is a photomicrograph of a fluorescence image after FISH of a normal cell
  • FIG. 11 (b) is a photomicrograph of a fluorescence image after FISH of a 21-chromosome trisomy cell, where X is the X chromosome, and Y is Y chromosome.
  • Example 2 If necessary, it is possible to collect nuclei after chromosome examination (FISH) and perform genetic examination using a DNA microarray. By using a DNA microarray, a single cell can be used to test for many diseases. The basic operation procedure will be described in detail.
  • NRBC-immobilized slides after chromosome examination (FISH) as in [1] to [7] of Example 1 were used.
  • Target cell test process (gene test: DNA microarray method) From the NRBC-immobilized slide after FISH, NRBC or NRBC nuclei were collected in a microtube using a micromanipulator attached to an inverted microscope.
  • Random primers SP180-1 manufactured by Operon Biotechnology Co., Ltd.
  • PCR enzyme EX Taq DNA Polymerase RR001A manufactured by Takara Bio Co., Ltd.
  • buffer attached to the PCR enzyme
  • dNTPPmix dNTPPmix
  • the amplified gene is purified using a PCR product purification kit (Qiagen 28104), and PCR reaction is performed using UULISYS I Nucleic Acid Labeling Kits and Alexa Flour 546 (Molecular Probe U-21652)
  • the gene amplified in (1) was fluorescently labeled and purified with Microcon® YM-30 (Millipore® 42409) to obtain a fluorescently labeled PCR product.
  • Cy3-dUTP GE Healthcare PA55021
  • Equal amounts of this fluorescently labeled PCR product and 2x hybridization buffer (12 x SSC, 0.4% SDS, 10 x hard Denhardt's solution, 0.2 mg / ml denatured salmon sperm DNA) are heat-denatured at 94 ° C for 1 minute. And then cooled rapidly on ice.
  • DNA microarrays also called DNA chips made by Takara Bio Co., Ltd. DNA chip slide glass Takara-Hubble Slide Glass TX720 and Sumitomo Bakelite Co., Ltd. Oligo Oligo 10 ⁇ l was dropped on a DNA immobilization substrate BS-11607 on which probe DNA was immobilized at SC World Co., Ltd. and covered with a cover glass.
  • a hybridization mask seal manufactured by SC World Co., Ltd. was attached on the DNA microarray, and 2 ⁇ l of the hybridization solution was dropped into the well and sealed.
  • the DNA microarray was placed in a humidified tapper in an incubator set at a hybridization temperature (45 to 60 ° C.), and hybridization was performed for 4 to 18 hours.
  • DNA corresponding to the DNA spotted on the DNA microarray is present in the hybridization solution, a DNA hybridization reaction occurs at that location, and thus fluorescence labeled on the DNA in the hybridization solution is observed.
  • a 0.15M KCl low expansion solution was added to the concentrated blood sample obtained by the Percoll method, and erythrocytes without nuclei were destroyed at room temperature for 10 minutes (some nucleated erythrocytes remained unbroken). ). Next, the supernatant was removed by centrifugation at 1500 rpm for 5 minutes. Furthermore, the operation of adding PBS, centrifuging, and removing the supernatant was repeated three times to obtain a concentrated blood sample in which the amount of nucleated red blood cells was reduced.
  • the target cells were collected in the same manner as in [2] to [6] of Example 1 except that this concentrated blood sample with a reduced amount of nucleated red blood cells was used to obtain an NRBC suspension.
  • PBS was added thereto, passed through a leukocyte removal filter, or a concentrated blood sample from which leukocytes were removed using CD45-labeled magnetic beads was injected into the biochip on the biochip reaction detection apparatus shown in FIG.
  • the liquid flow path 101 is indicated by a solid line
  • the control pattern wiring of the open / close control valve 102 disposed in the middle of each liquid flow path 101 is indicated by a broken line.
  • the biochip reaction detection apparatus has a FISH chamber 103 containing a cell immobilizing filter 104, a PCR chamber 105, and a DNA microarray chamber (gene testing chamber) 106.
  • 5-10 ⁇ l of the concentrated blood sample solution that has passed through the leukocyte removal filter or 5 ⁇ l of the NRBC suspension recovered from the blood smear sample slide is sent to the FISH chamber 103 in the biochip, and the cells are placed on the cell immobilization filter 104. Immobilized.
  • Washing was performed by flowing 20 ⁇ l of 2 ⁇ SSC into the FISH chamber 103 at room temperature.
  • the cells immobilized in the chamber were observed and imaged.
  • FIG. 13 (a) is a photomicrograph of NRBC, which is a target cell.
  • FIG. 13 (b) is a micrograph of NRBC, which is a target cell after FISH, 21 is 21 chromosomes, X is X chromosome, and Y is Y chromosome.
  • the evaluated target NRBC was normal with no chromosome number abnormality.
  • reaction solution after the PCR reaction is sent to the genetic testing chamber 106 containing the DNA microarray in which the probe DNA to be tested is immobilized, 2 ⁇ l of 2 ⁇ hybridization buffer is added, and the biochip is heated to 94 ° C. with the apparatus. After heat denaturation for 2 minutes, the mixture was rapidly cooled to 37 ° C. and placed at 37 ° C. for 4 to 16 hours to allow hybridization reaction (vibration was applied as necessary to promote the hybridization reaction).
  • the gene testing chamber 106 is washed with 10 ⁇ l of 2 ⁇ SSC at room temperature, and then 10 ⁇ l of 2 ⁇ SSC / 0.2% SDS solution is injected and heated to 65 ° C. with the apparatus for 5 minutes. placed. Next, it was washed with 20 ⁇ l of 0.05 ⁇ SSC at room temperature.
  • the DNA microarray in the gene testing chamber 106 was observed with fluorescence, and an image was taken.
  • Table 1 shows the arrangement of DNA microarrays.
  • a 30 base synthetic oligo DNA (custom product) that does not match the human gene sequence at each concentration is used as a positive control, and X, Y, 13, 18, 21 manufactured by GSP Laboratory Co., Ltd. are used as test probes.
  • Trisomy 13 is a so-called Patou syndrome
  • Trisomy 18 is a so-called Edward syndrome
  • Trisomy 21 is a test for chromosomal abnormalities that cause so-called Down syndrome.
  • the fluorescence value of the chromosome 21 probe was about 1.5 times higher than that of the normal cells, and it was possible to determine the abnormal number of chromosomes.
  • the determination of the number of chromosomes is quantitatively compared with the magnitude of the fluorescence value, and the determination of genetic abnormality is based on the hybridization results with the probe. It was qualitatively shown to be used for diagnosis.
  • a specific target cell such as a fetus-derived cell is examined using the collected maternal blood to safely and non-invasively fetus Useful for prenatal diagnosis.
  • it is useful for diagnosis and treatment by examining abnormal target cells present in the blood of infants and adults.
  • the target cell search device of the present invention and the biochip attached thereto are used to accurately and quickly perform this method for testing target cells in blood.

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Abstract

Le but de la présente invention est de réaliser le diagnostic prénatal d'un fœtus de manière invasive, sûre, rapide et précise. L'invention concerne un procédé simple et efficace pour tester une cellule cible dans le sang, consistant à concentrer de manière appropriée une cellule cible tirée d'un fœtus qui est présente en quantité extrêmement faible dans le sang prélevé sur le corps de la mère, ou à concentrer de manière appropriée une cellule cible anormale qui est présente dans le sang prélevé sur un nourrisson ou sur un adulte humain, rechercher la cellule cible dans un délai court et avec une grande précision sur une plaque plane ou dans un trajet d'écoulement d'une biopuce, collecter la cellule cible de manière fiable après identification de l'emplacement de la cellule cible, et analyser avec précision le chromosome, l'ADN ou l'ARN dans le noyau de la cellule. Dans le présent procédé de test d'une cellule cible dans le sang, une cellule cible dans le sang peut être testée en exécutant une étape de production de dictionnaire cible et une étape de production de dictionnaire non cible à l'avance, puis en soumettant le sang à tester à une étape d'acquisition d'échantillon, une étape de marquage d'échantillon, une étape d'expression de marquage optique, une étape de commande de mise au point, une étape d'acquisition d'image en couleurs, une étape de traitement de binarisation, une étape de contraction/dilatation, une étape de recherche de candidat primaire, une étape de recherche de candidat secondaire, une étape de recherche de candidat tertiaire, une étape d'identification de cellule cible, une étape de collecte de cellule cible et une étape de test de cellule cible.
PCT/JP2011/080306 2010-12-28 2011-12-27 Procédé de test d'une cellule cible dans le sang, dispositif de recherche d'une cellule cible, et biopuce WO2012091056A1 (fr)

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JP7198577B2 (ja) 2017-11-17 2023-01-04 シスメックス株式会社 画像解析方法、装置、プログラムおよび学習済み深層学習アルゴリズムの製造方法
JP7076698B2 (ja) 2017-11-17 2022-05-30 国立研究開発法人国立がん研究センター 画像解析方法、画像解析装置、プログラム、学習済み深層学習アルゴリズムの製造方法および学習済み深層学習アルゴリズム
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JP2022058244A (ja) * 2020-09-30 2022-04-11 富佳生技股▲ふん▼有限公司 核酸検出箱及び核酸検出デバイス
KR102651120B1 (ko) * 2021-11-18 2024-03-25 한국과학기술원 표적 물질에 대한 정량 정보 제공 방법 및 이를 이용한 디바이스

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05249103A (ja) * 1991-12-06 1993-09-28 Cell Analysis Syst Inc 血液細胞分析器
JP2000501859A (ja) * 1995-07-19 2000-02-15 モルフォメトリックス テクノロジーズ インク． 画像解析のためのウィンドウ・テクスチャ抽出
JP2011179924A (ja) * 2010-02-26 2011-09-15 Olympus Corp 顕微鏡システム、標本観察方法およびプログラム

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05249103A (ja) * 1991-12-06 1993-09-28 Cell Analysis Syst Inc 血液細胞分析器
JP2000501859A (ja) * 1995-07-19 2000-02-15 モルフォメトリックス テクノロジーズ インク． 画像解析のためのウィンドウ・テクスチャ抽出
JP2011179924A (ja) * 2010-02-26 2011-09-15 Olympus Corp 顕微鏡システム、標本観察方法およびプログラム

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104487564B (zh) * 2012-08-23 2017-06-16 富士施乐株式会社 图像处理装置、图像处理方法以及图像处理系统
WO2014030380A1 (fr) * 2012-08-23 2014-02-27 富士ゼロックス株式会社 Dispositif de traitement d'image, programme de traitement d'image, procédé de traitement d'image, milieu pouvant être lu par informatique, et système de traitement d'image
JP2014039504A (ja) * 2012-08-23 2014-03-06 Fuji Xerox Co Ltd 画像処理装置、プログラム及び画像処理システム
US9934571B2 (en) 2012-08-23 2018-04-03 Fuji Xerox Co., Ltd. Image processing device, program, image processing method, computer-readable medium, and image processing system
JP5333635B1 (ja) * 2012-08-23 2013-11-06 富士ゼロックス株式会社 画像処理装置、プログラム及び画像処理システム
CN104487564A (zh) * 2012-08-23 2015-04-01 富士施乐株式会社 图像处理装置、程序、图像处理方法、计算机可读介质以及图像处理系统
JP2014235494A (ja) * 2013-05-31 2014-12-15 富士ゼロックス株式会社 画像処理装置及びプログラム
WO2014192184A1 (fr) * 2013-05-31 2014-12-04 富士ゼロックス株式会社 Dispositif de traitement d'image, procédé de traitement d'image, programme et support de stockage
US10395091B2 (en) 2013-05-31 2019-08-27 Fujifilm Corporation Image processing apparatus, image processing method, and storage medium identifying cell candidate area
JP2016533475A (ja) * 2013-09-30 2016-10-27 ベンタナ メディカル システムズ， インコーポレイテッド 適応的病理組織画像分解のためのシステム及び方法
US10395371B2 (en) 2013-09-30 2019-08-27 Ventana Medical Systems, Inc. Systems and methods for adaptive histopathology image unmixing
US10783641B2 (en) 2013-09-30 2020-09-22 Ventana Medical Systems, Inc. Systems and methods for adaptive histopathology image unmixing
CN105214744A (zh) * 2015-10-26 2016-01-06 深圳华迈兴微医疗科技有限公司 一种磁微粒化学发光微流控芯片
JP2018068169A (ja) * 2016-10-26 2018-05-10 ヨダカ技研株式会社 細胞搬送システム
CN109136060A (zh) * 2017-06-15 2019-01-04 德诺杰亿（北京）生物科技有限公司 一种细胞列阵原位杂交的系统及方法
CN112927216A (zh) * 2021-03-22 2021-06-08 苏州恩可医药科技有限公司 一种nk细胞活性快速检测方法及系统
CN112927216B (zh) * 2021-03-22 2023-10-03 苏州恩可医药科技有限公司 一种nk细胞活性快速检测方法及系统
CN116106525A (zh) * 2023-04-13 2023-05-12 深圳市帝迈生物技术有限公司 血液分析仪
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