JP2016186454A - Detection method of rare cell in blood - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of heightening detection accuracy of a rare cell by discriminating a rare cell from a non-objective cell showing a false positive reaction to a rare cell identification marker.SOLUTION: Blood from which erythrocyte is removed is added to a substrate for cell expansion, and stained by a rare cell identification marker (marker A), a non-objective cell identification marker (marker B), and a marker (marker C) for such non-objective cells that a reaction to a cell stained by the maker A is different from a reaction to a rare cell, to thereby acquire respective fluorescent images. Thus, the rare cell can be identified from the fluorescent images.SELECTED DRAWING: None

Description

  The present invention relates to a method for detecting rare cells in blood with high accuracy.

  The blood usually contains blood cells such as red blood cells and white blood cells (neutrophils, eosinophils, basophils, lymphocytes, monocytes), but also circulating tumor cells (CTCs: Circulating Tumor Cells). In some cases, rare cells such as circulating vascular endothelial cells (CECs), circulatory endothelial progenitor cells (CEPs), and other progenitor cells may be included. In addition, the cultured cell population may include stem cells, specific differentiated cells, and other characteristic cells.

  CTC, one of the rare cells, is a cell found in the blood of patients with breast cancer, lung cancer, prostate cancer, pancreatic cancer, etc., and its number reflects the metastatic nature of the cancer. It has been attracting attention as it becomes useful information. However, the density of CTC in blood is extremely low (in the case of low, about 1 to 10 per 10 mL of whole blood), and its detection and counting are not easy.

  For example, with existing flow cytometers, it is theoretically possible to simultaneously identify, count and analyze rare cells such as CTC and leukocytes, but the number of rare cells in a blood sample is so small that there is no detection failure. There is a fear, and it is difficult to accurately perform analysis involving rare cells using this system.

  As a method for detecting a rare cell in a large amount of non-target cells (mainly white blood cells) with high sensitivity, a method of performing nuclear staining or fluorescent staining is known. At that time, in order to make it easy to perform microscopic observation and staining, for example, it was produced by installing a flow channel forming frame on a substrate having a microchamber or groove on the surface that can contain and hold cells. A system for expanding cells in a cell suspension using a cell expansion substrate is used.

When immunostaining is performed to identify rare cells and non-target cells, the most used method is an anti-cytokeratin (CK) antibody and an anti-CD45 antibody.
For example, the anti-CK antibody is a marker for identifying CTC, and the anti-CD45 antibody is a marker for identifying non-target cells. As an example, a cell that shows reactivity to anti-CK antibody (CK positive) and does not react to anti-CD45 antibody (CD45 negative) is determined as CTC, and reacts to anti-CK antibody. Cells that do not show sex (CK negative) and are reactive with anti-CD45 antibodies (CD45 positive) can be determined not to be CTCs.

  By using such means, by identifying the number of rare cells and non-target cells and measuring the number thereof, for example, the ratio of the number of CTCs to the total number of cells and the concentration of CTC in the blood sample are calculated, Analysis of CTC contained in a blood sample derived from a patient such as cancer can be performed.

  However, CTCs with low CK expression have recently been reported (Non-patent Document 1). In order to detect such a CTC having a low CK expression, a method for lowering the positive threshold in a signal caused by CK can be considered. However, as a result of lowering the positive threshold value for CK, there is a false positive group that becomes CK-positive and CD45-negative in a part of multinuclear cells that are one type of non-target cells that have been negative for CK according to the conventional positive threshold criteria. Appears and becomes an obstacle to accurately identifying and quantifying rare cells such as CTC. Since the number of polynuclear cells is considerably larger than the number of rare cells, even if a small percentage of the cells are false positives, the influence on the quantitative result of the number of rare cells is large. The reason why some of the polynuclear cells are CK-positive is that those cells express some CK, or that are not CK but express a protein having a structure similar to CK. It is conceivable that the anti-CK antibody reacts.

Special table 2013-508729 gazette

Low cytokeratin- and low EpCAM-expressing circulating tumor cells in pancreatic cancer. (Daniel Adams et al.) JOURNAL OF CLINICAL ONCOLOGY, 2013. Vol 31 (15)

  In order to solve the above-described problems, an object of the present invention is to provide a method for improving the detection accuracy of rare cells by discriminating non-target cells that show a false positive reaction with a rare cell identification marker. .

  As a result of intensive studies to solve the above problems, the present inventor has identified rare cells by using a third marker having different responses to non-target cells and rare cells when identifying rare cells and non-target cells. The present inventors have found that it is possible to discriminate between non-target cells that show false positives for markers and rare cells, and have completed the present invention.

  In addition, Patent Document 1 describes that three or more types of markers are used to distinguish rare cells from other cells, but this determines the expression level of protein in rare cells and non-target cells. Thus, it is used to promote detection of rare cells, and it does not avoid false positive determination for markers using the difference in reactivity between rare cells and non-target cells as in the present invention.

That is, the present invention provides the following method for simultaneous detection of blood cells.
[Claim 1]
A method for detecting rare cells in blood comprising the following steps (a) to (e):
(A) a step of removing red blood cells from blood (specimen treatment step);
(B) a step of expanding cells contained in a predetermined amount of blood treated in step (a) on a cell expansion substrate (cell expansion step);
(C) Marker A among cells contained in a predetermined amount of blood treated in step (a), a rare cell identification marker (marker A), a non-target cell identification marker (marker B), and a non-target cell A step of reacting a fluorescently labeled antibody that is a marker (marker C) having a different reactivity to a stained cell and a reactivity to a rare cell (marker C);
(D) a step of acquiring a fluorescent image of each marker stained in step (c) (image acquisition step);
(E) The process of identifying a rare cell using the fluorescence image of each marker acquired at the process (d).

[Section 2]
As the step (e), using the fluorescence image of each marker acquired in the step (d), the reactivity with respect to the marker A exceeds the positive threshold and the reactivity with respect to the marker B is less than the positive threshold Item 2. The method for detecting a rare cell according to Item 1, comprising a step of identifying a rare cell by determining whether a positive reaction to the marker A is a false positive using the marker C.

[Section 3]
Item 3. The method for detecting a rare cell according to Item 1 or 2, wherein a marker is used as the marker C, wherein a rare cell is negative and a non-target cell stained with the marker A is positive.

[Claim 4]
Item 4. The method for detecting a rare cell according to Item 1 or 2, wherein a marker is used as the marker C, wherein a rare cell is positive and a non-target cell stained with the marker A is negative.

[Section 5]
Among the non-target cells, cells stained with marker A are polynuclear cells, and the marker C is selected from the group consisting of anti-CD15, anti-CD16, anti-CD4, anti-CD11b, anti-CD44, anti-CD123, and anti-CD61 antibodies. Item 4. The rare cell detection method according to Item 3, which is a multinucleated cell marker using a primary antibody.

[Claim 6]
Cancer cell marker using a primary antibody wherein the rare cell is CTC and the marker C is selected from the group consisting of anti-Her2, anti-EGFR, anti-ER, anti-PgR, anti-ALK, anti-c-met and anti-S100 antibodies Item 5. The rare cell detection method according to Item 4.

[Claim 7]
Item 7. The fluorescent labeling antibody corresponding to the marker A has a fluorescent substance in which the peak of the emission wavelength when irradiated with predetermined excitation light is in a range exceeding 600 nm. Rare cell detection method

[Section 8]
The marker A is anti-cytokeratin, anti-EpCAM, anti-Vimentin, anti-E-cadherin, anti-Zonula occludens, anti-ESPR1, anti-N-cadherin, anti-Twist1, anti-Akt, anti-Pi3K, anti-Zeb1, anti-Plastin-3, anti-ALDH1 The method for detecting a rare cell according to any one of claims 1 to 7, which is a marker using a primary antibody selected from the group consisting of an anti-Gangliosides and an anti-ABC antibody.

[Claim 9]
Item 9. The rare cell detection method according to any one of Items 1 to 8, wherein the marker B is a marker using an anti-CD45 antibody as a primary antibody.

  By the method of the present invention, it becomes possible to accurately distinguish non-target cells and rare cells that show a false positive reaction with a rare cell identification marker in the detection of rare cells, and it is possible to detect rare cells with high accuracy. It becomes.

FIG. 1 is a schematic diagram of classification ratios based on leukocyte markers according to an embodiment of the present invention. In this specification, when the fluorescence intensity for each marker fluorescently labeled antibody exceeds the positive threshold, it is positive (+), and when the fluorescence intensity for each marker fluorescently labeled antibody is less than the positive threshold Indicates negative (−) (eg, CK (+) indicates that the luminance of fluorescence with respect to the anti-CK fluorescently labeled antibody exceeds the positive threshold). In total leukocytes (including both mononuclear cells and polynuclear cells), CK (−) is 94%, CK (+) and CK45 (+) is 5%. Further, leukocytes classified as CTC false positives that are CK (+) and CK45 (−) are 1%, and these leukocytes are CD16 (+). FIG. 2 is a plot diagram showing the results of the reference example. FIG. 3 is a fluorescence image acquired by the same method as in the embodiment of the present invention. The left figure is a polynuclear sphere, the middle figure is a mononuclear sphere, and the right figure is an image of MDA-MB231. When this image is viewed in color, CK is red, CD45 is green, and the nucleus is blue. In the left figure, it can be seen that the polynuclear sphere is stained with red representing CK together with blue representing nucleus and green representing CD45.

Hereinafter, although the form for implementing this invention is demonstrated, this invention is not limited to these.
(blood)
“Blood” as described herein is intended to include any component of whole blood, including red blood cells, white blood cells, platelets, endothelial cells, mesothelial cells or epithelial cells. Blood is also a component of plasma such as proteins, lipids, nucleic acids and carbohydrates, and any other cells that may be present in the blood due to pregnancy, organ transplantation, infection, trauma or disease, such as CTC, CEC, CEP Including rare cells such as other progenitor cells.

(Red blood cells)
As used herein, “erythrocytes” are erythrocytes, particularly non-nucleated red blood cells.

(White blood cells)
“Leukocytes” as described herein are leukocytes or cells of the hematopoietic lineage that are not reticulocytes or platelets. Leukocytes include lymphocytes such as natural killer cells (NK cells), B cells and T cells. Leukocytes also include phagocytic cells and mast cells such as monocytes, macrophages, granulocytes.

(Mononuclear sphere)
The “mononuclear cell” described in the present specification is a type of white blood cell, and occupies 22 to 46% of white blood cells in a normal person. Mononuclear cells include monocytes and lymphocytes. In 1 μL of blood, 100 to 600 monocytes (2 to 6% of total leukocytes) and 1000 to 3600 lymphocytes (20 to 20 of total leukocytes) are contained. 40%) is present.

(Polynuclear sphere)
The “polynuclear sphere” described in the present specification is a kind of white blood cell, and occupies 55 to 80% of white blood cells in a normal person. Polynuclear cells include neutrophils, eosinophils and basophils, which are further subdivided into rod-shaped and segmented nuclei. There are 150-400 neutrophils (spheroids) (3-5% of total leukocytes) and 2500-6300 neutrophils (segmental nuclei cells) (50-70% of total leukocytes) in 1 μL of blood. And occupy the majority of polynuclear spheres. On the other hand, 100 to 360 eosinophils (2 to 4% of all leukocytes) and 0 to 50 basophils (0 to 1% of all leukocytes) are present.
The types of leukocytes, the number of cells and the cell ratio are shown below.

− Rare cell detection method −
The rare cell detection method in the present invention includes at least (a) a specimen processing step, (b) a cell expansion step, (c) a staining step, (d) an image acquisition step, and (e) an identification step. In one embodiment of the present invention, the steps are performed in the order of (a) → (b) → (c) → (d) → (e), and (a) → (c) → (b) in another embodiment. → (d) → (e) The process order is performed.

-Process (a): Sample processing process-
The sample processing step (a) is a step of removing red blood cells from the blood of the patient. When preparing a specimen to be used in the present invention, it is appropriate to remove erythrocytes and perform other necessary pretreatments in advance. In this specification, a patient includes not only a subject suffering from cancer or the like but also a subject suspected of suffering from the disease.

(Anti-coagulation treatment)
The blood collected and taken out of the body (whole blood) coagulates as time passes when exposed to air as it is, and the cells contained therein cannot be collected and observed. Therefore, it is preferable that the collected blood is immediately subjected to anticoagulation treatment.

  Various anticoagulants for whole blood are known and can be used in accordance with conditions such as general concentration and treatment time. For example, anticoagulant of the type that inhibits coagulation by binding to calcium ion by chelating action and removing it from the reaction system, represented by ethylenediaminetetraacetic acid (EDTA) and citric acid (including salts such as sodium salt) And anticoagulant of the type that inhibits coagulation by forming a complex with antithrombin III in plasma, represented by heparin, and suppressing the production of thrombin. A blood collection tube in which such an anticoagulant is previously stored may be used.

  In addition, for cells, immobilization treatment that delays cell autolysis and decay and retains its morphology and antigenicity; improves the permeability of the cell membrane and allows substances (from outside the cell to inside the cell) Permeabilization treatment for facilitating penetration of fluorescently labeled antibodies that target antigens in cells, nuclear stains, etc.); Sites involved in immobilization of cells such as microchambers on cell development substrates In addition to preventing cells from adsorbing to other areas, it is necessary to perform blocking treatment to prevent non-specific adsorption of fluorescently labeled antibodies to sites other than the target antigen in immunostaining performed as necessary Depending on When the immobilization process, the permeabilization process, and the blocking process are not performed at the same time as the specimen processing process, these processes may be performed at the same time as the staining process, as will be described later.

(Removal of red blood cells)
When observing the expanded cells, the cells must be expanded so that the target cells do not overlap each other in the observation region. For this purpose, it is necessary to remove from the specimen red blood cells that account for about 96% of the formed components in the blood. By removing red blood cells, unnecessary overlapping of cells can be suppressed, and target cells can be efficiently developed to detect rare cells without reducing detection sensitivity.

  The means for removing red blood cells is not particularly limited, but for example, red blood cells may be hemolyzed using ammonium chloride or the like. Further, the cell fraction containing at least rare cells and non-target cells may be purified by separating erythrocytes and other cells by centrifugation. These techniques are known and can be performed using appropriate centrifuges and centrifugation conditions.

  Here, the density gradient centrifugation method is known as a method capable of fractionating components in blood containing various cells according to specific gravity. In particular, it is preferable to use a density gradient centrifugation method in order to remove red blood cells contained in a large amount in blood and use only a cell fraction containing rare cells and non-target cells for the cell expansion step.

  The separation liquid used for the density gradient centrifugation method may have any specific gravity suitable for the fractionation of cells in blood and an osmotic pressure and pH that do not destroy the cells. For example, commercially available Ficoll (trademark, GE Healthcare Bioscience), Percoll (trademark, GE Healthcare Bioscience), Polymorphprep (Trademark, Polymorphprep, Cosmo Bio), etc. The sucrose solution can be used. When density gradient centrifugation is performed after adjusting the specific gravity of this separated liquid to be smaller than the specific gravity of red blood cells and larger than the specific gravity of white blood cells, the blood sample is divided into `` a fraction rich in red blood cells '' and `` other than red blood cells '' Can be separated into at least two layers.

  For example, in one embodiment of the present invention, when the specific gravity of the separation liquid is 1.119 or less, preferably 1.113 or less, more preferably 1.085 or less, nucleated cells other than erythrocytes (rare cells and non-target cells) ) In the fraction containing a large amount of red blood cells can be suppressed to 2 to 6% or less. Furthermore, when the specific gravity of the separation liquid is about 1.077, rare cells such as CTC can be separated from erythrocytes and many non-target cells (in this case, however, rare cells such as CTC and non-nuclear cells such as polynuclear cells can be separated). The target cells cannot be completely separated beforehand, and there is a possibility of false positives).

-Process (b): Cell expansion process-
The cell spreading step (b) is a step of spreading and collecting cells contained in a prescribed amount of blood (cell suspension) that has undergone the specimen processing step (a) on a cell spreading substrate. You may perform a cell expansion | deployment process (b) after the dyeing | staining process (c) mentioned later.

(Cell development substrate)
The cell expansion substrate in the present invention typically corresponds to a device substrate (channel substrate) used in a cell observation method described later, and forms a channel in combination with a channel forming member. By introducing the cell suspension into the channel, the cells in the cell suspension are used on the surface of the cell development substrate. However, the substrate for cell deployment is not limited to such a substrate, and various members on the substrate that can be arranged without overlapping cells can be used as the substrate for cell deployment.

  The cell development substrate in the present invention is preferably formed with a fine chamber (microchamber) or groove so that cells in the cell suspension can be collected in a state suitable for fluorescence observation.

  When the substrate for cell deployment has a groove, it is preferable that the groove width of the groove is in the range of 1 to 100 μm and the groove depth is set in the range of 1 to 100 μm. If the dimensions are within such a range, the cells can be reliably deployed in the groove. The cross-sectional shape of the groove is not particularly limited, but a V-shaped cross section is preferable because cells can be arranged closely. In addition to the V shape, for example, only one side surface of the groove may be inclined, the top and bottom of the groove may be rounded, or a U-shaped groove. Moreover, it is preferable that the groove | channel of the said dimension makes the groove width and groove depth the same dimension. If it sets in this way, while it becomes difficult for the cell which once entered in the groove | channel to move, it can also prevent that the cell which entered the groove | channel dams another cell.

  The groove can be processed by a known fine processing technique, but a commercially available prism sheet, for example, can be substituted. In general, the prism sheet is a plate in which grooves of the same size are arranged in a plurality of rows on the upper surface of a flat plate, and the groove width and depth of the grooves that meet the above preferred ranges are commercially available.

  When the cell development substrate has a microchamber, the shape of the microchamber is not particularly limited, but for example, an inverted frustoconical shape having a flat bottom surface and a tapered side surface is preferable. The diameter and depth of the bottom surface of the microchamber can be adjusted as appropriate so that a suitable number of cells can be collected and accommodated for nucleic acid analysis and staining image observation. For example, it is preferable that the diameter of the bottom surface is 20 to 500 μm and the depth is 20 to 500 μm so that 1 to 100 cells can be accommodated per microchamber. The diameter of various cells in blood (excluding erythrocytes) is generally 5 to 100 μm, and the diameter of rare cells such as CTC is said to be about 10 to 100 μm. When a laser diode is used as the excitation light source when observing a fluorescent dyed image, the size of the irradiation region (spot) is, for example, an ellipse having a major axis of about 100 μm and a minor axis of about 10 μm. The size of the bottom surface of the microchamber is larger than the size of the irradiation area of the laser diode, and when one microchamber is irradiated with light, the light is irradiated across the adjacent microchambers. It is appropriate that no fluorescence is emitted from the microchambers simultaneously.

  The arrangement of the plurality of microchambers on the surface of the cell development substrate is not particularly limited, but the cell recovery rate (the ratio of cells recovered in the microchamber out of all the cells in the suspension) ) Is preferably adjusted so that the orientation of the array and the spacing between the microchambers are as high as possible. For example, it is preferable to arrange the microchambers so that the cells settle in the microchamber at at least one location from the inlet to the outlet when the cell suspension is fed into the flow path.

  Various matrix materials are well known in the art, such as glass; organic plastics such as glass and polycarbonate and polymethyl methacrylate, polyolefins; polyamides; polyesters; silicones; polyurethanes; epoxies; Polyacrylate; Polyester; Polysulfone; Polymethacrylate; Polycarbonate; PEEK; Polyimide; Polystyrene; and one or more of fluoropolymers may be included.

  The cell deployment substrate may be made of a material that allows cells to adhere easily. In this case, as described above, it is desirable to perform a surface treatment with a blocking agent or the like on the inner surface of the microchamber of the cell development substrate or a portion other than the inside of the groove.

(Cell immobilization method: material and structure of substrate for cell development)
In order to increase the detection efficiency of rare cells, it is necessary to immobilize the cells, that is, to prevent the position of the cells on the cell development substrate from moving. By immobilizing the cells, it becomes easy to specify the position of the cells to be observed, and the target cells detected can be collected as necessary.

  Cell immobilization methods include a method (structure method) that limits the range in which cells can move depending on the structure of the surface of the cell deployment substrate, and physics generated by the surface properties of the cell deployment substrate. It can be broadly classified into a method (interaction method) in which cells are adsorbed by dynamic interaction so as not to move.

  As a structural method for solid phase immobilization, for example, as described above, by forming a plurality of microchambers or grooves on the surface of the cell deployment substrate, the movement of the cells is restricted to only inside the chambers or grooves. It is done.

  As an interactive method of solid phase immobilization, the properties of the inner surface of the microchamber, particularly the bottom surface or the inside of the groove, are made into a property that cells can be easily adsorbed by the interaction, that is, cell adhesion. Specifically, for example, cells are easily adsorbed by physical (intermolecular) interaction, such as plastic or glass such as polystyrene, polymethylmethacrylate, or polycarbonate, on the inner wall surface of the microchamber or groove or the entire cell development substrate. There is a method of manufacturing with a hydrophobic material. When the entire substrate is made of such a material, the cell itself is blocked to lose its adsorption capacity so that it is not hindered from being collected and collected in the microchamber or groove during feeding. Alternatively, it is preferable that the property that cells are easily adsorbed is limited to only necessary portions such as a micro chamber or an inner wall surface of a groove.

  Moreover, the method of coating the inner wall surface of a micro chamber or a groove | channel with the molecule | numerator which improves the adhesiveness of a cell like poly-L-lysine is mentioned. The surface of the substrate for cell development other than the inside of the microchamber or groove is treated with a blocking agent to prevent the cells from adsorbing, or moderately hydrophilic by UV ozone treatment or oxygen plasma treatment in which UV irradiation is performed in an air atmosphere. There is also a way to make it. Inside the microchamber or groove, a material that is easy to adsorb cells is exposed, so that the cells are adsorbed there and do not go out of the microchamber or groove again by the flow of the cell suspension. Can be.

(Pre-wet liquid feeding process)
In the cell spreading step (b), a step of feeding a pre-wet solution for the purpose of improving the wettability of the surface of the cell spreading substrate may be provided prior to feeding the cell suspension. Good. If the cell deployment substrate is made of a hydrophobic material such as polystyrene, even if the cell suspension is introduced into the channel, bubbles remain in the microchamber or groove due to surface tension. Thus, the cell suspension may not be able to fill all the microchambers or grooves. In order to suppress such problems, an organic solvent having a low surface tension, such as an aqueous solution of ethanol, is introduced into the flow path as a pre-wet liquid in advance before the cell suspension is spread on the surface of the cell spreading substrate. By setting it, the wettability of the surface of the cell development substrate can be improved. After treatment with pre-wet liquid in this way, physiological saline or pure water such as PBS is passed to replace and wash the pre-wet liquid, and then the cell suspension is passed. The suspension enters the interior of almost any microchamber or groove, allowing the cells to be collected efficiently.

  The pre-wet solution is not particularly limited as long as it is an aqueous solution having a surface tension lower than that of water. For example, an aqueous solution containing alcohols such as ethanol, methanol, isopropyl alcohol, preferably 30% (w / v ) An aqueous ethanol solution having the following concentration, or an aqueous solution containing 0.01 to 1% (w / v) of a surfactant such as Tween (registered trademark) 20, Triton X (registered trademark) or SDS can be used.

-Process (c): Dyeing process-
The cell expansion step (c) is a step of staining cells contained in the cell suspension that has undergone the specimen processing step (a) using a fluorescently labeled antibody that is a marker, and before the cell expansion step (b). You may do it later.

  In the staining step (c), at least a fluorescently labeled antibody comprising an antibody (primary antibody) that specifically binds to a molecule expressed by a rare cell and a fluorescent substance that directly or indirectly labels it. Fluorescence containing an antibody (primary antibody) that specifically binds to a molecule expressed by a non-target cell and a fluorescent substance that directly or indirectly labels it, as a rare cell identification marker (marker A) A labeled antibody is used as a non-target cell identification marker (marker B).

  In the present invention, the molecules to which markers A and B (and marker C described below) bind can be any cell present in a sample that can be identified by known microscopic, histological, or molecular biological techniques. Components, including surface antigens, transmembrane receptors or co-receptors, proteins bound or aggregated on the surface, macromolecules such as carbohydrates, and intracellular components such as cytoplasmic or nuclear components However, it is not limited to these.

  For example, proteins expressed in epithelial CTC and hardly expressed in leukocytes are known as cancer cell markers, such as EpCAM (Epithelial cell adhesion molecule) expressed on the cell surface, Examples include cytokeratin (CK) that is expressed inside cells. On the other hand, examples of the protein that is expressed in leukocytes and hardly expressed in CTC include CD45 expressed on the cell surface, which is known as a leukocyte marker. Therefore, when the rare cell is CTC and the non-target cell is leukocyte, an anti-EpCAM antibody or anti-CK antibody can be used as the primary antibody of marker A, and an anti-CD45 antibody or the like can be used as the primary antibody of marker B Can be used.

  Further, in the staining step in the present invention, a marker (marker C) having different reactivity to cells stained with marker A among non-target cells and reactivity to rare cells is used. Here, the marker C is used for distinguishing cells stained with the marker A among non-target cells, that is, non-target cells showing a false positive reaction with the marker A and rare cells.

  The marker C is negative for rare cells, negative for non-target cells, and positive for non-target cells stained with marker A (referred to herein as marker C1). Or a marker that is positive for rare cells, positive for non-target cells, and negative for cells that are stained with marker A among the non-target cells (marker C2 in the present specification). May be called).

  For example, when the rare cell is CTC, the non-target cell is a leukocyte, and the non-target cell stained with the marker A is a polynuclear cell, the primary antibody of the marker C1 is anti-CD15, anti-CD16, anti-CD4, Multinuclear cell specific markers such as anti-CD11b, anti-CD44, anti-CD123, and anti-CD61 antibodies can be used, and the primary antibody of marker C2 is anti-Her2, anti-EGFR, anti-ER, anti-PgR, anti-ALK, anti-ALK, Cancer cell specific markers such as c-met and anti-S100 antibodies can be used.

(Fluorescent labeled antibody)
The fluorescently labeled antibody used as a marker in the present invention may be a primary antibody that specifically recognizes the target protein and a fluorescent substance described later bound thereto (direct labeling method), or such It may be a combination (indirect labeling method) of a primary antibody and a secondary antibody using this as an antigen to which a fluorescent substance is bound. As used herein, “using a specific antibody as a primary antibody” includes use of both the direct labeling method and the indirect labeling method described above.

  These fluorescently labeled antibodies can be prepared by a known method. For example, using various commercially available antibodies and fluorescent labeling kits, the predetermined functional groups of the antibody and the fluorescent dye are reacted with each other in the presence of a predetermined reagent and bonded according to the attached protocol. By using a biotin-avidin bond, a desired fluorescently labeled antibody can be prepared.

  Both the primary antibody and the secondary antibody may be polyclonal antibodies, but from the viewpoint of quantitative stability, monoclonal antibodies are preferred. The type of animal that produces the antibody (immunized animal) is not particularly limited, and can be selected from mice, rats, guinea pigs, rabbits, goats, sheep, etc., as in the past. When the bound antibody is used as a fluorescently labeled antibody, antibodies derived from different animal species must be used as the primary antibody used for each marker.

  When using a fluorescent antibody bound to a primary antibody as a fluorescent-labeled antibody, the marker is directly brought into contact with the cell contained in the cell suspension that has undergone the specimen treatment step (a) to react the cell with the marker. . In addition, when using a primary antibody and a secondary antibody having this as an antigen and a fluorescent substance bound thereto as a fluorescently labeled antibody, the primary antibody is brought into contact with the cell (primary reaction), and then the secondary antibody labeled with a fluorescent substance. The reaction is performed by contacting the antibody (secondary reaction). The primary reaction and the secondary reaction may be performed sequentially or simultaneously.

(Fluorescent substance)
The fluorescent substance used for the fluorescently labeled antibody is not particularly limited as long as it can determine whether each marker is positive or negative based on the fluorescence intensity in the acquired fluorescent image. It is preferable to use various fluorescent substances (fluorescent dyes). Here, the fluorescent substances used for the fluorescently labeled antibodies as the marker B and the marker C may be different substances (one whose fluorescence wavelength emission peak is separated to some extent and can be distinguished as a different color by image processing software). Alternatively, the same type (the emission peak of the fluorescence wavelength is close and can be determined to be the same color by image processing software or the like) may be used (see Example 2 below). Moreover, it is necessary to use different fluorescent substances for the fluorescently labeled antibodies that are marker A and marker B and marker A and marker C, respectively.

  As the fluorescent substance used for the fluorescently labeled antibody that is the marker A, it is preferable to select a fluorescent substance having a peak emission wavelength exceeding 600 nm when irradiated with predetermined excitation light. This is because fluorescence having a peak emission wavelength in the range exceeding 600 nm is relatively less susceptible to autofluorescence and is preferably used for detecting weak fluorescence emitted from a few rare cells.

  Considering the relationship between the excitation light wavelength and fluorescence wavelength of the fluorescent materials of each marker, and the fluorescence wavelength of nuclear staining, use appropriate multiple cut filters to appropriately measure each fluorescence. A fluorescent material having a suitable excitation light wavelength and fluorescence wavelength may be selected and used.

  In the case where cell immobilization treatment, permeabilization treatment, blocking treatment and the like are not performed in the sample treatment step, those treatments may be performed simultaneously in the staining step as necessary. That is, an aqueous solution in which a necessary fixative, penetrant, or blocking agent is dissolved together with a fluorescently labeled antibody or a nuclear staining agent is fed, and the cells are allowed to react with the cells by filling the flow path for a predetermined time. Also good.

  For example, a staining solution, which is an aqueous solution in which each marker or nuclear stain is dissolved, is sent to a flow path provided on a cell development substrate, fills the flow path for a predetermined time, and then a microchamber on the cell development substrate. Alternatively, the fluorescently labeled antibody may be bound to the antigen of the cell by contacting the cell contained in the groove with the staining solution.

  In addition, when (c) the staining step is performed before (b) the cell expansion step, the cells are reacted with the staining solution for a predetermined time in advance, washed, suspended in an appropriate buffer, and then expanded on the cell expansion substrate. You can do it.

  The time for the staining step, that is, the time for contacting the cells with the fluorescently labeled antibody solution can be adjusted appropriately so that immunostaining is sufficiently performed according to the case of performing general fluorescent immunostaining. It may be about 5 minutes to half a day.

  The temperature of the staining step, that is, the reaction temperature, can be appropriately adjusted so that immunostaining is sufficiently performed in accordance with the case of performing general fluorescent immunostaining, but it is usually about 4 to 37 ° C. .

In the present invention, staining for nuclear markers (nuclear staining) may be further performed. For nuclear staining, it is preferable to use a fluorescent dye (molecule) that emits fluorescence by intercalating into double-stranded DNA. Such fluorescent dyes are known as “nuclear dyes”, and for example, Hoechst dyes (Hoechst 33342, Hoechst 33258, etc.) that are permeable to cell membranes and capable of nuclear staining of living cells can be used. In addition, DAPI (4 ', 6-diamidino-2-phenylindole) is capable of nuclear staining of dead cells that have become permeabilized because the cell membrane has been altered because it is not permeable to cell membranes. ) Can also be used.
After performing the staining step, before taking an observation image of the cells stained in the image acquisition step, a washing solution may be added as necessary to remove unreacted fluorescently labeled antibodies.

-Process (d): Image acquisition process-
In the image acquisition step (d), a fluorescence image of each marker and / or nuclear marker stained in the staining step (c) is acquired.

  The fluorescent image can be taken by a method similar to the conventional method. For example, in order to take a fluorescent image, sequentially irradiate excitation light with an appropriate wavelength corresponding to the fluorescent substance bound to each marker, and cut an unnecessary wavelength component using an appropriate filter. What is necessary is just to image, observing the fluorescence emitted from a fluorescent substance.

In addition, for the purpose of confirming cell morphology, observation in bright field and image photographing may be performed together.
Each image acquired in this step can be used in the subsequent identification step after being integrated or corrected using an appropriate image processing means (software or the like) as necessary.

-Process (e): Identification process-
In the identification step (e), rare cells (and non-target cells) are identified from the fluorescence image of each marker acquired in the image acquisition step (d). Typically, for cells where the reactivity to marker A exceeds the positive threshold and the reactivity to marker B is below the positive threshold, whether the positive response to marker A is false positive using marker C Based on the above, rare cells (and non-target cells) are identified.

  For example, in one embodiment of the present invention, when the rare cell is CTC and the non-target cell is a leukocyte, the non-target cell that may be stained with the marker A is a polynuclear cell. In this case, for example, the primary antibody of marker A is an anti-CK antibody, the primary antibody of marker B is an anti-CD45 antibody, and the primary antibody of marker C is positive for polynuclear cells and leukocytes other than CTC and polynuclear cells. Anti-CD16 antibodies that are negative can be used.

  Here, if a marker A is positive and a marker B is negative in a cell where the marker C is negative, the positive of the marker A is true, so that the cell can be identified as CTC. On the other hand, if marker C is positive, the positive of marker A is a false positive, so that the cell can be identified as a polynuclear cell rather than a CTC.

  The cell analysis apparatus described below and the flow channel device and reagent container attached thereto, in other words, the cell observation system including them are suitable means that can be used to carry out the rare cell detection method of the present invention. It is an example. However, the present invention is not limited to the embodiment using such a cell analysis device or the like, and may be implemented using other means.

<Cell analyzer>
The cell analysis device is used for at least a liquid feeding system mechanism for feeding various liquids such as a cell suspension in a cell expansion process and a staining liquid in a staining process to the flow path of the flow path device, and those processes. A flow path device holder for holding a flow path device, a reagent container holder for holding a reagent container, and a control mechanism for controlling the behavior of various devices included in the cell analysis apparatus are provided. The cell analyzer further observes the fluorescence-stained cells collected on the cell development substrate of the flow channel device, and the optical system mechanism for capturing the fluorescence images is also in the same apparatus as the liquid feeding system mechanism. It is preferable to provide. If necessary, the cell analyzer can further include a sample processing mechanism that can remove red blood cells and the like by density gradient centrifugation in the same apparatus as the optical system mechanism.

(Sample processing mechanism)
The sample processing mechanism corresponds to means for removing red blood cells from blood collected from a patient by lysis, centrifugation, or the like, and preferably further removing unnecessary white blood cells such as polynuclear cells according to the embodiment. The sample processing mechanism may further have a function for performing various processes that can be performed simultaneously with the sample processing, such as cell immobilization processing, permeabilization processing, and blocking processing.

  The specimen processing mechanism for removing red blood cells and the like by centrifugation can be constructed according to a conventional centrifuge that can perform centrifugation using a predetermined method such as density gradient centrifugation. In addition, when removing red blood cells by lysis, the specimen processing mechanism when performing cell immobilization treatment, permeabilization treatment, blocking treatment, etc. as necessary can add reagents necessary for blood, It can be constructed using pipettes and tips.

  When the sample processing mechanism is provided in a cell analyzer equipped with a liquid delivery system or the like, for example, the sample processing is automatically performed by setting a blood collection tube containing blood in the cell analyzer. It is preferable to make it. On the other hand, the treatment of removing erythrocytes by lysis and the cell immobilization treatment that is performed as needed, just add the patient's blood into the blood collection tube containing the reagent for it (simultaneously with the blood coagulation treatment) When it is possible to reduce the size of a cell analyzer equipped with a liquid delivery system, etc., the sample processing mechanism does not need to be incorporated into the cell analyzer equipped with a liquid delivery system, etc. The sample processing apparatus (general centrifuge, etc.) may be used. In such a case, the processed specimen is set in a cell analyzer equipped with a liquid feeding mechanism or the like.

(Liquid feeding mechanism)
Under the control of the control means, the liquid feeding system sucks and discharges the cell suspension, staining liquid, washing liquid, and other reagents used as necessary, under the control of the control container. Is a mechanism for feeding the liquid into the flow path.

  The liquid delivery system moves, for example, in a plane direction and a vertical direction with respect to the flow channel device (substrate for cell deployment) to enable suction and discharge of the liquid at an arbitrary position, such as a syringe pump, a replaceable chip It can be constructed using possible actuators. The syringe pump has a capability of sucking and discharging the liquid used in each step at a desired flow rate.

  Specifically, it is possible to use a liquid feeding system mechanism that individually includes a supply unit and a liquid feeding unit, and a flow path device that includes a reservoir on the inlet side. In this embodiment, the supply means aspirates a predetermined amount of liquid such as a cell suspension stored in the reagent container and then discharges the liquid in the reservoir, and the liquid is temporarily stored in the reservoir. . The liquid feeding means is connected to the outlet and introduces the liquid in the reservoir from the inlet to the flow path at a predetermined flow rate by suction.

  Also, a liquid supply system mechanism that has both a supply means and a liquid supply means, and a flow path device that has a reservoir on the outlet side can be used. In this embodiment, the liquid feeding system mechanism sucks a predetermined amount of liquid such as a cell suspension stored in the reagent container, and then discharges the liquid at a predetermined flow rate at the inflow port. Introduce directly. Liquid that has flowed out of the outlet through the flow path can be temporarily stored in the reservoir. After the predetermined process is completed by the liquid feeding, the liquid feeding system mechanism sucks and discharges the liquid that has filled the flow path from the inlet. The discharged liquid may be discharged from the liquid feeding system mechanism in the waste liquid storage unit of the reagent container.

(Optical system mechanism)
The optical system mechanism includes a member such as a laser diode (LD) serving as a light source, a condensing lens, a pinhole member, a dichroic mirror, and a fluorescent filter to excite a fluorescent substance to emit fluorescence. The LD is adapted to the excitation wavelength of the fluorescent substance possessed by the fluorescently labeled antibody to be observed, and a plurality of LDs are prepared as necessary so that an appropriate one is irradiated by the dichroic mirror. The fluorescent filter corresponds to the fluorescent wavelength of the fluorescent substance used for the fluorescently labeled antibody. If necessary, a plurality of fluorescent filters are prepared and used while being replaced with a filter switch.

  The optical system mechanism further includes a member such as an objective lens, an eye lens, a CCD camera, etc. that allows the fluorescently stained cells to be visually observed, and a member for photographing those fluorescent images. A member for measuring fluorescence intensity such as a photomultiplier tube (PMT) may be included.

  The optical system mechanism including these members can be constructed in a form conforming to a fluorescence microscope, and is preferably incorporated into the cell analysis apparatus together with the liquid feeding system mechanism. In such an embodiment, after performing the cell expansion step and the staining step, the image acquisition step can be performed to smoothly observe and image fluorescent cells. In addition, the optical system mechanism is provided in an apparatus based on a fluorescence microscope that is separate from the apparatus centering on the liquid feeding system mechanism, and the flow path device that has performed the cell expansion process and the staining process by the former apparatus is used in the latter apparatus. It is also possible to move the image acquisition process.

  When analyzing a cell using a cell development substrate equipped with a microchamber, the optical system mechanism further specifies the position of the microchamber to be observed and the position of the cell accommodated in the macrochamber. Means may be provided. For example, by providing a reference point (reticle mark) on a cell development substrate, information on the position of the target microchamber (microchamber that emits fluorescence labeled with the target cell) or the target cell itself Can be obtained as coordinates from the reference point. If information based on such reference points is used, even if the cell deployment substrate is moved from the cell analyzer to another observation / imaging device, the target microchamber or The cells can be observed immediately.

(Data processing mechanism)
The data processing mechanism is a mechanism capable of integrating and correcting data such as fluorescence images and fluorescence intensities acquired by the optical system mechanism in the image acquisition means and the detection means. The data processing program (software), a storage medium storing the program in a computer-executable format, and a computer for executing the program can be constructed. Such a data processing mechanism may be provided as a separate device (such as a personal computer) that is used by being connected to a cell analysis device including a liquid feeding system mechanism and an optical system mechanism. In addition, it may be incorporated in a cell analysis apparatus equipped with an optical system mechanism or the like.
The data processing mechanism has at least a function for identifying rare cells and non-target cells from the fluorescence image acquired by the image acquisition means.

  For example, in the same visual field, a fluorescence image representing fluorescence emitted from a fluorescently labeled antibody that is marker A, a fluorescence image representing fluorescence emitted from a fluorescently labeled marker that is marker B, and a fluorescence image that represents fluorescence emitted from a fluorescently labeled marker that is marker B A process for integrating the fluorescence images representing the fluorescence to be generated; a process for identifying a region having pixel characteristics (brightness, color) having a predetermined characteristic in the integrated image as a rare cell or a non-target cell; It is preferable to use a program that can automatically execute processing for measuring the number of each cell; processing for sequentially performing all of the visual fields obtained by imaging these processing.

  In particular, for example, in one aspect, fluorescence emitted from a fluorescently labeled antibody that is marker A is detected in a specific visual field, while cells that cannot detect fluorescence from a fluorescently labeled antibody that is marker B are rare cells. It is estimated to be. Furthermore, in such a cell, if fluorescence from the fluorescently labeled antibody that is marker C cannot be detected, the cell is identified as a rare cell, whereas if fluorescence from marker C is detected, the cell is rare. It is not a cell but is identified as a non-target cell that is non-specifically stained with marker A.

  The program in the embodiment of the present invention preferably compares, for example, the number of measured rare cells and non-target cells so that the ratio of the rare cells to the total number of cells can be automatically calculated.

(Control mechanism)
The control mechanism is a mechanism that is connected to various devices of the cell analysis apparatus and can control the behavior of these devices so that the cell detection method can be performed according to a predetermined process or procedure. Can be constructed by such a control program, a storage medium storing the program in a computer-executable manner, and a computer capable of executing the program. Such a control means may be provided as a separate device (such as a personal computer) that is used by being connected to a cell analyzer equipped with a liquid feeding system mechanism, an optical system mechanism, etc., or may be fed like a microcomputer. It may be incorporated in a cell analysis apparatus equipped with a liquid system mechanism and an optical system mechanism. The control program may be stored in a storage medium built in the computer, or may be placed in a state where the personal computer can be used via a network or a removable storage medium.

  The control program can automate operations relating to cell deployment means (process), staining means (process), image acquisition means (process), and the like. For example, with a predetermined time schedule, the liquid supply system mechanism is operated so as to supply the cell suspension in the cell expansion process or the staining liquid in the staining process, or the predetermined excitation corresponding to the fluorescent substance contained in the staining agent. The optical system mechanism can be operated to irradiate light and take a fluorescent image.

  In addition, the control mechanism stores data acquired regarding cell staining, for example, a captured fluorescent image, reads out the data in an appropriate process, and passes the function to an appropriate mechanism, and a program for that. It is preferable to have.

<Flow channel device>
The channel device is constructed by a cell deployment substrate and a channel forming member, and a space closed by these becomes a channel that can be filled with a liquid such as a cell suspension. Yes. From the viewpoint of ease of observation and maintenance, the cell deployment substrate and the flow path forming member may be attachable / detachable by means such as engagement, screw fixation, and adhesion. On the ceiling side near the upstream and downstream ends of the flow path, that is, on the flow path forming member, an inflow port and a discharge port for allowing the various liquids to flow in and out are formed.

  The height of the flow path (the distance between the cell development substrate and the top plate member, that is, the thickness of the frame member) is preferably 50 μm to 500 μm. When the height of the flow path is within such a range, the cell suspension (cells contained therein) in the flow path can be easily moved by the force of liquid feeding, and depending on the cells in the flow path. Since clogging is less likely to occur, the cells can be expanded smoothly.

  The flow path forming member is mounted on a frame member that forms a side wall of the flow path that creates a space for giving the flow path a predetermined height and forms a planar range of the flow path, and a frame member. It can construct | assemble by the top-plate member which forms the ceiling of a flow path. The top plate member may include a space (reservoir) that temporarily communicates a liquid such as a cell suspension that communicates with the outflow port. In addition, the ceiling of the flow path may have an uneven structure so that the cells can flow into the microchamber easily. The position of the unevenness can be adjusted, for example, so that the center of the microchamber and the center of the concave portion and the convex portion are shifted (does not come on the vertical line). The flow changes toward the microchamber with the concave and convex portions as a boundary, and cells easily enter.

  The cell development substrate and the flow path forming member are preferably made of a transparent material such as plastic such as polystyrene, polymethylmethacrylate, polycarbonate, cycloolefin copolymer, or glass such as quartz glass. Cell analysis systems usually observe and image fluorescence-stained cells, so that at least the top plate member of the flow path forming member can measure fluorescence labeled with specific cells by the optical detection system of the cell analyzer. The corresponding part must be made of a transparent material. The frame member may be made of a material such as silicon resin (polydimethylsiloxane: PDMS) having appropriate elasticity and adhesiveness, and the frame member is sandwiched between the cell deployment substrate and the top plate member. Thus, the flow channel device may be constructed.

<Reagent container>
In the reagent container, a cell suspension, a staining solution (fluorescence-labeled antibody solution, nuclear staining agent solution, or a mixed solution thereof), a washing solution, and other necessary solutions are used for carrying out the cell detection method. Various liquids that need to be sent to the path are stored. For example, a liquid having a relatively high storage stability such as a washing solution can be stored in a predetermined part of the reagent container in a sealed state in advance, and cells such as a cell suspension and a staining solution are stained. The liquid that needs to be prepared immediately before is prepared so that it can be added to and stored in a predetermined part of the reagent container after preparation. Solutions that are used multiple times, such as cleaning solutions, may contain a volume of solution corresponding to each process in separate parts, or when solutions of the same composition are used repeatedly in each process The total amount of liquid may be contained in one site. In addition, the reagent container is provided with a part for storing waste liquid sucked and discharged from the flow path after feeding, as necessary.

[Example 1]
(A) Specimen treatment step Polymorphprep (trademark: Cosmobio) (sodium diatrizoate 13.8 (w / v)%, Dextran 500 8.0 (w / v)%) was collected containing the anticoagulant EDTA2Na. Erythrocytes, mononuclear cells, and polynuclear cells were fractionated from blood samples from healthy individuals collected by blood vessels (Terumo Benogect II vacuum blood collection tubes), and the mononuclear cell fraction and the multinucleated cell fraction were isolated. Were mixed. The mixture was reacted with 4 (w / w)% formaldehyde for 20 minutes at room temperature to immobilize cells and prepare a white blood cell suspension.

  Similarly, MDA-MB231 strain, which is a cancer cell known to have weak CK expression, was fixed with 4 (w / w)% formaldehyde and washed with PBS to prepare a MDA-MB231 cell suspension. did.

(C) Staining step (a) Each of the white blood cell suspension and MDA-MB231 cell suspension obtained in the specimen treatment step was centrifuged at 300 G for 1 minute, and the supernatant was removed. Staining solutions prepared by adding PE-labeled anti-cytokeratin (CK) antibody, APC-labeled anti-CD45 antibody and FITC-labeled anti-CD16 antibody to PBS (containing 1% Tween 20 and 3 (w / v)% BSA) After adding to the cell sample and reacting for 30 minutes, it was washed 3 times with PBS (containing 0.1% BSA).

(B) Cell development step The leukocyte cell suspension and MDA-MB231 cell suspension stained in step (c) were each developed on a hemocytometer (NanoEnTek C-Chip DHC-F01).

(D) Image acquisition process The image of the cell developed on the hemocytometer in the above process was taken under the wavelength corresponding to each excitation light of FITC, PE, and APC, and from the obtained photographed image, respectively. The brightness of was measured. Images were taken with a Carl Zeiss erecting fluorescence microscope Axio imager. The FITC dye was a Carl Zeiss 38 HE fluorescent filter, the PE dye was a Carl Zeiss 48 HE fluorescent filter, and the APC dye was a Carl Zeiss 50 fluorescent filter. The luminance was measured by image analysis, and Carl Zeiss Zen2012 (Blue edition) software was used for the image analysis.

(E) Identification Step In the MDA-MB231 strain, a positive threshold value for each marker was set based on the luminance obtained as a result of performing the steps (a) to (d). Specifically, in the anti-CK fluorescently labeled antibody (PE), in the luminance value (luminance ≧ 800) capable of detecting 99% or more of MDA-MB231 cells (CK weak cancer cells), in the anti-CD45 fluorescently labeled antibody (APC) Is the maximum luminance value (luminance ≧ 150) detected with MDA-MB231 cells, and the maximum luminance value detected with MDA-MB231 cells in anti-CD16 antibody (FITC) was the respective positive threshold (luminance ≧ 540) ( Round up the 10th place of the threshold value for each luminance).

  Based on the set positive threshold value and the luminance value acquired from the image, positive / negative of CK, CD45 and CD16 in each of leukocytes and MDA-MB231 cells was determined respectively (from the above positive threshold setting method, anti-CD45 Regarding the brightness of the fluorescently labeled antibody and the anti-CD16 antibody, all the MDA-MB231 strains do not exceed the positive threshold value, and are naturally determined to be negative). Table 1 shows the respective ratios in the total number of cells. Based on markers A and B alone, when a positive threshold is set so that 99% of MDA-MB231 cells can be detected (CK (+) / CD45 (−)), multinucleated cells that show false positives appear. I understand that. However, by using the marker C, out of cells showing CK (+) / CD45 (−), the CD16 (+) cell is excluded as such a false positive cell, so that CK (+) / Only CD45 (−) / CD16 (−) cells can be correctly identified as MDA-MB231 cells.

From the results of Example 1 above, even when patient-derived blood is used as a specimen, for example, only CK (+) / CD45 (−) / CD16 (−) cells are used based on the rare cell detection method of the present invention. It was shown that accurate quantification can be performed by determining CTC as CTC.

[Example 2]
(C) The FITC-labeled anti-CD45 antibody is used in place of the APC-labeled anti-CD45 antibody in the staining step (that is, all cells that react with at least one of the anti-CD45 antibody or the anti-CD45 antibody are labeled with FITC, and the amount of fluorescence is a threshold value) This was performed in the same manner as in Example 1 except that CD45 · CD16 (+) is determined if larger.

  Table 2 shows the proportion of positive / negative cells of each marker in the total number of cells of MDA-MB231 and leukocytes. When a positive threshold was set as in Example 1, the CK (+) and CD45 / CD16 (−) cell group contained only MDA-MB231 cells and no leukocytes. Therefore, it can be seen that even in the method of Example 2 in which the cell group of CK (+) and CD45 / CD16 (−) is determined as CTC, leukocytes showing a false positive for the CK marker can be excluded and only CTC can be detected correctly.

[Example 3]
(C) The procedure was the same as in Example 1 except that a labeled secondary antibody was used instead of the labeled antibody in the staining step.
Table 3 shows the proportion of positive / negative cells of each marker in the total number of cells of MDA-MB231 and leukocytes. When a positive threshold value is set as in Example 1, it can be seen that multinucleated cells showing false positives appear in the cell group of CK (+) and CD45 (−). However, by using the marker C (CD16), out of the cells showing CK (+) / CD45 (−), the cells of CD16 (+) are excluded as such false positive cells. It can be seen that method 3 can also eliminate white blood cells that are falsely positive for the CK marker and correctly detect only CTC.

[Comparative Example 1]
(C) In the staining step, the same procedure as in Example was carried out except that the FITC-labeled anti-CD16 antibody was not added to the staining solution.
Table 4 shows the ratio of positive / negative cells of each marker in the total number of cells of MDA-MB231 and leukocytes. The cell group of CK (+) and CD45 (−) contained not only MDA-MB231 cells but also 1% of leukocytes. Therefore, in the method of Comparative Example 1 in which the cell group of CK (+) and CD45 (−) is determined as CTC, leukocytes showing false positives for the CK marker are mixed in such a cell group, and only CTC is detected. It turns out that it cannot detect correctly.

[Reference Example 1]
Only cells that were stained in the same manner as in Example 1 for cancer cells (MDA-MB231), leukocytes (mononuclear cells) and leukocytes (polynuclear cells) and were in the CK (+) group (PE value ≧ 800) Were extracted, and their FITC (CD16) luminance was plotted on the horizontal axis and APC (CD45) luminance was plotted on the vertical axis. It can be seen that cells using CK (+) when using the above threshold, which is slightly lower than normal, include a certain number of polynuclear cells as well as cancer cells (almost no mononuclear cells). . On the other hand, as a result of qualitative evaluation, as shown in Table 5 for the staining properties of markers for CK, CD45, and CD16 in polynuclear cells and mononuclear cells, the polynuclear cells can be detected as CK (+), and It was found that the staining for CD45 was weaker than that of mononuclear cells. From the results of the reference examples, it can be seen that CK (+) polynuclear cells are a group with a weaker CD45 than mononuclear cells. Like CTC, CK (+) and CK45 (−) Some suggest that it is easy to be determined as a false positive (in contrast, a mononuclear cell is CD45 (+) under an appropriate threshold even if it is CK (+), and is unlikely to be false positive. ). However, the expression level of CD16 is different between the cancer cell to be detected and the polynuclear cell that can be false positive, suggesting that it can be discriminated by setting an appropriate threshold value.

Claims (9)

A method for detecting rare cells in blood comprising the following steps (a) to (e):
(A) a step of removing red blood cells from blood (specimen treatment step);
(B) a step of expanding cells contained in a predetermined amount of blood treated in step (a) on a cell expansion substrate (cell expansion step);
(C) Marker A among cells contained in a predetermined amount of blood treated in step (a), a rare cell identification marker (marker A), a non-target cell identification marker (marker B), and a non-target cell A step of reacting a fluorescently labeled antibody that is a marker (marker C) having a different reactivity to a stained cell and a reactivity to a rare cell (marker C);
(D) a step of acquiring a fluorescent image of each marker stained in step (c) (image acquisition step);
(E) The process of identifying a rare cell using the fluorescence image of each marker acquired at the process (d).   As the step (e), using the fluorescence image of each marker acquired in the step (d), the reactivity with respect to the marker A exceeds the positive threshold and the reactivity with respect to the marker B is less than the positive threshold The method for detecting a rare cell according to claim 1, further comprising the step of identifying a rare cell by determining whether a positive reaction to the marker A is a false positive using the marker C.   The method for detecting a rare cell according to claim 1 or 2, wherein a marker is used as the marker C, wherein a rare cell is negative and a non-target cell stained with the marker A is positive.   The method for detecting a rare cell according to claim 1 or 2, wherein a marker is used as the marker C, wherein a rare cell is positive and a non-target cell stained with the marker A is negative.   Among the non-target cells, cells stained with marker A are polynuclear cells, and the marker C is selected from the group consisting of anti-CD15, anti-CD16, anti-CD4, anti-CD11b, anti-CD44, anti-CD123, and anti-CD61 antibodies. The method for detecting a rare cell according to claim 3, which is a multinuclear cell marker using a primary antibody.   Cancer cell marker using a primary antibody wherein the rare cell is CTC and the marker C is selected from the group consisting of anti-Her2, anti-EGFR, anti-ER, anti-PgR, anti-ALK, anti-c-met and anti-S100 antibodies The method for detecting a rare cell according to claim 4, wherein   The fluorescently labeled antibody corresponding to the marker A has a fluorescent substance whose emission wavelength peak exceeds 600 nm when irradiated with predetermined excitation light. Rare cell detection method   The marker A is anti-cytokeratin, anti-EpCAM, anti-Vimentin, anti-E-cadherin, anti-Zonula occludens, anti-ESPR1, anti-N-cadherin, anti-Twist1, anti-Akt, anti-Pi3K, anti-Zeb1, anti-Plastin-3, anti-ALDH1 The method for detecting a rare cell according to any one of claims 1 to 7, which is a marker using a primary antibody selected from the group consisting of an anti-Gangliosides and an anti-ABC antibody.   The method for detecting a rare cell according to any one of claims 1 to 8, wherein the marker B is a marker using an anti-CD45 antibody as a primary antibody.