WO2014185151A1 - 細胞染色方法及びその方法に使用する検体採取管 - Google Patents
細胞染色方法及びその方法に使用する検体採取管 Download PDFInfo
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- WO2014185151A1 WO2014185151A1 PCT/JP2014/058029 JP2014058029W WO2014185151A1 WO 2014185151 A1 WO2014185151 A1 WO 2014185151A1 JP 2014058029 W JP2014058029 W JP 2014058029W WO 2014185151 A1 WO2014185151 A1 WO 2014185151A1
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/305—Fixative compositions
Definitions
- the present invention relates to a method for staining cells and a sample collection tube used in the method. More specifically, the present invention relates to a method for cell immobilization, permeabilization and / or labeling of cells in cell staining, and a specimen collection tube used for the method of immobilization, permeabilization and / or labeling of the cells.
- Cytology is performed to determine the presence or absence of lesions by observing cells collected from human or animal bodies.
- blood contains blood cells, and organs' mucous membranes, mucus, sputum, ascites, gastric fluid, urine, and the like are mixed with cells from which the organ has been detached.
- these cells are collected, stained, and observed with a microscope or the like.
- CTC circulating tumor cells
- vascular endothelial cells vascular endothelial progenitor cells
- various stem cells hereinafter referred to as “rare cells” are cells that are extremely rare in peripheral blood depending on the pathological condition.
- the detection of these rare cells is clinically useful.
- rare cells contain very small amounts of specimens, it is often still difficult to detect rare cells from blood-derived specimens.
- cytodiagnosis In cytodiagnosis, generally, collected cells are fixed and permeabilized and then stained and observed with a microscope or the like. Usually, this procedure involves sample collection, cell suspension preparation (mainly in the case of liquid samples such as blood, urine (including separation of target cell fraction)), cell immobilization, washing, cell A plurality of steps such as permeabilization, cell staining, and washing are sequentially performed, and the stained cells are observed with a microscope or the like.
- a staining method for detecting a specific cell for example, a dye staining method or an antigen-antibody method that specifically reacts with a specific substance expressed in a detection target cell is used.
- Patent Document 1 for the purpose of detecting cells in blood, after reacting an anticoagulant and blood, erythrocyte lysis is performed, and the remaining cell suspension is used as an adhesive support. Incubate at 37 ° C. for 40 minutes to allow cells to adhere, then fix cells with paraformaldehyde, wash twice, perform cell permeabilization, wash, and then react with antibody (Patent Document 1) Paragraph number 0075).
- Patent Document 1 Paragraph number 0075.
- such a procedure is complicated and includes a plurality of times of centrifugation, washing, etc., so that rare cells in the specimen may be damaged or flow out.
- Patent Document 2 lysis of cells (including erythrocytes) of a prescribed type in a sample (including in a blood sample), staining of intracellular nucleic acid, and fixation of nucleated cells are performed in a single stage. It describes that the cost and time of analysis are reduced (claims of Patent Document 2).
- red blood cells that are cells other than the detection target cells are lysed by the cell lysing agent, and staining is performed by staining the entire nucleic acid in the cells with a nucleic acid staining dye. It does not specifically stain the specific substance that is expressed.
- the present invention prevents damage and loss (outflow) of cells in a collected specimen when specifically staining a specific substance expressed in a detection target cell in the liquid specimen, and It is an object of the present invention to provide a simple cell staining method with reduced time in the cell staining procedure. Furthermore, this invention makes it a subject to provide the sample collection tube suitable for using for said cell staining method.
- the present inventors examined the procedure for staining cells in a liquid specimen. After cell immobilization treatment, the cells were not permeabilized and the subsequent cell permeabilization treatment or identification of the cells was performed. The present inventors have found that there is no problem in the labeling treatment of the substance, and found that the cell immobilization treatment step and the cell permeabilization treatment step can be performed in one step. As a result, the number of centrifugation and washing of the cells is reduced, and damage and loss (outflow) of the cells in the specimen can be prevented, and the time in the cell staining procedure can be shortened and simplified. It came to complete. Furthermore, the present inventors have found that the cell staining procedure can be carried out more easily by carrying out the labeling step of a specific substance possessed by cells in one step in addition to the above steps. It came to complete.
- the present invention includes the following matters. [1] A method of specifically staining a specific substance possessed by cells in a specimen, (A) A method of carrying out the step of immobilizing the cells and (B) a step of permeabilizing the cells in one step.
- a sealable sample collection tube having an openable / closable mouth for storing a collected sample, and simultaneously immobilizing and permeabilizing cells contained in the sample and labeling a specific substance possessed by the cells.
- a flexible plate-like body is stored in a state of being sandwiched between inner walls of an intermediate portion of the sample collection tube, (2) Specimen collection tube accommodated in the sample collection tube so that the cell immobilizing agent, the cell permeabilizing agent, and the substance that specifically labels a specific substance possessed by the cell do not contact each other .
- the method of the present invention in the case of specifically staining a specific substance expressed in a detection target cell in a liquid sample, damage and loss of the cell in the sample collected by reducing the number of cell washings (Outflow) can be prevented, and as a result, the accuracy of cytodiagnosis can be improved. Furthermore, according to the method of the present invention, it is possible to provide a simple cell staining method in which time is shortened in the procedure for staining cells in a liquid specimen.
- the cells contained in the sample can be immobilized and permeabilized and the specific substance possessed by the cells can be simultaneously and easily labeled in a short time. Cell damage and loss (outflow) can also be prevented.
- blood anticoagulation treatment can be simultaneously performed on a blood sample.
- FIG. 1 is a cross-sectional view showing an embodiment of the sample collection tube of the present invention.
- FIG. 2 is an image in which cells in blood to which MCF-7 cells (breast cancer cells) were added were fluorescently stained and observed under a microscope in a comparative example (conventional cell staining method).
- FIG. 3 shows cells in blood to which MCF-7 cells (breast cancer cells) were added in Example 1 (cell staining method in which anticoagulation treatment, immobilization treatment, permeabilization treatment and labeling treatment were performed in one step). Is an image obtained by performing fluorescence observation under a microscope.
- FIG. 4 is an image obtained by fluorescently staining cells in blood to which MCF-7 cells (breast cancer cells) were added in Example 2 (an example in which density gradient centrifugation was combined) and performing fluorescence observation under a microscope. is there.
- Cell staining method of the present invention “A method of specifically staining a specific substance possessed by cells in a specimen, (A) A method of carrying out the step of immobilizing the cells and (B) a step of permeabilizing the cells in one step. ] It is.
- the specimen targeted by the present invention is a specimen containing cells collected from a human or an animal.
- a preferred sample targeted by the present invention is a liquid sample.
- Typical examples of such specimens are blood (including serum and plasma) and urine.
- a cell suspension can be prepared according to a predetermined method to obtain a liquid specimen.
- an operation for obtaining a fraction containing cells to be detected from the specimen as necessary (referred to as “acquiring operation of the fraction to be detected”). May be performed.
- the detection target cell fraction is obtained by, for example, performing an anticoagulation treatment, followed by density gradient centrifugation to detect the detection target cell fraction, for example, a fraction not containing red blood cells. Acquisition and the like.
- the detection of the cell fraction to be detected is performed before the cell immobilization treatment, but if necessary, it is performed between the cell immobilization treatment and the cell permeabilization treatment. It is also possible to do.
- the staining method of the present invention is a method of specifically staining a specific substance possessed by a detection target cell, for example, determining whether a detection target cell is contained in a specimen or observing cells contained in the specimen. Can be implemented.
- the specific substance to be dyed may be appropriately selected according to the detection target cell, as is usually done.
- PSA prostate specific antigen
- the specific cytokeratin selected accordingly can be stained specifically.
- a method of specifically staining a specific substance possessed by cells in a specimen is a single step of immobilizing and permeabilizing cells in a collected specimen.
- the specimen is blood
- the blood is first immobilized and permeabilized in one step, and then the specific substance is specifically labeled. Then, it can be washed and observed with a microscope or the like.
- the anticoagulation treatment of blood is performed first, and then the above-described immobilization treatment and permeabilization treatment can be performed in one step.
- Anticoagulation treatment, cell immobilization treatment, and cell permeabilization treatment can also be performed in one step. Furthermore, as one of the preferred embodiments of the present invention, the cell immobilization treatment, the cell permeabilization treatment, and the specific labeling step for the specific substance can be performed in one step (further, The blood anticoagulation treatment can also be performed in one step).
- performing in one step means “performing a series of treatments in that step without performing washing without removing the added fixing agent” or “adding fixing agent and permeation”. This means that a series of treatments in the process is carried out without carrying out washing without removing the agent.
- washing washing to remove the fixing agent
- the process proceeds to the labeling process.
- cleaning method in this case is based on adding the phosphate buffered saline solution and other washing
- the washing for removing the fixing agent as described above is not performed between these treatments. .
- the immobilizing agent as described above between these treatments. Also, no cleaning is performed except for the penetrant.
- the acquisition operation of the cell fraction to be detected may be performed within the above-described one step, for example, between the cell immobilization process and the cell permeabilization process, if necessary. As described above.
- the specific substance labeling treatment and blood anticoagulation treatment can be carried out in the same one step.
- Noise can be reduced.
- the above-described cell immobilization treatment and cell permeabilization treatment are performed by adding a fixing agent and a permeabilizing agent to a specimen, and usually allowing to stand at room temperature for 10 minutes to 1 hour.
- a substance that specifically captures the specific substance and a label for example, a label Conjugated antibody
- an anticoagulant can be added together, and the treatment in this case is usually carried out by leaving at room temperature for 10 minutes to 1 hour.
- Anticoagulant When blood goes out of the body, the liquid turns into a gel and the blood clots. Therefore, when the sample is blood, if it is necessary to prevent blood coagulation depending on the purpose of the test, an anticoagulant is added to the sample after the sample is collected.
- the anticoagulant used in the present invention is not particularly limited, and commonly used ones such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,2-diamino Examples include cyclohexanetetraacetic acid (DCTA), ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), heparin species such as heparin, heparin sulfate, and low molecular weight heparin, citric acid, and oxalic acid.
- the concentration of these blood coagulants may be as usual.
- the cell immobilization treatment is a treatment performed for the purpose of delaying the self-decomposition and decay of cells and maintaining the form and antigenicity.
- the immobilizing agent used in the present invention is not particularly limited, and for example, aldehydes (for example, formaldehyde, glutaraldehyde, glyoxal, etc.) and alcohols (for example, ethanol, methanol, etc.) that are commonly used are used. it can.
- aldehydes for example, formaldehyde, glutaraldehyde, glyoxal, etc.
- alcohols for example, ethanol, methanol, etc.
- it does not act directly as a fixing agent, for example, it undergoes hydrolysis or the like to release a fixing agent such as formaldehyde, for example, a formaldehyde donor (formaldehyde donor or FA). It can also be used as a fixing agent.
- Aldehydes are cross-linking agents that form a covalent bond between an aldehyde group and a specific amino acid, and can stabilize protein structure and gel cell protoplasts to suppress enzyme activity. Alcohols can be precipitated by denaturing proteins.
- the concentration of these immobilizing agents may be as usual, but when the sample is blood and formaldehyde is used as the immobilizing agent, the volume of the sample should be kept to prevent changes in blood properties. It is preferable to perform the immobilization treatment by adding formaldehyde to the specimen so that the concentration is 0.01 to 1.0% by volume.
- the cell permeabilization treatment means, for example, the permeability of a cell membrane in order to bring a specific substance (for example, an intracellular antigen) possessed by a cell into contact with a substance (for example, an antibody) that captures the specific substance. It is a process to raise.
- a specific substance for example, an intracellular antigen
- a substance for example, an antibody
- the penetrant used in the present invention Commonly used surfactants such as Triton X-100, Tween 20, Saponin, Digitoni, Leucoperm, NP-40, etc., are usually used at concentrations of, for example, 0.01 to 0.5 with respect to the sample volume.
- the permeabilization treatment can be performed by adding to the specimen so that the volume%.
- the method for labeling a specific substance possessed by a cell in the present invention is not particularly limited as long as it is a suitable method according to the cell and the specific substance to be labeled, and usually the specific substance is specifically captured. Performed with substance and label.
- a method for capturing a specific substance for example, an antigen antibody method using an antibody that specifically binds to a specific substance possessed by a cell as an antigen, or a specific substance having a sugar chain
- Examples include a method using a lectin that specifically binds to the sugar chain.
- a method of labeling a specific substance using an antibody to which a label is bound for example, a method of labeling a specific substance using an antibody to which a label is bound (primary antibody method), or a specific substance and a primary antibody are first bound, and then a label is bound to the primary antibody.
- a method of binding a secondary antibody (sandwich method) or the like can be used.
- the labeling body is not particularly limited as long as it is suitable for the inspection, and commonly used fluorescent dyes, enzymes / coenzymes, chemiluminescent substances, and the like can be used.
- a fluorescent dye as a label.
- the labeled body not bound to the specific substance is removed by centrifugal washing or the like, and then observed with a microscope or the like.
- Sample collection tube of the present invention Sample collection tube of the present invention, “A sealable specimen collection tube with an openable / closable mouth for storing collected specimens, and simultaneously immobilizing and permeabilizing cells contained in the specimen and labeling specific substances possessed by the cells. And (1) A flexible plate-like body is stored in a state of being sandwiched between inner walls of an intermediate portion of the sample collection tube, (2) Specimen collection tube accommodated in the sample collection tube so that the cell immobilizing agent, the cell permeabilizing agent, and the substance that specifically labels a specific substance possessed by the cell do not contact each other . ] It is.
- a fixing agent, a penetrating agent, and a substance that specifically labels a specific substance are mixed, That is, they are not sealed in a mixed state but are separately stored.
- an anticoagulant is further separated and stored separately from other treatment agents (this book)
- the treatment agent or the like means “an immobilizing agent, a penetrating agent, and a substance that labels a specific substance” or “an anticoagulant, an immobilizing agent, a penetrating agent, and a substance that labels a specific substance”. Means).
- the sample collection tube of the present invention After collecting the liquid sample, open the openable / closable mouth, put the sample into the sample collection tube of the present invention, close the mouth, and then turn the sample collection tube several times to bring the sample and the above-mentioned treatment agent, etc.
- the cells in the specimen When mixed and usually left at room temperature for 10 minutes to 1 hour, the cells in the specimen can be immobilized, permeabilized, and labeled with a specific substance in one step.
- the sample is blood
- blood coagulation is also combined. It can be implemented in the process.
- polyesters such as polyethylene terephthalate and copolymerized polyethylene terephthalate, polyacrylonitrile, polymethylmethacrylate.
- acrylic resins such as acrylate and polymethacrylic acid
- polyolefins such as polypropylene and polyethylene
- polyamides such as polyvinyl chloride and nylon
- thermoplastic resins such as polystyrene
- inorganic materials such as glass can be used.
- the sample collection tube of the present invention is a flexible plate-like body that is sandwiched between the inner walls of the intermediate portion of the sample collection tube so that the flexible plate-like body does not play inside the sample collection tube due to vibration or the like. Is stored. By this flexible plate-like body, the inside of the specimen collection tube is separated into two spaces.
- at least a part of the processing agent or the like is further attached to the flexible plate or the inner wall of the sample collection tube as will be described later.
- Can store individual immobilizing agents such as treatment agents, penetrating agents, and substances for labeling specific substances (preferably further anticoagulants), not in a mixed state, but separately. .
- the flexible plate is obtained by placing a liquid sample into the sample collection tube of the present invention and inverting the sample collection tube several times so that the sample is mixed with the treatment agent. It is preferable that the specific gravity of the flexible plate-like body is larger than the specific gravity of the specimen so as to sink inside.
- the flexible plate may be any plastic film, for example, stretched PET film, nylon film, filler-filled PP film, filler-filled PE film, plastic laminated aluminum foil film, etc. Accordingly, surface treatment such as plasma treatment or embossing may be performed.
- a non-woven fabric processed into a plate-like body may be used, and as the material of the nonwoven fabric, polyester, nylon, rayon and a combination thereof are used. Furthermore, what laminated
- the shape of the flexible plate-like body can be clamped on the inner wall according to the shape of the specimen collection tube. For example, in the case of a cylindrical sample collection tube, it has a circular shape that can be held between the inner walls.
- the flexible plate-like body is used by allowing the treatment agent or the like to be stored in the sample collection tube by adhering to the inner wall or the flexible plate-like body, and then using the flexibility. Can be easily inserted into the specimen collection tube.
- the flexible plate-like body is sandwiched away from the permeabilizing agent. The penetrant and the flexible plate are not in contact with each other.
- the treatment agent or the like is preferably attached to the inner wall of the sample collection tube or the flexible plate-like body so as to be stored separately.
- the treatment agent or the like is attached after mixing a water-soluble polymer exhibiting adhesiveness.
- part as long as the vicinity of a pipe opening is avoided, you may make it adhere to any site
- the whole surface or a part of the inner wall of the sample collection tube or the flexible plate-like body may be used.
- two kinds of materials such as a fixing agent and an anticoagulant are attached to the flexible plate-like body, they are attached to each other.
- the water-soluble polymer include water-soluble substances such as polyvinyl pyrrolidone and polyvinyl alcohol, water-soluble substances such as carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, ethyl hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl ethyl cellulose, and hydroxypropyl methyl cellulose.
- water-soluble acrylic acid derivatives such as 2-hydroxyethyl acrylate and 2-hydroxypropyl acrylate
- mixed substances of various water-soluble proteins such as gelatin and starch.
- a substance that labels a specific substance a substance that specifically captures a specific substance and a labeled body, for example, a labeled antibody
- ELISA enzyme immunoassay
- a commonly used method of adsorbing an antibody to a plastic plate using a hydrophobic interaction can be used. Specifically, for example, a labeled antibody diluted to 2 to 5 ⁇ g / ml with a sodium bicarbonate buffer (0.1 M, pH 9.6) is brought into contact with the coating region (the region of the inner wall to be attached), and is allowed to come to room temperature for 3 hours. (Alternatively overnight at 4 ° C).
- the labeled antibody solution that has not been adsorbed is collected, washed with a phosphate buffered saline (PBS) containing 0.05% Tween, and further washed with PBS.
- PBS phosphate buffered saline
- sodium chloride or the like is further added after the sample is placed in the sample collection tube as necessary.
- the salt concentration may be increased.
- the flexible plate-like body is placed in a place away from the permeabilizing agent as described above, as it is in the sample collection tube. To pinch.
- the tubes were layered and centrifuged at 400 g for 40 minutes at room temperature. After centrifugation, all layers other than the erythrocyte layer were collected, PBS was newly added, centrifugal washing was performed three times, and immobilization was performed using 20% neutral buffered formalin solution (Wako Pure Chemical Industries). The 20% neutral buffered formalin solution was diluted with PBS to 4% formaldehyde and reacted at room temperature for 10 minutes.
- the plate was centrifuged 3 times with PBS, then permeabilized and blocked with PBS containing 0.1% Tween 20 and 3% BSA, and FITC (fluorescein isothiocyanate) labeled anti-cytokeratin antibody (Becton Dickinson) PE (phycoerythrin) -labeled anti-CD45 antibody (Beckman Coulter) was added and allowed to react for 1 hour.
- FITC fluorescein isothiocyanate labeled anti-cytokeratin antibody
- PE phytoerythrin
- -CD45 antibody Bosed anti-CD45 antibody
- Example 1 Cell staining method in which anticoagulation treatment, immobilization treatment, permeabilization treatment and labeling treatment are performed in one step
- a Cyto-chex blood collection tube manufactured by Streck
- EDTA anticoagulant
- a cell fixing agent estimated as formaldehyde donors such as diazolidinyl urea and imidazolidinyl urea from the analysis results.
- MCF-7 cells breast cancer cells were added to the blood and used as a model of blood circulating cancer cells.
- Tween 20 was added to a portion of 200 ⁇ L so as to be 0.1%, and then FITC-labeled anti-cytokeratin antibody, PE-labeled anti-CD45 antibody, and Hoechst were added and reacted for 30 minutes. A part of the sample was observed with a hemocytometer under a microscope.
- Example 2 Example in which density gradient centrifugation is combined
- a blood collection tube manufactured by Terumo
- EDTA as an anticoagulant was used.
- MCF-7 cells breast cancer cells
- the 20% neutral buffered formalin solution was diluted with PBS, added to 3 mL of blood so that the final concentration of formaldehyde was 0.08%, and mixed by inverting 10 times.
- This blood was diluted 2-fold with PBS containing 0.5% BSA, 4 mL of which was layered on a tube containing 3 mL of density gradient centrifugation medium (Ficoll, specific gravity 1.077) in advance, and 400 g at room temperature. For 40 minutes. After centrifugation, all layers other than the erythrocyte layer are collected, and Tween 20 is added to a part of the layer, and then FITC-labeled anti-cytokeratin antibody, PE-labeled anti-CD45 antibody, and Hoechst are added. The reaction was carried out for 20 minutes, and a part of the sample was observed with a hemocytometer under a microscope.
- density gradient centrifugation medium Ficoll, specific gravity 1.077
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Abstract
Description
[1]
検体中の細胞が有する特定の物質を特異的に染色する方法であって、
(A)前記細胞の固定化処理をする工程、及び
(B)前記細胞の浸透化処理をする工程を
一つの工程で実施する方法。
採取した検体を保存し、該検体に含まれる細胞の固定化及び浸透化並びに該細胞が有する特定の物質の標識を同時に行うための、開閉可能な口を有する密封可能な検体採取管であって、
(1)可撓性板状体が、該検体採取管の中間部分の内壁に挟持された状態で収納され、
(2)該細胞の固定化剤、該細胞の浸透化剤及び該細胞が有する特定の物質を特異的に標識する物質が、それぞれ接触しないように該検体採取管に収納されている
検体採取管。
本発明の細胞染色方法は、
『検体中の細胞が有する特定の物質を特異的に染色する方法であって、
(A)前記細胞の固定化処理をする工程、及び
(B)前記細胞の浸透化処理をする工程を
一つの工程で実施する方法。』
である。
本発明が対象とする検体は、ヒトや動物から採取された、細胞を含む検体である。本発明の効果の点から、本発明が対象とする好ましい検体は、液状の検体である。このような検体の例として代表的なものは、血液(血清、血漿を含む)及び尿である。また、生体から剥離した細胞を対象とする場合も、所定の方法に従って細胞懸濁液を作製して、液状の検体とすることもできる。
本発明の染色方法は、検出対象細胞が有する特定の物質を特異的に染色する方法であり、例えば、検体に検出対象細胞が含まれているかの判定や検体に含まれている細胞を観察するために実施することができる。この場合、染色する特定の物質は、通常行われているように、検出対象細胞に応じて合目的的に選定すればよい。例えば、前立腺癌細胞を検出するためには前立腺特異抗原(prostate specific antigen、PSA)を特異的に染色することができ、また、癌細胞を対象として観察するためにはその癌細胞での発現に応じて選定した特定のサイトケラチンを特異的に染色することができる。
本発明における、検体中の細胞が有する特定の物質を特異的に染色する方法(以下、単に細胞染色方法という)は、採取した検体中の細胞の固定化処理と浸透化処理を一つの工程で実施する。具体的には、例えば検体が血液である場合、まず該血液の固定化処理と浸透化処理を一つの工程で行い、次いで、前記特定の物質を特異的に標識処理する工程を行い、好ましくは、その後洗浄し、顕微鏡等で観察することができる。この場合、検体採取後、最初に、血液の抗凝固処理を実施した上で、次に、上記の固定化処理と浸透化処理を一つの工程で行うこともでき、また、この場合の血液の抗凝固化処理、細胞の固定化処理及び細胞の浸透化処理を一つの工程で行うこともできる。更に、本発明の好ましい態様の一つとして、前記細胞の固定化処理、前記細胞の浸透化処理及び前記特定の物質を特異的に標識処理する工程を一つの工程で行うこともできる(更に、前記の血液の抗凝固化処理も一つの工程で行うこともできる)。
血液は体外に出すと液体がゲル状に変わり、血液凝固する。従って、検体が血液の場合は、検査の目的によって血液凝固を防ぐ必要がある場合は、検体採取後、抗凝固剤を検体に添加する。検体が血液である場合に本発明で使用する抗凝固剤は、特に制限はなく、通常用いられているもの、例えば、エチレンジアミン四酢酸(EDTA)、ジエチレントリアミン五酢酸(DTPA)、1,2-ジアミノシクロヘキサン四酢酸(DCTA)、エチレンビス(オキシエチレンニトリロ)四酢酸(EGTA)やヘパリン、ヘパリン硫酸、低分子ヘパリン等のヘパリン種、クエン酸、シュウ酸等が挙げられる。これらの血液凝固剤の添加濃度も通常行われている通りでよい。
細胞の固定化処理とは、細胞の自己分解や腐敗を遅延させるとともに、形態及び抗原性を保持する目的で行われる処理である。本発明で用いる固定化剤は、特に制限はなく、通常用いられている、例えば、アルデヒド類(例えば、ホルムアルデヒド、グルタルアルデヒド、グリオキサール等)やアルコール類(例えば、エタノール、メタノール等)を用いることができる。また、それ自体が固定化剤として直接作用するものではないが、それ自体が加水分解等を受けることによって、例えばホルムアルデヒド等の固定化剤を遊離するような、例えばホルムアルデヒド供与体(ホルムアルデヒドドナー又はFAドナーともいう。)等も固定化剤として使用可能である。
細胞の浸透化処理とは、例えば、細胞が有する特定の物質(例えば、細胞内の抗原)とこの特定の物質を捕捉する物質(例えば、抗体)とを接触させるために、細胞膜の透過性を上げる処理である。本発明で使用する浸透化剤は、特に制限はない。通常用いられているTritonX-100、Tween20、Saponin、Digitoni、Leucoperm、NP-40等の界面活性剤を、通常行われている濃度で、例えば、検体の容量に対して0.01~0.5容量%となるように、検体に添加して、浸透化処理を行うことができる。
本発明における細胞が有する特定の物質の標識処理の方法は、細胞及び標識する特定の物質に応じた合目的的な方法であれば特に制限はなく、通常、特定の物質を特異的に捕捉する物質と標識体を用いて行う。この場合の特定物質の捕捉方法としては、例えば、細胞が有する特定の物質を抗原として、これと特異的に結合する抗体を用いる抗原抗体法や、特定の物質が糖鎖を有する物質の場合、その糖鎖と特異的に結合するレクチンを用いる方法等が挙げられる。
上記の標識処理を行った後は、好ましくは、遠心洗浄等によって、上記の特定の物質と結合していない標識体を除いた後、顕微鏡等で観察する。
本発明の検体採取管は、
『採取した検体を保存し、該検体に含まれる細胞の固定化及び浸透化並びに該細胞が有する特定の物質の標識を同時に行うための、開閉可能な口を有する密封可能な検体採取管であって、
(1)可撓性板状体が、該検体採取管の中間部分の内壁に挟持された状態で収納され、
(2)該細胞の固定化剤、該細胞の浸透化剤及び該細胞が有する特定の物質を特異的に標識する物質が、それぞれ接触しないように該検体採取管に収納されている
検体採取管。』
である。
本発明の検体採取管の材質としては、採取する検体に応じて、通常使用されているものを使用することができ、例えば、ポリエチレンテレフタレート、共重合ポリエチレンテレフタレート等のポリエステル、ポリアクリロニトリル、ポリメチルメタアクリレート、ポリメタアクリル酸等のアクリル樹脂、ポリプロピレン、ポリエチレン等のポリオレフィン、ポリ塩化ビニル、ナイロン等のポリアミド、ポリスチレン等の熱可塑性樹脂のほか、ガラス等の無機材質が使用可能である。
本発明の検体採取管は、振動等によって可撓性板状体が検体採取管内部で遊ぶことがないように、検体採取管の中間部分の内壁に挟持された状態で可撓性板状体が収納されている。この可撓性板状体によって、検体採取管の内部が2つの空間に分離されている。そして、本発明では、このような構造を有する検体採取管に、更に、処理剤等の少なくとも一部を後述するように可撓性板状体又は検体採取管の内壁に付着させて収納することによって、処理剤等の個々の固定化剤、浸透化剤、及び特定の物質を標識する物質を(好ましくは、更に抗凝固剤を)、混合状態ではなく、それぞれ分離して収納することができる。
処理剤等は、それぞれ分離して収納するために、検体採取管の内壁又は可撓性板状体へ付着されていることが好ましい。処理剤等を内壁等へ付着させるには、接着性を示す水溶性高分子を混在させた後に付着させることが好ましい。また、付着部位としては、管口近傍を避けていれば、どの部位にどのように付着させてもかまわない。検体採取管の内壁又は可撓性板状体の全面でも一部でもかまわない。可撓性板状体に固定化剤と抗凝固剤等のように2種類を付着させる場合は、それぞれが離れた所に付着させる。付着方法としてはスプレーコート、ディッピング等の方法により塗布し、後に風乾、熱乾燥、減圧乾燥等の方法により乾燥させる。前記水溶性高分子としては、例えば、ポリビニルピロリドン、ポリビニルアルコール等の水溶性物質、カルボキシメチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース、エチルヒドロキシエチルセルロース、エチルセルロース、メチルセルロース、カルボキシメチルエチルセルロース、ヒドロキシプロピルメチルセルロース等の水溶性セルロース誘導体、2-ヒドロキシエチルアクリレート、2-ヒドロキシプロピルアクリレート等の水溶性アクリル酸誘導体、ゼラチン、でんぷん等の水溶性多種タンパク質の混合物質等が挙げられる。
[比較例1]
(従来法による細胞染色法)
抗凝固剤としてEDTA(エチレンジアミン四酢酸)を含む採血管を用いた。採取した血液にMCF-7細胞(乳癌細胞、ATCC HTB-22)を添加し、血液循環がん細胞のモデルとした。その血液を0.5%BSA(ウシ血清アルブミン)を含むPBS(リン酸緩衝食塩水)溶液で2倍希釈し、そのうち4mLをあらかじめ密度勾配遠心用分離媒体(Ficoll、比重1.077)3mLを入れたチューブに重層し、室温にて400gで40分遠心分離を行った。遠心後、赤血球層以外の全層を採取し、新たにPBSを加えて遠心洗浄を3回行い、20%中性緩衝ホルマリン液(和光純薬)を用いて固定化を行った。20%中性緩衝ホルマリン液は4%ホルムアルデヒドとなるようにPBSにて希釈を行い、10分間室温で反応させた。反応後、PBSで3回遠心洗浄を行った後、0.1%Tween20と3%BSAを含むPBSで浸透化処理とブロッキングをし、FITC(フルオレセインイソチオシアネート)標識抗サイトケラチン抗体(ベクトンディッキンソン)、PE(フィコエリスリン)標識抗CD45抗体(ベックマンコールター)を加えて1時間反応させた。0.1%Tween20を含むPBSで3回遠心洗浄後、Hoechst(ヘキスト33342(同仁化学研究所))による核染色を5分行い、再び0.1%Tween20を含むPBSで遠心洗浄を行った。この細胞懸濁液の一部を血球計算盤にて顕微鏡下で蛍光観察を行った。
(抗凝固化処理、固定化処理、浸透化処理及び標識化処理を一工程で行う細胞染色法)
抗凝固剤(EDTA)と細胞固定化剤(分析結果から、ジアゾリジニル尿素やイミダゾリジニル尿素等のホルムアルデヒド供与体と推定される)を含む採血管としてCyto-chex採血管(Streck製)を用い、採取した血液にMCF-7細胞(乳癌細胞)を添加し、血液循環がん細胞のモデルとした。その一部200μLに対し0.1%となるようにTween20を加え、続いて、FITC標識抗サイトケラチン抗体、PE標識抗CD45抗体、及びHoechstを加えて30分間反応させた。その一部を血球計算盤にて顕微鏡下で蛍光観察を行った。
(密度勾配遠心法を組み合わせた実施例)
抗凝固剤としてEDTAを含む採血管(テルモ製)を用いた。採取した血液にMCF-7細胞(乳癌細胞)を添加し、血液循環がん細胞のモデルとし、20%中性緩衝ホルマリン液(和光純薬)を用いて固定化を行った。20%中性緩衝ホルマリン液はPBSにて希釈を行い、血液3mLに対し、ホルムアルデヒドの終濃度が0.08%になるように加え、10回転倒して混和させた。この血液を、0.5%BSAを含むPBSで2倍に希釈し、そのうち4mLをあらかじめ密度勾配遠心用分離媒体(Ficoll、比重1.077)3mLを入れたチューブに重層し、室温にて400gで40分遠心分離を行った。遠心後、赤血球層以外の全層を採取し、その一部に0.1%となるようにTween20を加え、続いて、FITC標識抗サイトケラチン抗体、PE標識抗CD45抗体、及びHoechstを加えて20分間反応させ、一部を血球計算盤にて顕微鏡下で蛍光観察を行った。
2 開閉可能な口
3 可撓性板状体
4 固定化剤(可撓性板状体に付着)
5 浸透化剤(液体)
6 特定の物質を標識する物質(内壁に付着)
7 抗凝固剤(可撓性板状体に付着)
Claims (11)
- 検体中の細胞が有する特定の物質を特異的に染色する方法であって、
(A)前記細胞の固定化処理をする工程、及び
(B)前記細胞の浸透化処理をする工程を
一つの工程で実施する方法。 - 前記工程(A)及び前記工程(B)に加えて、更に、
(C)前記細胞が有する前記特定の物質の標識処理をする工程を
一つの工程で実施する請求項1に記載の方法。 - 前記検体が血液である請求項1又は2に記載の方法。
- 前記工程(A)において、ホルムアルデヒドを固定化剤として用い、その濃度が前記検体の容量に対して0.01~1.0容量%で前記細胞を固定化処理する請求項1~3のいずれか一項に記載の方法。
- 前記工程(B)において、界面活性剤を浸透化剤として用いて前記細胞を浸透化処理する請求項1~4のいずれか一項に記載の方法。
- 前記界面活性剤が、Triton―100、Tween20、Saponin、Digitonin、Leucoperm及びNP-40からなる群から選ばれる少なくとも一つを含み、かつ、その濃度が前記検体の容量に対して0.01~0.5容量%で処理する請求項5に記載の方法。
- 前記工程(C)において、前記特定の物質を特異的に捕捉する物質及び蛍光標識体を用いて前記細胞が有する前記特定の物質の標識処理をする請求項1~6のいずれか一項に記載の方法。
- 前記特定の物質を特異的に捕捉する物質が抗体である請求項7に記載の方法。
- 採取した検体を保存し、該検体に含まれる細胞の固定化及び浸透化並びに該細胞が有する特定の物質の標識を同時に行うための、開閉可能な口を有する密封可能な検体採取管であって、
(1)可撓性板状体が、該検体採取管の中間部分の内壁に挟持された状態で収納され、
(2)該細胞の固定化剤、該細胞の浸透化剤及び該細胞が有する特定の物質を特異的に標識する物質が、それぞれ接触しないように該検体採取管に収納されている
検体採取管。 - 前記固定化剤が前記可撓性板状体上の少なくとも一部に付着され、前記特定の物質を特異的に標識する物質が前記検体採取管の内壁の少なくとも一部に付着され、かつ前記浸透化剤が前記検体採取管内に収納されている請求項9に記載の検体採取管。
- 前記検体が血液であり、更に抗凝固剤が、前記可撓性板状体上の少なくとも一部に、前記固定化剤、前記浸透化剤及び前記特定の物質を特異的に標識する物質と接触しないように付着された請求項9又は10に記載の検体採取管。
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