JPH10332688A - Slide for separating cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To separate a cell without requiring any skill by adhering the cell to a slide and cutting a part where at least one cell is adhered. SOLUTION: A cell is adhered to a slide, for example, by a method for dropping the cell floating liquid that is diluted to a proper concentration on the slide, and using a site spin and a method for applying the floating liquid. For example, the cell that is adhered is observed, for example, by a microscope, a target cell is selected out of a plurality of types of cells and the position is confirmed, that part is cut, for example, by a surgical knife, and a target cell is separated. The slide is preferably made of a transparent material to verify the adhesion position of the cell under the inspection by a microscope and is preferably made of a material that can be easily cut, for example, by the surgical knife, thus examining the presence or absence of contamination by a virus and variation of cancer genes and cancer inhibitors.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a slide for separating cells of interest. More specifically, the present invention relates to a slide used for separating one or a plurality of cells of interest when performing a molecular pathological diagnosis.

[0002]

2. Description of the Related Art In recent years, when performing pathological diagnosis of various diseases,
Emphasis is placed on molecular pathological diagnosis at the gene level as well as on morphological diagnosis of tissues. Molecular pathological diagnosis is a diagnostic method in which a gene is isolated from an extirpated specimen of a diseased tissue and the presence or absence and pattern of the abnormality are examined. At this time, if the amount of the test gene is small, the PCR method (Sugita et al., Clinical Immunity, 2
2 , 283-289, 1990) and the like. However, in general, various cells are mixed in diseased tissue in addition to disease-related cells. Therefore, when analyzing a gene, there is a possibility that a gene derived from a cell other than the target is also analyzed at the same time, which is a factor that hinders accurate diagnosis. Therefore, only cells with abnormal morphological or immunohistological differences were isolated,
Analyzing genes for one or more abnormal cells,
A so-called single-cell analysis has been devised. Traditional single-cell analysts
The micromanipulation method (Ca) in which one cell is lifted using a capillary pipette under microscopic observation
therine, EE, et al., Biochimica et Biophysica A
cta, 1181 , 77-82, 1993) and the cell sorter method (Hans,
PB, et al., J. Immunology, 155 , 190-202, 1995)
There is. However, these methods require special instruments such as a micromanipulator, a glass microneedle making device, and a cell sorter, and their handling requires skill, so it is difficult to generalize them. There is no technique for separating one or more target cells, particularly only morphologically abnormal cells, and the development of a simple separation method is expected.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to use a device existing in a general blood test room to separate cells without skill and to carry out gene analysis. To provide slides.

[0004]

Means for Solving the Problems As a result of intensive studies, the present inventors have dropped a cell suspension on the slide of the present invention, and attached the cells to the slide using cytospin, or blood. After smearing on a slide, selecting the target cells under microscopy, cutting out only the portion to which the cells adhered, found that the target cells could be easily separated, and completed the present invention. . That is, the present invention
A slide for separating one or more cells of interest and a method for separating the slide. More specifically, the present invention relates to a slide for separating cells by attaching a cell and then cutting off a portion to which one or a plurality of cells are attached, and a method for separating target cells using the slide.

[0005] In order to separate cells, it is first necessary to attach the cells to a slide. Examples of a method for attaching the suspension include a method in which a cell suspension diluted to an appropriate concentration is dropped on a slide and cytospin is used, and a method in which the suspension is smeared. After attaching the cells to the slide, observe the attached cells with a microscope, for example, select the target cells from multiple types of cells, confirm the position of attachment, and cut out this part with a scalpel etc. One or more cells can be separated.

[0006] For this purpose, the material of the slide is not particularly limited, but a transparent material is preferable since the position of cell attachment is usually confirmed under a microscope, and the target cell is separated. For this reason, a material that can be easily cut out with scissors or a scalpel is preferable. However, even if it is non-transparent, any material may be used as long as the position of cell attachment can be confirmed by, for example, staining, reflected light, or a label. Furthermore, since the separated cells are often subjected to gene analysis by the PCR method, etc., the material must be able to withstand heat of about 100 ° C. and be capable of maintaining the sinking state even in such a high-temperature reaction solution. Is preferred.
As those satisfying these conditions, polyvinylidene chloride, polyvinyl chloride, polyester, polypropylene and the like can be mentioned, but polyester is preferable. The size of the slide is not particularly limited, but 5 to 50 mm x 30 to
Anything that can be easily operated within a range of about 150 mm may be used. The interval between lines applied to the slide varies depending on the target cells, but is about 0.1 to 5 mm. For example, when separating lymphocytes, the interval is preferably 0.5 to 1 mm. The test cells are not particularly limited, and if they are floating cells such as blood cells and bone marrow cells, they can be subjected to enzymatic treatment with trypsin or the like for solid cancer or organ tissue, and the cells can be separated and subjected to the test. The concentration of cell fluid varies depending on the type of cells,
It is about 1000 / ml, preferably 50-200 / ml.

By using the slide of the present invention, 1) it is possible to examine the presence or absence of viral infection, the mutation of oncogenes and tumor suppressor genes, the reconstitution of T cell receptor genes and immunoglobulin genes, etc. for cells having abnormal morphology. Yes, 2) Even pathological specimens with low content of abnormal cells such as lymph nodes, bone marrow, and peripheral blood, isolate only abnormal cells by microscopic observation,
Genetic diagnosis is possible, and 3) various cell specimens of virus-infected persons are prepared, and the effects of identification of infected cells, morphological observation, immunohistochemical search, etc. can be obtained.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
As an example of a method for separating cells, a method using cytospin and a method using smear will be described. The cytospin method is based on the following procedure. 1) Prepare a 25 mm x 40 mm slide. A line or a grid is formed on the slide at a substantially equal interval with a thin pen or a syringe needle. Further, if addresses are assigned to the grid by numbers, symbols, or the like, it is easy to identify a target portion. 2) Dilute the test cells with phosphate buffer, cell culture, etc. to an appropriate concentration. The slide is set on an instrument for a cytospin device, and a test cell solution is added. 3) Centrifuge under appropriate conditions to attach cells to slides. 4) Remove the slide from the cytospin device and stain appropriately. 5) Observe with a microscope and find the target cells. 6) Select a grid containing one cell and cut it out with scissors, scalpel or the like. 7) Transfer the cut lattice pieces to the microtube. 8) Extract nucleic acid by treating with proteinase K. 9) Amplify and analyze the gene by PCR or the like.

When a blood sample is used, it can be performed by the following smear method. 1) Prepare the same slide as above. 2) Drop the blood on the slide and prepare a blood smear according to the usual method. At this time, an anticoagulant such as EDTA may be used. 3) May
Perform Giemsa staining. 4) Observe with a microscope and find the target cells. 5) Select a grid containing one cell and cut it out with scissors, a scalpel or the like. 6) Transfer the cut lattice pieces to the microtube. 7) Extract nucleic acid by treating with proteinase K. 8) Amplify and analyze the gene by PCR or the like.

[0010]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Example 1 HTLV-I and HTLV-II were superinfected
Virus in guinea pig T cell line (GP-1)
Diagnosis of infection condition SureSeal (manufactured by HYBAID) was performed using a 23G
Marked grids at mm intervals were prepared. GP-1
Cells were suspended in cell culture to a concentration of about 100 / ml. 0.5 ml of suspension, SureSeal with other necessary instruments, cytospin device (Sakura Seiki Co., Ltd., auto smear
(CF-12D), and centrifuged at 1200 rpm for 10 minutes to attach the cells to SureSeal. Observe SureSeal with a microscope,
Five grids containing one cell were selected and cut out with a scalpel. The cells are placed in a microtube with the SureSeal lattice attached, treated with proteinase K according to a conventional method, and DN
A was extracted. SK-54, SK-55 which are primers specific to the pol gene of HTLV-I, and SK-58 and SK-59 which are primers specific to the pol gene of HTLV-II,
The pol gene of each provirus was amplified by PCR. After electrophoresis, the amplified product was stained with ethidium bromide to detect the pol gene. Table 1 shows the results of detection of the pol gene. Cell A is HTLV-II and cell D is HTLV-II.
It was confirmed that cells B, C and E were infected with VI.

[0011]

[Table 1]

Example 2 Adult T-cell leukemia (ATL)
-1 in abnormal lymphocytes in peripheral blood of the elderly
Detection of Viral DNA Peripheral blood was collected from an ATL patient and smeared on a polyester slide (20 × 40 mm) on which a grid was formed at intervals of about 1 mm. The specimen was observed with a microscope, and five lattices containing one lymphocyte with a leaf-shaped nucleus were selected and cut out with ophthalmic scissors. Cells attached to each slide lattice piece were treated with proteinase K. HTLV-I
Were prepared (primers having HTLV-I nucleotide sequence numbers of 7341 to 7360 and primers of 7460 to 7383), and the pX gene was amplified by PCR. After electrophoresis, the amplified product was stained with ethidium bromide to detect the pX gene. Table 2 shows the results of pX gene detection.
It was shown to. HTLV from 2 out of 5 abnormal lymphocytes
The -I pX gene was detected.

[0013]

[Table 2]

[0014]

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing an example of a slide for site spin. Symbols and numbers for grid identification are attached. The black dots indicate the cells, and the central bold square frame indicates the position where the cell suspension is dropped.

Claims (9)

[Claims] 1. A slide for separating one or more cells of interest. 2. A slide for separating cells by adhering cells and cutting off a portion to which one or more cells have adhered. 3. The slide according to claim 1, wherein a plurality of lines are provided. 4. The slide according to claim 1, wherein grid lines are provided. 5. The slide according to claim 1, wherein the material is polyester. 6. A method for separating one or more cells using a slide. 7. A method for separating cells by attaching cells to a slide and then cutting off a portion to which one or more cells are attached. 8. A method for separating cells by attaching cells to a slide using cytospin and then cutting off a portion to which one or more cells are attached. 9. A method in which cells are attached to a slide by smearing a cell suspension, and the cells are separated by cutting off a portion where one or more cells are attached.