JP2006242588A - Manufacturing method of cell specimen for fish - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a manufacturing method of a cell specimen for FISH (fluorescence in situ hybridization) capable of examining FISH simply and sufficiently without requiring the skill of an examiner. <P>SOLUTION: The manufacturing method of the cell specimen for FISH includes a process (1) for treating a cell to be examined with paraformaldehyde, a process for hypotonic treatment and a process for dehydration and fixation. The examination of FISH can be performed simply and efficiently without using the skill of the examiner. The specimen manufactured by this manufacturing method is characterized in that segmented neurophil is not destructed, the cell is uniformly fixed without being overlapped, has a sufficient expanse and is suitable for the examination of FISH. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for preparing a cell specimen for FISH.

  FISH (fluorescence in situ hybridization) is a method for directly locating and distributing target genes by hybridizing a fluorescently labeled nucleic acid probe with a sample to which chromosomal DNA is exposed, while maintaining the morphology of cells and chromosomes. Is observed under a fluorescence microscope. Neutrophil FISH test that performs FISH on neutrophils is widely used for diagnosis of chronic myelogenous leukemia, monitoring of molecular target therapy such as IFN-α therapy and Gleevec (GLIVEC (STI571)) (Non-Patent Documents 1 to 3).

  The conventional preparation method for peripheral blood neutrophil FISH is prepared by diluting EDTA-collected venous blood with phosphate buffered saline (PBS), removing the erythrocyte layer with dextran sulfate, and separating the supernatant by Ficoll specific gravity centrifugation. After the neutrophil layer is washed with PBS, a specimen is prepared using cytospin (Non-patent Documents 3 and 4).

Takahashi N, et al: Lineage Involvement of Stem Cells Bearing the Philadelphia Chromosome in Chronic Myeloid Leukemia in the Chronic Phase as Shown by a Combination of Fluorescence-Activated Cell Sorting and Fluorescence in Situ Hybridizatin. Blood 92: 4758-4763-1998 Miura I, et al: Molecular Cytogenetics of Stem Cell: Target Cells of Chromosome Aberrations as Revealed by the Application of Fluorescence In Situ Hybridization to Fluorescence-Activated Cell Sortion. Int J Haematol. 72: 310-317,2000 Naoto Takahashi, Takehisa Miura: Clinical application of CML molecular markers Monitoring of therapeutic effects of INF-α therapy using FISH method. Clinical blood 41: 272-274, 2000 Inspection and Technology 28: 1343, 2000

  The conventional neutrophil FISH test is cumbersome to operate, and the segmental nucleus that is characteristic of neutrophils is likely to be destroyed, so that the skill of the inspector is required to identify neutrophils. is there.

  An object of the present invention is to provide a method for preparing a cell specimen for FISH, which enables FISH examination simply and efficiently without requiring the skill of an examiner.

  As a result of earnest research, the inventor of the present application treated the test cells with paraformaldehyde, and then performed hypotonic treatment and dehydration fixation treatment, so that the cells of the neutrophils were uniformly divided without breaking them. It was found that the cells can be fixed without overlapping, and the cells have a sufficient spread, and a good specimen for FISH examination can be prepared, and the present invention has been completed.

  That is, the present invention provides a cell sample for FISH, comprising: (1) a step of treating a test cell with paraformaldehyde; next, (2) a hypotonic treatment step; and (3) a step of dehydration fixation. A manufacturing method of is provided.

  According to the present invention, a method for preparing a cell specimen for FISH is provided, which enables FISH inspection simply and efficiently without requiring the skill of an examiner. The specimen prepared by the method of the present invention does not destroy the neutrophil lobule nucleus, is uniformly fixed without overlapping cells, and has a sufficiently wide cell, which is good for FISH examination. It is a specimen.

  The test cells used in the method of the present invention are not particularly limited as long as they are test cells for FISH examination, but neutrophils and lymphocytes are preferable. In particular, since the method of the present invention has a remarkable effect of not destroying the nuclei of neutrophils, neutrophils are particularly preferable as the test cells.

  In the method of the present invention, first, (1) a test cell is treated with paraformaldehyde. As paraformaldehyde, paraformaldehyde commonly used for fixation of tissue specimens can be used. Paraformaldehyde is preferably in the form of a dilute aqueous solution, preferably an aqueous solution having a concentration of 0.2% to 1% (v / v), particularly preferably about 0.5% (v / v). The aqueous solution preferably contains a buffer in addition to paraformaldehyde. The temperature of the paraformaldehyde treatment is not particularly limited, and it is convenient and preferable to carry out at room temperature. The treatment time is not particularly limited, but is preferably about 5 to 20 minutes, particularly about 10 minutes. The amount of the paraformaldehyde aqueous solution used is not particularly limited, but it is suitably about 500 to 20000 times the cell volume.

  The (1) paraformaldehyde treatment may be performed on the collected test cells by separating the test cells from whole blood or the like, or may be performed on a sample containing the test cells and cells other than the test cells. Alternatively, paraformaldehyde treatment may be performed, and test cells may be collected in a later step. In the following examples, (1) formaldehyde treatment is performed on the whole blood from which red blood cells have been removed, and then neutrophils and lymphocytes are collected.

  When paraformaldehyde treatment is performed on a sample containing a test cell and cells other than the test cell, it is preferable to sort the test cell prior to the next (2) hypotonic treatment. Since the accuracy of sorting using a cell sorter tends to decrease if the sorting of the test cells is after (2) hypotonic treatment, the sorting of the test cells is prior to (2) hypotonic treatment. It is preferable to carry out. The cell sorting method itself is well known in this field, and a cell sorter, particularly a method using FACS (fluorescence activated cell sorter) is preferable. The FACS method is well known in this field, and FACS devices are also commercially available, so FACS can be easily performed using commercially available devices. When performing FACS, it is necessary to label the test cell with a fluorescently labeled antibody against a specific antigen expressed by the test cell. For example, when neutrophils are collected by FACS, fluorescently labeled anti-CD16b antibody or the like is used. When lymphocytes are sorted by FACS, anti-CD19 antibody or anti-CD3 antibody is used. It is done. Since these fluorescently labeled antibodies are also commercially available, commercially available products can be used. The test cell and the fluorescently labeled antibody may be reacted at any stage before the sorting step, or may be reacted before the step (1). In the following examples, a fluorescently labeled antibody is allowed to act at the stage of whole blood before step (1).

  Next, (2) a hypotonic treatment process is performed. Hypotonic treatment is treatment of cells with a hypotonic solution, ie, a solution having a lower osmotic pressure than physiological saline. The hypotonic solution is not particularly limited, but an aqueous solution of a salt such as potassium chloride or sodium citrate can be preferably used. The salt concentration is not particularly limited, but is preferably about 0.05M to 0.1M, and particularly preferably about 0.075M. The temperature of the hypotonic treatment is not particularly limited, and it is convenient and preferable to carry out at room temperature. The treatment time is not particularly limited, but is preferably about 5 to 20 minutes, particularly about 10 minutes. The amount of the hypotonic solution is not particularly limited, but is usually about 50,000 to 20000 times the volume of the cell. By performing the hypotonic treatment, the test cells can be spread widely, and false positives caused by overlapping fluorescent signals can be prevented.

  Next, the test cells are placed on the substrate, and (3) dehydration fixation treatment is performed. Here, as the substrate, a glass plate such as a slide glass can be used as in the conventional method. Although it is possible to place one sample on one glass slide and perform (3) dehydration fixation treatment, in a preferred embodiment of the method of the present invention, the test cells are provided on one plate, It is preferable to place in a plurality of wells arranged and perform step (3) in each well. Test cells derived from one or a plurality of samples can be placed in the plurality of wells, but it is preferable to insert test cells derived from a plurality of samples in order to improve examination efficiency and reduce costs. In other words, when a plurality of samples are placed in a plurality of wells provided on one substrate, the FISH hybridization step can be performed simultaneously on a plurality of samples accommodated in the plurality of wells. And the cost per sample is reduced. In addition, semi-automated testing can be facilitated by placing test cells in the aligned wells. That is, an image analysis system that automatically captures the coordinates and images of cells within a range arbitrarily set under a fluorescence microscope is commercially available. If it is possible to align multiple samples in a narrow area without having to transfer specimens for each sample, the inspector instantly calls the stored coordinates and images and counts the signals on the cells to calculate the positive ratio can do. In addition, the necessary image can be reported as it is to the client of the inspection.

  The size of the well is not particularly limited, but a diameter of about 1 mm to 3 mm is suitable, and a diameter of about 2 mm is particularly preferable. The height is suitably about 0.1 mm to 1.0 mm, particularly about 0.1 mm. The distance between the wells is not particularly limited, but is preferably about 1 mm to 2 mm. Such a well can also be formed by cutting a slide glass. However, by placing a flexible pad having a plurality of aligned through holes on a flat substrate, it is easier and less expensive. Wells can be formed. That is, the planar substrate located under the through hole is the bottom surface of the well, and the side wall of the through hole is the side wall of the well. The flexible pad is preferably a silicone rubber pad, but is not limited thereto. If the flexible pad is strongly pressed against the substrate with a finger, the liquid does not leak from the well even if the liquid containing the test cells is put into the well. A preferred example of a substrate in which a plurality of wells are aligned is shown in FIG. In FIG. 1, 10 is a slide glass, 12 is a silicone rubber pad, and 14 (16 circles in total) are through holes (wells) having a diameter of 2 mm opened in the silicone rubber pad. Of course, the number and arrangement of wells are not limited to the example of FIG. The number of test cells accommodated in each well is not particularly limited, but is preferably about 2000 to 3000 cells.

  When performing a FISH test to diagnose chronic myelogenous leukemia, perform FISH on each of neutrophils and lymphocytes to determine the percentage of cells with positive Philadelphia (Ph) chromosomes that are characteristic of chronic myelogenous leukemia. taking measurement. In this case, it is preferable to place neutrophils in a part of the plurality of wells described above, and to put lymphocytes in the other part of the wells for observation at the same time because the accuracy of the examination is improved. For example, neutrophils derived from 8 specimens are placed in the upper 8 wells of a plurality of wells shown in FIG. 1, and the 8 wells obtained from the lower stage are collected from 8 specimens from which these neutrophils are derived. Each can contain lymphocytes. Chronic leukemia is clinically acute. It will have the same clinical symptoms as so-called acute lymphoblastic leukemia. By aligning lymphocytes, it can be roughly classified whether it has caused an acute transformation to myeloid (Myeloid system) or an acute transformation to lymphoid. In this method, when T and B cells are sorted and aligned as lymphocytes, it is possible to obtain more detailed information as to whether T lymphocytes or B lymphocytes are acutely changed.

  The use of wells is not always necessary, and a plurality of sample test cells can be spotted on a single substrate using an auto spotter. For example, (2) after the low-tone treatment, centrifuge to remove the supernatant to 3 μl, and develop 0.7 μl on a slide glass. This makes it possible to produce a spot with a diameter of just 2 mm.

  (3) The dehydration fixation process itself can be performed in the same manner as conventional tissue specimen preparation. That is, it can be performed by treating with an organic solvent mixed with water such as methanol or ethanol at an arbitrary ratio. However, formalin is preferably not used because it may destroy chromosomal DNA. In order to destroy the protein in the cell membrane and make it easier for the probe to enter the cell in the subsequent FISH, it is preferable to add acetic acid to the organic solvent, and a Carnoy's solution (methanol: Acetic acid = 3: 1 (v / v) mixed solution) can be particularly preferably used. The temperature of the dehydration and fixing treatment is not particularly limited, and it is convenient and preferable to carry out at room temperature. The treatment time is not particularly limited, but is preferably about 5 to 20 minutes, particularly about 10 minutes. Although the method of a process is not specifically limited, For example, it can carry out by immersing the whole board | substrate in an organic solvent.

  The FISH cell specimen prepared as described above can be subjected to a well-known FISH method in the same manner as a conventional FISH cell specimen.

The method of the present invention or the preferred embodiment described above has the following advantages.
1) Inspection accuracy is improved.
Objective data that does not depend on the skill of the inspector can be obtained. In addition, by preparing a sample using lymphocytes as test cells, it is possible to accurately determine the transition to lymphoid blast crisis.
2) High value added reporting is possible.
By attaching and reporting both the scattergram and the FISH image to the examination client, the whole image of the peripheral blood sample can be grasped and more objective data interpretation becomes possible.
3) The number of analysis steps can be reduced.
It is not necessary to find and analyze the segmental nucleus cells from the blood cells, and the Ph chromosome positive ratio can be easily calculated.
4) FISH can be automated.
Since neutrophil cells and lymphocyte cells sorted by CD16b are spot-aligned within a circle with a diameter of 2 mm, FISH image analysis can be semi-automated easily. Moreover, this method is applicable not only to neutrophil SORTER FISH but also to all SORTER FISH.
5) Inspection costs can be reduced.
In the case of the sorter FISH [CD16b (neutrophil) FISH], the use of the well shown in FIG. 1 makes it possible to hybridize 8 specimens together, so that the cost of the probe used can be reduced to 1/8.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

I. Materials and Methods Scattergram analysis of neutrophil fraction 1) Scattergram analysis of changes in neutrophil fraction after blood collection was performed using blood collected from anticoagulant parin.
2) After collecting heparin, the scattergrams after 48 hours and 72 hours after being left at room temperature were compared.

2. Selection of timing and fixing method for low tone processing 1) We examined the low tone processing by 0.075MKCL before and after fixing.
2) Regarding fixation methods, Carnoy, 0.5% paraformaldehyde / PBS, and formalin fixation were compared.

3. Protocol (Example of the method of the present invention)
1) Add 5 μl of anti-CD16b antibody to a Spitz tube.
2) Add 100 μl of whole blood to a test tube and incubate for 15 minutes at room temperature. (In a dark room)
3) Add 10ml of ammonium chloride hemolytic agent and incubate for 10 minutes. (At room temperature in a dark room)
4) Using a swing rotor centrifuge, centrifuge at 1500 rpm to remove the supernatant. At this time, the red blood cell residue is completely sucked by tilting the Spitz tube.
5) Add 5 ml of PBS to wash the cells.
6) Centrifuge at 1500rpm to remove the supernatant. Also at this time, the red blood cell residue is completely sucked by tilting the Spitz tube.
7) Add 500 μl of 0.5% paraformaldehyde (paraformaldehyde for tissue fixation manufactured by Wako Pure Chemical Industries, Ltd.) / PBS fixing solution, and stir for 10 minutes at room temperature.
8) Transfer cells to FACS test tube.
9) Sort each cell fraction (neutrophils, lymphocytes, etc.) by FACS. Approximately 10,000 cells (100-200 μl).
10) Add an equal amount of 0.075MKCL to the collected cells and perform a low-temperature treatment for 10 minutes at room temperature.
11) Centrifuge to remove the supernatant to about 4 μl.
12) Matsunami white green frost (with MSA coat) Place a silicone rubber pad on the slide glass, and remove the air between the rubber pad and the slide glass to bring them into close contact.
13) Drop 1 μl of white green polished frost (with MSA coat) glass slide with a silicon pad and air dry.
14) CD16b neutrophils and lymphocytes are aligned and spot-fixed on a glass slide. At this time, adjust so that there are about 2000 cells per spot.
15) The air-dried aligned specimen is immersed in a Carnoy (methanol: acetic acid = 3: 1) solution and fixed again for 10 minutes.
16) Air dry and use for FISH method.

II. Results
1. Scattergram analysis of neutrophil fraction
1) Scattergram analysis of changes in neutrophil fraction after blood collection was performed using blood collected from anticoagulant parin. As a result, the fraction of neutrophils and lymphocytes in mononuclear cells 48 hours after blood collection was 30% for neutrophils and 19.8% for lymphocytes. The fraction at 72 hours after blood collection was 4.6% for neutrophils and 27.1% for lymphocytes. From this, it was found that neutrophils in peripheral blood after blood collection are rapidly destroyed. Therefore, it was suggested that neutrophil FISH needs to be prepared within at least 48 hours after blood collection.
2) Therefore, a SORTER FISH specimen preparation method was developed using peripheral blood collected from heparin within 48 hours after blood collection. In the case of peripheral blood collected with heparin within 48 hours, it was possible to sort FACS-sorted neutrophils and lymphocytes of more than 10,000 cells sufficiently necessary from a blood volume of 100 μl in a short time.

2. Selecting the timing of the low tone processing and fixing method 1) If the low tone processing is performed before the FACS sort, accurate Scattergram analysis cannot be performed. 0.075M KCL equivalent to the amount of the sorted cells was added, and the mixture was subjected to low-pressure treatment for 10 minutes at room temperature.
2) Fixation with Carnoy's solution, similar to chromosome preparation, proved unsuitable for preparation of aligned specimens due to the risk of contamination due to cell diffusion. In addition, fixation with formalin was not adopted because it leads to the destruction of intracellular nucleic acids. First, before the FACS sort, the aligned specimens, which were fixed with 0.5% paraformaldehyde / PBS fixative solution at room temperature for 10 minutes and then air-dried, were soaked in Carnoy (methanol: acetic acid = 3: 1) solution and fixed again for 10 minutes. As a result, a good specimen could be produced. That is, the cells could be fixed without overlapping the cells uniformly without breaking the lobular nucleus. The cells also had a sufficient spread.

3. Establishment of specimen preparation protocol Based on the timing of the above-mentioned undertone treatment and the selection of the fixing method, the protocol described in I. Material and Methods was established. Moreover, in order to produce the aligned specimen, the silicone rubber pad shown in FIG. 1 was used. By using this silicone rubber pad, it became possible to prepare a specimen while preventing cell contamination.

It is a figure which shows the state formed by mounting the silicone rubber pad on the slide glass used in the preferable aspect of this invention to accommodate the several well which accommodates a test cell.

Explanation of symbols

10 Slide glass 12 Silicone rubber pad 14 Silicon rubber pad through-hole (well)

Claims (10)

  A method for preparing a cell specimen for FISH, comprising: (1) a step of treating a test cell with paraformaldehyde; next, (2) a hypotonic treatment step; and (3) a step of dehydrating and fixing.   The method according to claim 1, wherein the step (1) is performed by treating a test cell with a 0.2% to 1% aqueous solution of paraformaldehyde.   The method according to claim 1 or 2, wherein the step (3) is performed by treating the test cells after the hypotonic treatment with methanol or a mixed solution of ethanol and acetic acid.   The method according to any one of claims 1 to 3, wherein the test cells are neutrophils and / or lymphocytes.   The method according to claim 4, wherein the test cell is a neutrophil.   6. The method according to claim 1, further comprising a step of performing the step (1) on a sample containing the test cells and cells other than the test cells, and sorting the test cells before the step (2). The method according to any one of the above.   After the step (2), the test cells derived from one or a plurality of samples are placed in a plurality of wells provided on one plate and arranged, and the step (3) is performed in each well. Item 7. The method according to any one of Items 1 to 6.   The method according to claim 7, wherein neutrophils as test cells are placed in a part of the plurality of wells arranged, and lymphocytes as test cells are placed in another well.   9. The method according to claim 7, wherein the well is formed by placing a flexible pad having a plurality of aligned through holes on a flat substrate. The method of claim 9, wherein the planar substrate is a glass plate and the flexible pad is made of silicone rubber.

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