JP2007202472A - Cell fixation solution, method and kit for cell fixation using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell fixation solution for the preparation of specimen having little peeling and deformation trouble of the cell and provide a method for the cell fixation using the cell fixation solution. <P>SOLUTION: The invention provides a cell fixation solution containing ethanol, isopropanol and PEG, a cell fixation kit containing the solution as a constitution component and a cell fixation method comprising the treatment of a cell with the cell fixation solution. An excellent specimen absolutely free from the problems of conventional specimen preparation method can be prepared by using the cell fixed by the cell fixation solution. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell fixing solution used in the field of, for example, clinical examinations, a cell fixing method and a kit using the cell fixing solution.

  In clinical laboratory studies and the like, observation of cells using a microscope is widely performed to observe the morphology and structure of cells, or to examine the number and types of cells.

  Especially in the field of clinical testing, there are few restrictions on sample collection, it can be applied to a wide range of subjects, and the burden on the patient is light, and sample preparation is simple and quick. In a wide range of examinations and the like, examinations using a microscope (microscopy) are performed for differentiating various diseases, determining the degree of diseases, determining therapeutic effects, and the like.

  In diseases such as kidney and urinary tract infections, inflammatory lesions, degenerative lesions, stone disease, tumors, etc., depending on the respective disease, urine, for example, red blood cells, white blood cells, epithelial cells, cylinders, bacteria, fungi, Various formed components such as crystals and mucus yarn appear. In particular, analysis of urinary components is particularly important for early detection of renal / urinary tract diseases and estimation of abnormal sites.

  For example, analysis of urinary red blood cells is important in determining the presence or absence of bleeding in the pathway from the glomerulus of the kidney to the urethra, and the origin site is identified by examining the morphology of the red blood cells and the type of column. You can also Analysis of leukocytes in urine is important for early detection of inflammation and infection in the kidney and urinary tract. Furthermore, detection of malignant cells (cancer cells) is important in the early detection of urinary malignant tumors and the determination of their presence.

  Conventionally, as a urine specimen processing method, it is common to draw a sediment obtained by centrifuging fresh urine and prepare a specimen by a glass method (direct coating method), but cell detachment tends to occur, or There is a drawback that there are individual differences in the procedure. Therefore, there are many facilities that try to prevent cell detachment when preparing a specimen by adding a commercially available cell fixing solution (cell preservation solution) to the urine sample.

  In addition, after centrifuging the urine specimen, the sediment is applied to an anti-peeling coated slide glass, fixed in 95% ethanol, a commercially available cell fixing solution is added to the sediment, and then 5% polyethylene glycol (hereinafter “ Abbreviated as “PEG”), and a method for obtaining a smear after fixing with 95% ethanol (Non-patent Document 1). Cells contained in urine are treated with a first fixative solution (0.2% sodium azide, 0.8%). 50% ethanol containing EDTA-3K) and a second fixative (95% isopropyl alcohol containing 2% PEG) and then obtaining a smear (Non-Patent Document 2) A method of fixing cells by adding an aqueous alcohol solution containing diol and PEG to a liquid specimen such as urine (Patent Document 1) has been studied. Alternatively, there are a spray method in which powder is applied to a slide glass, spray-fixed and dried, a method of collecting cells using an apparatus such as an auto smear, a method of collecting cells through a filter, and the like.

  However, the method of treating a urine sample by the above-described method has a drawback that cell degeneration is strong, although cell detachment is less at the time of sample preparation than the direct smearing method. In addition, since morphological changes of cells and erythrocytes occur, the actual situation is that a satisfactory specimen is not obtained.

  Furthermore, cell fixation at the time of cytological examination including urine is often performed using 95% ethanol. However, only the fixation with 95% ethanol has a problem that the cells are detached in the subsequent staining process after fixation. In particular, even when liquid specimens such as urine and ascites were fixed with 95% ethanol, it was difficult to attach cells to the slide glass. In addition, when the slide glass soaked in a 95% ethanol fixing solution is taken out of the fixing solution, all the cells are dried.

Japanese Examined Patent Publication No. 6-52264 Hiroyuki Osaki, 3 others, “Examination of specimen processing method for urine cytology”, Journal of Japanese Society for Clinical Cytology, 2005, Vol. 44, No. 4, p. 215-218 Yuko Yamamoto, 4 others, “New urine cytological specimen preparation method-Kure Kyosai Hospital Method”, Journal of Japanese Society for Clinical Cytology, 1998, Vol. 37, No. 3, p. 292-297

  The present invention has been made in view of the situation as described above, and it is an object of the present invention to provide a cell fixing solution for obtaining a specimen with less cell detachment and deformation, and a cell fixing method using the cell fixing solution. .

In order to achieve the above object, the present invention comprises the following arrangement.
(1) A cell fixing solution comprising ethanol, isopropanol and PEG.
(2) A method for fixing cells, which comprises treating cells with a cell fixing solution containing ethanol, isopropanol and PEG.
(3) A cell fixing kit comprising a cell fixing solution containing ethanol, isopropanol and PEG as constituent components.

  That is, the present inventors conducted extensive research to develop a method for obtaining a specimen without cell detachment or drying, and as a result, fixed cells using a cell fixing solution containing ethanol, isopropanol and PEG. It has been found that if a sample is prepared by using this method, an excellent sample can be prepared that solves all the problems described above that have occurred in the conventional sample preparation. In addition, the cell fixing solution and the fixing method using the cell fixing solution can be applied not only to urine specimens but also to preparation of specimens of all specimens, and are excellent methods that can be applied in fields other than clinical diagnosis. The headline and the present invention were completed.

  If cells in a sample are fixed with the cell fixing solution of the present invention and a specimen is prepared using this, exfoliation, drying, deformation, and overstaining of nuclei can be prevented, and an excellent specimen can be obtained.

  Conventionally, in order to prevent the cells from drying and peeling, commercially available spray fixation is used, but the powdered specimen prepared by applying powder on a slide glass and fixing with the cell fixing solution of the present invention is completely dried. There is no detachment of cells and almost no drying after staining, so no spray treatment is necessary. In addition, since this smear has good cell retention and can be mailed, it can be used in a wide range of fields including clinical examinations.

  Furthermore, in the fixing method of the present invention, it is only necessary to perform the fixing treatment using only the cell fixing solution of the present invention, so that it is not necessary to purchase a commercially available cell fixing solution, which is economical.

  In addition, when gynecological specimens and respiratory specimens are fixed by spraying, depending on the spraying method, cells and dryness are severe, and there is a problem that they do not become specimens. All problems can be solved. In particular, the present invention is the first idea to fix these specimens with an alcohol solution containing PEG.

  The cell fixing solution of the present invention comprises ethanol, isopropanol and PEG.

  The PEG used in the cell fixing solution of the present invention has a weight average molecular weight of 400 to 4,000, preferably a weight average molecular weight of 1,000 to 2,000, more preferably a weight average molecular weight of about 1,540. It is done. If the molecular weight is too small, the sample cells may be detached. On the other hand, if the molecular weight is too large, the viscosity of the obtained cell fixing solution becomes high and difficult to handle, and the cells fixed using the cell fixing solution do not maintain the moisture retention of the cells and are dried. .

  Moreover, the cell fixing solution of the present invention may contain an aqueous solution such as water or physiological saline, if necessary.

  That is, PEG becomes cloudy and crystallizes at low temperature (5 ° C. or less) without water. Therefore, it is more preferable to further contain water in the cell fixing solution of the present invention, since the cell fixing solution can be prevented from becoming clouded and crystallized during low-temperature storage.

  In order to allow the cell fixing solution of the present invention to contain water, for example, water (preferably distilled water), an appropriate aqueous solution such as physiological saline, distilled water, etc. (these are collectively referred to as “physiological saline, etc. In the cell fixing solution of the present invention.

  The buffer is not particularly limited as long as it is usually used in this field and is isotonic with the cell solution and does not cause deformation of the cell. Specifically, for example, Tris Buffer solution, phosphate buffer solution, veronal buffer solution, borate buffer solution, Good buffer solution and the like can be mentioned. The concentration used is usually 0.1 mM to 10 M, preferably 1 mM to 5 M, more preferably 5 mM to 1 M. is there. The pH of the buffer solution is not particularly limited as long as it does not adversely affect the separation of substances, but is usually 2 to 13, preferably 4 to 11, and more preferably 5 to 9.

  Among these, use of physiological saline is preferable because it has a function of preventing degeneration such as cell contraction.

  As a method for preparing the cell fixing solution of the present invention, first, a lysate containing ethanol and isopropanol, and if necessary, an aqueous solution such as water or physiological saline is prepared. Next, there is a method in which PEG is dissolved in this solution so as to obtain a desired concentration.

  The content of PEG in the cell fixing solution is 2 to 10 w / v%, preferably 5 to 10 w / v%, more preferably 7 to 10 w / v%, and still more preferably 8 to 9 w / v%.

  The total amount of alcohol in the solution is 70-100 v / v%, preferably 90-100 v / v%, more preferably 93-95 v / v%.

  The content of ethanol with respect to the total amount of alcohol in the solution is 70 to 95 v / v%, preferably 75 to 90 v / v%, more preferably 80 to 90 v / v%.

  The content of isopropanol with respect to the total amount of alcohol in the solution is 30 to 5 v / v%, preferably 25 to 10 v / v%, more preferably 20 to 10 v / v%.

  The content of water or an aqueous solution such as physiological saline contained in the solution is 0 to 10 v / v%, preferably about 5 to 7 v / v%, in the solution.

  As a specific method for preparing the cell fixing solution of the present invention, for example, first, a lysate containing 70 to 95 v / v% ethanol and 30 to 5 v / v% isopropanol is prepared. Subsequently, PEG is added to the lysate and dissolved, and the PEG is added so that the final concentration of PEG is 2 to 10 w / v%, whereby the cell fixing solution of the present invention is obtained.

  In order to prepare the cell fixing solution of the present invention further containing an aqueous solution, for example, first, an alcohol mixture containing 70 to 95 v / v% ethanol and 30 to 5 v / v% isopropanol is prepared. Next, 5 to 7 parts by volume of an aqueous solution such as water or physiological saline is added to 95 to 93 parts by volume of the alcohol mixture to prepare a solution. What is necessary is just to prepare so that PEG may be added and dissolved in the obtained solution and the final concentration of PEG may be 2-10 w / v%.

  In order to carry out the cell fixing method of the present invention, the cells need only be treated with the cell fixing solution of the present invention. As a simple method, a sample or sediment containing the cells to be fixed is suspended in the cell fixing solution of the present invention, or the suspension containing the cells to be fixed and the cell fixing solution of the present invention are mixed. There are ways to do this.

  When fixing the cells, the amount of the cell fixing solution of the present invention is usually at least twice the volume of the cell (or sediment) volume, but too much is a waste of reagents. The amount is 2 to 30 times the volume, preferably 3 to 20 times, more preferably 5 to 10 times.

  The treatment time is not particularly limited, but it is usually sufficient if it takes about 10 to 60 minutes.

  More specifically, for example, the following may be performed.

  That is, for example, when using a liquid specimen such as a urine sample, first, the collected urine is sufficiently mixed so that the components in the urine are uniform. Next, 5 to 50 ml is dispensed into a centrifuge tube and centrifuged at about 3000 rpm (1500 G) for 2 to 5 minutes. After centrifugation, the supernatant is removed to obtain a urine sediment. To the obtained urinary sediment, the amount of the cell fixing solution of the present invention as described above relative to the amount of urine sediment is added and mixed thoroughly, and then left to stand for about 10 to 60 minutes. Can be fixed.

  The method for preparing a specimen using the cells fixed by the fixing method of the present invention suffices to be carried out in accordance with a known method for preparing a pathological tissue specimen, which is usually performed in the field of clinical laboratory science or the like.

  For example, after fixing the cells by the fixing method of the present invention, the cells fixed by centrifugation or the like are collected, smeared on, for example, a slide glass, and then naturally dried to produce a smear.

  The obtained smear may be subjected to a staining process known per se such as Papanicolaou staining appropriately selected according to the properties of the cells. In addition, reagents used in the field may be appropriately selected from those known per se in this field.

  In addition, the smear prepared using the cells fixed by the fixing method of the present invention can be stained immediately after drying, but it is the same as that used when fixing the cells before staining. Refixing with the cell fixing solution of the present invention having a composition is preferable because a clearer cell image can be obtained.

  That is, the fixing method of the present invention is a method using only the cell fixing solution of the present invention. In the case of re-fixation, it is not necessary to prepare and use 95% ethanol which has been used conventionally, and it is only necessary to re-fix using the cell fixing solution of the present invention used for cell fixation. The complexity is reduced.

  The cell fixing kit of the present invention comprises the cell fixing solution of the present invention comprising ethanol, isopropanol and PEG as constituent components, and preferred embodiments and specific examples of each component have been described above. Street.

  The kit may be further combined with a cell staining reagent known per se.

  The cell fixing solution of the present invention is not limited to pathological use, and can be used for a wide range of samples such as animal and plant cell tissues and microorganisms.

  For example, examples of the sample to which the present invention is applied include body fluids such as blood, serum, plasma, ascites, pleural effusion, pericardial fluid, bile, spinal fluid, synovial fluid, lymph fluid, excrement such as urine and feces, Puncture of sputum, pharyngeal mucus, gastric juice, bronchial lavage fluid, transbronchial collection, gynecological vaginal plasters, neck, intima, urine, stomach lavage fluid, bladder lavage fluid, pus, skin-derived material, mammary gland and thyroid gland And biological samples such as needle washing liquids and tissue seals.

  Since the cell fixing solution of the present invention has a high cell retention rate, when a specimen is prepared using cells fixed with the cell fixing solution, cell detachment, drying, deformation, and nuclear overstaining can be prevented. And an excellent specimen can be obtained. In addition, since red blood cells can be stored well, if a specimen is prepared using cells treated with the cell fixing solution of the present invention, clinical diagnosis can be performed using only this specimen.

  Conventionally, in order to fix cells in a liquid sample, it was necessary to fix the cells with a commercially available fixing solution, prepare a powdered sample, dry the sample, and then store the sample in 95% ethanol. This is because the ethanol concentration in the commercially available fixative is often low, and fixing with a commercially available fixative is not sufficient, and refixation with 95% ethanol is necessary. On the other hand, if the cells are fixed using the cell fixing solution of the present invention, the refixation treatment with 95% ethanol is not essential. Even in the case of re-fixation, it may be re-fixed with the cell fixing solution of the present invention, and there is no need to fix with 95% ethanol. In addition, it is economical because the cells can be fixed without purchasing a commercially available fixative. In this case, cell fixation using the cell fixing solution of the present invention is possible, but over-staining of nuclear hematoxylin can be suppressed compared to commercially available fixing solution or 95% ethanol, but if light staining is preferred. Hematoxylin may be halved.

In addition, those fixed with the cell fixing solution of the present invention show excellent staining properties even in special staining such as PAS staining and Alcian blue staining, and are more positive than those fixed with 95% ethanol, which makes it difficult to judge. There is no. In addition, the immunohistochemical staining also showed excellent staining properties and proved to be a very excellent fixing solution.

  Hereinafter, the present invention will be described in more detail with reference to experimental examples and examples. However, the present invention is not limited to these examples.

Experimental Example 1 Setting of PEG addition amount [sample]
As a result of pathological diagnosis, among the natural urine of a patient diagnosed with bladder cancer (G2 to G3), two types of urine were used, which had many inflammatory cells and the cells seem to be easily detached when a specimen was prepared.
(Preparation of cell fixative)
A solution in which polyethylene glycol 1540 (hereinafter referred to as PEG1540) was dissolved in a 95 w / v% aqueous ethanol solution so as to be 2 to 14 w / v% was prepared as a cell fixing solution.
[Fixed processing]
A sample (natural urine) was taken into a 50 mL centrifuge tube and centrifuged at 3000 rpm (1500 G) for 2 minutes. After removing the supernatant and sufficiently removing the water from the sediment, 10 mL of the cell fixing solution prepared above was added and mixed well. The mixture was allowed to stand for 30 minutes and fixed, and then similarly centrifuged at 3000 rpm (1500 G) for 2 minutes, and the precipitate was smeared onto a slide glass by a pipette method to prepare a specimen. The smear was air-dried until the next day, completely dried, stored in a 95% aqueous ethanol solution (no PEG), and then subjected to normal Papanicolaou staining.
In the following examples, silane-coated glass was used for all slide glasses.

[result]
A photograph of the resulting paint preparation is shown in FIG.
In FIG. 1, the upper row shows the results when a cell fixative containing 2, 4, 6, 8, 10, 12, 14 w / v% PEG1540 from the left is used. The lower row shows the results when using a cell lysate containing 2, 4, 6, 8, 10, 12 w / v% PEG1540 from the left, using natural urine specimens with few sediments as the upper row as samples. Respectively.

  As is clear from FIG. 1, when the concentration of PEG1540 is higher than 10 w / v%, the cells tend to detach and the cells tend to aggregate. This is because the concentration of PEG1540 exceeded 10w / v% and the viscosity of the solution became so high that the cells were solidified and could not be fixed on the slide glass. Conceivable. Furthermore, when the concentration of PEG1540 is 14 w / v%, it can be seen that the cells are clearly detached.

  Moreover, although the micrograph is not shown, it was confirmed by optical microscope observation that the cell was noticeably dried when the concentration of PEG1540 was 2 to 4 w / v%.

  From the above, it can be seen that the concentration of PEG1540 in the cell fixing solution is preferably 10 w / v% or less.

Example 1. Cell fixing solution of the present invention [sample]
The same natural urine as that used for the specimen in the upper part of FIG.
(Preparation of cell fixative)
(1) Cell fixative of the present invention
An alcohol mixture of 86 v / v% ethanol and 14 v / v% isopropanol was prepared. Prepare a solution by adding 5 parts by volume of physiological saline to 95 parts by volume of this alcohol mixture, and dissolve PEG1540 at 10 w / v% or 12 w / v% in the solution. Thus, the cell fixing solution of the present invention was used.
(2) 95% ethanol cell fixative
A solution in which PEG1540 was dissolved in 95 w / v% ethanol aqueous solution so as to be 8 w / v% or 10 w / v% was prepared, respectively, to prepare a 95% ethanol cell fixative.
[Fixed processing]
As a cell fixing solution, except that the cell fixing solution of the present invention or the 95% ethanol cell fixing solution prepared above was used, a smear sample was prepared after treatment of the urine sample and fixing treatment as in Experimental Example 1, The powdered specimen was naturally dried until the next day and completely dried. The smear of cells fixed with the cell fixative of the present invention is the cell fixative of the present invention (PEG 10 w / v%), and the smear of cells fixed with the 95% ethanol cell fixative is 95% ethanol ( After storage in the absence of PEG, normal Papanicolaou staining was performed.

〔result〕
A photograph of the resulting paint preparation is shown in FIG.
In FIG. 2, from the left, when using a 95% ethanol cell fixative containing 8 w / v% PEG1540, using a 95% ethanol cell fixative containing 10 w / v% PEG1540, 10 w / v% PEG1540 When the cell fixing solution of the present invention is used, the results of using the cell fixing solution of the present invention containing 12 w / v% PEG1540 are shown.

  As is clear from FIG. 2, when the 95% ethanol cell fixative was used, the cells were stained black regardless of the concentration of PEG1540, but when the cell fixative of the present invention was used, Thus, overstaining of the cells was suppressed, and a preferable stained specimen was obtained. However, when the concentration of PEG1540 was 12 w / v%, the cells tended to detach.

  In addition, it was confirmed by observation using a microscope that when using a 95% ethanol cell fixative, the cells were strongly atrophy and the nucleus was stained black.

  From the above, it can be seen that the cell fixing solution of the present invention containing ethanol, isopropanol and PEG is clearly superior to the cell fixing solution containing only ethanol and PEG.

Example 2 Examination of Cell Fixation Solution-PEG Concentration [Sample]
The same natural urine as that used in the upper sample of FIG.
[Preparation of cell fixing solution of the present invention]
An alcohol mixture of 86 v / v% ethanol and 14 v / v% isopropanol was prepared. To 95 parts by volume of this alcohol mixture, 5 parts by volume of physiological saline is added to prepare a solution, and PEG1540 is added to the solution at 4, 6, 7, 8, 9, 10, 12 w / v%. Each dissolved product was prepared and used as a cell fixative.
[Fixed processing]
Except that the cell fixing solution prepared above was used as the cell fixing solution, after the urine sample treatment and fixation treatment as in Experimental Example 1, the powdered specimen was naturally dried until the next day and completely dried. After storing in the cell fixing solution of the present invention (containing PEG 10 w / v%), normal Papanicolaou staining was performed.

〔result〕
A photograph of the obtained paint finish is shown in FIG.
In FIG. 3, the results when using a cell fixative containing PEG1540 so as to be 4, 6, 7, 8, 9, 10, 12 w / v% from the left are shown.

  As is clear from FIG. 3, when the cells are fixed with a cell fixing solution containing 4 w / v% to 6 w / v% PEG1540, the cells are atrophied and are gathered in a lump. This is considered to be related to the fact that the cell itself did not grow well at this PEG concentration, and it was difficult to smear even when preparing a smear. Moreover, when the concentration of PEG1540 is 4 w / v%, a small number of cell detachments are observed. Although the photomicrograph is not shown, when observed under a microscope, when the concentration of PEG1540 was 4 w / v% to 6 w / v%, the cells were stained red and the cells were dried.

  On the other hand, when the concentration of PEG1540 is 7 w / v% to 10 w / v%, it can be seen that the cells grow well and adhere to the slide glass. According to microscopic observation, almost no cell drying was observed.

  However, when the concentration of PEG1540 is 12 w / v%, it can be seen that the cells cannot adhere to the slide glass and have spread. Perhaps, this is because the PEG1540 concentration in the cell fixative solution has become excessive, resulting in insufficient cell fixation.

  From the above, it can be seen that the PEG concentration in the cell fixing solution of the present invention is preferably 7 to 10 w / v%.

Example 3
〔sample〕
The same natural urine as that used in the upper sample of FIG.
(Preparation of cell fixative)
(1) Cell fixative of the present invention
An alcohol mixture of 86 v / v% ethanol and 14 v / v% isopropanol was prepared. A cell was prepared by adding 5 parts by volume of physiological saline to 95 parts by volume of this alcohol mixture, and preparing a solution in which PEG1540 was dissolved to 9 w / v% in the solution. A fixative was used.
(2) 95% ethanol cell fixative
A solution in which PEG 1540 was dissolved in a 95 w / v% ethanol aqueous solution so as to be 9 w / v% was prepared and used as a 95% ethanol cell fixing solution.
[Fixed processing]
As a cell fixing solution, except that the cell fixing solution of the present invention or the 95% ethanol cell fixing solution prepared above was used, a smear sample was prepared after treatment of the urine sample and fixing treatment as in Experimental Example 1, The powdered specimen was naturally dried until the next day and completely dried. The smear of cells fixed with the cell fixative of the present invention is the cell fixative of the present invention (containing PEG 9 w / v%), and the smear of cells fixed with the 95% ethanol cell fixative is 95% ethanol. After storing each in a cell fixative, normal Papanicolaou staining was performed.

〔result〕
An optical micrograph of the obtained smear is shown in FIG.
In FIG. 4, (1) shows the results when the cell fixing solution of the present invention is used, and (2) shows the results when the 95% ethanol cell fixing solution is used. The photograph on the upper left of FIG. 4 shows the denatured and dried cells of the same specimen as (2).

  From the results of microscopic observation, it can be seen that when the 95% ethanol cell fixing solution is used, the cells are stained red and the cells are dried. On the other hand, when the cell fixing solution of the present invention is used, it can be seen that a preferable specimen was obtained without drying the cells.

  From the above, it can be seen from microscopic observation that the cell fixing solution of the present invention containing ethanol, isopropanol and PEG is clearly superior to the cell fixing solution containing only ethanol and PEG.

Example 4
〔sample〕
Ascites from patients diagnosed with ovarian cancer as a result of pathological diagnosis was used.
(Preparation of cell fixative)
(1) Cell fixative of the present invention
An alcohol mixture of 86 v / v% ethanol and 14 v / v% isopropanol was prepared. A cell was prepared by adding 5 parts by volume of physiological saline to 95 parts by volume of this alcohol mixture, and preparing a solution in which PEG1540 was dissolved to 9 w / v% in the solution. A fixative was used.
(2) Mixed alcohol cell fixative
An alcohol mixture of 86 v / v% ethanol and 14 v / v% isopropanol was prepared. A mixed solution in which 5 parts by volume of water was added to 95 parts by volume of this alcohol mixed solution was prepared as a mixed alcohol cell fixing solution.
[Fixed processing]
A sample (ascites) was taken in a 50 mL centrifuge tube and centrifuged at 3000 rpm (1500 G) for 2 minutes. After removing the supernatant and sufficiently removing the water from the sediment, the sediment was smeared on a slide glass to prepare a specimen. The smear is air-dried until the next day and completely dried, and the smear of cells fixed with the cell fixative of the present invention is mixed with the cell fixative of the present invention (containing PEG9w / v%) and mixed alcohol cell fixative. The smears of the cells fixed with the above were each stored in a mixed alcohol cell fixative and then subjected to normal Papanicolaou staining.

〔result〕
An optical micrograph of the obtained smear is shown in FIG.
In FIG. 5, (1) shows the results when the cell fixative of the present invention is used, and (2) shows the results when the mixed alcohol cell fixative is used.

  From the results of microscopic observation, it can be seen that when the cell fixing solution of the present invention was used, a preferable specimen was obtained that was very three-dimensional and free from cell drying and nucleus deformation.

  From the above, it can be seen that the cell fixing solution of the present invention containing ethanol, isopropanol and PEG is clearly superior to the mixed alcohol cell fixing solution containing only ethanol and isopropanol.

  In addition, the specimen obtained by fixing with the cell fixing solution of the present invention for 30 minutes and completely dried was very good in cell retention, so the possibility of mailing the specimen obtained in a medical examination etc. This suggests that the cell fixing solution of the present invention is an excellent cell fixing solution in this respect as well.

Example 5 FIG. Comparison with conventional direct smearing method [sample]
As a result of pathological diagnosis, natural urine (2 cases) of patients diagnosed with bladder cancer (G2 to G3) was used.
[Preparation of cell fixing solution of the present invention]
An alcohol mixture of 86 v / v% ethanol and 14 v / v% isopropanol was prepared. A cell was prepared by adding 5 parts by volume of physiological saline to 95 parts by volume of the alcohol mixture, and preparing a solution in which PEG1540 was dissolved to 10 w / v% in the solution. A fixative was used.
[Fixed processing]
A sample (natural urine) was taken into a 50 mL centrifuge tube and centrifuged at 3000 rpm (1500 G) for 2 minutes. After removing the supernatant and sufficiently removing the water from the sediment, remove a portion of the sediment with a Pasteur pipette and prepare two specimens applied on the slide glass by the pulling glass method. Was subjected to normal Papanicolaou staining, and the other specimen was subjected to normal Giemsa staining. To the remaining sediment, 10 mL of the cell fixing solution prepared above was added and mixed well. The mixture was allowed to stand for 30 minutes and fixed, and then similarly centrifuged at 3000 rpm (1500 G) for 2 minutes, and the precipitate was smeared onto a slide glass by a pipette method to prepare a specimen. The smear was air-dried for 30 minutes, stored in the cell fixative of the present invention, and then subjected to normal Papanicolaou staining.

〔result〕
A photograph of the obtained paint finish is shown in FIG.
In FIG. 6, (1) is the case where the conventional direct smear / Papanicolaou staining is performed, (2) is the case where the Papanicolaou staining is performed after fixation using the cell fixing solution of the present invention, and (3) is the conventional direct The results of smearing and Giemsa staining are shown.

  As is clear from FIG. 6, the cells were detached in the conventional direct smearing method (FIGS. 6 (1) and (3)), and the slide glass was smeared on the slide glass. It can be seen that the cells are gathered and set at the end of the pulling end (the position indicated by the arrows in FIGS. 6 (1) and (3)).

  In contrast, when the cells were fixed using the cell fixing solution of the present invention and then stained with Papanicolaou (FIG. 6 (2)), the cells spread uniformly on the slide glass and no cell detachment was observed. (The range enclosed by the arrow in Fig. 6 (2)).

  Further, FIG. 7 shows a photograph of a powdered specimen prepared by using a sample with a small amount of sediment processed in the same manner as the sample used in FIG.

  7. In FIG. 7, (1) is the case where the conventional direct smear / Papanicolaou staining is performed, (2) is the case where the Papanicolaou staining is performed after fixation using the cell fixing solution of the present invention, and (3) is the conventional direct The results of smearing and Giemsa staining are shown.

  As is apparent from FIG. 7, it can be seen that the cells are detached by the conventional direct smearing method (FIGS. 7 (1) and (3)). On the other hand, when the cells were fixed using the cell fixing solution of the present invention and stained with Papanicolaou (FIG. 7 (2)), the cells spread uniformly on the slide glass without detachment of the cells ( It can be seen that a good specimen was obtained even when the number of samples was small.

  In addition, in the case of direct smearing and Papanicolaou staining (FIG. 6 (1)), and the case of staining with Papanicolaou after fixation using the cell fixing solution of the present invention (FIG. 6 (2)) Optical micrographs are shown in FIG.

  In FIG. 8, (1) shows the results when conventional direct smearing / Papanicolaou staining is performed, and (2) shows the results when Papanicolaou staining is performed after fixation using the cell fixing solution of the present invention. The upper micrograph is an enlarged version of the lower micrograph.

  As is clear from FIG. 8, the sample fixed with the cell fixing solution of the present invention (FIG. 8 (2)) has a higher cell yield than the sample obtained by the direct smearing method (FIG. 8 (1)). Is good and is observed in three dimensions.

  In addition, when the above-mentioned sample with little sediment is directly smeared and stained with Papanicolaou (FIG. 7 (1)), and after fixing with the cell fixing solution of the present invention, it is stained with Papanicolaou (FIG. 7 (2)). An optical micrograph of () is shown in FIG.

  In FIG. 9, (1) shows the results when conventional direct smearing and Papanicolaou staining are performed, and (2) shows the results when Papanicolaou staining is performed after fixation using the cell fixing solution of the present invention. The upper micrograph is an enlarged version of the lower micrograph.

  As can be seen from FIG. 9, when the cell fixing solution of the present invention is used for fixation, even if there are few samples, the yield of cells is good, so that it is extremely effective when performing cytodiagnosis. It can also be seen that a good moisturizing effect and stainability were obtained even for specimens containing large cell clumps.

Example 6 Comparison with conventional cell fixatives [sample]
The same natural urine as in Example 5 was used.

(Preparation of cell fixative)
(1) Cell fixative of the present invention
An alcohol mixture of 86 v / v% ethanol and 14 v / v% isopropanol was prepared. A volume of physiological saline is added to 95 parts by volume of this alcohol mixture to prepare a lysate, and PEG1540 is dissolved in the lysate so as to have a concentration of 10 w / v%. did.
(2) Commercially available cell fixatives The following 4 types of commercially available cell fixatives were used. The composition of each cell fixing solution was as described in the instruction manual of each product.
(A) Commercial cell fixative A
Composition: 50% ethanol, 2% PEG1540.
(B) Commercial cell fixative B
Composition: 50% ethanol, PEG1540 (concentration unknown), dithiothreitol, rifampicin.
(C) Commercial cell fixative C
Composition: Ethanol 50%, PEG1540 several%, mucus melting agent contained in trace amount.
(D) Commercial cell fixative D
Composition: Contains 25% ethanol, toluene, and citric acid.

[Fixed processing]
A sample (natural urine) was taken into a 50 mL centrifuge tube and centrifuged at 3000 rpm (1500 G) for 2 minutes. After removing the supernatant and sufficiently removing the water from the sediment, a portion of the sediment is removed with a Pasteur pipette, and a specimen is applied on the slide glass by the pulling glass method. Went. The remaining sediment was divided into 5 test tubes, and 10 mL each of the cell fixative of the present invention prepared above or a commercially available cell fixative was added and mixed well. After fixing for 30 minutes, centrifuge for 2 minutes at 3000rpm (1500G) in the same way, and apply the sediment to the slide glass using a slide rack (manufactured by Medical and Biological Laboratories). A sample was made by spraying. The smear was air-dried for 30 minutes, stored in the cell fixative of the present invention, and then subjected to normal Papanicolaou staining.

〔result〕
A photograph of the obtained paint finish is shown in FIG.
In FIG. 10, (1) is when the direct smearing method is performed, (2) is when fixed with the cell fixing solution of the present invention, (3) is when fixed with the commercially available cell fixing solution A, (4) Shows the results when fixed with a commercially available cell fixative B, (5) shows the results when fixed with a commercially available cell fixative C, and (6) shows the results when fixed with a commercially available cell fixative D.

  As is apparent from the results of FIG. 10, when the smear is observed with the naked eye, it is fixed with the cell fixing solution of the present invention (FIG. 10 (2)) and when it is fixed with a commercially available cell fixing solution A. In FIG. 10 (3), the cells were not detached. When the direct smearing method is used (FIG. 10 (1)) and when fixed with other commercially available cell fixing solutions (FIGS. 10 (4) to (6)), the cells are detached. I understand.

  Moreover, the optical microscope photograph of the obtained coating powder sample is shown in FIG.11 and FIG.12.

  In FIG. 11, (1) is the case of FIG. 10 (1) in which the direct smearing method was performed, (2) is the case of FIG. 10 (2) fixed with the cell fixing solution of the present invention, and (3) is a commercially available product. In the case of FIG. 10 (3) fixed with the cell fixing solution A, (4) shows micrographs of the smears obtained in the case of FIG. 10 (4) fixed with the commercially available cell fixing solution B, respectively.

  When the smear sample was observed with the naked eye when fixed with a commercially available cell fixing solution A, the cells were not peeled off as in the case of fixing with the cell fixing solution of the present invention. (FIG. 10 (3)), but when observed with a microscope, it can be seen that the cells have expanded (cells indicated by arrows in FIG. 11 (3)). Moreover, when it fixed with the commercially available cell fixing solution B, the nucleus of the cell was dyed black, and the nucleolus could not be observed (FIG. 11 (4)). Furthermore, in FIGS. 11 (1), (3) and (4), only large cells, ie, cancer cells, are observed, but when fixed with the cell fixing solution of the present invention, red blood cells are also stained beautifully with orange G. Were observed (cells indicated by arrows in FIG. 11 (2)). It can also be seen that a very good specimen was obtained in which no cell expansion was observed and the shape of the nucleus was not deformed.

  In FIG. 12, (1) is the case of FIG. 10 (1) where direct smearing is performed, (2) is the case of FIG. 10 (2) fixed with the cell fixing solution of the present invention, and (3) is a commercially available product. In the case of FIG. 10 (5) fixed with the cell fixing solution C, (4) shows the micrographs of the smears obtained in the case of FIG. 10 (6) fixed with the commercially available cell fixing solution D.

  As can be seen from FIG. 12, when fixed with a commercially available cell fixative C, the cells expand (cells indicated by arrows in FIG. 12 (3)). It can also be seen that when the cells are fixed with a commercially available cell fixing solution D, the shape of the cell nucleus is distorted (cells indicated by arrows in FIG. 12 (4)). Furthermore, in FIG. 12 (1), (3) and (4), only large cells, that is, cancer cells are observed. On the other hand, when fixed with the cell fixing solution of the present invention, erythrocytes are also clearly stained with orange G and observed (cells indicated by arrows in FIG. 12 (2)), no cell expansion is observed, and nuclei are observed. It can be seen that a very good specimen was obtained, in which the shape of was not deformed.

Example 7
〔sample〕
As a result of pathological diagnosis, bile from a patient diagnosed with bile duct cancer was used.
[Preparation of cell fixing solution of the present invention]
An alcohol mixture of 86 v / v% ethanol and 14 v / v% isopropanol was prepared. A volume of physiological saline is added to 95 parts by volume of this alcohol mixture to prepare a lysate, and PEG1540 is dissolved in the lysate so as to have a concentration of 10 w / v%. did.
[Fixed processing]
Bile was collected in a 50 mL centrifuge tube and centrifuged at 3000 rpm (1500 G) for 2 minutes. After removing the supernatant and sufficiently removing the water from the sediment, 10 mL of the cell fixing solution prepared above was added and mixed well. The sample was allowed to stand for 30 minutes for fixation, and centrifuged at 3000 rpm (1500 G) for 2 minutes in the same manner. The sediment was smeared on a slide glass to prepare a specimen. The smear was air-dried for 30 minutes, stored in the cell fixative of the present invention, and then subjected to normal Papanicolaou staining.

〔result〕
An optical micrograph of the obtained smear sample is shown in FIG.
As can be seen from FIG. 13, when the cell fixing solution of the present invention is used, even when a bile sample is used, an extremely excellent specimen with little cell detachment is obtained, which is observed three-dimensionally and easily determined by cytodiagnosis. It turns out that it is.

FIG. 3 is a photograph of a smear prepared by using fixed urine sediment of a bladder cancer patient obtained in Experimental Example 1. FIG. FIG. 2 is a photograph of a smear prepared using a fixed urine sediment of a bladder cancer patient obtained in Example 1. FIG. FIG. 3 is a photograph of a smear prepared using a fixed urine sediment of a bladder cancer patient obtained in Example 2. FIG. FIG. 4 is an optical micrograph of a smear prepared by using a fixed urinary sediment of a bladder cancer patient obtained in Example 3. FIG. FIG. 4 (1) shows the case where the cell fixing solution of the present invention is used, and (2) shows the case where the 95% ethanol cell fixing solution is used. The photograph at the upper left of FIG. 4 is an optical micrograph of denatured and dried cells of the same specimen as (2). It is an optical micrograph of the smear sample produced using what fixed the ascites of the patient diagnosed with ovarian cancer obtained in Example 4. FIG. FIG. 5 (1) shows the case where the cell fixing solution of the present invention is used, and (2) shows the case where the mixed alcohol cell fixing solution is used. It is the photograph of the powdered end specimen produced using what fixed in the natural urine of the patient diagnosed with the bladder cancer obtained in Example 5. FIG. Fig. 6 (1) shows a conventional direct smear and Papanicolaou staining, (2) shows a Papanicolaou staining after fixation using the cell fixative of the present invention, and (3) shows a conventional direct smear.・ This is when Giemsa staining is performed. It is the photograph of the powdered end specimen produced using what fixed in the natural urine (sample with few sediments) of the patient diagnosed with the bladder cancer obtained in Example 5. FIG. Fig. 7 (1) is the case of performing conventional direct smearing and Papanicolaou staining, (2) is the case of performing Papanicolaou staining after fixation using the cell fixative of the present invention, and (3) is the conventional direct smearing・ This is when Giemsa staining is performed. FIG. 6 is an optical micrograph of a smear prepared by using the natural urine of a patient diagnosed with bladder cancer obtained in Example 5 and fixed. FIG. FIG. 8 (1) shows the case where conventional direct smearing / Papanicolaou staining is performed, and (2) is the case where Papanicolaou staining is performed after fixing using the cell fixing solution of the present invention. The upper micrograph is an enlarged version of the lower micrograph. FIG. 6 is an optical micrograph of a smear prepared using a sample obtained by fixing the natural urine (a sample with a small amount of sediment) of a patient diagnosed with bladder cancer obtained in Example 5. FIG. FIG. 9 (1) shows the case where conventional direct smearing / Papanicolaou staining is performed, and (2) is the case where Papanicolaou staining is performed after fixation using the cell fixing solution of the present invention. The upper micrograph is an enlarged version of the lower micrograph. It is the photograph of the smear sample produced using what fixed in the natural urine of the patient diagnosed with the bladder cancer obtained in Example 6. FIG. Fig. 10 (1) shows a direct smearing method, (2) shows a case of fixing with the cell fixing solution of the present invention, (3) shows a case of fixing with a commercially available cell fixing solution A, and (4) shows a commercially available case. When (5) is fixed with a commercially available cell fixative C, (6) is when fixed with a commercially available cell fixative D. It is an optical microscope photograph of the smear sample produced using what fixed the natural urine of the patient diagnosed with bladder cancer obtained in Example 6. FIG. FIG. 11 (1) shows a direct smearing method, (2) shows a case of fixing with the cell fixing solution of the present invention, (3) shows a case of fixing with a commercially available cell fixing solution A, and (4) shows a commercially available product. It is a case where it fix | immobilizes with the cell fixing solution B of. It is an optical microscope photograph of the smear sample produced using what fixed the natural urine of the patient diagnosed with bladder cancer obtained in Example 6. FIG. Fig. 12 (1) shows a direct smearing method, (2) shows a case of fixing with the cell fixing solution of the present invention, (3) shows a case of fixing with a commercially available cell fixing solution C, and (4) shows a case of commercially available. It is a case where it fix | immobilizes with the cell fixing solution D of. It is an optical micrograph of a smear prepared from a patient diagnosed with cholangiocarcinoma obtained in Example 7 using a fixed bile.

Claims (14)

A cell fixing solution comprising ethanol, isopropanol and polyethylene glycol. The cell fixing solution according to claim 1, wherein the cell fixing solution is prepared by dissolving 2 to 10 w / v% of polyethylene glycol in a lysis solution containing ethanol and isopropanol. The cell fixing solution according to claim 2, wherein the lysing solution further contains water or physiological saline. The cell fixing solution according to claim 2, wherein the total amount of alcohol is 70 to 100 v / v% in the lysis solution. The cell fixing solution according to claim 4, comprising 70 to 95 v / v% ethanol and 30 to 5 v / v% isopropanol with respect to the total amount of alcohol. The cell fixing solution according to claim 4, comprising 75 to 90 v / v% ethanol and 25 to 10 v / v% isopropanol with respect to the total amount of alcohol. The cell fixing solution according to claim 1, wherein the polyethylene glycol has a molecular weight of 400 to 4000. The cell fixing solution according to claim 1, wherein the polyethylene glycol has a molecular weight of 1000 to 2000. The cell fixative according to claim 1, wherein the content of polyethylene glycol in the cell fixative is 2 to 10 w / v%. The cell fixative according to claim 1, wherein the content of polyethylene glycol in the cell fixative is 5 to 10 w / v%. The cell fixing solution according to claim 1, wherein the cells are contained in a liquid specimen. The cell fixing solution according to claim 1, wherein the cells are contained in urine sediment. A method for fixing cells, which comprises treating cells with a cell fixing solution comprising ethanol, isopropanol and polyethylene glycol. A cell fixing kit comprising a cell fixing solution containing ethanol, isopropanol and polyethylene glycol as a constituent component.

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