JP2007132776A - Cell-immobilizing solution, and container containing it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provided a cell-immobilizing solution for preparing a sample for cell diagnostic inspection quickly and easily even when a specimen includes blood, a container containing the cell-immobilizing solution, and a preparation method of the sample for cell diagnostic inspection using the cell-immobilizing solution. <P>SOLUTION: The cell-immobilizing solution includes a hemolytic agent, a cell-immobilizing agent and glycerol as effective components. A glycerol amount in the cell-immobilizing solution is adjusted so that an immobilization action by the immobilizing agent is generated after a hemolytic action by the hemolytic agent is completed. The container containing the cell-immobilizing solution is also provided, and cell immobilization is performed quickly and easily by using one cell-immobilizing solution in one container. Since transfer operation is not required, possibility of infection to an operator is greatly reduced. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell fixing solution. More specifically, a cell fixing solution for fixing cells to prepare a specimen for cytodiagnosis examination, a container containing the cell fixing solution, and a method for preparing a specimen for cytodiagnosis examination using the cell fixing solution About.

  Conventionally, cytological examinations have been performed mainly by a slide direct smear method such as a smear specimen. However, in the smear method, the collected material adhering to the collection device is directly smeared on the slide glass, so that it was difficult to smear all the cells contained in the collected material on the slide glass. Therefore, in the collected cells, important cells may be discarded, and an accurate diagnosis cannot be made, leading to misdiagnosis.

  Furthermore, since the collected material is smeared directly on the slide glass, for example, when a collected material to which blood adheres is used, this blood may accidentally adhere to an operator's finger or the like. That is, the worker was exposed to the risk of blood infection.

  Thus, in recent years, liquid fixed cytology (LBC) has begun to be introduced, and needs from medical institutions are increasing. In LBC, since the instrument attached to the sample is directly immersed in the cell fixing solution, all cells contained in the sample can be used, and the sample is not smeared directly on the slide glass. The risk of infection is also reduced. In addition, for biological specimens with cell degeneration and puncture aspirate specimens, conventionally, the specimen had to be smeared on a slide glass on the day of collection and until the fixing process was performed. A specimen can be prepared after fixed storage (for example, see Non-Patent Documents 1 to 5).

However, when an LBC cell fixing solution that is currently marketed is used for a sample mixed with blood, the cells and red blood cells are fixed together to form a blood clot. Therefore, it becomes difficult to perform microscopic examination of the target cell. Therefore, when using a specimen mixed with blood, the blood removal process must be performed in advance before fixing the cells, and it takes a long time to prepare the specimen. Further, during the blood removal process, important cells may be destroyed or removed together with the blood.
"Gynecological cytology examination" brochure, Institute of Medical Biology "Matsunami Post Sampler", Matsunami Glass Industry Co., Ltd. Medical Division Product Catalog Yasuyoshi Ishii et al., “Lung Cancer Positive Rate by Tokyo Cancer Type Animal Slaughter Method Using YM Type Koji Fixing Solution”, 1986, Mutoh Chemical Co., Ltd. Nakamaru Ikuyuki et al., “Effectiveness and safety of YM-type cocoon fixative”, 1985, Mutoh Chemical Co., Ltd. Pamphlet Yasuaki Hirooka, “Intra-peritoneal lavage cytology for gastric cancer”, [online], August 26-27, 2000, 40th Cytologist Educational Seminar, [searched November 8, 2005], Internet, <URL: http://home.hiroshima-u.ac.jp/hiroct/seminar/gastricca.html>

  An object of the present invention is to use a cell fixing solution, a container containing the cell fixing solution, and the cell fixing solution for quickly and easily preparing a specimen for cytodiagnosis even when the specimen contains blood. It is to provide a method for preparing a specimen for cytodiagnosis.

  The present invention provides a cell fixing solution containing a hemolytic agent, a cell fixing agent, and glycerol as active ingredients.

  In one embodiment, the amount of the glycerol in the cell fixing solution is adjusted such that the fixing action by the cell fixing agent occurs after the hemolytic action by the hemolytic agent is completed.

  In one embodiment, the hemolytic agent is at least one selected from the group consisting of sodium lauryl sulfate and ammonium chloride, and the cell fixative is at least one selected from the group consisting of ethanol and 2-propanol. One.

  In a further embodiment, the cell fixing solution contains 0.5 to 6 g of the hemolytic agent and 100 to 800 mL of the fixing agent with respect to 100 mL of the glycerol.

  The present invention also provides a cell fixing container containing any of the cell fixing solutions described above.

  The present invention further provides a cell fixing kit comprising any one of the cell fixing solutions described above and a container for loading the cell fixing solution.

  In one embodiment, in the cell fixing kit, the cell fixing solution is loaded in the container.

  The present invention provides a method for fixing cells, comprising the step of introducing a specimen containing cells into any of the above cell fixing solutions.

In one embodiment, the cell fixation method of the present invention comprises:
A step of centrifuging the cell fixing solution containing the sample immediately after the completion of the adding step to obtain a sediment; and a step of introducing any of the cell fixing solutions described above into the sediment;
Further included.

The present invention also provides a method for preparing a specimen for cytological examination, the method comprising:
Placing a sample containing cells into any of the cell fixatives described above and allowing to stand for at least 30 minutes;
Centrifuging the left cell fixing solution to obtain a sediment; and smearing the sediment on a slide glass;
Is included.

The present invention further provides a method for preparing another specimen for cytodiagnosis, which comprises:
Introducing a specimen containing cells into any of the above cell-fixing solutions;
A step of centrifuging the cell fixing solution containing the sample immediately after the completion of the adding step to obtain a sediment;
A step of adding any of the above cell fixatives to the sediment and allowing to stand for at least 30 minutes;
Centrifuging the left cell fixative to obtain a sediment; and smearing the sediment on a slide glass;
Is included.

  According to the present invention, a cell fixing solution containing a hemolytic agent, a cell fixing agent, and glycerol as active ingredients is provided. When this cell fixative is used, even if it is a specimen containing blood, there is no need for a step of removing blood, and in this cell fixative, erythrocytes are hemolyzed and blood clots are not formed. Only cells are fixed. Moreover, cell fixation can be performed quickly and easily by using one cell fixing solution in one container. Therefore, it is possible to easily produce a specimen for cytological examination that is easy to microscopically in a short time.

(I) Cell fixing solution The cell fixing solution of the present invention contains a hemolytic agent, a cell fixing agent, and glycerol as active ingredients. According to the present invention, since the action speed of both of the hemolytic agent and the cell fixing agent is almost inversely proportional to the dissolution concentration of glycerol, the hemolytic action and the cell fixing action are determined depending on the amount of glycerol in the cell fixing solution. Based on finding out that it can control the speed of.

  Specifically, the rate of hemolysis decreases as the glycerol concentration increases at a constant hemolytic agent concentration, and similarly, the rate of cell fixation also decreases the glycerol concentration at a constant cell fixing agent concentration. As you go up, the speed decreases. The rate of action of each is inversely proportional to the glycerin concentration. In practice, the rate of action refers to the delay in the start time of each action and the extension of the action time from start to completion. In addition, the different rates of action of each of these drugs (hemolytic agent >> cell fixative) can be achieved by increasing the concentration of glycerol under a certain concentration ratio of hemolyzer and cell fixative. Due to the difference in the rate of speed reduction, there is a time difference in action between the two (see FIG. 1). For this reason, in the present invention, hemolysis and cell fixation can be performed with one cell fixing solution. Hereinafter, the cell fixing solution will be described in detail.

(1) Hemolytic agent The hemolytic agent contained in the cell fixing solution of the present invention is not particularly limited as long as it is a substance capable of destroying red blood cells and causing hemolysis. For example, saponin, ammonium chloride, sodium lauryl sulfate (SDS) and the like can be mentioned. These may be used alone or in combination of two or more. For example, a combination of sodium lauryl sulfate (SDS) and ammonium chloride is used.

(2) Cell fixative Examples of the cell fixative contained in the cell fixative of the present invention include fixatives commonly used in LBC, such as ethanol, methanol, 2-propanol, and the like. These may be used alone or in combination of two or more. For example, a combination of ethanol and 2-propanol is used. The hemolytic agent described above causes hemolysis and destruction of erythrocytes and at the same time denatures and destroys other nucleated cells. However, in cells completely fixed with alcohol or the like, even if a hemolytic agent is added in excess (for example, several tens of times the usual amount), there is no change in the morphology of cells containing erythrocytes. That is, the fixing action by a cell fixing agent such as alcohol can also act as a hemolytic action stop agent at the same time.

(3) Glycerol Glycerol contained in the cell fixing solution of the present invention adjusts the hemolysis rate and cell fixing rate, prevents cell drying and maintains its shape, enhances the adhesion of cell slide glass smears, It has effects such as a decrease in the sedimentation rate of cells by increasing the specific gravity. Glycerol used in the cell fixing solution of the present invention is not particularly limited, and commercially available products can be used as appropriate.

(4) Cell fixative Since the cell fixative of the present invention contains glycerol, the viscosity of the cell fixative is high. Therefore, the hemolysis rate and the cell fixation rate are decreased, respectively, whereby the hemolysis rate and the cell fixation rate can be adjusted. That is, under a certain concentration of hemolytic agent and cell fixing agent, the rate of action of both decreases as the glycerol dissolution concentration increases, so that by adjusting the amount of glycerol in the cell fixing solution, hemolysis and Control the rate of cell fixation.

  In the present invention, by adding glycerol that does not affect the substance and has high specific gravity, high viscosity, water solubility and transparency, the hemolysis action is completed first, then the fixation action occurs, and nucleated cells Each amount is adjusted so as to minimize the effects of hemolytic denaturation. Therefore, a time difference can be provided between the end of hemolysis and the start of cell fixation. Red blood cells with weak cell membranes are destroyed without being fixed, and nucleated cells other than red blood cells are fixed without being destroyed. In addition, by providing a time difference, it is possible to separate and remove most of the broken and hemolyzed red blood cells floating in the high specific gravity cell fixing solution by centrifugation or the like.

  Therefore, in the cell fixing solution of the present invention, the amounts of the hemolytic agent, the cell fixing agent, and glycerol are adjusted so that the fixing action by the cell fixing agent occurs after the hemolytic action by the hemolytic agent is completed. These amounts can be appropriately determined depending on the types of hemolytic agent and cell fixing agent. The amount of the hemolytic agent contained in the cell fixing solution of the present invention is an amount sufficient to hemolyze erythrocytes that can be mixed in the sample, and the amount of the cell fixing agent is used to fix the cells contained in the sample. It is a sufficient amount.

  For example, the hemolytic agent is contained in an amount of preferably 0.5 to 6 g, more preferably 1 to 5 g, and still more preferably 1.5 to 4 g with respect to 100 mL of glycerol. When the hemolytic agent is a combination of SDS and ammonium chloride, the total amount of SDS and ammonium chloride may preferably be 2.5-3.5 g per 100 mL of glycerol. In this case, the mass ratio of SDS to ammonium chloride is preferably 1: 1 to 1:30, more preferably 1: 1.5 to 1:10, and still more preferably 1: 2 to 1: 6.

  For example, the cell fixing agent is contained in a proportion of preferably 100 to 800 mL, more preferably 200 to 700 mL, and still more preferably 300 to 600 mL with respect to 100 mL of glycerol. When the cell fixing agent is ethanol and 2-propanol, the total amount of ethanol and 2-propanol can be preferably 350 to 500 mL with respect to 100 mL of glycerol. In this case, the mass ratio of ethanol to 2-propanol is preferably 1: 0.2 to 1: 8, more preferably 1: 0.3 to 1: 3, and still more preferably 1: 0.4 to 1: 1.

  Furthermore, the mass ratio of the hemolytic agent to the cell fixing agent is preferably 1:20 to 1: 800, more preferably 1:50 to 1: 600, and still more preferably 1:80 to 1: 400. For example, when the hemolytic agent is SDS and the cell fixing agent is ethanol, the ratio is preferably 1: 100 to 1: 200.

  In general, the cell fixing solution of the present invention may contain media and additives commonly used by those skilled in the art, in addition to the components described above. The medium is used for the purpose of adjusting the viscosity of the cell fixing solution itself. Examples of such a medium include physiological saline and phosphate buffered physiological saline (pH 7.4). The additive is used for the purpose of preventing and protecting the cells with glycerol, enhancing the smearing (attaching) of the cells on the slide glass, and increasing the specific gravity of the cell fixing solution. Examples of such additives include polyethylene glycol.

  For example, the medium is contained in a proportion of preferably 100 to 800 mL, more preferably 200 to 700 mL, and still more preferably 300 to 600 mL with respect to 100 mL of glycerol. When the medium is physiological saline, the physiological saline can be preferably 350 to 500 mL with respect to 100 mL of glycerol.

  For example, the additive is contained in an amount of preferably 0.01 to 15 g, more preferably 0.03 to 10 g, and still more preferably 0.05 to 8 g with respect to 100 mL of glycerol. When the additive is polyethylene glycol, the polyethylene glycol can preferably be 0.08-4 g per 100 mL of glycerol.

  The amount of cell fixative used is appropriately determined according to the amount of specimen. For example, when the amount of the sample is 0.1 to 0.4 mL, the amount of the cell fixative is 5 to 10 mL, and when the amount of the sample is 0.4 to 2.0 mL, the amount of the cell fixative is When the amount is 10 to 20 mL and the amount of the specimen is 0.5 to 20 mL, the amount of the cell fixing solution is 20 to 30 mL.

(II) Cell fixing container The cell fixing container of the present invention contains the cell fixing solution. As described later, in consideration of separating fixed cells, hemolyzed red blood cells, and the like by centrifugation, the cell fixing container of the present invention is preferably a container capable of performing centrifugation.

  The material for the cell fixing container of the present invention is not particularly limited. For example, Preferably, polyethylene (PE), polypropylene (PP), polystyrene (PS), polycarbonate (PC), etc. are mentioned. More preferably, it is a material that can be centrifuged, and examples of such a material include polypropylene (PP) and polystyrene (PS). Further, the shape and size of the container are not particularly limited, and may be any shape and size that can be mounted on a centrifuge normally used in the field.

  The amount of the cell fixing solution contained in the cell fixing container of the present invention is appropriately determined according to the amount of the specimen as described above. Further, the size (capacity) of the cell fixing container is appropriately determined according to the amount of the cell fixing solution. The cell fixing solution is preferably contained in a proportion of 40 to 80% by volume, more preferably 60 to 75% by volume with respect to the capacity of the cell fixing container.

(III) Cell fixing kit The cell fixing kit of the present invention comprises a cell fixing solution and a container for loading the cell fixing solution. Preferably, the container may be capable of performing centrifugation, and includes a cell fixing container loaded with a cell fixing solution as described in (II) above. If necessary, for example, collection devices, additional cell fixatives, instructions for use, and the like may be included. The cell fixing container may be a combination of several cell fixing containers having different sizes.

(IV) Cell immobilization method The cell immobilization method of the present invention includes a step of introducing a specimen containing cells into the cell fixing solution of the present invention (hereinafter sometimes referred to as “input step”). When a lot of blood is mixed in the specimen, a step of centrifuging the cell fixing solution containing the specimen immediately after the completion of the adding step to obtain a sediment; and the cell fixing solution of the present invention is introduced into the sediment The method further includes a step.

  In the present invention, the specimen containing cells refers to a specimen for general LBC cytodiagnosis examination. Such specimens include gynecological specimens (eg, vaginal neck collection with cotton swabs, brushes, endometrial scrapings with special instruments), secretions (milk, nasal discharge, etc.), exudates, scrapes ( Bronchi, skin, etc.), puncture (lung, breast, thyroid, parotid, subcutaneous mass, etc.), lavage fluid (bronchial, intraperitoneal, intravesical, etc.), digestive fluid, sputum (3-day accumulation, etc.), urine Examples include sediment.

  In the loading step, the sample is loaded into the cell fixing solution of the present invention. The amount of the cell fixing solution to be used can be appropriately determined according to the amount of the specimen as described above. For example, in the case of a collected material using a collection tool such as a cotton swab or a brush, the portion to which the collected material is attached may be immersed in an arbitrary amount of cell fixing solution. In the case of secretion fluid, exudate, puncture, etc., these may be directly put into an arbitrary amount of cell fixing solution. After the addition, the specimen and the cell fixing solution are sufficiently stirred and allowed to stand until the cells are fixed. In this case, hemolysis occurs first in the cell fixing solution, and then cell fixation starts. Cell fixation is usually complete in about 30 minutes. In the present invention, even when a small amount of blood (for example, up to about 50 μL) is mixed in the sample, the cells can be fixed sufficiently in a microscopic manner by the above operation.

  On the other hand, when a lot of blood is mixed in the specimen, it is preferable to remove red blood cells in advance. When the specimen is put into the cell fixing solution, the red blood cells are immediately hemolyzed and destroyed. Thereafter, when centrifugation is performed during the time until cell fixation is started, the hemolyzed erythrocytes and the cells to be examined are separated, and the erythrocytes that have undergone hemolysis destruction in the high-density cell fixing solution float in the supernatant. Therefore, hemolyzed red blood cells can be removed by removing the supernatant. In the present invention, the centrifugation is performed immediately after the sample is introduced, preferably within at least 120 seconds. Centrifugation is performed at 1600 G for 20 to 50 seconds, for example. After removing the supernatant, the cell fixing solution of the present invention is poured into the sediment containing the cells to be examined, and after stirring well, it is allowed to stand until the cells are fixed (for example, at least 30 minutes). The amount of the cell fixing solution additionally added here is appropriately determined according to the amount of sediment, and can be usually the same amount as the cell fixing solution used first.

  After the cells are fixed, the cells can be separated into fixed cells and a cell fixing solution by centrifugation or the like. The obtained sediment can be used for preparing a specimen for cytological examination. Alternatively, the fixed cells can be stored with the cell fixative, for example, at room temperature for about 2-4 weeks and stored in a cool dark place for about 2-6 months.

(V) Method for Preparing Specimen for Cytological Examination The method for preparing the specimen for cytological examination of the present invention is the above-described method for fixing cells, and then, the sediment (fixed cells) obtained by the method is used as a slide glass. Including the step of smearing. For example, a specimen can be prepared by the following method.

  The fixed cells are usually in the form of a paste, which is collected using, for example, a pipette and placed on one slide glass. Stack another glass slide and rub the two glass slides together. Next, the two slide glasses are peeled off to obtain two slide glasses in which cells are smeared with a uniform thickness. A sample for cytological examination can be obtained by evaporating and drying excess moisture, alcohol, and the like of the smear cells on the slide glass by, for example, blowing air, and strengthening the sticking of the cells on the slide glass. The obtained specimen for cytodiagnosis is stained by means usually used by those skilled in the art. In LBC, Papanicolaou staining is common. In addition, when alcohol is used as a cell fixing agent, it is unsuitable for Giemsa staining.

  As a slide glass used for specimen preparation, a slide glass generally used may be used, but it is preferable to use a protein-coated slide glass having an increased adhesiveness on the surface of the slide glass. Furthermore, you may use together spray fixing agent (The cell fixing solution for LBC which sprays directly on the cell mounted on the slide glass, and fixes a cell). By using the protein-coated glass slide and the spray fixing agent in combination, it is possible to further strengthen the attachment of the cells to the glass slide.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to this range.

(Example 1: Preparation of cell fixative 1)
Sodium lauryl sulfate (1st grade: Wako Pure Chemical Industries, Ltd.) 1.33 g as a hemolytic agent, ethanol (19.5 grade: Wako Pure Chemical Industries, Ltd.) 416.67 mL, glycerol (1) Class: SAJ Co., Ltd.) 100 mL, additive polyethylene glycol 4000 (Class 1: Wako Pure Chemical Industries, Ltd .; average molecular weight 3,000) 0.83 g, and physiological saline 416.67 mL were mixed, and cell fixation Liquid 1 was prepared.

(Example 2: Preparation of cell fixative 2)
6.67 g of ammonium chloride (1st grade: Wako Pure Chemical Industries, Ltd.) as a hemolytic agent, 458.33 mL of 2-propanol (19.5% or higher grade: SAJ Ltd.) as a cell fixing agent, glycerol (1st grade) : SAJ Co., Ltd.) 100 mL, polyethylene glycol 4000 (1st grade: Wako Pure Chemical Industries, Ltd .; average molecular weight 3,000) 2.5 g as an additive, and physiological saline 375 mL are mixed, and cell fixative 2 is prepared. Prepared.

(Example 3: Preparation of cell fixative 3)
First, 50 mL of physiological saline, 0.16 g of sodium lauryl sulfate (1st grade: manufactured by Wako Pure Chemical Industries, Ltd.) as a hemolytic agent, and ethanol (1st grade 99.5%: manufactured by Wako Pure Chemical Industries, Ltd.) as a cell fixing agent ) 50mL was mixed and A liquid was prepared. Separately, 45 mL of physiological saline, 0.8 g of ammonium chloride (first grade: Wako Pure Chemical Industries, Ltd.) as a hemolytic agent, and 2-propanol (first grade 99.5% or higher: SAJ stock as a cell fixing agent) (Manufactured) 55 mL was mixed to prepare B liquid. Next, liquid A and liquid B were mixed at a ratio of 7: 3, and 100 mL thereof was mixed with 12 mL of glycerol (first grade: manufactured by SAJ) and polyethylene glycol 4000 (first grade: manufactured by Wako Pure Chemical Industries, Ltd .; average) A cell fixing solution 3 was prepared by adding 0.3 g of molecular weight 3,000) and stirring.

(Example 4: Production of cell fixation container)
6 mL of the cell fixing solution 1 was put into a container (capacity: 10 mL) made of PP (polypropylene) and capable of being centrifuged to produce a cell fixing container 1.

  This cell fixing container 1 is used when the volume of the collected material is within 0.4 mL.

(Example 5: Production of cell fixation container)
A cell fixing vessel 2 was manufactured by putting 15 mL of the cell fixing solution 1 into a vessel (capacity 20 mL) made of PP (polypropylene) and capable of being centrifuged.

  This cell fixing container 2 is used when the volume of the collected material is within 1.0 mL.

(Example 6: Production of cell fixation container)
20 mL of the cell fixing solution 1 was placed in a container (capacity: 30 mL) made of PP (polypropylene) and capable of being centrifuged to produce a cell fixing container 3.

  This cell fixing container 3 is used when the volume of the collected material is 0.8 to 1.8 mL.

(Example 7: Preparation of specimen 1)
Using the cell fixing container 2, a specimen for cytological examination was prepared by the following procedure.

  First, 0.6 mL of a sample containing a trace amount of blood was obtained by endometrial intima scraping. The obtained collected material was put into the cell fixing container 2 and allowed to stand. Immediately after the addition, the cell fixing solution in the container was muddy red. One to three seconds after the collected material was added, the cell fixing solution in the container became transparent reddish brown and hemolysis was observed. Furthermore, from about 2 to 3 minutes after the collected material was added, the cell fixative began to change to a turbid brown color, which is a sign of the start of fixation, and protein coagulation was observed. Next, 30 minutes after the collected material was added, the whole cell-fixed container was centrifuged (1000 G, 5 minutes), and the supernatant was removed. The amount of red beans in the obtained sediment was placed on a slide glass with a pipette, covered with another slide glass, and the two slide glasses were rubbed together and smeared uniformly. Next, the two slide glasses were peeled off, and air was blown for about 15 minutes to evaporate and dry excess water and alcohol to obtain a specimen for staining.

(Example 8: Preparation of specimen 2)
Using the cell fixing vessel 3, a specimen for cytological examination was prepared by the following procedure.

  First, 1 mL of puncture aspiration sample was obtained from the subcutaneous mass. Since the obtained collected material had a lot of blood contamination, it was put into the cell fixing container 3 and mixed, and after 5 seconds, centrifugation (1600 G, 40 seconds) was performed. Subsequently, the supernatant (including floating hemolyzed erythrocytes) was removed, and the cell fixing solution 1 (20 mL) obtained in Example 1 was additionally added and mixed. 30 minutes after the addition, the whole cell-fixed container was centrifuged (1000 G, 5 minutes), and the supernatant was removed. The amount of red beans in the obtained sediment was placed on a slide glass using a pipette, covered with another slide glass, and the two slide glasses were rubbed together and smeared uniformly. Next, the two slide glasses were peeled off, and air was blown for about 15 minutes to evaporate and dry excess water and alcohol to obtain a specimen for staining.

(Example 9: Preparation of specimen 3)
A specimen was prepared in the same manner as in Example 7 except that a mixture of a small amount of oral mucosa and 40 μL of blood was used as the specimen material. FIG. 2 shows a photograph of the state of the cell fixing solution in the container and a cell image (micrograph) of the specimen.

  The cell fixing solution in the container became transparent reddish brown and hemolysis was observed. Also, as can be seen from the cell image, many red blood cells that hinder the observation of the morphology of epithelial cells (nucleated cells) were hemolyzed and broken down and scattered in the background. Therefore, the sample is in a state that is easy to determine and diagnose.

(Comparative Example 1: Preparation of specimen 4)
A mixture of a small amount of oral mucosa and 40 μL of blood was used as the specimen material. This specimen material was put into a container containing a conventionally used cell fixing solution (50% ethanol + 2% polyethylene glycol) and well stirred and mixed. After centrifugation (1000 G, 5 minutes), the amount of red beans in the upper part of the sediment (buffy coat part) of the nucleated cell layer is placed on a glass slide using a pipette and covered with another glass slide. The glass slides were rubbed together and smeared uniformly. Next, the two slide glasses were peeled off, and air was blown for about 15 minutes to evaporate and dry excess water and alcohol to obtain a specimen for staining. FIG. 3 shows a photograph of the state of the cell fixing solution in the container and a cell image (micrograph) of the specimen.

  Small clots were observed in the cell fixative in the container. Also, as can be seen from the cell image, a large amount of red blood cells became large and small clots and entangled with epithelial cells, making it difficult to observe the morphology of epithelial cells (nucleated cells).

(Example 10: Preparation of specimen 5)
A specimen was prepared in the same manner as in Example 8 except that a mixture of a small amount of oral mucosa and 300 μL of blood was used as the specimen material. FIG. 4 shows a photograph of the state of the cell fixing solution in the container and a cell image (micrograph) of the specimen.

  The cell fixative in the container was turbid red immediately after the sample material was added, but soon became transparent reddish brown and hemolysis was observed. After removing red blood cells by centrifugation, the cell fixative became translucent light yellow. In the cell image of the sediment obtained by centrifugation after the additional addition of the cell fixative 1, it can be seen that the red blood cells are almost removed and the morphology of the epithelial cells (nucleated cells) can be accurately observed. Thus, the sample was in a state that was very easy to determine and diagnose.

(Comparative Example 2: Preparation of specimen 6)
A sample for staining was obtained in the same manner as in Comparative Example 1 except that a mixture of a small amount of oral mucosa and 300 μL of blood was used as the sample material. FIG. 5 shows a photograph of the state of the cell fixing solution in the container and a cell image (micrograph) of the specimen.

  Large clots were observed in the cell fixative in the container. Also, as can be seen from the cell image, a large amount of red blood cells became large and small clots and entangled with epithelial cells, making it very difficult to observe the morphology of epithelial cells (nucleated cells).

  According to the present invention, by combining a hemolytic agent, a cell fixing agent, and glycerol, even if a specimen containing blood is used, a step of removing blood is not required, and one cell fixing solution is contained in one container. By using it, cell fixation can be performed quickly and easily. Therefore, a specimen for cytodiagnosis examination that can be easily microscopically can be easily produced, which is very useful. In addition, since there is no need to transfer the specimen, the risk of infection to the worker is greatly reduced.

It is a graph which shows the concept of the relationship between the hemolytic action and the cell fixing action in the cell fixing solution containing glycerol. It is the photograph of the state of the cell fixing solution in a container when the small amount of oral mucosa and 40 microliters of blood are processed with the cell fixing solution of this invention, and the microscope picture of a sample. It is the photograph of the state of the cell fixing solution in a container when the small amount of oral mucosa and 40 microliters of blood are processed with the conventional cell fixing solution, and the microscope picture of a sample. It is the photograph of the state of the cell fixing solution in a container when the small amount of oral mucosa and 300 microliters of blood are processed with the cell fixing solution of this invention, and the microscope picture of a sample. It is the photograph of the state of the cell fixing solution in a container when the small amount of oral mucosa and 40 microliters of blood are processed with the conventional cell fixing solution, and the microscope picture of a sample.

Claims (12)

  A cell fixing solution containing a hemolyzing agent, a cell fixing agent, and glycerol as active ingredients.   The cell fixing solution according to claim 1, wherein the amount of the glycerol is adjusted so that the fixing action by the cell fixing agent occurs after the hemolytic action by the hemolytic agent is completed.   The cell fixative according to claim 1 or 2, wherein the hemolytic agent is at least one selected from the group consisting of sodium lauryl sulfate and ammonium chloride.   The cell fixing solution according to any one of claims 1 to 3, wherein the cell fixing agent is at least one selected from the group consisting of ethanol and 2-propanol.   The cell fixing solution according to any one of claims 1 to 4, comprising 0.5 to 6 g of the hemolytic agent and 100 to 800 mL of the fixing agent with respect to 100 mL of the glycerol.   A cell fixing container comprising the cell fixing solution according to any one of claims 1 to 5.   A cell fixing kit comprising the cell fixing solution according to any one of claims 1 to 5 and a container for loading the cell fixing solution.   The cell fixing kit according to claim 7, wherein the cell fixing solution is loaded in the container.   A method for fixing a cell, comprising a step of introducing a specimen containing cells into the cell fixing solution according to any one of claims 1 to 5. A step of centrifuging the cell fixing solution containing the specimen immediately after the completion of the adding step to obtain a sediment; and a step of introducing the cell fixing solution according to any one of claims 1 to 5 to the sediment;
10. The method of claim 9, further comprising: A method for preparing a specimen for cytodiagnosis,
A step of putting a specimen containing cells into the cell fixing solution according to any one of claims 1 to 5 and allowing it to stand for at least 30 minutes;
Centrifuging the left cell fixative to obtain a sediment; and smearing the sediment on a slide glass;
Including the method. A method for preparing a specimen for cytodiagnosis,
A step of introducing a specimen containing cells into the cell fixing solution according to any one of claims 1 to 5;
A step of centrifuging the cell fixing solution containing the sample immediately after the completion of the adding step to obtain a sediment;
Putting the cell fixing solution according to any one of claims 1 to 5 into the sediment and allowing it to stand for at least 30 minutes;
Centrifuging the left cell fixative to obtain a sediment; and smearing the sediment on a slide glass;
Including the method.

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