KR101395081B1 - Gynecologic cell preserving solution - Google Patents

Abstract

The present invention relates to a cell preserving solution comprising: a solvent including 30-50 w/v% of alcohol and 50-65 w/v% of distilled water; 1.0-2.5 w/v% of a buffer agent; 0.1-1.5 w/v% of a blood remover; 0.1-1.5 w/v% of protein precipitant; 0.1-1.5 w/v% of an anticoagulant; 0.1-2.0 w/v% of an anti-drying agent; 0.1-1.5 w/v% of an osmoregulation agent; 0.1-1.5 w/v% of a preservative; and 0.1-1.5 w/v% of a mucolytic agent. By using the cell preserving solution of the present invention, cell degeneration during a retention period can be minimized; an optimal pH can be maintained; and cell shapes can be preserved to be close to native shapes by preventing the drying and decomposition of cells, for instance, being utilized as a cell preserving solution for a gynecologic clinical specimen.

Description

{GYNECOLOGIC CELL PRESERVING SOLUTION}

The present invention relates to a cell-preserving solution, and more particularly, to a gynecological cell-preserving solution capable of preserving cell morphology.

Various methods for identifying various diseases including cancer have been known. However, it is known that a so-called cytopathological method of collecting cells in a human body region suspected of having a disease and examining the pathological form thereof is the most reliable. For cytologic examination using cytopathologic methods, cells of the human body suspected of having a disease are collected and examined under a microscope.

In general, cell specimens are classified into gynecology specimens typified by cervical cells and non-gynecology specimens such as body fluids such as pleural fluids and pleural fluid, and bronchial washing fluids. The cells collected from these specimens are examined under a microscope to check for various diseases including the presence or absence of cancer cells.

Papanicolaou smear (abbreviated as Pap smear) is mainly used as an examination method using conventional cytopathologic methods, in which collected cells are stained on a slide and examined. However, in the case of using conventional cytological examination, cells were dried in the process of applying the cells on a slide, and mucous and erythrocytes remained, which made it difficult to accurately read or diagnose. In addition, most of the collected cells were not stained on the slide, so there was a limit in the reliability of the test.

Liquid based cytology (Liquid Based Cytology) has been developed to replace such conventional cytology. For cytologic diagnosis, liquid cells are stored in a preservative solution, and if necessary, the cells stored in the preservative solution are examined to check for the presence or absence of a cell, that is, whether a specific disease has occurred. With respect to liquid cell testing, techniques and apparatus have been developed for automatically screening and placing a cell sample containing a suitably stained, properly preserved cell monolayer on a standardized slide.

Compared with the conventional cytology, the liquid cell cytology shows that the collected specimen cells are uniformly distributed on the slide. In addition, as compared with cytological examination, liquid cell cytology improves cell fixation and decreases unclear factors. Especially, most of the collected cells, unlike the cytological examination that discards most of the collected specimens in relation to the diagnosis of cervical cancer, , It is known that it can improve the specificity and sensitivity of the test.

SurePath (TriPath Imaging, Inc, Burlington, NC, USA) and ThinPrep (Cytyc Corp, Marlborough, MA, USA) are among the most widely used liquid cytology. In these products, a sample collected from a human body is collected in one of the brush apparatuses, stored in a cell preservation solution, and then subjected to a cytological examination at a later stage. The specimens stored in the cell stock solution are layered or centrifuged according to the concentration gradient, separated by a filter, and stained and examined.

However, if a cell-preserving solution used for liquid-cell examination is used, not only cells can not be stored for a long period of time, but blood cells, mucous and proteinaceous substances remaining in cells can not be completely removed, and a separate processing step is required. In particular, currently widely used liquid cell tests require expensive equipment. Therefore, there is a need to develop a cell-preserving solution capable of performing liquid-cell examination at a lower cost and capable of storing cells for a long period of time while maintaining the cell shape.

Disclosure of the Invention The present invention has been proposed in order to overcome the above-described problems of the prior art. The basic object of the present invention is to provide a method and apparatus capable of preserving cells collected from a human body for a long time in an original form, And to provide a gynecological cell-preserving solution which can be used as a gynecological cell.

It is another object of the present invention to provide a gynecological cell preservation solution capable of performing cytopathological examination at low cost.

The cell preservation solution according to the present invention having the above-mentioned object comprises a solvent comprising 35 to 50 w / v% alcohol and 50 to 65 w / v% distilled water; Buffer 1.0 to 2.5 w / v%; 0.1 ~ 1.5 w / v% blood remover; 0.1-1.5 w / v% protein precipitant; 0.1 to 1.5 w / v% of a blood coagulation inhibitor selected from the group consisting of sodium citrate and sodium oxalate; 0.1 to 2.0 w / v% of an anti-dry agent; Osmolality adjusting agent 0.1 to 1.5 w / v%; Preservatives 0.1 to 1.5 w / v%; And 0.1 to 1.5 w / v% of a mucolytic agent.

The alcohol that can be used as a solvent may be selected from the group consisting of ethanol, isopropanol, and combinations thereof, and the buffer may be tris (hydroxymethyl) methylamine (Tris).

On the other hand, the blood remover may be acetic acid, and the protein precipitant may be acetone or ammonium sulfate.

In addition, the drying inhibitor may be glycerin, and the osmotic pressure regulator may be selected from the group consisting of dextrose, sodium chloride, sodium bicarbonate, calcium chloride, potassium chloride, sodium lactate, and Ringer's solution.

The preservative that can be used may be selected from the group consisting of sodium azide, sorbic acid, and sodium benzoate.

The mucolytic agent may be selected from the group consisting of acetylcysteine, carboxymethyl cysteine, Erdostein, letostein, and polyethylene glycol-p- (1,1,3,3-tetramethylbutyl ) -Phenyl ether (polyethylene glycol-p- (1,1,3,3-tetramethylbutyl) -phenyl ether, Triton X-100).

The cell-preserving solution according to the present invention enables the cells isolated from the human body to be stored in the liquid form for a long period of time, for example, for use in liquid-phase cytology, and can easily grasp the morphology of cells in cytopathological examination, Especially for gynecological examinations.

In addition, the preservative solution according to the present invention eliminates blood, proteinaceous substances and mucus which cause difficulties in the cytopathological diagnosis, and thereby, the specificity and sensitivity of the abnormality of the cell through cytopathological examination are improved, It is expected to improve.

In particular, it is possible to reduce solution turbidity and cell degeneration caused by xylene which has been used in conventional cell preservation solutions, and to prevent drying of cells when necessary, thereby preventing change in dyeing phase due to drying. Accordingly, in the conventional pap smears, most of the cells that have been discarded can be preserved, thereby greatly improving the accuracy of the diagnosis.

In addition, most of the cell-preserving liquids currently used are expensive products developed by foreign companies, while cell-preserving liquids according to the present invention can be used for cytopathological examination at a relatively low cost. In addition, there is an advantage that an immunohistological test or a molecular biological test can be additionally performed using the sample remaining in the preservative solution.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing the degree of staining and cell morphology of specimen cells stored in a gynecological cell preservative prepared according to an embodiment of the present invention and a cell preservative prepared in a comparative example; FIG.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings.

The present invention minimizes the protein denaturation of the cells to preserve the original shape of the cells collected from the human body, maintains the proper pH during the storage period, prevents cell drying and corruption after cell harvesting, Lt; RTI ID = 0.0 > preservation < / RTI > solution in its original form. The cell preservation solution of the present invention having such a purpose can be used, for example, as a cell preservation solution for a gynecology sample.

The cell preserving solution for gynecologic specimens according to the present invention may include a solvent, a buffer, a blood remover, a protein precipitator, a blood coagulation inhibitor, an anti-drying agent, an osmotic pressure regulator, an antiseptic, a mucolytic agent and the like.

The solvent which can be used for the cell preservation solution of the present invention is preferably a mixed solvent of alcohol and distilled water. Alcohol can play a role in increasing cell adhesion and cell adhesion, as well as cell permeability, dehydration and bacterial inhibition. For example, the alcohol usable as a solvent may be selected from the group consisting of methanol, ethanol, isopropanol, and combinations thereof, preferably ethanol.

The alcohol as a solvent is added to the cell suspension at a concentration of 30 to 50 w / v%, preferably 35 to 45 w / v%, more preferably 35 to 40 w / v% (for example, 38.60 w / v% . The distilled water, which is another solvent constituting the mixed solvent, may be, for example, primary distilled water and may be used in an amount of 50 to 65 w / v%, preferably 55 to 60 w / v% (for example, 57.90 w / ). ≪ / RTI >

In particular, according to the present invention, a buffer may be used in the above-mentioned solvent for the purpose of maintaining the pH of the cell preserving solution at a constant level. By using a buffer, the pH of the cell stock solution can be maintained in the neutral range, for example, in the range of pH 6.0 to 9.5, thereby preventing denaturation of stored cells. That is, the buffer used in the cell preserving solution of the present invention maintains the cell shape in the original normal form by keeping the pH of the cell preserving solution constant, and at the same time, .

The buffer used for the cell preservation solution of the present invention may have a pK _a value of 7.0 to 9.0 at room temperature (15 to 25 ° C). For example, buffering agents that may be used in the cell preservative solution of the present invention is tris (hydroxymethyl) methylamine (tris (hydroxymethyl) methylamine, Tris ; pK a 8.06), 3 - {[ tris (hydroxymethyl) methyl] amino} propane sulfonic acid (3 - {[tris (hydroxymethyl ) methyl] amino} propane sulfonic acid, TAPS; pK a 8.43), 3- [N- tris (hydroxymethyl) methylamino] -2-hydroxy-propane sulfonic acid (3- (2-tris (hydroxymethyl) methylamino] -2-hydroxypropane sulfonic Acid, TAPSO, pK _a 7.63), 2 - {[tris (hydroxymethyl) amino} ethane sulfonic acid, TES, pK _a 7.40), N, N-bis (2-hydroxyethyl) glycine, Bicine pK _a 8.35) and N-tris hydroxymethyl) methyl glycine (N-tris (hydroxymethyl) methyl glycine, Tricine; pK a 8.05) can be at least one selected from the group consisting of, but the invention is not limited to illustrated buffer is never. Tris, TAPS, TAPSO, TES and Tricine can be preferably used as the buffer in the cell preservation solution according to the present invention, and Tris is particularly preferable.

Such a buffer may be used in a proportion of 1.0 to 2.5 w / v%, preferably 1.0 to 2.0 w / v% (for example, 1.45 w / v%) in the cell preserving solution. If the amount of the buffer is less than the above range, it may be difficult to maintain the pH, and if it is more than this range, other components can not be used, so that cell degeneration may occur during cell storage.

Meanwhile, the cell preserving solution according to the present invention includes functional additives for preventing cell drying and corruption during denaturation or smear of specimen cells, in addition to the above-mentioned solvent and buffer components. As an example of such a functional additive, a blood removing agent capable of providing a hemolytic function can be used. An example of a blood remover that can be used is acetic acid. The blood remover can be added to the cell stock solution of the present invention in an amount of 0.1 to 1.5 w / v%, preferably 0.5 to 1.0 w / v% (e.g., 0.72 w / v% ). ≪ / RTI >

Also included are protein precipitants for removing protein components that may be contained in the sample cells. Examples of usable protein precipitants include ketones such as acetone or ammonium sulfate, but the present invention is not limited thereto. In particular, when acetone is used, the cell can be immobilized, and the cell preservation solution of the present invention can effectively penetrate into the specimen cell.

The protein precipitating agent may be contained in the cell preservation solution of the present invention at a concentration of 0.1 to 1.5 w / v%, preferably 0.1 to 0.5 w / v% (for example, 0.24 w / v%). If the content of the protein precipitating agent is less than the above-mentioned range, it is difficult to expect a protein removal effect that can be contained in the specimen cells, and if the amount exceeds the above range, cell degeneration may occur.

In addition, a blood coagulation inhibitor may be contained in the cell preservation solution of the present invention. The use of the anticoagulant agent may cause the blood to be solidified in the middle of the process of eliminating the blood contained in the specimen cells, which may cause errors in the diagnosis process. Examples of anticoagulants that can be used according to the present invention include at least one selected from the group consisting of sodium citrate, ethylenediamine tetra-acetic acid (EDTA), sodium oxalate, and heparin. The anticoagulant may be contained in the cell suspension of the present invention in an amount of 0.1 to 1.5 w / v%, preferably 0.1 to 0.5 w / v% (for example, 0.12 w / v%).

In addition, a drying preservative may be used in the cell preservation solution according to the present invention. The drying inhibitor which can be particularly preferably used is a transparent compound, preferably glycerin, preferably 0.1 to 2.0 w / v%, preferably 0.3 to 1.0 w / v% (for example, 0.48 w / v%) It is used in quantities.

Furthermore, the cell preservation solution of the present invention includes an osmotic pressure regulating agent, whereby the preservation performance of the specimen cells can be improved by keeping the osmotic pressure of the cell preservation solution appropriately. The cell preserving solution that can be used may be selected from the group consisting of dextrose, sodium chloride, sodium bicarbonate, calcium chloride, potassium chloride, sodium lactate and Ringer's solution. For example, when the osmotic pressure regulator is used at a concentration of 0.1 to 1.5 w / v%, preferably 0.1 to 0.5 w / v% (for example, 0.12 w / v%) in the cell preservation solution of the present invention, Thereby improving the permeability to the specimen cells.

On the other hand, the cell-preserving solution according to the present invention may contain a preservative in order to prevent corruption caused by microorganisms or the like of cells to be a specimen. Examples of preservatives that can be used include at least one selected from the group consisting of sodium azide, sorbic acid, sodium benzoate and formalin. The preservative may be contained in the cell stock solution according to the present invention in an amount of 0.1 to 1.5 w / v%, preferably 0.1 to 0.5 w / v% (for example, 0.12 w / v%). If the content of the preservative is less than the above-mentioned range, antiseptic effects such as inhibition of microbial growth can not be expected. If the content exceeds the above range, the viscosity of the cell stock solution may become excessively high.

In addition, a mucolytic agent may be used for the cell preservation solution according to the present invention, which can be used, for example, for gynecologic examination. Components that can be used as a mucolytic agent include acetylcysteine, preferably Carboxymethylcystein, S-carboxy methylcysteine (SCMC), Erdostein, Letostein, ) And polyethylene glycol-p- (1,1,3,3-tetramethylbutyl) -phenyl ether (Triton X-100) And at least one selected from the group consisting of In particular, in the case of Triton X-100, it can be suitably used to disperse the mucus as a non-ionic surfactant. The mucolytic agent may be used at a concentration of 0.1 to 1.5 w / v%, preferably 0.1 to 1.0 w / v% (for example, 0.24 w / v%) in the cell preservation solution of the present invention.

The cell preserving solution formulated with the above components and concentrations can be stored in a container such as an ampoule or cryo tube in a lyophilized state at room temperature (15 to 30 ° C) or, if necessary, at about -80 ° C. Meanwhile, the specimen cells to be stored in the cell stock solution according to the present invention can be collected from the human body or other animals using, for example, a brush, and the specimen cells can be separated through centrifugation or the like.

At this time, the specimen cell to be stored may be a gynecologic specimen. Examples of gynecologic specimens include squamous epithelial cells, flattened live cells and malignant cells collected from the uterine cervix, but the gynecologic specimens that can be stored in the cell preservation solution according to the present invention are not limited thereto.

When the specimen cells are stored using the cell preservation solution according to the present invention, even if prolonged periods such as one week, one month, six months, 12 months, 18 months, or 24 months or more elapse, As shown in FIG.

The appropriate sample cells can then be isolated and stored in a container in which the cell stock solution is stored, and used for subsequent cytological examination. For this purpose, a method of laminating the specimen cells stored in the cell stock solution on a slide can be used.

In particular, according to the present invention, the accuracy of diagnosis can be improved by removing blood, mucus, and protein components that can be stored for a long time in a liquid state after collecting specimen cells and which interfere with cell diagnosis, It is possible to prevent the dyeing phase from changing due to drying. In particular, it is possible to reduce the turbidity and cell degeneration of xylenes used in conventional cell preservation solutions, and can save the cells that have been mostly discarded in the cytogenetic test, thereby improving the accuracy of the diagnosis. In addition, an immunohistological test or a molecular biological test can be additionally performed on the specimen cells remaining in the preservative solution.

Hereinafter, the present invention will be described by way of illustrative examples, but the present invention is by no means limited to the following examples.

Example 1: Preparation of cell preservation solution for gynecological sample

Cell storage solutions for gynecologic specimens were prepared according to the ingredients and blending ratios shown in Table 1 below.

Ingredients and compounding of cell preservation solution for gynecologic specimens ingredient Formulation (w / v%) function Ethyl alcohol 30 to 50 menstruum Distilled water (primary) 50 to 65 menstruum Acetic acid 0.1 to 1.5 Blood remover Acetone 0.1 to 1.5 Protein precipitant Sodium citrate 0.1 to 1.5 Blood coagulation inhibitor glycerin 0.1 to 2.0 Drying agent Dextrose 0.1 to 1.5 Osmotic pressure Sodium azide 0.1 to 1.5 antiseptic Tris 1.0 to 2.5 Buffer Triton-x 100 0.1 to 1.5 Mucolytic agent

Comparative Examples 1 and 2: Preparation of cell preservation solution for gynecological sample

Cell storage solutions for gynecologic specimens were prepared according to the ingredients and blending ratios shown in Table 2 below.

Ingredients and compounding of cell preservation solution for gynecologic specimens ingredient Comparative Example 1 (w / v%) Comparative Example 2 (w / v%) Ethyl alcohol 30 to 50 40 to 50 (methyl alcohol) Distilled water (primary) 50 to 65 50 to 60 Acetic acid 0.1 to 1.5 0.5 to 1.0 Acetone 0.1 to 1.5 0.1 to 0.5 Sodium citrate 0.1 to 1.5 0.1 to 0.5 glycerin 0.1 to 2.0 0.3 to 1.0 Xylene 0.1 to 1.5 - Dextrose 0.1 to 1.5 0.1 to 0.5 Sodium azide 0.1 to 1.5 0.1 to 0.5 Tris 1.0 to 2.5 1.4 to 2.0 Triton X-100 0.1 to 1.5 0.1 to 0.9

Experimental Example 1: Measurement of cell preservation state using gynecological cell storage solution

The samples collected from the human body were stored using the cell preserving solutions prepared in Example 1 and Comparative Example 1-2, and the preservation state of the cells was measured. Cervical cells were used as gynecologic specimens, specifically squamous epithelial cells, flattened live cells and malignant cells. Each sample is collected using a brush, centrifuged from these specimens into a centrifuge, and centrifuged to centrifuge the specimen. The specimen is then centrifuged, the specimen is put in a specimen bottle in which each cell stock is stored, and the stored cells are examined for abnormalities Respectively.

3) background (erythrocyte, hemolysis, and mucus removal) were used as the evaluation indexes, 1) nuclear (size, dyeability, chromatin, nucleolus, nuclear membrane), 2) cytoplasm (shape, dyeability, inclusion body and contents preservation state) The scores were classified as 0 (poor), 1 (good, satisfactory), 2 (good), and 3 (excellent) Table 3 below shows the results of evaluation index of cervical cells. FIG. 1 is a photograph showing the degree of staining and cell morphology of specimen cells stored in each cell-preserving solution in order to examine the cell preservation state of the gynecologic specimen cell smears according to the present example.

Gynecological sample evaluation index cells (squamous epithelium, flattened live cells, malignant cells) Comparative Example 1 Comparative Example 2 Example 1 nucleus 2 2 3 cytoplasm 3 3 3 background 3 3 3 Sum 8 8 9

As shown in Table 3 and FIG. 1, the gynecologic specimen cells stored in the cell preservation solution prepared according to the example of the present invention exhibited a significant decrease in nuclear and cytoplasmic retention And the hemolytic state and erythrocyte / mucus clearance state were much better.

Although the present invention has been described based on the preferred embodiments of the present invention, the present invention is not limited to the embodiments described above. It will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. It will be apparent, however, that such modifications and variations are all within the scope of the present invention.

Claims (6)

A solvent comprising 30-50 w / v% alcohol and 50-65 w / v% distilled water; Buffer 1.0 to 2.5 w / v%; 0.1 ~ 1.5 w / v% blood remover; 0.1-1.5 w / v% protein precipitant; 0.1 to 1.5 w / v% of a blood coagulation inhibitor selected from the group consisting of sodium citrate and sodium oxalate; 0.1 to 2.0 w / v% of an anti-dry agent; Osmolality adjusting agent 0.1 to 1.5 w / v%; Preservatives 0.1 to 1.5 w / v%; And 0.1 to 1.5 w / v% of a mucolytic agent.
The method according to claim 1,
Wherein the alcohol is selected from the group consisting of ethanol, isopropanol, and combinations thereof, and wherein the buffer comprises tris (hydroxymethyl) methylamine (Tris).
The method according to claim 1,
Wherein the blood remover is acetic acid, and the protein precipitant is acetone or ammonium sulfate.
The method according to claim 1,
Wherein the drying inhibitor is glycerin, and the osmotic pressure regulator is selected from the group consisting of dextrose, sodium chloride, sodium bicarbonate, calcium chloride, potassium chloride, sodium lactate, and Ringer's solution.
The method according to claim 1,
Wherein the preservative is at least one selected from the group consisting of sodium azide, sorbic acid, and sodium benzoate.
The method according to claim 1,
The mucolytic agent is selected from the group consisting of acetylcysteine, carboxymethyl cysteine, Erdostein, letostein and polyethylene glycol-p- (1,1,3,3-tetramethylbutyl) - Wherein at least one selected from the group consisting of polyethylene glycol-p- (1,1,3,3-tetramethylbutyl) -phenyl ether and Triton X-100) is selected.

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