KR20140120640A - Liquefied cell inspection preserving solution - Google Patents

Abstract

The present invention relates to a liquefied cell inspection preserving solution for cytological diagnosis of collected cells. The solution stores collected cells while maintaining the collected cells an appropriate range of pH, maintains a morphological structure by suppressing protein denaturation of cells, and prevents the drying and decay of the cells. The liquefied cell inspection preserving solution of the present invention comprises 40 to 70 vol% of a buffer solution, 1.0 to 10.0 vol% of ketone, 1.0 to 10.0 vol% of a cell coating agent, 0.1 to 2.0 vol% polyol, 0.2 to 5.0 vol% of cross-linking agents, 0.1 to 0.5 vol% of a detergent, 0.1 to 2.0 vol% of a solubilizing agent, 0.1 to 2.0 vol% of a pH-retaining agent, 0.1 to 2.0 vol% of a blood removing agent, 0.1 to 0.5 vol% of a preservative, and a residual amount of solvent.

Description

[0002] Liquefied cell inspection preserving solution [

The present invention relates to a liquid cell cytological preservation liquid, and more particularly, to a liquid cell cytological preservation liquid for preventing the drying and denaturation of cells while maintaining the morphological structure of collected cells for a long period of time in order to assist cytological diagnosis.

Ancillary solution for cytological diagnosis is a solution for Liquid Based Cytology which preserve the cells collected from the human body and diagnose cytopathologically. Liquid cell cytology is used for cervical cells, gynecologic specimens, and non-gynecologic specimens such as sputum, body fluids, urine, and fine needle aspiration biopsy. The collected cells are examined with a microscope to check for the presence of cancer cells.

Conventional cytopathologic examination is performed by Conventional Pap smear, in which cells are stained on a slide.

However, in this case, when the cells are coated on the glass slide, the cells are dried, and the mucous and erythrocytes are not removed.

In addition, only 10% of the cells collected from the cervix through the brush are smeared on the slide, and all the remaining cells are discarded. Therefore, the cells that are important for diagnosis are not likely to be spread on the slide.

Recently, a liquid-based cytology (Liquid Based Cytology) method has been developed to solve the above problems.

This is designed to allow the collected tissue cells to be stored in an auxiliary solution for cytological diagnosis and to carry out a subsequent pathological examination. The collected cells are collected with a brush, stored in a solution for storing cells, It is a method to diagnose the abnormality of cells by examining the cells stored in the cell storage solution.

And comparing the accuracy of the cytological examination according to the conventional cytopathological examination method and the accuracy of the cytological examination according to the liquid cytological examination method.

According to the cytology, there is a risk that the test specimen is unevenly distributed on the slide, and therefore, the diagnosis is different depending on which part is observed. However, when the liquid cell test is performed, the accuracy of the diagnosis is high because the test specimen is uniformly distributed on the slide.

In order to carry out this liquid cytology, a solution that can store the collected cells is required. The solution used in conventional liquid cytology was 95% ethanol.

However, in this case, there is a disadvantage that the cells can not be stored for a long period of time (it can be stored for about one week), and the red blood cells are not removed, thus requiring a separate process for removing red blood cells. And there was a disadvantage that DNA extraction could not be done due to modification of nucleus.

It is an object of the present invention to provide a preservation liquid for cell cytological examination for cytological diagnosis by preventing the cells from drying out while improving the preservability so that the collected cells can be kept for a long time while maintaining the morphological structure thereof.

In order to attain the above object, the present invention provides a liquid cell cytotoxic agent according to the present invention which comprises 40 to 70% of a buffer solution, 1.0 to 10.0% of a ketone, 1.0 to 10.0% of a cell coating agent, 0.1 to 2.0% of a polyol, 0.1 to 0.5% of a detergent, 0.1 to 2.0% of a mucolytic agent, 0.1 to 2.0% of a PH preservative, 0.1 to 2.0% of a blood remover, and 0.1 to 0.5% of a preservative, Wherein at least one of sodium azide, citric acid, sorbic acid, sodium benzoate, and formalin is selected as the preservative, and the preservative includes a green tea plant solution, It is characterized in that 0.5-2.5 parts by weight of the green tea powder is added to 100 parts by weight of distilled water.

The present invention relates to a cell-preserving solution for enhancing preservation while maintaining the morphological structure of the collected cells and preventing the cells from being dried. According to this, the present invention can be stored for a long time. And prevents the cells from being dried, so that it has a useful effect of giving accuracy in the examination of a specimen such as blood.

In addition, since the liquid cell cytological preservative of the present invention can store a sample at room temperature for a long period of time, the cost incurred in storing the sample can be greatly reduced.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a staining image of a specimen stored for 1 month in a liquid cell examination storage liquid according to an embodiment of the present invention; FIG.
FIG. 2 is a view showing a staining image of a specimen stored for 18 months or longer in a liquid cell test preservation solution according to an embodiment of the present invention. FIG.
3 is a view showing a staining image of a sample stored in a solution corresponding to a comparative example for one year.

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

The present invention relates to a preservation solution for cytologic diagnosis of collected cells. The present invention relates to a preservation solution for the cytological diagnosis of collected cells. The collected cells are kept while maintaining the PH in an appropriate range to maintain the morphological structure by inhibiting protein denaturation of cells, It is to prevent corruption.

The liquid cell cytological examination liquid according to the present invention is characterized by containing 40 to 70% of a buffer solution, 1.0 to 10.0% of a ketone, 1.0 to 10.0% of a cell coating agent, 0.1 to 2.0% of a polyol, 0.1 to 2.0% of a binder, 0.1 to 0.5% of a detergent, 0.1 to 2.0% of a mucolytic agent, 0.1 to 2.0% of a PH preservative, 0.1 to 2.0% of a blood remover, 0.1 to 0.5% of a preservative and remaining balance.

More specifically, the buffer solution employs a buffer solution of phosphoric acid, in order to maintain nucleation and osmotic pressure.

The solvent employs ethyl alcohol for cell permeability, dehydration and bacterial inhibition.

Acetic acid (acetic acid) is used as the PH preservative, but the concentration of PH 2.0 to 6.5 should be maintained to maintain a weak acidity.

The blood-removing agent may employ acetic acid so as to function as a hemolytic agent.

The crosslinking agent employs formaldehyde as an example, and has functions such as molecular crosslinking and production of insoluble products.

The detergent employs Nonidet P40, a surfactant NP cell.

The mucolytic agent may employ dithiothreitol to disperse the mucus.

In addition, glycerol may be employed as a kind of polyol in order to prevent drying of collected cells.

The ketone for carrying out the function of the fixation agent and the cell infiltration of the collected cells employs acetone having three carbon atoms.

Polyethylene glycol (PEG) can be used as a cell coating agent for cell coating, anti-shrinkage and nuclear retention.

As the preservative, at least one of sodium azide, citric acid, sorbic acid, sodium benzoate and formalin may be selected or a mixture of two or more thereof may be adopted.

More preferably, the preservative may be one containing a green tea plant other than the above-mentioned kind.

Sodium azide can be adopted as an example of an antiseptic agent, and a green tea powder employs a green tea powder powder dissolved in distilled water.

The green tea powder may be added with 0.5-5.0 parts by weight of the green tea powder, which is powdered with green tea, called natural preservative, per 100 parts by weight of distilled water.

This is because, if the amount is less than 0.5 parts by weight, the effect is not expected to be expected. If the amount is more than 5.0 parts by weight, the viscosity may increase.

The liquid cell cytotoxic agent having such a constitution is used for pre-sample preservation treatment of the collected cells.

Solvents, ketones, cell coating agents, polyols, crosslinking agents, and detergents are preliminarily compounded among the components constituting the above-mentioned liquid cell cytological preservation solution.

Thereafter, a buffer solution, a mucolytic agent, a PH preservative, a blood remover, and an antiseptic are mixed and then mixed with the pre-blended solution.

At this time, when the pre-formulated solution is referred to as the "A" solution and the post-formulated solution is referred to as the "B" solution, the mixing sequence of the "A" It is preferable to mix them.

The mixed liquid cell test preserving solution is preferably maintained at room temperature (15 to 30 ° C).

On the other hand, as an example of the specimen work, a specimen is put into a centrifuge, centrifuged by a rotary motion, the supernatant of the centrifuged specimen is removed, MonoLex is added, and the specimen is centrifuged after mixing.

Then, after removing the supernatant of the centrifuged specimen, the separated specimen is put into a specimen bottle and the slides are manufactured by using the equipment.

Such a sample treatment method may show a slight difference depending on the blood or mucus.

In the present invention, after collecting cells with a brush, the collected cells are stored in a liquid cell test preservation solution, and if necessary, cells stored in a liquid cell test preservation solution can be examined to diagnose the presence or absence of a cell.

Hereinafter, examples prepared according to the liquid cell cytology preservative of the present invention will be described in comparison with Comparative Examples.

[Example 1]

40 parts by weight of PH 3 phosphoric acid buffer solution, 1.0 part by weight of acetone, 2.0 parts by weight of polyethylene glycol, 0.5 part by weight of glycerol, 0.2 part by weight of formaldehyde, 0.1 part by weight of Nonidet P40 as detergent, 0.2 parts by weight of dithiothreitol, Acetic acid), 0.5 parts by weight of acetic acid, 0.2 parts by weight of sodium azide, and 54.8 parts by weight of ethyl alcohol.

[Example 2]

40 parts by weight of PH 3 phosphoric acid buffer solution, 2.0 parts by weight of acetone, 5.0 parts by weight of polyethylene glycol, 1.0 part by weight of glycerol, 1.0 part by weight of formaldehyde, 0.3 part by weight of Nonidet P40 as detergent, 0.5 parts by weight of dithiothreitol, Acetic acid), 1.0 part by weight of acetic acid, 0.3 part by weight of sodium azide, and 47.9 parts by weight of ethyl alcohol.

[Example 3]

40 parts by weight of PH 3 phosphoric acid buffer solution, 5.0 parts by weight of acetone, 5.0 parts by weight of polyethylene glycol, 2.0 parts by weight of glycerol, 3.0 parts by weight of formaldehyde, 0.5 part by weight of Nonidet P40 as a detergent, 0.5 parts by weight of dithiothreitol, Acetic acid), 1.0 part by weight of acetic acid, 0.4 part by weight of sodium azide, 0.1 part by weight of a green tea plant and 37.0 parts by weight of ethyl alcohol.

[Example 4]

40 parts by weight of PH 3 phosphoric acid buffer solution, 10.0 parts by weight of acetone, 9.0 parts by weight of polyethylene glycol, 2.0 parts by weight of glycerol, 5.0 parts by weight of formaldehyde, 0.5 part by weight of Nonidet P40 as a detergent, 0.5 parts by weight of dithiothreitol, Acetic acid) 1.0 part by weight, acetic acid 1.0 part by weight, sodium azide 0.5 part by weight, and ethyl alcohol 30.5 parts by weight.

[Example 5]

40 parts by weight of PH 3 phosphoric acid buffer solution, 5.0 parts by weight of acetone, 5.0 parts by weight of polyethylene glycol, 2.0 parts by weight of glycerol, 3.0 parts by weight of formaldehyde, 0.5 part by weight of Nonidet P40 as a detergent, 0.5 parts by weight of dithiothreitol, Acetic acid), 1.0 part by weight of acetic acid, 0.4 part by weight of sodium azide, 0.1 part by weight of a green tea solution and a part by weight of ethyl alcohol of 41.5.

[Example 6]

40 parts by weight of PH 3 phosphoric acid buffer solution, 5.0 parts by weight of acetone, 5.0 parts by weight of polyethylene glycol, 2.0 parts by weight of glycerol, 3.0 parts by weight of formaldehyde, 0.5 part by weight of Nonidet P40 as a detergent, 0.5 parts by weight of dithiothreitol, Acetic acid), 1.0 part by weight of acetic acid, 0.3 part by weight of sodium azide, 0.2 part by weight of a green tea plant and 37.0 parts by weight of ethyl alcohol.

[Comparative Example 1]

95% of the solution was used.

[Comparative Example 2]

Phosphate buffered saline (PBS) is used.

The results of the morphological experiment, the erythrocyte removal, the staining test, and the genetic test according to the storage period according to the embodiments of the present invention and the comparative examples are as follows.

The cells used in the above experiment are cells obtained by using a brush to collect cells of the cervix.

First, the morphological observations according to the storage period are to test the cell preservation performance in the preserved solution by measuring the morphological changes according to the storage period when the collected cells are stored in the preservation solution.

The experimental method is to store the collected cells in a preservation solution at room temperature.

After one month, six months, twelve months, eighteen months, and twenty four months, the cells stored in the storage solution were attached to a filter membrane, and the adhered cells were moved on a glass slide .

The cells transferred to the slide are immobilized in 95% ethanol and stained by Pap stain, and the cells are observed under a microscope.

The erythrocyte elimination experiment and the staining experiment were performed to determine whether red blood cells on the collected cells were removed by the preservation solution.

The erythrocyte removal method also involves transferring the cervical cells buried in the brush to the inside of the container by bringing a brush scrubbing the cervix to the bottom of the container containing the preservative solution of the present invention or the container containing the solution of the comparative example.

Then, after 24 hours, 2 cc of the solution was collected from each container, adhered to the ultrafiltration polymer thin film, and the attached cells were moved on a glass slide and stained and observed with a microscope.

The staining test method is the same as the method of collecting the cells in the erythrocyte elimination test. However, after 1 month or 1 year, the solution contained in each container is filtered through the ultrafiltration polymer membrane, and the stored cells are subjected to ultrafiltration To the polymer thin film.

Subsequently, the polymer membrane on which the cervix cells are transferred is taken on a cell observation glass slide and moved to the side of the glass slide.

The slides on which the cells have been transferred are immersed in 95% ethanol, fixed, stained, and observed with a microscope.

The method for examining the possibility of genetic testing is such that the collected specimen cells are stored at room temperature for one month or more in a preservative solution corresponding to the embodiments of the present invention or a comparative example, After repeating PCR (Polymerase Chain Reaction) 25 to 30 times, electrophoresis pictures are taken.

The experimental results of the embodiments of the present invention and the comparative examples applying the above-described experimental methods are shown in Table 1 below.

Morphological findings according to storage period Staining phase Erythrocyte elimination degree Possible genetic testing 1 month 6 months 12 months 18 months 24 months Example 1 adequate adequate adequate adequate inappropriateness adequate 85% possible Example 2 adequate adequate adequate adequate adequate adequate 90% possible Example 3 adequate adequate adequate adequate adequate adequate 95% possible Example 4 adequate adequate adequate adequate adequate adequate 95% possible Example 5 adequate adequate adequate adequate adequate adequate 95% possible Example 6 adequate adequate adequate adequate adequate adequate 95% possible Comparative Example 1 inappropriateness inappropriateness inappropriateness inappropriateness inappropriateness Inappropriate for more than 1 week Impossible Some available Comparative Example 2 Can not store at room temperature Can not store at room temperature Can not store at room temperature Can not store at room temperature Can not store at room temperature inappropriateness Impossible Possible in refrigerated storage

As shown in Table 1, the specimens stored in the liquid cell cytotoxic solution corresponding to the examples of the present invention retained the original shape of the cells even after prolonged elapsed time, whereas in the case of Comparative Examples 1 and 2, It is impossible to diagnose the specimen because it is impossible or the cell deformation occurs and the morphological structure can not be maintained.

FIG. 1 is a view showing a staining image of a specimen stored for about one month in a storage solution according to an embodiment of the present invention, and FIG. 2 is a graph showing a staining image of a specimen stored for 18 months or more in a storage solution corresponding to the embodiment of the present invention FIG.

As shown in FIG. 1 and FIG. 2, the specimen stored and stored in the preservative solution corresponding to the example of the present invention is distinguishable in different colors depending on the cell characteristics so that normal cells and cancer cells can be distinguished have.

On the other hand, FIG. 3 is a diagram showing a staining image of a specimen stored in a solution for one year in a solution corresponding to a comparative example. It can be seen that cytologic diagnosis is impossible because each cell of the specimen is denatured or destroyed or unclearly entangled.

In addition, the specimen stored in the preservative solution according to the embodiment of the present invention can be subjected to gene testing by repeating the PCR reaction several times as described above without any other procedure.

In particular, genetic testing can be performed using cells stored at room temperature, and the rate of genetic testing reaches about 99.9%.

Therefore, not only is it possible to perform rapid screening, but also a cell stored at room temperature for a long period of time can be genetically inspected, thereby greatly reducing the cost of storing cells for cytological diagnosis.

Claims (1)

The composition of claim 1, wherein the composition comprises 40 to 70% of buffer solution, 1.0 to 10.0% of ketone, 1.0 to 10.0% of cell coating agent, 0.1 to 2.0% of polyol, 0.2 to 5.0% of crosslinking agent, 0.1 to 0.5% of detergent, 0.1 to 2.0% of a PH preservative, 0.1 to 2.0% of a blood remover, and 0.1 to 0.5% of a preservative,
The buffer solution employs a phosphate buffer, employs alcohol as a solvent,
Wherein the preservative is selected from at least one of sodium azide, citric acid, sorbic acid, sodium benzoate, and formalin,
Wherein the preservative comprises a green tea plant liquid, wherein the green tea plant powder is dissolved in distilled water and 0.5-5.0 parts by weight of the green tea powder is added to 100 parts by weight of distilled water. Preservative.

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