KR100642200B1 - Gel for pap smear and method for manufacturing the same - Google Patents

Abstract

Disclosed contents are gel preservatives that can mix high purity alcohol of 50 vol% or more in agar to uniformly distribute alcohol in gel mixture to inhibit bacterial growth and prevent cellular degeneration and obtain excellent cell sample retention. The manufacturing method and the cytology method using the same are related. The present invention is a first step of stirring while heating by mixing agar in powder form in distilled water; A second step of stopping heating and cooling when the mixed solution (agar solution) of distilled water and agar reaches a boiling point; A third step of mixing alcohol with the agar at a temperature where the agar is in a liquid state; And a fourth step of sequentially putting the liquid substance prepared by mixing the alcohol and the agar solution into a sealed container and cooling the gel to form a gel.

Cytology, cervical cancer, specimen, smear, specimen, preservative

Description

Gel Preservative and Manufacturing Method thereof {GEL FOR PAP SMEAR AND METHOD FOR MANUFACTURING THE SAME}

Detailed description of the embodiments of the present invention will be made with reference to the accompanying drawings, in which numerals designate corresponding parts in the drawings.

1 is a flow chart sequentially showing the manufacturing process of the gel preservative according to the present invention,

Figure 2 is a photograph showing a state of storing the sample collected in an airtight container containing a gel preservative of the present invention.

** Explanation of symbols for main parts of drawings **

10 container 12 lid

20: mechanism 30: preservative

The present invention relates to a preservative for preserving cytological specimens in an undenatured state, and more particularly, by mixing 50% by volume or more of high-purity alcohol with agar to allow alcohol to be present in the gelled mixture. The present invention relates to a gel-like preservative and a method for preparing the same, which can inhibit bacterial growth and prevent cellular degeneration by the function of alcohol volatilized in the container.

In general, the Pap smear for the screening of cervical cancer is a very important part of the pathological examination for morphological diagnosis of the disease. This test is an economical, simple, and highly efficient test method that has been instrumental in reducing deaths from cervical cancer in countries around the world since its introduction by Papanicolau in the 1940s.

However, in order to perform accurate diagnostic work in this cervical cancer cytology, it is important that the specimen be lightly smeared on the slide surface and secured with an appropriate fixative agent. Do. To this end, if possible, smear the glass slide thinly and immediately fix the sample with fixative solution. However, if the medical staff collecting the sample does not master the smearing technique or the method of handling the specimen, it may be difficult to obtain an appropriate cell specimen because of problems such as deterioration of fixation, drying in the air, or thick coating. Therefore, in order to obtain a better sample of the cell sample (sample), it is preferable not to smear the sample immediately on the spot, but to preserve the collected state as it is sent to the laboratory with the inspector who is experienced in handling the sample. However, this method must be able to prevent the denaturation of the cell sample that may occur during the transport period while keeping the sample intact. However, the sample can be preserved and sent to the laboratory in Pap smear. There is no known way.

According to domestic and international data, 1.5-60% of squamous epithelial lesions or invasive carcinomas of the cervix are not found and are false negative. Kim Hee-soo et al. Reported that 70% of false-negatives were reported in his thesis ("Herative Negative Rate of Cervical Pap smear Test in 1,000 Cases of Histologically Confirmed Endometrial Endothelial Tumors and Invasive Cancers") Abnormal sampling has been reported to occur and 64% of false negatives have been reported by Dodd et al. ("Quality-assurance study of simultaneously sampled, noncorrelating cervical cytology and biopsies", Diagn Cytopathol 9: 138-144, 1993). Is caused by a sampling error. As such, many errors occur in the process of sampling and smearing / fixing the sample, which may lead to a wrong diagnosis and a dangerous result for a patient who is diagnosed incorrectly. Recently, liquid-based cytology has produced relatively high quality sample slides by diluting the sample with liquid preservatives and sending it to the laboratory, but it requires time-consuming cell separation using expensive equipment. It is not widely implemented due to higher cost and higher price.

The simplest way to overcome the above problems is not to smear / fix the collected sample on site, but to send it to a skilled person so that smear / fixing can be done. This urgently demanded a preservative with excellent preservability to easily and easily preserve the collected samples and transport them to the laboratory.

Accordingly, an object of the present invention is to solve the above-mentioned defects and to meet the above expectations, and to store the sample in a container containing a gel-preserved material prepared by mixing high purity alcohol in agar (agar) In addition, the present invention provides a gel-like preservative that can obtain excellent preservation by inhibiting bacterial growth and preventing cellular degeneration by the function of alcohol volatilized in a container.

Another object of the present invention is to provide a method for preparing the preservative.

Another object of the present invention is to use a gel preservative to preserve the sample collected in the field as it is transported to the specialized pathology laboratory as it is to be able to make a sample by more accurate smear / fixation work to increase the test accuracy To provide a way.

Gel preservatives according to the present invention for achieving the above object agar; And alcohol mixed in the agar.

According to an aspect of the present invention, there is provided a method for preparing a gel preservative for preserving a cell sample, the method comprising: mixing agar in powder form with distilled water and stirring while heating; A second step of stopping heating and cooling when the mixed solution (agar solution) of distilled water and agar reaches a boiling point; A third step of mixing alcohol with the agar at a temperature where the agar is in a liquid state; And a fourth step of putting the liquid substance prepared by mixing the alcohol and the agar solution into a container, cooling the gel, and forming a gel.

Cell test method using a gel preservative according to the present invention for achieving the above another object is a first step of taking a sample and storing it in a preservative container containing a gel preservative; A second step of moving a sample stored in the preservative container into a laboratory; A third step of removing a sample from the preservative container in a laboratory and smearing / fixing it on a slide; A fourth step of manufacturing a sample slide readable by a microscope by staining a sample fixed to the slide; And a fifth step of diagnosing the sample slide by reading the microscope. Preferably, the method may further include a sixth step of capturing the specimen slide with a micrograph after the diagnosis.

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

The gel preservative of the present invention is in a gel form by mixing alcohol with agar. The alcohol dissolved in the agar is uniformly distributed, forming a fear filled with alcohol in the agar.

In this preservative, agar is a function of the basic structure containing alcohol in the solid state. It is transparent as polysaccharide, maintains solid at 85 ℃, and is not suitable for constituent of preservative because it is not decomposed by enzymes produced by bacteria. Agar concentrations range from 0.2% to 12% by volume. If the concentration of agar is less than 0.2% by volume, the preservative is almost liquid and the cell sample is mixed with the preservative, which makes it difficult to preserve the sample in its original form and prevents direct smearing of the sample. In addition, when the concentration of agar is more than 12% by volume, the agar solution has a thick porridge-like appearance, and it is difficult to form a gel when containing alcohol, and also has low volatility. This agar is a polysaccharide contained in cell walls matrix of various flushes, such as woodpecker or snails, obtained by hot water extraction, and classified into agarose without a sulfate group and agaropectin with a sulfate group. Since it is already widely used in foods and the like, the safety is guaranteed, so a detailed description of the manufacturing method is omitted here.

On the other hand, 50% or more of high purity alcohol (ethanol or methanol) is used as a functional material which exhibits a preservation effect. Alcohol is first prepared by dissolving agar in water (distilled water) to prepare agar solution, and then mixed in the agar solution, wherein the mixing ratio of alcohol to agar solution (volume ratio basis) is preferably about 0.3 to 1.5. Herein, if the mixing ratio of alcohol is 0.3 or less, cell preservation is inferior, and if it is 1.5 or more, the mixture becomes almost liquid and hardly gels. It is based on anhydrous alcohols of 99.9% purity and may be accompanied by some changes depending on alcohol purity. Since alcohol has strong volatility, it should be stored in an airtight container after manufacture.In case of preservation, the sample should be put in the airtight container and then closed and sealed.

The manufacturing method of this preservative of the structure demonstrated above is demonstrated in detail with reference to FIG. 1 is a flow chart sequentially showing the manufacturing process of the gel preservative according to the present invention, Figure 2 is a photograph showing a state of storing the sample collected in a sealed container containing the gel preservative of the present invention.

Agar in powder form is mixed with distilled water and stirred while heating (step S1). At this time, by adjusting the amount of agar powder mixed in distilled water can be variously adjusted the concentration of agar, agar concentration is suitable for 0.2% to 12% by volume.

When the mixed solution of distilled water and agar (hereinafter referred to as agar solution) reaches the boiling point, the heating is stopped and cooled (step S3). Then, while monitoring the temperature with a thermometer, when the temperature of the agar solution drops below 65 ° C, alcohol is slowly mixed into the agar solution (step S5). In this case, ethanol or methanol is used as the alcohol, and the mixing ratio of the alcohol to the agar solution (volume basis) is set within the range of 0.3 to 1.5.

The preservative in the liquid state thus prepared is put in a container and cooled in air, and as the temperature drops, the preservative forms a gel in the container. Thus, the present preservative contained in the sealed container for storage is shown in Figure 2, in Figure 2, the lid 12 of the sealed container 10 is opened and the sample (mechanical brush (brush) 20) with the collected sample is a transparent gel-like preservative ( 30 shows soaked and preserved.

Example.

100 cc of distilled water was poured into the flask, and the mixture was stirred while heating by mixing 4 g of agar powder. In this state, when the mixture of distilled water and agar reached the boiling point, the heating was stopped and the thermometer was cooled in the air with the thermometer in the mixture. At this time, the temperature of the agar solution fell to 65 ℃ or less while watching the thermometer 70cc of anhydrous alcohol (absolute ethanol) was mixed in the agar solution. The liquid substance thus prepared was placed in a container, cooled in air, and closed by a lid. As a result, the gel preservative was prepared in a sealed container for storage and usable anywhere.

Example of utility of this preservative.

1. Sample adequacy test.

After preserving 251 cervical cytoplasmic specimens in the present preservatives and transporting them to the laboratory, specimen slides were made in the laboratory and diagnostic tests of 251 cervical sinus cells were performed. In addition, the adequacy of the specimens was determined based on the sample adequacy provisions set forth in <Bethesda 2001>, an international diagnostic standard for cervical cytology.

For reference, the appropriate sampling standards prescribed by Bethesda 2001 are as follows, and all three conditions must be satisfied.

1) Squamous epithelial cells are well observed and the number should be more than 8,000-12,000.

2) There must be at least 10 well-preserved internal coronary cells or transitional cells.

3) The epithelial cells obstructed by inflammatory cells or blood should be 75% or less.

Of the 251 specimen slides, 192 specimen slides were produced within 3 days of sampling, and 59 specimen slides were made after 5 days of sampling. Is shown in.

Table 1. Adequacy test results (total 251 cases)

Criteria proper Inadequacy A. Number of squamous epithelial cells 242 9 (8 samples duplicated with B criteria) B. Endoscopic Cells / Transitional Cells 240 11 (8 specimens duplicated with A determination criteria) C. Epithelial Cells Occlusion 249 2 Sum 22 (14 samples except duplicates)

As can be seen in Table 1 above, the preservative was found to be ineligible in 22 cases (8.7%). In each sample, eight specimens with overlapping criteria A and B items were used. There were 14 cases, and the ratio of inadequate specimens to the total was 5.6%. In other words, the appropriate number of samples treated with this preservative was 94.4% of the total, so it was judged that the preservative had excellent preservation performance. For reference, the results are clear when compared to the applicant's usual Pap smear test samples during the same period (January to May 2004). During the same period, there were 464 cases of cervical cytology and the proportion of inadequate samples was 9.7% (45/464).

2. Sample adequacy testing over time until sample slide production date.

The specimens arrived at the laboratory were divided into two groups according to the time required from collection to preparation of the sample slides. Here, one group made a sample on a normal working schedule (within 3 days of collection) after the sample arrived, and the other group made a sample slide after 5 days of collection. In general, in case of cytological examination, the production of samples is rarely delayed by more than 5 days. Therefore, an unavoidable case was set by setting the delay date to 5 days.

Table 2. Number of ineligible samples due to the sampling period

Total number a. Squamous cell number b. Endoscopic Cells / Conjunctival Cells c. Epithelial cell obstruction The number of inappropriate Frequency of inadequate specimens relative to total cases Normal production group (within 3 days after collection) 192 6 (6) 9 (6) 2 17 (the number of rooms except duplicate: 11) 5.7% (11/192) Delayed production group (5 days after collection) 59 3 (2) 2 (2) 0 5 (excluding duplicates: 3) 5.1% (3/59) Sum 251 9 (8) 11 (8) 2 22 (the number of rooms except duplicate: 14) 5.6% (14/251)

In Table 2 above, the values in parentheses indicate the number of duplicated items in test items a and b.

As can be seen in Table 2 above, the specimens were prepared and inspected within 3 days after being collected in the preservative, or when the specimens were produced after 5 days after being preserved in the preservative. It was found that the number of inadequacies was very good, within 6%. Therefore, it was found that the preservative can effectively prevent the denaturation of sample cells even if stored for 5 days after sample collection. In addition, although the inadequateness was slightly lower in the delayed sample, this may be related to the total number of test cases less than that of the normal production group, and there may be environmental factors, where deviations within 1% are negligible, which can be changed at any time by external factors in the cytology. Judging by the level. Normal preservation laboratory work will be made within two to three days after sampling, so this preservative will satisfy this practical work.

3. Testing of denaturation according to existing cytology and the use of this preservative.

The cases of smearing and testing immediately after collection by conventional cytometry and smearing of preserved specimens in this preservative were tested by smearing in laboratory and classified according to the degree of denaturation. Materials were collected from Pap smears performed during the same period (January to May 2004) with this preservative.

Table 3. Comparison of denaturation in conventional cytology and cytology after treatment with this preservative.

Cytometry Total Classification according to cell degeneration I Ⅱ Ⅲ Ⅳ Conventional Inspection 464 261 104 44 35 This preservative application inspection method 251 245 6 0 0

Group I is a normal group where no degeneration was observed or below 5% of all smear cells. Group II is a case where degeneration is observed in 5 to 25% of all smear cells. Degeneration was observed in 25% to 50% of all smears and group Ⅳ was classified as more than 50% of all smears. In the above table, the sample using this preservative is significantly lower in sample denaturation compared to the existing test method, which is excellent as a test method. In the existing test method, group II or more was 39.4% (183/464), whereas only 6 cases (2.4%) belonged to group II, and none of group III or more occurred. Judging from the results, the preservatives had a very low rate of sample denaturation and a slight degree of degeneration, which was superior to conventional cytology in terms of denaturation. Thus, it can be seen that the inspection using this preservative can almost completely prevent the possibility of reading error due to sample denaturation.

Referring to the method of testing the cells using the gel-like preservatives described above is as follows.

1. Gel Preservative Treatment

The doctor takes the sample out of the country and puts it in a container with gel preservative and closes the lid. Here, in the foreign sense, it includes a doctor who does not reside in a skilled pathologist.

2. Sample transport

Samples stored in a preservative container are transferred to a pathology laboratory. At this time, the pathology laboratory may be its own pathology laboratory or distant consignment laboratory. As such, the specimens are stored in a preservative container without being directly smeared in the field and transported to a specialized pathology laboratory, thereby preventing the problem of breakage of the glass slide in the transportation stage.

3. Smear / Fixing Process

The specimens stored in the transported preservative container are fixed by smearing them on a glass slide after removing them from the container by a skilled pathologist in a pathology laboratory. In this case, 95% alcohol solution is used as a fixative. As such, the specimen stored in the preservative container is transported to the pathology laboratory to perform the smearing and fixation work by an experienced pathologist. You can prevent it. Furthermore, since it is carried out by an experienced pathologist in the pathology laboratory, homogeneous and excellent sample preparation is possible, thereby increasing the accuracy of the test in the next step.

4. Sample Slide Making

At the end of the fixation process, the pathologist completes the sample slides so that the slides can be stained and read by the microscope.

5. Sample Slide Reading

The pathologist makes a diagnosis by reading the microscope slide prepared in the above step under a microscope. Prior to this, if a necropsy is needed, the cell pathologist may undergo an examination.

6. Photomicrograph

At the end of the microscopic diagnosis, part of the specimen is taken by another cytopathologist under a microscope. In this way, since the slide search is performed once more at the stage immediately before the diagnosis result is taken in order to take a microscope picture, it is possible to reduce the possibility of misdiagnosis that may occur during the reading process at the previous stage. The sample photograph thus taken is reported with the diagnosis result. This has the effect of instilling the reliability of the cytology in the patient.

The embodiments disclosed herein are only presented by selecting the most preferred examples to help those skilled in the art from the various possible examples, the technical spirit of the present invention is not necessarily limited or limited only by this embodiment, the present invention Various changes and modifications are possible within the scope without departing from the spirit of the invention, as well as other equivalent embodiments.

As described above, the present invention does not need to smear / fix the sample cells immediately at the place where the sample is collected. There is an effect that can be produced.

In addition, since the preservative of the present invention is a gel rather than a liquid, it is possible to prevent the sample from being unwound and change the original shape, and to preserve the sample as it is close to the original, so that the smearing operation can be easily performed. Furthermore, the sample treated with the present preservative has a low degree of denaturation of cells, so that the nucleus and cytoplasm are stained vividly, which is advantageous for reading. In addition, the present invention can be stored in the original state for a long time by storing the collected sample, when the smear / fixation work is wrong or abnormal, the smeared again using the preserved sample to prepare a sample to be tested Can be. It is also possible to use it as a sample for other types of additional tests, such as the Human Papilloma Virus (HPV) test.

In particular, the cell test method of the present invention can prevent the breakage problem of the glass slide in the transport step because the specimen is stored in a preservative container and transported to a specialized pathology laboratory without smearing directly at the sample collection site. In addition, the specimens stored in the preservative container are transported to the pathology laboratory to perform the smearing and fixing work by skilled inspectors, thus preventing the problem of poor sample preparation caused by the smearing / fixing directly by inexperienced doctors or nurses. can do. Furthermore, since it is performed by a skilled pathologist in a pathology laboratory, it is possible to produce a homogeneous and excellent sample, thereby increasing the accuracy of the test. In addition, since the slide search is performed once more at the stage immediately before the diagnosis result report to take the microscopic findings, it is possible to reduce the possibility of misdiagnosis that may occur during the previous stage reading process. This has the effect of instilling the reliability of the cytology in the patient.

Claims (15)

A first step of mixing while stirring the powder agar in distilled water heating; A second step of stopping heating and cooling when the mixed solution (agar solution) of distilled water and agar reaches a boiling point; A third step of mixing alcohol with the agar at a temperature where the agar is in a liquid state; And And a fourth step of putting the liquid substance in which the alcohol and the agar solution are mixed into a closed container, and cooling the same to form a gel. The method of claim 1, wherein the concentration of the agar powder mixed in the distilled water in the first step is 0.2% by volume to 12% by volume. The method of claim 1, wherein the alcohol is mixed when the temperature of the agar solution falls below 65 ° C in the third step. 4. The method of claim 1 or 3, wherein ethanol or methanol having a purity of 50% or more is used as the alcohol in the third step. The method of claim 4, wherein the mixing ratio (volume ratio) of the alcohol to the agar solution is set in the range of 0.3 to 1.5. Agar; And an alcohol mixed in the agar. 7. The gel preservative according to claim 6, which is used for cell preservation for preserving the collected cell sample. 7. The gel preservative of claim 6, wherein the gel agar is dissolved in distilled water with a concentration of 0.2% by volume to 12% by volume. delete 7. The gel preservative of claim 6, wherein ethanol or methanol having a purity of 50% or more is used as the alcohol. The gel preservative according to claim 10, wherein the mixing ratio (volume ratio) of the alcohol to the gel agar is set in the range of 0.3 to 1.5. delete delete delete delete

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