CN114149960A - Sample density separating medium and cell separating method - Google Patents
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Abstract
The invention relates to the technical field of cytology slide preparation, in particular to a sample density separating medium and a cell separating method. The sample density separating medium provided by the invention consists of a solvent, ethylene diamine tetraacetic acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, dithiothreitol, tea saponin and sodium sulfite. The method can effectively soften and decompose mucus in a sample, the detection background is clear, and the observation of cell morphology is clear.
Description
Technical Field
The invention relates to the technical field of cytology slide preparation, in particular to a sample density separating medium and a cell separating method.
Background
The cervical cancer is the only current cause, and early-stage prevention cancer can be found, so that early-stage screening of the cervical cancer has important significance for preventing the cervical cancer. Liquid-based cytology examination, abbreviated as TCT, is a new cytology slide making technology which is proposed in recent years, and is a great innovation in the slide making field. Compared with the traditional smear, the liquid-based cytology preserves the specimen through the preservation solution, and then the specimen is treated through a series of early-stage technologies, so that impurity components in the specimen are removed, and a thin layer which is clearly observed under a microscope is manufactured, so that a reader can observe the specimen more easily, the diagnosis accuracy and the sensitivity of the smear are obviously improved compared with the traditional smear, and the effective collection and preservation of cervical exfoliated cells are the key technologies for screening cervical cancer.
The existing liquid-based cytology slide making technology comprises a membrane filtration method, a centrifugal sedimentation method and a centrifugal slide throwing method, wherein the automatic centrifugal sedimentation slide making technology is to add a collected sample into a sample density separation liquid, different cell types and components are utilized to separate different components in the sample under the action of centrifugal force, the first centrifugation is to enter normal cells in a preservation liquid into the density separation liquid, blood cells and leucocytes are divided into an upper layer and a lower layer due to the fact that the specific gravity of the blood cells and the leucocytes is smaller than that of the density separation liquid, so that the cells in the sample settle to the junction of the two layers, the cells with larger specific gravity are preferentially settled through secondary centrifugation, the enriched cells are transferred to a glass slide to be naturally settled, and finally observation and diagnosis are carried out under a microscope.
The main component of the existing sample density separation solution is a synthetic sucrose polymer, namely polysucrose for short, but after the density separation solution is added into a preservation solution, red blood cells which are not completely cracked by the preservation solution at the layered part of the preservation solution and the sample density separation solution are contacted with the polysucrose to be aggregated into serial money and deposited at the bottom of a tube to be mixed with sunken cells (epithelial cells and tumor cells) with diagnostic significance, so that the observation of a sample after subsequent preparation is influenced. In addition, the traditional sample density separating medium does not have the function of removing mucus, and the mucus wraps cells, so that the cells cannot be separated through density centrifugation, and the final flaking effect is influenced. Therefore, there is still a need in the art for further optimization of sample density separations.
Disclosure of Invention
In view of the above, the present invention provides a sample density separation solution and a cell separation method for improving the sheet preparation effect.
The sample density separating medium provided by the invention comprises a solvent, disodium ethylene diamine tetraacetate, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, dithiothreitol, tea saponin and sodium sulfite; the solvent consists of water, glycerol and phenoxyethanol.
The phenoxyethanol is used as a preservative, can deal with complex components in a cell sample, and can ensure that cells are preserved for a long time without morphological change; the disodium ethylene diamine tetraacetate has the cell anticoagulation effect, and is not easy to coagulate blood in cells; the potassium dihydrogen phosphate and the disodium hydrogen phosphate have a pH value buffering effect, and the potassium dihydrogen phosphate and the disodium hydrogen phosphate are matched with each other for use, so that the buffering effect can be improved; the sodium chloride and the potassium chloride are used for maintaining normal osmotic pressure of cells, keeping the cells not to swell or shrink, and keeping proper ion concentration to effectively keep the shape of the cells; dithiothreitol and tea saponin, which are helpful for cell mucus and blood to disperse and crack, so that cells are released and finally cells with diagnostic significance are collected; the antioxidant effect of the sodium sulfite protective agent can prevent the formation of crystalline substances during the long-term working process of automatic equipment.
In the solvent in the sample density separation liquid, the volume ratio of water, glycerol and phenoxyethanol is (70-90): (9-29): (0.5-2).
In some embodiments, the volume ratio of water, glycerol and phenoxyethanol in the solvent is 900:99:1, or 840:159:1, or 700:299: 1.
In some embodiments, the sample density separation fluid consists of a solvent and the following concentrations of components: 1-3 g/L of disodium ethylene diamine tetraacetate, 0.4-0.8 g/L of disodium hydrogen phosphate, 1-3 g/L of potassium dihydrogen phosphate, 0.1-0.3 g/L of potassium chloride, 6.5-8 g/L of sodium chloride, 0-0.2 g/L of dithiothreitol, 0-0.2 g/L of tea saponin and 0-0.1 g/L of sodium sulfite.
In some embodiments, the sample density separation fluid consists of a solvent and the following concentrations of components: 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of monopotassium phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.1g/L of dithiothreitol, 0.1g/L of tea saponin and 0.08g/L of sodium sulfite;
in other embodiments, the sample density separation fluid is comprised of a solvent and the following concentrations of components: 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of monopotassium phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.2g/L of dithiothreitol, 0.2g/L of tea saponin and 0.08g/L of sodium sulfite;
in other embodiments, the sample density separation fluid is comprised of a solvent and the following concentrations of components: 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of monopotassium phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.3g/L of dithiothreitol, 0.1g/L of tea saponin and 0.08g/L of sodium sulfite;
in other embodiments, the sample density separation fluid is comprised of a solvent and the following concentrations of components: 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of monopotassium phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.1g/L of dithiothreitol, 0.3g/L of tea saponin and 0.08g/L of sodium sulfite.
In the embodiment of the invention, the pH value of the sample density separation liquid is 6.8-7.2. In some embodiments, the sample density separation fluid has a pH of 6.8, 6.9, 7.0, 7.1, or 7.2.
In some embodiments, the sample density separation solution has a pH of 6.8, wherein the volume ratio of water, glycerol and phenoxyethanol in the solution is 900:99: 1; and contains 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of potassium dihydrogen phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.1g/L of dithiothreitol, 0.1g/L of tea saponin and 0.08g/L of sodium sulfite.
In some embodiments, the sample density separation fluid has a pH of 6.9, wherein the volume ratio of water, glycerol, and phenoxyethanol in the solution is 840:159: 1; and contains 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of potassium dihydrogen phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.1g/L of dithiothreitol, 0.1g/L of tea saponin and 0.08g/L of sodium sulfite.
In some embodiments, the sample density separation solution has a pH of 7.0, wherein the volume ratio of water, glycerol and phenoxyethanol in the solution is 700:299: 1; and contains 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of potassium dihydrogen phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.1g/L of dithiothreitol, 0.1g/L of tea saponin and 0.08g/L of sodium sulfite.
In some embodiments, the sample density separation fluid has a pH of 7.1, wherein the volume ratio of water, glycerol, and phenoxyethanol in the solution is 840:159: 1; and contains 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of potassium dihydrogen phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.1g/L of dithiothreitol, 0.3g/L of tea saponin and 0.08g/L of sodium sulfite.
In some embodiments, the sample density separation fluid has a pH of 7.2, wherein the volume ratio of water, glycerol, and phenoxyethanol in the solution is 840:159: 1; and contains 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of potassium dihydrogen phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.2g/L of dithiothreitol, 0.2g/L of tea saponin and 0.08g/L of sodium sulfite.
In some embodiments, the sample density separation fluid has a pH of 7.2, wherein the volume ratio of water, glycerol, and phenoxyethanol in the solution is 840:159: 1; and contains 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of potassium dihydrogen phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.3g/L of dithiothreitol, 0.1g/L of tea saponin and 0.08g/L of sodium sulfite.
The sample density separating medium has reasonable component proportion, can rapidly disperse cells, stabilize the pH value of the cells, prevent blood in a sample from coagulating, immediately crack red blood cells in the sample, dissolve mucin in mucus in the sample, and simultaneously carry out antiseptic sterilization, so that the sample can be stored for a long time, cell components with diagnostic significance are fully reserved, and the sample density separating medium has the advantages of simple preparation process, low operation difficulty, non-toxic and environment-friendly raw materials, low cost and easy realization of large-scale production.
The preparation method of the sample density separation liquid comprises the following steps:
dissolving sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate and disodium ethylene diamine tetraacetate in water, and stirring for 20min to obtain solution A;
adding dithiothreitol, tea saponin and sodium sulfite into the solution A, and stirring for 5min to obtain a solution B;
adding phenoxyethanol into the solution B, and stirring for 5min to obtain a solution C;
and adjusting the pH value to 6.8-7.2 by using a sodium hydroxide solution to prepare the sample density separation liquid.
The invention relates to application of a sample density separating medium in preparing a TCT smear.
The invention also provides a preparation method of the TCT smear, which comprises the following steps: and processing the sample by using the sample density separating medium disclosed by the invention, and then preparing the sample. In the present invention, the sample is a cervical swab.
The invention also provides a liquid-based cell examination method, which comprises the step of detecting after the sample is subjected to slice preparation by using the sample density separation liquid. The sample is a cervical swab.
The sample density separating medium provided by the invention consists of a solvent, ethylene diamine tetraacetic acid, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, dithiothreitol, tea saponin and sodium sulfite. The method can effectively soften and decompose mucus in a sample, the detection background is clear, and the observation of cell morphology is clear. Has good effect on cracking red blood cells and white blood cells, and can effectively avoid the interference on the observation result. And the sample density separation liquid has long storage time and stable detection effect. The components are environment-friendly and nontoxic, do not damage target cells, do not need a pretreatment step, and have the advantages of simple formula, simple and convenient operation and low storage cost. In addition, the antioxidant effect of the protective agent contained in the coating can prevent the formation of crystalline substances in the long-term working process of automatic equipment, and the coating is very suitable for being used on the automatic equipment.
Drawings
FIG. 1 shows the effect of flaking after treating cervical swabs with sample density separating medium (pH7.2) of test group 1;
FIG. 2 shows the effect of flaking after treating cervical swabs with sample density separating medium (pH6.5) of control group 1;
FIG. 3 shows the effect of flaking after cervical swabs were treated with sample density separating medium (pH7.2) of test group 2;
FIG. 4 shows the effect of flaking after treating cervical swabs with sample density separating medium (pH7.5) of control group 2.
Detailed Description
The invention provides a sample density separating medium and a cell separating method, and a person skilled in the art can realize the separation by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1
The separation solution was prepared as follows:
TABLE 1 Experimental group sample separation fluid formulation
TABLE 2 control sample isolate formulation
The preparation method of the sample density separation liquid comprises the following steps:
adding weighed sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate and disodium ethylene diamine tetraacetate into all purified water, and stirring for 20min to obtain a solution A.
② adding weighed dithiothreitol, tea saponin and sodium sulfite into the solution A and stirring for 5min to obtain solution B.
And thirdly, adding the weighed phenoxyethanol into the solution B, mixing and stirring for 5min to obtain a solution C.
Adding sodium hydroxide to make the PH value between 6.8 and 7.2.
And fifthly, the preparation environment is normal temperature and normal pressure.
Effect verification
The reagent of sample density separation liquid l liter is prepared according to the formulas described in the table 1 and the table 2, the pH value of the experimental group is adjusted according to the table 1, the pH value of the comparison group separation liquid is adjusted to 6.5 or 7.5, and 500ml of the sample density separation liquid is filled in each bottle.
Samples are respectively taken and put into centrifuge tubes filled with density separating medium of each experimental group or control group (6 mL of sample and 4mL of separating medium are put into each centrifuge tube), and the dyeing effect is observed after flaking and dyeing are carried out by a manual or automatic flaking dyeing machine (figures 1-4). The evaluation of the effect after dyeing is divided into three grades, the evaluation index is judged as shown in table 3, and the evaluation results are shown in tables 4 and 5;
TABLE 3 evaluation index judgment
Table 4 evaluation results
As shown in the experimental results of tables 4 and 5 and the figures 1 to 4, after the sample is added into the sample density separation liquid, the density separation effect of the experimental groups 1 to 6 is obviously better than that of the control groups 1 to 6, which shows that the formulas of the experimental groups 1 to 6 are obviously better than that of the comparative groups 1 to 6, the pH value is in the range of 6.8 to 7.2, the cell amount meets the requirements, the background is clean, and the number of blood cells, mucus cells and white blood cells does not influence the diagnosis requirement.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. The sample density separating medium is characterized by comprising a solvent, disodium ethylene diamine tetraacetate, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, dithiothreitol, tea saponin and sodium sulfite; the solvent consists of water, glycerol and phenoxyethanol.
2. The sample density separation solution according to claim 1, wherein the volume ratio of water, glycerol and phenoxyethanol in the solvent is (700-900): (99-299): (0-2).
3. The sample density separation solution of claim 1, wherein the volume ratio of water, glycerol and phenoxyethanol in the solvent is 900:99:1, or 840:159:1, or 700:299: 1.
4. The sample density separation liquid according to any one of claims 1 to 3, characterized by consisting of a solvent and the following components in concentrations:
1-3 g/L of disodium ethylene diamine tetraacetate, 0.4-0.8 g/L of disodium hydrogen phosphate, 1-3 g/L of potassium dihydrogen phosphate, 0.1-0.3 g/L of potassium chloride, 6.5-8 g/L of sodium chloride, 0-0.2 g/L of dithiothreitol, 0-0.2 g/L of tea saponin and 0-0.1 g/L of sodium sulfite.
5. The sample density separation liquid according to claim 4,
consists of a solvent and the following components in concentration: 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of monopotassium phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.1g/L of dithiothreitol, 0.1g/L of tea saponin and 0.08g/L of sodium sulfite;
or consists of a solvent and the following components in concentration: 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of monopotassium phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.2g/L of dithiothreitol, 0.2g/L of tea saponin and 0.08g/L of sodium sulfite;
or consists of a solvent and the following components in concentration: 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of monopotassium phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.3g/L of dithiothreitol, 0.1g/L of tea saponin and 0.08g/L of sodium sulfite;
or consists of a solvent and the following components in concentration: 1.86g/L of disodium ethylene diamine tetraacetate, 0.6g/L of disodium hydrogen phosphate, 1.44g/L of monopotassium phosphate, 0.2g/L of potassium chloride, 7g/L of sodium chloride, 0.1g/L of dithiothreitol, 0.3g/L of tea saponin and 0.08g/L of sodium sulfite.
6. The sample density-separated liquid according to any one of claims 1 to 5, wherein the pH value is 6.8 to 7.2.
7. The method for preparing a sample density separation solution according to any one of claims 1 to 6, comprising:
dissolving sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate and disodium ethylene diamine tetraacetate in water, and stirring for 20min to obtain solution A;
adding dithiothreitol, tea saponin and sodium sulfite into the solution A, and stirring for 5min to obtain a solution B;
adding phenoxyethanol into the solution B, and stirring for 5min to obtain a solution C;
and adjusting the pH value to 6.8-7.2 by using a sodium hydroxide solution to prepare the sample density separation liquid.
8. Use of the sample density isolate of any of claims 1 to 6 in the preparation of a TCT smear.
9. A method for preparing a TCT smear, comprising: processing a sample with the sample density separating medium of any one of claims 1 to 6 and then tabletting.
10. The method of claim 8, wherein the sample is a cervical swab.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106047795A (en) * | 2016-06-22 | 2016-10-26 | 杭州海世嘉生物科技有限公司 | Sample density separation liquid and preparation method thereof |
| CN107509722A (en) * | 2017-08-30 | 2017-12-26 | 迈克生物股份有限公司 | Sample preservation liquid |
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| CN113729009A (en) * | 2021-10-14 | 2021-12-03 | 山东高创医疗器械国家研究院有限公司 | Cervical cell preservation solution and preparation method and preservation method thereof |
| CN113945440A (en) * | 2021-10-14 | 2022-01-18 | 山东高创医疗器械国家研究院有限公司 | Papanicolaou staining kit, preparation method thereof and staining method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047795A (en) * | 2016-06-22 | 2016-10-26 | 杭州海世嘉生物科技有限公司 | Sample density separation liquid and preparation method thereof |
| CN107509722A (en) * | 2017-08-30 | 2017-12-26 | 迈克生物股份有限公司 | Sample preservation liquid |
| CN109797130A (en) * | 2019-01-18 | 2019-05-24 | 孝感宏翔生物医械技术有限公司 | A kind of cell extract and its application |
| CN110742059A (en) * | 2019-11-27 | 2020-02-04 | 武汉医尔特科技有限公司 | Liquid-based cell preservation solution and preparation method thereof |
| CN113729009A (en) * | 2021-10-14 | 2021-12-03 | 山东高创医疗器械国家研究院有限公司 | Cervical cell preservation solution and preparation method and preservation method thereof |
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