CN113973804A

CN113973804A - Cervical cell preservation solution

Info

Publication number: CN113973804A
Application number: CN202111226657.6A
Authority: CN
Inventors: 张珺; 邹亚平; 狄媛媛; 胡鹏高
Original assignee: Wuhan Zhongji Biotechnology Co ltd
Current assignee: Wuhan Zhongji Biotechnology Co ltd
Priority date: 2021-10-21
Filing date: 2021-10-21
Publication date: 2022-01-28

Abstract

The invention relates to the technical field of cell preservation, and more particularly relates to cervical cell preservation solution, which comprises the following components: alcohol solution, buffer solution, acid solution, surfactant, sodium chloride, anticoagulant and metal ions. The cell preservation solution does not contain components harmful to human bodies, and is safe and environment-friendly; the sample containing blood and mucus has good processing capability; denaturalizing protein in the exfoliated cervical cells, stopping cell movement, and keeping the cell morphology in the state of the cells when the cells are sampled; can be preserved and sterilized, and can ensure that cervical cells preserved in the cervical cells are not decomposed by external microorganisms; the cells can be preserved for a longer time.

Description

Cervical cell preservation solution

Technical Field

The invention relates to the technical field of cell preservation, and particularly relates to cervical cell preservation solution.

Background

Both pap smear and TCT are cytological examinations of exfoliated cervical cells. The pap smear method is to scrape off cervical exfoliated cells with a vaginal speculum and observe whether the cells are abnormal or not under a microscope. The pap smear method is low in price and convenient for general survey, is widely used for cervical cancer screening since the advent of 1941, and makes an important contribution to reducing the incidence and mortality of cervical cancer. The pap smear method is cheap and convenient for general survey, but has the defects that the sample cells prepared by the method are piled together, so that the method is inconvenient to observe, the diagnosis accuracy is low, and certain misdiagnosis and missed diagnosis rate exist. The TCT technology is characterized in that cells are brushed out by a small brush, then the cells are brushed into a small bottle filled with a fixing liquid, a high-quality smear is obtained through the steps of centrifugation, flaking, dyeing and the like, and the screening rate is high, and the missed diagnosis rate is low. At present, TCT technology has largely replaced traditional pap smears for cervical cancer screening. According to cervical cancer diagnosis and treatment specifications (2018) in China, TCT screening is mainly adopted for women in China, HPV detection can be used as effective supplement of TCT, and the combination of the TCT and the HPV detection is favorable for improving screening efficiency.

The cell preservation solution is a key reagent for determining the quality of TCT detection. The collected cervical cell sample often contains blood, mucus and other components, which can shield cells with diagnostic significance and influence subsequent diagnosis. A cell preservation solution having excellent performance is required to have the ability to handle blood-containing and mucus-containing samples.

At present, the cell preservation solution on the market absolutely causes pollution to the environment and poses certain threat to the health of detection personnel; the shape of the cell is not well fixed, and the shapes of the nucleus and cytoplasm cannot be well reserved; the lack of ability to properly process samples containing mucus and blood has potential implications for subsequent diagnosis; or contain some unnecessary components, which significantly increases the cost.

Disclosure of Invention

In order to solve the above-mentioned technical problems, the present invention provides a method,

the cervical cell preservation solution has the following advantages:

1. the composition does not contain components harmful to human bodies, and is safe and environment-friendly;

2. the sample containing blood and mucus has good processing capability;

3. can denature protein in cervical exfoliated cells, stop cell movement, and maintain cell morphology in the state of the cells during sampling;

4. can be preserved and sterilized, and can ensure that cervical cells preserved in the cervical cells are not decomposed by external microorganisms;

5. the cells can be preserved for a longer time;

6. can avoid generating precipitate and prevent cervical cells from aggregating to form clusters to influence observation;

7. the cost is low.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a cell diagram of example 1 of the present invention;

FIG. 2 is a cell map of example 2 of the present invention;

FIG. 3 is a cell map of example 3 of the present invention;

FIG. 4 is a cell map of example 4 of the present invention;

FIG. 5 is a cell map of example 5 of the present invention;

FIG. 6 is a cell diagram of comparative example 1 of the present invention;

FIG. 7 is a cell diagram of comparative example 2 of the present invention;

FIG. 8 is a cell diagram of comparative example 3 of the present invention;

FIG. 9 is a cell diagram of comparative example 4 of the present invention;

FIG. 10 is a cell diagram of comparative example 5 of the present invention;

FIG. 11 is a cell diagram of comparative example 6 of the present invention;

FIG. 12 is a cell diagram of comparative example 7 of the present invention;

FIG. 13 is a cell diagram of comparative example 8 of the present invention;

FIG. 14 is a cell diagram of comparative example 9 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings of the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.

An embodiment of the present invention provides a cervical cell preservation solution, including: alcohol solution, buffer solution, acid solution, surfactant, sodium chloride, anticoagulant and metal ions.

The alcohol solution plays a role of a fixing agent, so that on one hand, protein in cervical exfoliated cells can be denatured, cell activity is stopped, and the cell morphology is kept in the state of the cells during sampling; on the other hand, alcohols also have the function of antisepsis and sterilization, can ensure that cervical cells preserved in the alcohols are not decomposed by foreign microorganisms, and the application does not need to additionally add preservatives and can save the cost.

The secondary structure of the protein is mainly maintained by hydrogen bonds, and the original intermolecular hydrogen bonds of the protein can be changed by the great change of the pH value, so that the secondary structure of the protein is damaged, and the cell morphology is changed unpredictably. The buffer solution is introduced into the cell preservation solution, so that a pH environment similar to the living state of the cervical cells preserved in the cell preservation solution can be provided, and the function of maintaining the cell morphology is achieved.

The cells have certain ion concentration, and if the ion concentration in the solution in which the cells are positioned is too low, the cell morphology can expand due to osmosis, and the cells can be broken when the cell morphology is serious. Therefore, the cell preservation solution needs to have certain ionic strength to maintain the osmotic pressure balance inside and outside the cells, and sodium chloride is a good osmotic pressure regulator.

Cervical cell proteins in the cell preservation solution are inactivated by the action of the alcohol solution, so that the ion pump on the cell membrane is disabled, the concentration of metal ions for maintaining the protein structure in the cells is reduced by gradual penetration into the solution, the protein structure is unstable, and the cell nucleus is mainly disintegrated macroscopically. The metal ions are added into the cell preservation solution to maintain the structure of cell nucleus, which is beneficial to long-term preservation of cervical cell samples.

Therefore, the alcohol solution, the buffer solution, the sodium chloride and the metal ions can jointly keep the shape of the cells, so that the cell deformation is avoided, and the subsequent detection accuracy is influenced.

Anticoagulants prevent blood coagulation and produce red precipitates, which can further affect the diagnosis of the blood sample.

The surfactant can promote mucolysis and prevent cervical cells from aggregating and agglomerating to influence observation

In another embodiment of the present invention, the alcohol solution is one or more of methanol, ethanol, and isopropanol.

The alcohols have reasonable price and no harm to cells and environment.

In another embodiment of the present invention, the metal ion is one or more of Ca2+, Cu2+, Fe2+, Mn2+, Zn2 +.

The metal ions have low price and easy acquisition, and can better maintain the structure of the cell nucleus.

In another embodiment of the invention, the anticoagulant is one or more of sodium ethylene diamine tetracetate, potassium ethylene diamine tetracetate, sodium citrate, potassium citrate, lithium heparin and sodium heparin.

The anticoagulant has good anticoagulation effect, and red precipitate is not generated after the anticoagulant is used.

In another embodiment of the present invention, the buffer is one of disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydroxide, Tris-hydrochloric acid, HEPES, and barbituric acid.

The buffer solutions are easy to purchase, low in price and low in cost, and can keep the cervical cell preservation solution in a relatively stable pH range.

In another embodiment of the present invention, the pH of the buffer is 6 to 8.

When the pH is more than 8 or less than 6, the pH of the formed cervical cell preservation solution is greatly different from the pH of human cervical cells, and the stability of the cells is influenced.

In another embodiment of the present invention, the acid solution is acetic acid.

The major diagnostic factor in blood samples is erythrocytes, which have fragile cell membranes, and acetic acid breaks the cell membranes of erythrocytes, but not cervical cells. The cell membrane of a ruptured red blood cell is not easily observed under a microscope. The addition of acetic acid eliminates the effect of red blood cells on the diagnosis.

In another embodiment of the present invention, the surfactant is a nonionic surfactant, which can prevent the electrolyte in the solution from affecting the performance of the surfactant.

In another embodiment of the present invention, the surfactant is one or more of long-chain fatty alcohol polyoxyethylene ether, alkylphenol polyoxyethylene, fatty acid polyoxyethylene ester, polyoxyethylene alkylamine, and polyoxyethylene alkylolamide.

The nonionic surfactants have the advantages of low cost, good mucus dissolving effect, and no aggregation.

In another embodiment of the present invention, the pH of the preservation solution is 6 to 8. When the pH of the preservation solution is more than 8 or less than 6, the pH value of the preservation solution is greatly different from that of cervical cells of a human body, and the stability of the cervical cells is damaged.

In another embodiment of the present invention, the alcohol solution, the buffer solution, the acid solution, the surfactant, the sodium chloride, the sodium ethylene diamine tetracetate, and the metal ions are in a mass ratio of: 20-50: 1-5: 0.1-1: 0.01-0.5: 0.1-1: 0.1-1: 0.01-0.1.

The cervical cell preservation solution obtained by adopting the proportion has stable pH and better mucus dissolving effect, prevents red blood cell precipitation, and can better maintain the cell morphology and the sterilization effect.

In order to make the presentation of the solution of the present application clearer, it is explained by the following examples.

Example 1

Adding 400g of ethanol, 21.85g of disodium hydrogen phosphate, 6.09g of sodium dihydrogen phosphate, 5g of acetic acid, 2g of alkylphenol polyoxyethylene, 9g of sodium chloride, 2g of sodium ethylene diamine tetracetate and 1.3g of calcium chloride into 500g of pure water in sequence, and continuously supplementing the pure water to 1000g after all the substances are completely dissolved to obtain the cervical cell preservation solution; the pH of the cell preservation solution was adjusted to 7 with sodium hydroxide. The cell preservation solution has good antibacterial property, and has no phenomenon of cell aggregation and conglomeration caused by microorganism growth, good cell morphology maintenance, red precipitate generated by blood coagulation, and mucus.

Example 2

Sequentially adding 200g of methanol, 10g of HEPES, 1g of acetic acid, 0.1g of long-chain fatty alcohol-polyoxyethylene ether, 1g of sodium chloride, 1g of sodium citrate and 0.21g of copper chloride into 300g of pure water, and continuously supplementing the pure water to 1000g after all substances are completely dissolved to obtain the cervical cell preservation solution; the pH of the cell preservation solution was adjusted to 6 with hydrochloric acid. The cell preservation solution has good antibacterial property, no microorganism growth, good cell morphology maintenance, no red precipitate generated by blood coagulation, and no aggregation and agglomeration of cervical cells.

Example 3

Sequentially adding 500g of isopropanol, 36g of monopotassium phosphate, 14g of sodium hydroxide, 10g of acetic acid, 5g of polyoxyethylene fatty acid ester, 10g of sodium chloride, 10g of heparin sodium and 2.1g of zinc chloride into 380g of pure water, and continuously supplementing the pure water to 1000g after all substances are completely dissolved to obtain the cervical cell preservation solution; the pH of the cell preservation solution was adjusted to 8 with sodium hydroxide. The cell preservation solution has good antibacterial property, and has no phenomenon of cell aggregation and conglomeration caused by microorganism growth, good cell morphology maintenance, red precipitate generated by blood coagulation, and mucus.

Example 4

Sequentially adding 100g of methanol, 100g of ethanol, 100g of isopropanol, 30g of barbituric sodium-hydrochloric acid, 6g of acetic acid, 1g of polyoxyethylene alkylolamide, 2g of polyoxyethylene alkylamine, 6g of sodium chloride, 3g of heparin lithium, 2g of ethylene diamine tetraacetic acid and 1.8g of manganese chloride into 420g of pure water, and continuously supplementing the pure water to 1000g after all substances are completely dissolved to obtain the cervical cell preservation solution; the pH of the cell preservation solution was adjusted to 7 with sodium hydroxide. The cell preservation solution has good antibacterial property, and has no phenomenon of cell aggregation and conglomeration caused by microorganism growth, good cell morphology maintenance, red precipitate generated by blood coagulation, and mucus.

Example 5

Sequentially adding 120g of methanol, 230g of ethanol, 18g of Tris-hydrochloric acid, 14g of barbital sodium-hydrochloric acid, 7g of acetic acid, 1g of polyoxyethylene alkylolamide, 7g of polyoxyethylene alkylolamide, 3g of heparin lithium, 2g of sodium ethylene diamine tetracetate and 0.9g of ferrous chloride into 420g of pure water, and continuously supplementing the pure water to 1000g after all substances are completely dissolved to obtain the cervical cell preservation solution; the pH of the cell preservation solution was adjusted to 7 with sodium hydroxide. The cell preservation solution has good antibacterial property, and has no phenomenon of cell aggregation and conglomeration caused by microorganism growth, good cell morphology maintenance, red precipitate generated by blood coagulation, and mucus.

Comparative example 1

The procedure of example 1 was repeated except that calcium chloride was not added. The cell preservation solution has good antibacterial property, and has no phenomenon of cell aggregation and conglobation caused by microorganism growth, red precipitate generated by blood coagulation and mucus, but abnormal cell nucleus morphology and cell nucleus loss of some cells.

Comparative example 2

The same procedure as in example 1 was repeated except that alkylphenol ethoxylates were not added. The cell preservation solution has good antibacterial property, no microorganism growth, good cell morphology maintenance, and no red precipitate generated by blood coagulation, but has cell aggregation and agglomeration phenomenon caused by mucus. (FIG. 5)

Comparative example 3

The procedure of example 1 was repeated except that sodium chloride was not added. . The cell preservation solution has good antibacterial property, and has no phenomenon of cell aggregation and conglobation caused by microorganism growth, red precipitate generated by blood coagulation and mucus, but has poor cell morphology, and cell rupture caused by water absorption and expansion of cells.

Comparative example 4

The procedure of example 1 was repeated except that no sodium ethylenediaminetetraacetate was added. The cell preservation solution has good antibacterial property, no microorganism growth, good cell morphology maintenance, and no cervical cell aggregation, but has red precipitate generated by blood coagulation.

Comparative example 5

Adding 400g of ethanol, 21.85g of disodium hydrogen phosphate, 6.09g of sodium dihydrogen phosphate, 5g of acetic acid, 2002g of tween-2002, 9g of sodium chloride, 2g of sodium ethylene diamine tetracetate and 25g of calcium chloride into 500g of pure water in sequence, and continuously supplementing the pure water to 1000g after all the substances are completely dissolved to obtain the cervical cell preservation solution; the pH of the cell preservation solution was adjusted to 7 with sodium hydroxide. The cell preservation solution has good antibacterial property, no microorganism growth, no red precipitate generated by blood coagulation, cell aggregation and agglomeration caused by mucus, poor cell morphology, partial cell membrane damage and unclear cell boundary.

Comparative example 6

The procedure of example 1 was repeated except that the pH of the cell sap was not adjusted with sodium hydroxide and was measured with a pH strip to obtain a pH of 5. The cell preservation solution has good antibacterial property, no microorganism growth, and poor cell morphology.

Comparative example 7

The metal ions were added as magnesium chloride, and the rest was the same as in example 1. The cell preservation solution has abnormal cell morphology, and a small amount of cell nuclei are lost.

Comparative example 8

The acetic acid of the example was replaced with hydrochloric acid, and the rest was the same as in example 1. The cell preservation solution has severe rupture of cell membrane.

Comparative example 9

The same procedure as in example 1 was repeated except that acetic acid was not added. The cell preservation solution contains a large amount of red blood cells, which affects diagnosis.

Although the embodiments of the present invention have been described above, the above description is only for the convenience of understanding the present invention, and is not intended to limit the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A cervical cell preservation solution, comprising: alcohol solution, buffer solution, acid solution, surfactant, sodium chloride, anticoagulant and metal ions.

2. The cervical cell preservation solution according to claim 1, wherein the alcohol solution is one or more of methanol, ethanol and isopropanol.

3. The cervical cell preservation solution according to claim 1, wherein the metal ions are one or more of Ca2+, Cu2+, Fe2+, Mn2+, Zn2 +.

4. The cervical cell preservation solution according to claim 1, wherein the anticoagulant is one or more of sodium edetate, potassium edetate, sodium citrate, potassium citrate, heparin lithium and heparin sodium.

5. The cervical cell preservation solution according to claim 1, wherein the buffer is one of disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, potassium dihydrogen phosphate-sodium hydroxide, Tris-hydrochloric acid, HEPES, and barbital sodium-hydrochloric acid.

6. The cervical cell preservation solution according to claim 1, wherein the pH of the buffer solution is 6 to 8.

7. The cervical cell preservation solution according to claim 1, wherein the acid solution is acetic acid.

8. The cervical cell preservation solution according to claim 1, wherein the surfactant is a nonionic surfactant.

9. The cervical cell preservation solution according to claim 1, wherein the pH of the preservation solution is 6 to 8.

10. The cervical cell preservation solution according to claim 1, wherein the alcohol solution, the buffer solution, the acid solution, the surfactant, the sodium chloride, the anticoagulant, and the metal ions are in a mass ratio of: 20-50: 1-5: 0.1-1: 0.01-0.5: 0.1-1: 0.1-1: 0.01-0.1.