CN116649330B - Liquid-based exfoliated cell preservation liquid, kit and application - Google Patents

Liquid-based exfoliated cell preservation liquid, kit and application Download PDF

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CN116649330B
CN116649330B CN202310874604.8A CN202310874604A CN116649330B CN 116649330 B CN116649330 B CN 116649330B CN 202310874604 A CN202310874604 A CN 202310874604A CN 116649330 B CN116649330 B CN 116649330B
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exfoliated
cell
cell preservation
preservation solution
exfoliated cell
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CN116649330A (en
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黄小军
朱峻
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Shenzhen Senying Intelligent Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Dispersion Chemistry (AREA)
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  • Analytical Chemistry (AREA)
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  • General Physics & Mathematics (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The invention provides a liquid-based exfoliated cell preservation solution, a kit and application, wherein the exfoliated cell preservation solution comprises the following components: p-hydroxyacetophenone, N-ethylethanolamine, paraformaldehyde, cysteine, gamma-aminobutyric acid, naCl, KCl, citric acid, sodium citrate and water. The components in the exfoliated cell preservation solution are matched in a synergistic way, so that exfoliated cells can be preserved for a long time while impurities such as blood cells are removed effectively, cell forms are maintained not to shrink or expand, cell proteins are fixed simultaneously, and the reagents in the exfoliated cell preservation kit are matched with each other, thereby realizing the effect of quickly preparing cell smears and being beneficial to improving the accuracy of cell detection.

Description

Liquid-based exfoliated cell preservation liquid, kit and application
Technical Field
The invention belongs to the technical field of medicine, and relates to liquid-based exfoliated cell preservation liquid, a kit and application.
Background
Shed cells are mucosal epithelial cells shed from the inner surface of natural luminal organs and are of diagnostic value in the clinic. When the cells are not shed, the cells have inherent cell morphology, structure and biological activity, and when the cells are shed from the original living environment, the morphology, physiological functions and the like of the cells are changed, and the common preservation methods such as freezing preservation have damage to the cells, so that the accuracy of diagnosis is affected.
The liquid-based thin-layer cytology detection technology (Thinprep Cytology Test, TCT) is a technology widely used at present, and the key steps are that the sample is immediately put into a cell preservation solution after being taken out, the original form of the cells is maintained, non-diagnostic impurities are removed, and then a thin-layer smear is manufactured.
However, the conventional exfoliated cell preservation solution has problems such as an influence on cell morphology and cell protein, incomplete impurity treatment, and high cost, for example, 4% paraformaldehyde can be used for cell fixation, but has a high environmental temperature requirement, and a preservation time is short, and the influence on cell morphology and cell protein is generally more than 48 hours, so that it is necessary to develop an exfoliated cell preservation solution with good preservation effect and long preservation time.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a liquid-based exfoliated cell preservation solution, a kit and application, wherein the exfoliated cell preservation solution can rapidly remove blood cells and mucus while maintaining the intact form of exfoliated cells, and can keep the cell structure and fixed cell proteins for a long time, and the exfoliated cell preservation solution and reagents such as adhesive are matched with each other, so that the preparation method of the exfoliated cell smear is simple to operate, high in efficiency and high in success rate.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a liquid-based exfoliated cell preservation solution comprising:
p-hydroxyacetophenone, N-ethylethanolamine, paraformaldehyde, cysteine, gamma-aminobutyric acid, naCl, KCl, citric acid, sodium citrate and water.
In the invention, the p-hydroxyacetophenone and N-ethylethanolamine exert synergistic effect, on one hand, the p-hydroxyacetophenone and N-ethylethanolamine are matched with paraformaldehyde to slowly and fully fix the structure of an exfoliated cell membrane, and on the other hand, the phenomenon that cells are aggregated due to the hydroformylation of cell surface proteins by the paraformaldehyde is avoided, so that exfoliated cells are fully dispersed, and the cell tabletting of a thin layer is convenient to prepare; cysteine and gamma-aminobutyric acid have the synergistic effects of improving the permeability of the shed cell membrane, expanding and cracking red blood cells and degrading mucus, so that the shed cell skeleton is conveniently fixed by the p-hydroxyacetophenone, N-ethylethanolamine and paraformaldehyde, and impurity components are effectively removed by matching with NaCl and KCl, the preparation of a cell sheet with clear background is ensured, and the detection accuracy is improved; meanwhile, citric acid and sodium citrate are matched with each other to serve as buffer solution of the exfoliated cell preservation solution, so that the pH value of the exfoliated cell preservation solution is maintained at a relatively stable level.
The components of the exfoliated cell preservation solution exert synergistic interaction, provide a suitable environment for preserving exfoliated cells for a long time, ensure that the exfoliated cells can be preserved in the exfoliated cell preservation solution for a long time without cell shrinkage/expansion and protein change, and are favorable for the subsequent development of cytological detection tests.
Preferably, the final concentration of the p-hydroxyacetophenone in the exfoliated cell preservation solution is 10-20 mM, for example, 10mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16mM, 17 mM, 18 mM, 19 mM or 20mM, preferably 13-16 mM.
Preferably, the final concentration of the N-ethylethanolamine in the exfoliated cell preservation solution is 1-10 mM, for example, 1 mM, 2 mM, 3 mM, 4mM, 5mM, 6mM, 7 mM, 8 mM, 9mM or 10mM mM, preferably 2-4 mM.
Preferably, the final concentration of paraformaldehyde in the exfoliated cell preservation solution is 10-20 mM, for example, 10mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM, 15 mM, 15.5 mM, 16mM, 16.5 mM, 17 mM, 17.5mM, 18 mM, 18.5 mM, 19 mM, 19.5 mM or 20mM, preferably 14-17.5 mM.
In the invention, the p-hydroxyacetophenone, N-ethylethanolamine and paraformaldehyde in the exfoliated cell preservation solution play a synergistic interaction under the low concentration ratio, so that a better cell fixing effect than that of the conventional exfoliated cell preservation solution is realized, and the problems of incomplete impurity treatment, poor preservation solution stability, false negative detection result and the like caused by high concentration alcohols in the existing commercially available exfoliated cell preservation solution are effectively avoided.
Preferably, the final concentration of cysteine in the exfoliated cell preservation solution is 1 to 5mM, for example, 1 mM, 2 mM, 3 mM, 4mM or 5mM, preferably 2 to 4mM.
Preferably, the final concentration of the gamma-aminobutyric acid in the exfoliated cell preservation solution is 1-10 mM, for example, 1 mM, 2 mM, 3 mM, 4mM, 5mM, 6mM, 7 mM, 8 mM, 9mM or 10mM, preferably 5-9 mM.
Preferably, the final concentration of NaCl in the exfoliated cell preservation solution is 50-100 mM, for example, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM, preferably 60-70 mM.
Preferably, the final concentration of KCl in the exfoliated cell preservation solution is 20-30 mM, for example, 20mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM or 30 mM, preferably 20-25 mM.
In the invention, cysteine, gamma-aminobutyric acid and NaCl are matched together, so that the effect of expanding and cracking red blood cells is realized while the structural integrity of the exfoliated cells is not influenced.
Preferably, the final concentration of the citric acid in the exfoliated cell preservation solution is 1-10 mM, for example, 1 mM, 2 mM, 3 mM, 4mM, 5mM, 6mM, 7 mM, 8 mM, 9mM or 10mM, preferably 2-4 mM.
Preferably, the final concentration of the sodium citrate in the exfoliated cell preservation solution is 1-20 mM, for example, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4mM, 4.5 mM, 5mM, 5.5 mM, 6mM, 6.5 mM, 7 mM, 7.5mM, 8 mM, 8.5 mM, 9mM, 9.5 mM, 10mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM, 15 mM, 15.5 mM, 16mM, 16.5 mM, 17 mM, 17.5mM, 18 mM, 18.5 mM, 19 mM, 19.5 mM or 20mM, and preferably is 2.5-10 mM.
In the invention, the p-hydroxyacetophenone, N-ethylethanolamine, paraformaldehyde, cysteine, gamma-aminobutyric acid, naCl, KCl, citric acid, sodium citrate and water are fully mixed under a reasonable proportion to form the exfoliated cell preservation solution, so that the method has the effects of preserving exfoliated cells for a long time, maintaining cell morphological structures and fixing cell proteins, and effectively removing blood cells and mucus impurities, and has beneficial effects on preparing thin-layer exfoliated cell smears and ensuring the accuracy of cell detection.
Preferably, the exfoliated cell preservation solution is formulated at a final concentration from:
10-20 mM of p-hydroxyacetophenone
N-ethylethanolamine 1-10 mM
Paraformaldehyde 10-20 mM
Cysteine 1-5 mM
Gamma-aminobutyric acid 1-10 mM
NaCl 50~100 mM
KCl 20~30 mM
Citric acid 1-10 mM
Sodium citrate 1-20 mM
The balance being water.
Preferably, the exfoliated cell preservation solution is formulated at a final concentration from:
13-16 mM of p-hydroxyacetophenone
N-ethylethanolamine 2-4 mM
Paraformaldehyde 14-17.5 mM
Cysteine 2-4 mM
Gamma-aminobutyric acid 5-9 mM
NaCl 60~70 mM
KCl 20~25 mM
Citric acid 2-4 mM
Sodium citrate 2.5-10 mM
The balance being water.
In a second aspect, the present invention provides a liquid-based thin-layer exfoliated cell preservation kit comprising the exfoliated cell preservation solution of the first aspect.
Preferably, the exfoliated cell preservation kit further comprises any one or a combination of at least two of an adhesive, a diluent or a solid carrier.
Preferably, the adhesive comprises a first treatment agent and/or a second treatment agent;
the first treating agent is a chromium gelatin solution, and comprises chromium potassium sulfate and gelatin;
the second treatment agent comprises polylysine.
Preferably, the first treatment agent consists of the following components: 0.05% of chromium potassium sulfate, 0.5% of fish skin gelatin and deionized water.
Preferably, the second treatment agent consists of the following components: 0.55% poly L-lysine and deionized water.
Preferably, the diluent comprises disodium hydrogen phosphate and potassium dihydrogen phosphate, preferably consisting of: 0.15% disodium hydrogen phosphate, 0.025% potassium dihydrogen phosphate, and deionized water.
In a third aspect, the present invention provides a method for preparing a liquid-based thin-layer exfoliated cell smear using the exfoliated cell preservation solution of the first aspect and/or the exfoliated cell preservation kit of the second aspect, comprising:
placing the exfoliated cell sample into an exfoliated cell preservation solution for preservation;
treating the solid support with an adhesive;
and placing the exfoliated cell sample preserved by the exfoliated cell preservation solution on a solid phase carrier treated by an adhesive, and manufacturing a cell smear.
Preferably, the storage time is 15 min-30 days, for example, 15min, 30 min, 1 h, 3h, 6 h, 9 h, 12 h, 18 h, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 15 days, 18 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days or 30 days.
Preferably, the temperature of the preservation is 0 to 4 ℃, for example, 0 ℃, 1 ℃,2 ℃,3 ℃ or 4 ℃.
Preferably, the treating the solid support with the adhesive comprises treating the solid support with a first treating agent and treating the solid support with a second treating agent, followed by drying the solid support.
Preferably, the first treatment agent is used to treat the solid support for 3-5 hours, for example, 3h, 3.5 h, 4 h, 4.5 h or 5 h.
Preferably, the time for treating the solid phase carrier by the second treating agent is 1-2 hours.
Preferably, the drying time is 1-3 hours, for example, 1 h, 1.5 h, 2h, 2.5 h or 3h may be used.
Preferably, the temperature of the drying is 35 to 50 ℃, and may be 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃, 42 ℃, 43 ℃, 44 ℃, 45 ℃, 46 ℃, 47 ℃, 48 ℃, 49 ℃, or 50 ℃, for example.
Preferably, after the step of storing the exfoliated cell sample in the exfoliated cell storing liquid, the step of adding a diluent for adjusting pH is further included.
Preferably, the preparation method of the liquid-based thin-layer exfoliated cell smear comprises the following steps:
placing the exfoliated cell sample in an exfoliated cell preservation solution, adding a diluent to adjust the pH, and preserving at 0-4 ℃ for no more than 30 days;
uniformly mixing chromium potassium sulfate, fish skin gelatin and deionized water to obtain a first treating agent; uniformly mixing poly-L-lysine and deionized water to obtain a second treating agent;
soaking the glass slide for 3-5 hours by using a first treating agent, soaking the glass slide for 1-2 hours by using a second treating agent after cleaning, taking out, and drying for 1-3 hours at 35-50 ℃ in an oven;
the treated slide was prepared into a smear of exfoliated cells, and the cells were observed under a microscope.
In a fourth aspect, the invention provides the use of the shed cell preservation solution of the first aspect and/or the shed cell preservation kit of the second aspect in the preparation of a liquid-based thin-layer shed cell smear.
Preferably, the exfoliated cells comprise thyroid exfoliated cells.
Compared with the prior art, the invention has the following beneficial effects:
(1) The components of the exfoliated cell preservation solution are matched with each other to play a synergistic effect, so that the permeability of cell membranes is improved, cellular protein skeletons are fully fixed on the premise of not damaging the morphological structure of exfoliated cells, blood cells are effectively ruptured, and impurity components such as mucus are removed, so that an exfoliated cell sample can be preserved in the exfoliated cell preservation solution for a long time without breakage or agglomeration;
(2) The kit for preserving the exfoliated cells is used for preparing the liquid-based thin-layer cell smear, is simple and convenient to operate, high in success rate and good in accuracy, and has wide application prospects.
Drawings
FIG. 1 is a staining result of a thyroid desquamation cell sample of example 2;
FIG. 2 is a staining result of the thyroid desquamation cell sample of example 3;
FIG. 3 is a staining result of the thyroid desquamation cell sample of example 5;
FIG. 4 is a staining result of the thyroid desquamation cell sample of example 7;
FIG. 5 is a staining result of the thyroid desquamation cell sample of example 8;
FIG. 6 is a staining result of the thyroid desquamation cell sample of example 12;
FIG. 7 is a staining result of a sample of thyroid desquamation cells of comparative example 1;
FIG. 8 is a staining result of a sample of thyroid desquamation cells of comparative example 3.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Reagent name Manufacturer' s CAS number/lot number/good number
Para hydroxy acetophenone Alatine 99-93-4
N-ethylethanolamine Makeup M033314
Cysteine (S) Alatine C108237
Gamma-aminobutyric acid Alatine A424756
Citric acid Soy pal C8610
Sodium citrate Soy pal S8220
Example 1 this example provides a liquid-based thin-layer exfoliated cell preservation kit containing exfoliated cell preservation solution, an adhesive, a diluent and a slide.
Wherein, the exfoliated cell preservation solution is prepared by deionized water according to the final concentration: p-hydroxyacetophenone 14.5 mM, N-ethylethanolamine 3 mM, paraformaldehyde 15 mM, cysteine 3 mM, gamma-aminobutyric acid 7.5mM, naCl 65 mM, KCl 22.5 mM, citric acid 3 mM, sodium citrate 5 mM;
the adhesive comprises a first treating agent and a second treating agent, wherein the first treating agent consists of the following components: 0.05% of chromium potassium sulfate, 0.5% of fish skin gelatin and deionized water, wherein the second treating agent consists of the following components: 0.55% poly-L-lysine and deionized water;
the diluent consists of the following components: 0.15% disodium hydrogen phosphate, 0.025% potassium dihydrogen phosphate, and deionized water.
The thyroid exfoliated cell smear prepared by using the exfoliated cell preservation kit comprises the following steps:
placing a thyroid desquamation cell sample of a donor in 10 mL desquamation cell preservation solution, and shaking and mixing at 0 ℃ for 10 min; after the supernatant is removed by centrifugation, 10 mL exfoliated cell preservation solution is added into a thyroid exfoliated cell sample, and diluent is added to adjust the pH value to 7, and the thyroid exfoliated cell sample is preserved at 0 ℃;
soaking a slide 4 h by using a first treating agent, soaking the slide 1 h by using a second treating agent after cleaning, taking out, and drying at 40 ℃ in an oven for 2h;
and (3) making a smear of the detached cells from the treated slide, and observing the cell sample under a microscope.
Example 2 this example provides a liquid-based thin-layer exfoliated cell preservation kit containing exfoliated cell preservation solution, an adhesive, a diluent and a slide.
Wherein, the exfoliated cell preservation solution is prepared by deionized water according to the final concentration: p-hydroxyacetophenone 13 mM, N-ethylethanolamine 4mM, paraformaldehyde 14 mM, cysteine 4mM, gamma-aminobutyric acid 9mM, naCl 60 mM, KCl 25 mM, citric acid 4mM, sodium citrate 10 mM;
the formulation of the adhesive and the diluent was the same as in example 1.
The thyroid exfoliated cell smear prepared by using the exfoliated cell preservation kit comprises the following steps:
placing a thyroid desquamation cell sample of a donor in 10 mL desquamation cell preservation solution, and shaking and mixing at 0 ℃ for 10 min; after the supernatant is removed by centrifugation, 10 mL exfoliated cell preservation solution is added into a thyroid exfoliated cell sample, and diluent is added to adjust the pH value to 7, and the thyroid exfoliated cell sample is preserved at 0 ℃;
soaking a glass slide in 3.5 h by using a first treating agent, soaking the glass slide in 1.5 h by using a second treating agent after cleaning, taking out, and drying in an oven at 40 ℃ for 1.5 h;
and (3) making a smear of the detached cells from the treated slide, and observing the cell sample under a microscope.
Example 3 this example provides a liquid-based thin-layer exfoliated cell preservation kit containing exfoliated cell preservation solution, an adhesive, a diluent and a slide.
Wherein, the exfoliated cell preservation solution is prepared by deionized water according to the final concentration: p-hydroxyacetophenone 16mM, N-ethylethanolamine 2 mM, paraformaldehyde 17.5mM, cysteine 2 mM, gamma-aminobutyric acid 5mM, naCl 70 mM, KCl 20mM, citric acid 2 mM, sodium citrate 2.5 mM;
the formulation of the adhesive and the diluent was the same as in example 1.
The thyroid exfoliated cell smear prepared by using the exfoliated cell preservation kit comprises the following steps:
placing a thyroid desquamation cell sample of a donor in 10 mL desquamation cell preservation solution, and shaking and mixing for 10 min at 2 ℃; after the supernatant is removed by centrifugation, 10 mL exfoliated cell preservation solution is added into a thyroid exfoliated cell sample, and a diluent is added to adjust the pH value to 7, and the thyroid exfoliated cell sample is preserved at 2 ℃;
soaking a slide 4.5 h by using a first treating agent, soaking the slide 1.2 h by using a second treating agent after cleaning, taking out, and drying at 45 ℃ in an oven for 1.5 h;
and (3) making a smear of the detached cells from the treated slide, and observing the cell sample under a microscope.
Example 4 this example provides a liquid-based thin-layer exfoliated cell preservation kit containing exfoliated cell preservation solution, an adhesive, a diluent and a slide.
Wherein, the exfoliated cell preservation solution is prepared by deionized water according to the final concentration: p-hydroxyacetophenone 12 mM, N-ethylethanolamine 6mM, paraformaldehyde 12 mM, cysteine 4.5 mM, gamma-aminobutyric acid 10mM, naCl 80 mM, KCl 24.5 mM, citric acid 6mM, sodium citrate 13 mM;
the formulation of the adhesive and the diluent was the same as in example 1.
The thyroid exfoliated cell smear prepared by using the exfoliated cell preservation kit comprises the following steps:
placing a thyroid desquamation cell sample of a donor in 10 mL desquamation cell preservation solution, and mixing with shaking at 4deg.C for 8 min; after the supernatant is removed by centrifugation, 10 mL exfoliated cell preservation solution is added into a thyroid exfoliated cell sample, and diluent is added to adjust the pH value to 7, and the thyroid exfoliated cell sample is preserved at 4 ℃;
soaking a slide 4.8 h by using a first treating agent, soaking the slide 1 h by using a second treating agent after cleaning, taking out, and drying at 48 ℃ in an oven for 1 h;
and (3) making a smear of the detached cells from the treated slide, and observing the cell sample under a microscope.
Example 5 this example provides a liquid-based thin-layer exfoliated cell preservation kit containing exfoliated cell preservation solution, an adhesive, a diluent and a slide.
Wherein, the exfoliated cell preservation solution is prepared by deionized water according to the final concentration: p-hydroxyacetophenone 10mM, N-ethylethanolamine 10mM, paraformaldehyde 20mM, cysteine 1 mM, gamma-aminobutyric acid 1 mM, naCl 50 mM, KCl 27.5 mM, citric acid 10mM, sodium citrate 20 mM;
the formulation of the adhesive and the diluent was the same as in example 1.
The thyroid exfoliated cell smear prepared by using the exfoliated cell preservation kit comprises the following steps:
placing a thyroid desquamation cell sample of a donor in 10 mL desquamation cell preservation solution, and shaking and mixing for 8 min at 3 ℃; after the supernatant is removed by centrifugation, 10 mL exfoliated cell preservation solution is added into a thyroid exfoliated cell sample, and diluent is added to adjust the pH value to 7, and the thyroid exfoliated cell sample is preserved at 3 ℃;
soaking a slide 5h by using a first treating agent, soaking the slide 1 h by using a second treating agent after cleaning, taking out, and drying at 35 ℃ in an oven for 3h;
and (3) making a smear of the detached cells from the treated slide, and observing the cell sample under a microscope.
Example 6 this example provides a liquid-based thin-layer exfoliated cell preservation kit containing exfoliated cell preservation solution, an adhesive, a diluent and a slide.
Wherein, the exfoliated cell preservation solution is prepared by deionized water according to the final concentration: p-hydroxyacetophenone 20mM, N-ethylethanolamine 1 mM, paraformaldehyde 10mM, cysteine 5mM, gamma-aminobutyric acid 6mM, naCl 100 mM, KCl 30 mM, citric acid 1 mM, sodium citrate 1 mM;
the formulation of the adhesive and the diluent was the same as in example 1.
The thyroid exfoliated cell smear prepared by using the exfoliated cell preservation kit comprises the following steps:
placing a thyroid desquamation cell sample of a donor in 10 mL desquamation cell preservation solution, and mixing with shaking at 4deg.C for 8 min; after the supernatant is removed by centrifugation, 10 mL exfoliated cell preservation solution is added into a thyroid exfoliated cell sample, and diluent is added to adjust the pH value to 7, and the thyroid exfoliated cell sample is preserved at 4 ℃;
soaking a slide 3h by using a first treating agent, soaking the slide 2h by using a second treating agent after cleaning, taking out, and drying at 50 ℃ in an oven for 1 h;
and (3) making a smear of the detached cells from the treated slide, and observing the cell sample under a microscope.
In example 7, the final concentration of p-hydroxyacetophenone in the detached cell storage fluid was 5mM, the final concentration of N-ethylethanolamine was 15 mM, and the other conditions were the same as in example 5, as compared with example 5.
In example 8, the final concentration of p-hydroxyacetophenone in the detached cell storage fluid was 25 mM, the final concentration of N-ethylethanolamine was 0.8. 0.8 mM, and the other conditions were the same as in example 5, as compared with example 5.
In example 9, the final concentration of gamma-aminobutyric acid in the detached cell storage solution of this example was 15 mM, the final concentration of cysteine was 0.15 mM, and the other conditions were the same as in example 5.
In example 10, the final concentration of p-hydroxyacetophenone in the detached cell storage fluid was 18 mM as compared with example 1, and the other conditions were the same as in example 1.
In example 11, the final concentration of N-ethylethanolamine in the detached cell preservation solution was 8 mM as compared with example 2, and the other conditions were the same as in example 2.
In example 12, the final concentration of cysteine in the exfoliated cell preservation solution was 2.5. 2.5 mM as compared with example 2, and the other conditions were the same as in example 2.
In example 13, the final concentration of gamma-aminobutyric acid in the detached-cell storage solution was 2 mM as compared with example 4, and the other conditions were the same as in example 4.
In comparative example 1, the detached cell storage solution of this example was free of p-hydroxyacetophenone, and the final concentration of N-ethylethanolamine was 17.5. 17.5mM, as compared with example 1, except that the conditions were the same as in example 1.
In comparative example 2, the detached cell storage solution of this example was free of N-ethylethanolamine, and the final concentration of p-hydroxyacetophenone was 17.5. 17.5mM, in comparison with example 1, except that the conditions were the same as in example 1.
In comparative example 3, the detached cell storage solution of this example did not contain cysteine, and the final concentration of gamma-aminobutyric acid was 10.5. 10.5 mM, as compared with example 1, except that the conditions were the same as in example 1.
In comparative example 4, the detached cell storage solution of this example did not contain γ -aminobutyric acid, and the final concentration of cysteine was 10.5. 10.5 mM, as compared with example 1, except that the conditions were the same as in example 1.
Cell morphology detection
On days 0, 1, 4, 7, 10, 14, 21, 28 and 30 of the thyroid exfoliated cell samples stored in the exfoliated cell preservation solution, 10 mu L of the thyroid exfoliated cell samples are dripped on the glass slide treated by the first treatment agent and the second treatment agent, and after standing for 10 min, the glass slide is covered by dripping the staining agent for staining;
and placing the prepared cell smear under a microscope, and observing the cell form integrity and the cell definition, wherein the cell form integrity is evaluated as yes or no, the yes represents the cell form integrity, the no represents the cell form incompleteness, the cell definition score is 1-5, and the higher the score is, the higher the cell film-making definition is and the better the film-making effect is.
The cell morphology integrity results are shown in Table 1. The exfoliated cell preservation solution of examples 1 to 13 was used to preserve thyroid exfoliated cells at 0 to 4 ℃ and the exfoliated cells maintained morphological integrity within 30 days without rupture or degradation of cell membranes. The results show that the exfoliated cell preservation solution can preserve thyroid exfoliated cells for up to 30 days at 0-4 ℃ and the cells still have complete cell morphology.
The exfoliated cell preservation solution of comparative examples 1 to 4 lacks key components, and thus the exfoliated thyroid cells preserved at 0 to 4 ℃ are broken within 1 to 2 weeks, and the cell morphology is not complete.
TABLE 1
Numbering device Day 0 Day 1 Day 4 Day 7 Day 10 Day 14 Day 21 Day 28 Day 30
Example 1 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 2 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 3 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 4 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 5 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 6 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 7 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 8 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 9 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 10 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 11 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 12 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Example 13 Is that Is that Is that Is that Is that Is that Is that Is that Is that
Comparative example 1 Is that Is that Whether or not - - - - - -
Comparative example 2 Is that Is that Whether or not - - - - - -
Comparative example 3 Is that Is that Is that Is that Whether or not - - - -
Comparative example 4 Is that Is that Is that Is that Whether or not - - - -
The cell definition results are shown in Table 2, and according to the staining results of the thyroid exfoliated cell samples shown in the examples 2, 3, 5, 7, 8 and 12 in FIG. 1-6, the thyroid exfoliated cell samples are preserved for 30 days by using the exfoliated cell preservation solution in the examples 1-3, the cell staining results are clear, no cell aggregation phenomenon exists, and the cell membrane and the cell nucleus are completely visible; the exfoliated cell preservation solution of the examples 4-6 is used for preserving thyroid exfoliated cell samples for 30 days, the cell staining result is clear, the cell nucleus is complete and visible, and the cell membrane of a small part of cells is slightly shrunken at the 4 th week; the exfoliated cell preservation solution of examples 7-9 is used for preserving thyroid exfoliated cell samples for 30 days, the cell staining result is clear, the cell nucleus is complete and visible, and part of cells have shrinkage of cell membranes at week 2, but the integrity of cell morphology is still maintained; the exfoliated cell preservation solution of examples 10-13 was used to preserve thyroid exfoliated cell samples for 30 days, the cell staining results were clear, the cell membranes and nuclei were completely visible, and only a few cells had slightly shrunken cell membranes at week 4. The results show that the cell structure is clear and visible, and the accuracy of cytological detection is ensured by adopting the exfoliated cell preservation solution to preserve thyroid exfoliated cells for 30 days at 0-4 ℃ and then performing cell staining to prepare smears.
The exfoliated cell preservation solution of comparative examples 1 to 4 lacks key components, and thus the exfoliated thyroid cells preserved at 0 to 4 ℃ are broken within 1 to 2 weeks, and no clearly visible cell structure can be observed. The staining results of the thyroid desquamation cell samples of comparative example 1 and comparative example 3 are shown in fig. 7 to 8, respectively.
TABLE 2
Numbering device Day 0 Day 1 Day 4 Day 7 Day 10 Day 14 Day 21 Day 28 Day 30
Example 1 5 5 5 5 5 5 5 5 5
Example 2 5 5 5 5 5 5 5 5 5
Example 3 5 5 5 5 5 5 5 5 5
Example 4 5 5 5 5 5 5 5 4 4
Example 5 5 5 5 5 5 5 4 4 4
Example 6 5 5 5 5 5 5 5 4 4
Example 7 5 5 5 5 5 4 4 3 3
Example 8 5 5 5 5 5 4 4 3 3
Example 9 5 5 5 5 5 5 4 3 3
Example 10 5 5 5 5 5 5 5 5 5
Example 11 5 5 5 5 5 5 5 5 5
Example 12 5 5 5 5 5 5 5 5 5
Example 13 5 5 5 5 5 5 5 4 4
Comparative example 1 5 2 1 - - - - - -
Comparative example 2 5 3 1 - - - - - -
Comparative example 3 5 3 2 1 1 - - - -
Comparative example 4 5 4 2 1 1 - - - -
Blood cell removal rate detection
Treating a slide by using a first treating agent and a second treating agent, then dropwise adding 10 mu L of thyroid exfoliated cell samples subjected to shock treatment by using an exfoliated cell preservation solution, standing for 10 min, dropwise adding a coloring agent for dyeing, and covering the cover glass;
the prepared cell smear is placed under a microscope, and the blood cell removal rate is calculated according to the following formula, wherein the initial blood cell number is the blood cell number of the collected thyroid gland abscission cell sample:
blood cell removal rate = (initial blood cell number-number of blood cells after shaking)/initial blood cell number×100%
The results of the blood cell removal rate are shown in Table 3. The exfoliated cell preservation solution of the embodiment 1-3 is used for carrying out shock treatment on a thyroid exfoliated cell sample for 10 min, and the blood cell removal rate is more than 90%; the exfoliated cell preservation solution of the examples 4-6 is used for carrying out shock treatment on a thyroid exfoliated cell sample for 10 min, and the blood cell removal rate reaches 88%; the exfoliated cell preservation solution of the examples 7-9 is used for carrying out shock treatment on a thyroid exfoliated cell sample for 10 min, so that the blood cell removal rate is slightly reduced to more than 80%; the exfoliated cell preservation solution of examples 10-13 is used for carrying out oscillation treatment on thyroid exfoliated cell samples for 10 min, and the blood cell removal rate is similar to the effect of examples 1-6 and reaches more than 89%. The results show that more than 80% of blood impurities can be removed by adopting the exfoliated cell preservation solution to treat the exfoliated cell sample for only 10 minutes under the shaking condition.
The exfoliated cell preservation solution used in comparative example 1 does not contain p-hydroxyacetophenone, the exfoliated cell preservation solution used in comparative example 2 does not contain N-ethylethanolamine, the exfoliated cell preservation solution used in comparative example 3 does not contain cysteine, the exfoliated cell preservation solution used in comparative example 4 does not contain gamma-aminobutyric acid, the blood cell removal effect is affected, the blood cell removal rate is 65% -75%, and the blood cell removal rate is obviously lower than that of examples 1-13.
TABLE 3 Table 3
Numbering device Blood cell removal Rate (%)
Example 1 90.12
Example 2 90.08
Example 3 90.10
Example 4 88.74
Example 5 89.03
Example 6 88.28
Example 7 82.47
Example 8 83.59
Example 9 84.94
Example 10 89.84
Example 11 90.02
Example 12 90.04
Example 13 89.03
Comparative example 1 67.46
Comparative example 2 68.05
Comparative example 3 73.61
Comparative example 4 72.25
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.

Claims (14)

1. A liquid-based exfoliated cell preservation solution, which is characterized in that the exfoliated cell preservation solution is prepared from the following substances according to the final concentration:
10-20 mM of p-hydroxyacetophenone
N-ethylethanolamine 1-10 mM
Paraformaldehyde 10-20 mM
Cysteine 1-5 mM
Gamma-aminobutyric acid 1-10 mM
NaCl 50～100mM
KCl 20～30mM
Citric acid 1-10 mM
Sodium citrate 1-20 mM
The balance being water.
2. The exfoliated cell preservation solution according to claim 1, wherein the final concentration of the p-hydroxyacetophenone in the exfoliated cell preservation solution is 13 to 16mM.
3. The exfoliated cell preservation solution of claim 1, wherein the final concentration of the N-ethylethanolamine in the exfoliated cell preservation solution is 2 to 4mM.
4. The exfoliated cell preservation solution of claim 1, wherein the final concentration of paraformaldehyde in the exfoliated cell preservation solution is 14 to 17.5mM.
5. The exfoliated cell preservation solution of claim 1, wherein the final concentration of cysteine in the exfoliated cell preservation solution is 2 to 4mM.
6. The exfoliated cell preservation solution of claim 1, wherein the final concentration of the γ -aminobutyric acid in the exfoliated cell preservation solution is 5 to 9mM.
7. The shed cell preservation solution according to claim 1, wherein the shed cell preservation solution is formed by formulating the following substances in a final concentration:
13-16 mM of p-hydroxyacetophenone
N-ethylethanolamine 2-4 mM
Paraformaldehyde 14-17.5 mM
Cysteine 2-4 mM
Gamma-aminobutyric acid 5-9 mM
NaCl 60～70mM
KCl 20～25mM
Citric acid 2-4 mM
Sodium citrate 2.5-10 mM
The balance being water.
8. A liquid-based thin-layer exfoliated cell preservation kit, characterized in that the exfoliated cell preservation kit includes the exfoliated cell preservation liquid of any one of claims 1 to 7.
9. The shed cell preservation kit according to claim 8, further comprising any one or a combination of at least two of an adhesive, a diluent or a solid carrier;
the adhesive comprises a first treatment agent and/or a second treatment agent;
the first treating agent is a chromium gelatin solution, and comprises chromium potassium sulfate and gelatin;
the second treatment agent comprises polylysine;
the diluent comprises disodium hydrogen phosphate and potassium dihydrogen phosphate.
10. A method for preparing a liquid-based thin-layer exfoliated cell smear, characterized in that the method uses the exfoliated cell preservation solution of any one of claims 1 to 7 and/or the exfoliated cell preservation kit of claim 8 or 9, comprising:
placing the exfoliated cell sample into an exfoliated cell preservation solution for preservation;
treating the solid support with an adhesive;
and placing the exfoliated cell sample preserved by the exfoliated cell preservation solution on a solid phase carrier treated by an adhesive, and manufacturing a cell smear.
11. The method of claim 10, wherein the time of preservation is 15min to 30 days;
the preservation temperature is 0-4 ℃.
12. The method of claim 10, wherein treating the solid support with the adhesive comprises treating the solid support with a first treatment agent and treating the solid support with a second treatment agent, followed by drying the solid support;
the time for treating the solid phase carrier by the first treating agent is 3-5 hours;
the time for treating the solid phase carrier by the second treating agent is 1-2 hours;
the drying time is 1-3 h;
the temperature of the drying is 35-50 ℃.
13. The method of claim 10, wherein the step of adding a diluent is further included after the step of storing the exfoliated cell sample in an exfoliated cell preservative solution.
14. Use of the shed cell preservation solution according to any one of claims 1-7 and/or the shed cell preservation kit according to claim 8 or 9 for the preparation of a liquid-based thin-layer shed cell smear;
the exfoliated cells include thyroid exfoliated cells.
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101485303A (en) * 2009-02-26 2009-07-22 广州鸿琪光学仪器科技有限公司 Exfoliated cell preservative fluid
CN101914483A (en) * 2010-09-02 2010-12-15 福建新大陆生物技术股份有限公司 Liquid-based thin-layer cell preservation solution and use thereof
CN103120153A (en) * 2012-11-26 2013-05-29 刘召宏 Exfoliative cells preserving fluid
CN104642300A (en) * 2015-03-13 2015-05-27 上海御康医院有限公司 Exfoliative cell sap base preserving fluid, method thereof for flaking and kit
CN108770836A (en) * 2018-08-21 2018-11-09 生工生物工程（上海）股份有限公司 Liquid based cell preservative fluid and its preparation method and application
CN110037013A (en) * 2019-06-06 2019-07-23 江苏立峰生物科技有限公司 A kind of Thinprep pap test saves liquid and preparation method thereof
CN115176798A (en) * 2022-08-16 2022-10-14 苏州康唯纳思生物科技有限公司 Liquid-based cell preservation solution and preparation method and application thereof
WO2022242398A1 (en) * 2021-05-21 2022-11-24 深圳安侣医学科技有限公司 Pretreatment reagent, preparation method, cell staining method and pretreatment method

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101485303A (en) * 2009-02-26 2009-07-22 广州鸿琪光学仪器科技有限公司 Exfoliated cell preservative fluid
CN101914483A (en) * 2010-09-02 2010-12-15 福建新大陆生物技术股份有限公司 Liquid-based thin-layer cell preservation solution and use thereof
CN103120153A (en) * 2012-11-26 2013-05-29 刘召宏 Exfoliative cells preserving fluid
CN104642300A (en) * 2015-03-13 2015-05-27 上海御康医院有限公司 Exfoliative cell sap base preserving fluid, method thereof for flaking and kit
CN108770836A (en) * 2018-08-21 2018-11-09 生工生物工程（上海）股份有限公司 Liquid based cell preservative fluid and its preparation method and application
CN110037013A (en) * 2019-06-06 2019-07-23 江苏立峰生物科技有限公司 A kind of Thinprep pap test saves liquid and preparation method thereof
WO2022242398A1 (en) * 2021-05-21 2022-11-24 深圳安侣医学科技有限公司 Pretreatment reagent, preparation method, cell staining method and pretreatment method
CN115176798A (en) * 2022-08-16 2022-10-14 苏州康唯纳思生物科技有限公司 Liquid-based cell preservation solution and preparation method and application thereof

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