CN113424819A - Non-gynecological cell sample preservation solution and application thereof - Google Patents
Non-gynecological cell sample preservation solution and application thereof Download PDFInfo
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- CN113424819A CN113424819A CN202110875176.1A CN202110875176A CN113424819A CN 113424819 A CN113424819 A CN 113424819A CN 202110875176 A CN202110875176 A CN 202110875176A CN 113424819 A CN113424819 A CN 113424819A
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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
- A01N1/0231—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
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Abstract
The invention discloses a non-gynecological cell sample preservation solution and application thereof, wherein the cell sample preservation solution comprises the following components: according to the mass percentage, 30 to 50 percent of fixing agent, 0.1 to 0.3 percent of sodium dihydrogen phosphate, 0.5 to 0.9 percent of inorganic salt, 0.3 to 3 percent of formaldehyde, 0.01 to 0.05 percent of ProClin and the balance of water; the fixing agent is selected from at least three of methanol, ethanol, glycol, isopropanol or glycerol; the inorganic salt is selected from at least two of sodium chloride, sodium acetate, sodium bicarbonate, sodium sulfate or disodium ethylene diamine tetraacetate. After the sample preserving fluid is used for treating a blood sample, red blood cells can be effectively removed, so that the prepared slide has a clean background, is uniform in cell distribution, has sufficient cell components, is small in distribution range on the slide, is easy to read, has a clear cell structure, is very bright under a mirror, is convenient to observe and diagnose, and can obviously improve the positive rate.
Description
Technical Field
The invention relates to the technical field of biology, in particular to preservation solution for a non-gynecological cell sample and application thereof.
Background
In recent years, the detection rate of abnormal cells has been remarkably improved compared with the past due to the development and application of cytology. The thin-layer liquid-based cytology detection technology has the advantages of large cell collection amount, clean and clear background, uniform cell distribution, good dyeing effect, easy observation of cells under a mirror and the like, thereby being widely applied to the screening of gynecological and non-gynecological tumors. In the past, the main method of smear is direct smear, but smear is to pick up a small part of specimen for inspection by the experience of the producer, and the specimen often contains a lot of mucus, even pus, blood, and epithelial cells which are overlapped excessively to cover the abnormal cells. Resulting in difficulty in observation, poor diagnosis, increased false negatives, and a certain degree of influence on the accuracy of examination.
If the fixation is not timely after the cells or tissues are separated from the body, lysosomal enzymes can be released to dissolve the cells, so that the tissues are autolyzed and lose the original structures. Therefore, after the cells are collected, a proper sample preserving fluid is selected and fixed, so that the cells keep the original morphological structure and various contained substance components as much as possible. The quality of cell fixation directly affects subsequent slide production and staining, and further affects the accuracy of cytological diagnosis.
After the blood specimen is stained in the preparation, the background usually contains a large amount of red blood cells or blood sample debris, and for the specimens with small cell amount such as: cyst fluid, urine, urethral washes, red blood cells under the background of the slide can affect the interpretation of effective cells in the specimen, are difficult to observe, and affect the diagnosis.
How to develop a sample preservation solution which can make the sample observed more clearly under the microscope and avoid the influence of blood cells is a technical problem to be solved urgently.
Disclosure of Invention
Therefore, the invention provides a non-gynecological cell sample preservation solution and application thereof.
In order to achieve the above purpose, the invention provides the following technical scheme:
the embodiment of the invention provides a non-gynecological cell sample preservation solution, which comprises the following components: according to the mass percentage, 30 to 50 percent of fixing agent, 0.1 to 0.3 percent of sodium dihydrogen phosphate, 0.5 to 0.9 percent of inorganic salt, 0.3 to 3 percent of formaldehyde, 0.01 to 0.05 percent of ProClin and the balance of water;
the fixing agent is selected from at least three of methanol, ethanol, glycol, isopropanol or glycerol;
the inorganic salt is selected from at least two of sodium chloride, sodium acetate, sodium bicarbonate, sodium sulfate or disodium ethylene diamine tetraacetate.
In one embodiment of the present invention, the sample preservation solution comprises the following components: 34% of fixing agent, 0.15% of sodium dihydrogen phosphate, 0.82% of inorganic salt, 0.4% of formaldehyde, 3000.05% of ProClin and the balance of water.
In one embodiment of the invention, the fixative is methanol 14%, isopropanol 12% and ethylene glycol 8%;
the inorganic salt is 0.50 percent of sodium chloride and 0.32 percent of sodium acetate.
In one embodiment of the invention, the pH value of the preservation solution is 7.20-7.60.
In one embodiment of the invention, the pH of the preservation solution is 7.50.
In one embodiment of the present invention, the non-gynecological cell sample comprises: body cavity fluid, lavage fluid, cyst fluid, urine, cerebrospinal fluid, and nipple discharge.
The application of the non-gynecological cell sample preservation solution in biological sample preservation also belongs to the protection scope of the invention.
In one embodiment of the present invention, the biological sample is a hemoglobin-containing biological sample.
The invention has the following advantages:
after the sample preserving fluid is used for treating a blood sample, red blood cells can be effectively removed, so that the prepared slide has a clean background, is uniform in cell distribution, has sufficient cell components, is small in distribution range on the slide, is easy to read, has a clear cell structure, is very bright under a mirror, is convenient to observe and diagnose, and can obviously improve the positive rate.
The specimen treated by the specimen preserving fluid has long preserving time, does not generate flocculent precipitate, and can be subjected to specimen slice staining after long-term preservation.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
Fig. 1 shows a case where a whole blood sample is stored in the sample storage solution 1 to 5 according to the embodiment of the present invention for 72 hours after hemolysis, a: sample preservation solution 1; b: sample preservation solution 2; c: a sample preservation solution 3; d: sample preservation solution 4; e: sample preservation solution 5; f: the specimen preservation solution according to embodiment 1 of the present invention.
FIG. 2 shows the bottom red blood cells of a hemorrhoidal pleural effusion sample after centrifugation according to an embodiment of the present invention;
FIG. 3 shows the bottom red blood cells after centrifugation of a blood pleural effusion sample treated with a sample preservation solution according to example 1;
FIG. 4 shows the background of the specimen (100 Xtimes mirror) treated with the specimen preservation solution of example 1 according to the present invention;
FIG. 5 shows the background of the specimen (400 Xlens) treated with the specimen preservation solution of example 1 according to the present invention;
FIG. 6 shows a specimen background (100x times mirror) after treatment with a brand preservation solution according to an embodiment of the present invention;
FIG. 7 shows the background of a specimen (400 Xmirror) treated with a brand preservation solution according to an embodiment of the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a non-gynecological cell sample preservation solution, the solvent of the sample preservation solution is water, and the sample preservation solution comprises the following components in percentage by mass: 6% of methanol, 7% of ethylene glycol, 20% of isopropanol, 0.15% of sodium dihydrogen phosphate, 0.60% of sodium chloride, 0.22% of sodium acetate, 0.7% of formaldehyde and 3000.05% of ProClin, wherein the pH value of the preservation solution is 7.40.
The preparation method of the non-gynecological cell sample preservation solution in the embodiment comprises the following steps:
adding sodium chloride into purified water to obtain a sodium chloride solution with the mass percent of 0.60%, then adding sodium acetate and sodium dihydrogen phosphate into the sodium chloride solution, wherein the mass percent of the sodium acetate is 0.22%, and the mass percent of the sodium dihydrogen phosphate is 0.15%, and uniformly mixing to obtain a mixed solution; adding isopropanol, methanol, glycol, formaldehyde and ProClin 300(sigma) into the mixed solution, wherein the mass percent of the isopropanol is 20%, the mass percent of the methanol is 6%, the mass percent of the glycol is 7%, the mass percent of the formaldehyde is 0.7% and the mass percent of the ProClin 300 is 0.05%, stirring and uniformly mixing to obtain the non-gynecological cell sample preservation solution.
Example 2
The embodiment provides a non-gynecological cell sample preservation solution, the solvent of the sample preservation solution is water, and the sample preservation solution comprises the following components in percentage by mass: 14% of methanol, 12% of isopropanol, 8% of ethylene glycol, 0.15% of sodium dihydrogen phosphate, 0.50% of sodium chloride, 0.32% of sodium acetate, 0.4% of formaldehyde and 3000.05% of ProClin, wherein the pH value of the preservation solution is 7.5.
The preparation method of the non-gynecological cell sample preservation solution in the embodiment comprises the following steps:
adding sodium chloride into purified water, wherein the mass percent of the sodium chloride is 0.50%, then adding sodium acetate and disodium hydrogen phosphate into the sodium chloride solution, the mass percent of the sodium acetate is 0.32%, and the mass percent of the sodium dihydrogen phosphate is 0.15%, and uniformly mixing to obtain a mixed solution; adding isopropanol, methanol, glycol, formaldehyde and ProClin 300 into the mixed solution, wherein the mass percent of the isopropanol is 12%, the mass percent of the methanol is 14%, the mass percent of the glycol is 8%, the mass percent of the formaldehyde is 0.4%, and the mass percent of the ProClin 300 is 0.05%, stirring, and uniformly mixing to obtain the non-gynecological cell sample preservation solution.
Example 3
The embodiment provides a non-gynecological cell sample preservation solution, the solvent of the sample preservation solution is water, and the sample preservation solution comprises the following components in percentage by mass: 9% of methanol, 18% of isopropanol, 10% of ethylene glycol, 0.2% of sodium dihydrogen phosphate, 0.40% of sodium chloride, 0.30% of sodium acetate, 0.8% of formaldehyde and 3000.03% of ProClin. The pH of the preservation solution was 7.2.
The preparation method of the non-gynecological cell sample preservation solution in the embodiment comprises the following steps:
adding sodium chloride into purified water, wherein the mass percent of the sodium chloride is 0.40%, adding sodium acetate and sodium dihydrogen phosphate into a sodium chloride solution, the mass percent of the sodium acetate is 0.30%, and the mass percent of the sodium dihydrogen phosphate is 0.2%, and uniformly mixing to obtain a mixed solution; adding isopropanol, methanol, glycol, formaldehyde and ProClin 300 into the mixed solution, wherein the mass percent of the isopropanol is 18%, the mass percent of the methanol is 9%, the mass percent of the glycol is 10%, the mass percent of the formaldehyde is 0.8% and the mass percent of the ProClin 300 is 0.03%, stirring and uniformly mixing to obtain the non-gynecological cell sample preservation solution.
Example 4
The embodiment provides a non-gynecological cell sample preservation solution, the solvent of the sample preservation solution is water, and the sample preservation solution comprises the following components in percentage by mass: 8% of methanol, 7% of ethylene glycol, 19% of isopropanol, 0.15% of sodium dihydrogen phosphate, 0.62% of sodium chloride, 0.2% of sodium acetate, 2% of formaldehyde and 3000.03% of ProClin, wherein the pH value of the preservation solution is 7.3.
The preparation method of the non-gynecological cell sample preservation solution in the embodiment comprises the following steps:
adding sodium chloride into purified water, wherein the mass percent of the sodium chloride is 0.62%, adding sodium acetate and sodium dihydrogen phosphate into a sodium chloride solution, the mass percent of the sodium acetate is 0.2%, and the mass percent of the sodium dihydrogen phosphate is 0.15%, and uniformly mixing to obtain a mixed solution; adding isopropanol, methanol, glycol, formaldehyde and ProClin 300 into the mixed solution, wherein the mass percent of the isopropanol is 19%, the mass percent of the methanol is 8%, the mass percent of the glycol is 7%, the mass percent of the formaldehyde is 2% and the mass percent of the ProClin 300 is 0.03%, stirring and uniformly mixing to obtain the non-gynecological cell sample preservation solution.
Example 5
The embodiment provides a non-gynecological cell sample preservation solution, the solvent of the sample preservation solution is water, and the sample preservation solution comprises the following components in percentage by mass: 23% of methanol, 8% of isopropanol, 8% of ethylene glycol, 0.15% of sodium dihydrogen phosphate, 0.50% of sodium chloride, 0.22% of sodium acetate, 4% of formaldehyde and 3000.05% of ProClin. The pH of the preservation solution was 7.4.
The preparation method of the non-gynecological cell sample preservation solution in the embodiment comprises the following steps:
adding sodium chloride into purified water, wherein the mass percent of the sodium chloride is 0.50%, adding sodium acetate and sodium dihydrogen phosphate into a sodium chloride solution, the mass percent of the sodium acetate is 0.22%, and the mass percent of the sodium dihydrogen phosphate is 0.15%, and uniformly mixing to obtain a mixed solution; adding isopropanol, methanol, ethylene glycol, formaldehyde and ProClin 300 into the mixed solution
The non-gynecological cell sample preservation solution is obtained by uniformly mixing 8% by mass of isopropanol, 23% by mass of methanol, 8% by mass of ethylene glycol, 4% by mass of formaldehyde and 0.05% by mass of ProClin 300.
Non-gynecological samples that can be preserved with the sample preservation solutions of examples 1 to 5 include: body cavity fluid, lavage fluid, cyst fluid, urine, urethral lavage, cerebrospinal fluid, nipple discharge.
Test example 1
Sample preservation solution of example 1: the solvent of the sample preservation solution is water, and the sample preservation solution comprises the following components in percentage by mass: 6% of methanol, 7% of ethylene glycol, 20% of isopropanol, 0.15% of sodium dihydrogen phosphate, 0.60% of sodium chloride, 0.22% of sodium acetate, 0.7% of formaldehyde and 3000.05% of ProClin, wherein the pH value of the preservation solution is 7.40.
The solvents from the sample preservation solution 1 to the sample preservation solution are all water.
The sample preservation solution 1 contains the following components: the mass percent of the sodium chloride is 0.55 percent of solution, the mass percent of the sodium acetate is 0.23 percent, and the mass percent of the sodium dihydrogen phosphate is 0.20 percent; the mass percent of methanol is 20%, the mass percent of glycol is 6.5%, the mass percent of ProClin 300 is 0.05%, and the sample preservation solution 1 is obtained after uniform stirring.
The sample preservation solution 2 contains the following components: 0.60 percent of sodium chloride, 0.15 percent of sodium acetate, 0.13 percent of sodium dihydrogen phosphate, 24 percent of ethanol, 7 percent of glycol and 0.05 percent of ProClin 300.
The sample preservation solution 3 contains the following components: 0.65% by mass of sodium chloride, 0.15% by mass of sodium acetate, 0.15% by mass of sodium dihydrogen phosphate, 20% by mass of isopropanol, 6.5% by mass of ethylene glycol and 0.05% by mass of ProClin 300.
The sample preservation solution 4 contains the following components: 0.60 percent of sodium chloride, 0.22 percent of sodium acetate, 0.15 percent of sodium dihydrogen phosphate, 20 percent of isopropanol, 6 percent of methanol, 7 percent of glycol and 0.05 percent of ProClin 300.
The sample preservation solution 5 contains the following components: 0.65% by mass of sodium chloride, 0.18% by mass of sodium acetate, 0.13% by mass of sodium dihydrogen phosphate, 23% by mass of ethanol, 5% by mass of isopropanol, 7% by mass of ethylene glycol and 0.05% by mass of ProClin 300; the solution was stirred well.
A whole blood sample hemolysis test was performed using the sample preservation solutions 1 to 5 and the sample preservation solution of example 1, and 100. mu.L of the whole blood sample was added to each 2mL of the sample preservation solution, and the time for clarifying the solution and the clarity and floc formation after 72 hours of storage were observed, as shown in Table 1.
TABLE 1
As shown in Table 1 and FIG. 1, in the present invention, when the whole blood sample is hemolyzed using the sample preservation solution of example 1 and the sample preservation solutions 1 to 5 and left for 72 hours after the hemolysis of the whole blood sample, the sample preservation solution 1, the sample preservation solution 2, the sample preservation solution 3, the sample preservation solution 4, the sample preservation solution 5, and the sample preservation solution of example 1 are respectively provided from left to right,
after the whole blood sample is added into the sample preservation solution 1, the hemolysis effect can be achieved even if the sample preservation solution is kept for a long time, floccules of hemoglobin are generated in the sample preservation solutions 1 to 5 after hemolysis, and substances which can stabilize proteins after red blood cell disruption are not available in the sample preservation solutions 1 to 5. The sample preservation solution of example 1 can keep the solution clear and free of floc after being left for 72 hours, so that the sample preservation solution of example 1 of the present invention can stabilize proteins after red blood cells are broken, can store whole blood hemolysis samples for a long time, and can keep the hemolysis samples from deteriorating.
Test example 2
The sample preservation solution of a certain brand has the production lot number of 2020-11-01 and the validity period of 2022-10-31.
A blood pleural effusion sample was treated with the sample preservation solution of example 1 and a sample preservation solution of a certain brand, and the microscopic image of the sample after slide staining was observed.
FIG. 2 shows the condition of the bottom red blood cells after centrifugation of a blood pleural effusion sample, such as FIG. 3 shows the condition of hemolysis of the sample by the sample preservation solution of example 1; FIG. 4 shows the background of the specimen after treatment with the specimen preservation solution of example 1 (100 Xfold mirror); FIG. 5 shows the background of the specimen after treatment with the specimen preservation solution of example 1 (400 Xlens); FIG. 6 shows the background of a specimen treated with a sample preservation solution of a certain brand (100 Xmirror); FIG. 7 shows the background of a specimen (400 Xmirror) after treatment with a sample preservation solution of a certain brand.
In a microscope, the blood viscosity lumps in the sample are not seen in the blood specimen of the chest and abdominal water treated by the preservation solution of the embodiment 1, but the blood viscosity lumps background exists in the blood specimen of the chest and abdominal water treated by the preservation solution of a certain brand, so that the hemolysis effect of the sample preservation solution of the embodiment 1 of the invention on the sample is more obvious.
The specimen preserved by the sample preservative fluid has complete cell shape, uniform distribution, proper quantity and clean background, particularly has good treatment effect on blood cells, is beneficial to observation and diagnosis, and can prolong the specimen storage time.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. The non-gynecological cell sample preservation solution is characterized by comprising the following components: according to the mass percentage, 30 to 50 percent of fixing agent, 0.1 to 0.3 percent of sodium dihydrogen phosphate, 0.5 to 0.9 percent of inorganic salt, 0.3 to 3 percent of formaldehyde, 0.01 to 0.05 percent of ProClin and the balance of water;
the fixing agent is selected from at least three of methanol, ethanol, glycol, isopropanol or glycerol;
the inorganic salt is selected from at least two of sodium chloride, sodium acetate, sodium bicarbonate, sodium sulfate or disodium ethylene diamine tetraacetate.
2. The non-gynecological cell sample preservation solution according to claim 1,
the sample preservation solution comprises the following components: 34% of fixing agent, 0.15% of sodium dihydrogen phosphate, 0.82% of inorganic salt, 0.4% of formaldehyde, 3000.05% of ProClin and the balance of water.
3. The non-gynecological cell sample preservation solution according to claim 2,
the fixing agent is 14% of methanol, 12% of isopropanol and 8% of ethylene glycol;
the inorganic salt is 0.50 percent of sodium chloride and 0.32 percent of sodium acetate.
4. The non-gynecological cell sample preservation solution according to claim 1,
the pH value of the preservation solution is 7.20-7.60.
5. The non-gynecological cell sample preservation solution according to claim 1,
the pH value of the preservation solution is 7.50.
6. The non-gynecological cell sample preservation solution according to claim 1,
the non-gynecological cell sample comprises: body cavity fluid, lavage fluid, cyst fluid, urine, cerebrospinal fluid, and nipple discharge.
7. Use of the non-gynecological cell sample preservation solution according to any one of claims 1 to 6 for preserving biological samples.
8. The use of claim 7, wherein the biological sample is a biological sample containing hemoglobin.
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Citations (5)
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| CN102258003A (en) * | 2010-05-28 | 2011-11-30 | 孝感市中心医院 | Liquid based cell preserving fluid |
| CN105409925A (en) * | 2014-09-19 | 2016-03-23 | 孝感宏翔生物医械技术有限公司 | Liquid-based cell preservation solution |
| CN107509722A (en) * | 2017-08-30 | 2017-12-26 | 迈克生物股份有限公司 | Sample preservation liquid |
| CN111134109A (en) * | 2019-12-19 | 2020-05-12 | 苏州浚惠生物科技有限公司 | Urine single cell preservation solution and preparation method thereof |
| CN111328798A (en) * | 2020-04-29 | 2020-06-26 | 大连德泰克森生物医药有限公司 | Urine stabilization protective agent and preparation method thereof |
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2021
- 2021-07-30 CN CN202110875176.1A patent/CN113424819A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102258003A (en) * | 2010-05-28 | 2011-11-30 | 孝感市中心医院 | Liquid based cell preserving fluid |
| CN105409925A (en) * | 2014-09-19 | 2016-03-23 | 孝感宏翔生物医械技术有限公司 | Liquid-based cell preservation solution |
| CN107509722A (en) * | 2017-08-30 | 2017-12-26 | 迈克生物股份有限公司 | Sample preservation liquid |
| CN111134109A (en) * | 2019-12-19 | 2020-05-12 | 苏州浚惠生物科技有限公司 | Urine single cell preservation solution and preparation method thereof |
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Application publication date: 20210924 |