CN112741080B - Liquid-based cell preservation treatment liquid and preparation method and application thereof - Google Patents

Abstract

The application relates to a cell preservation solution, in particular to a liquid-based cell preservation treatment solution and a preparation method and application thereof. A liquid-based cell preservation treatment liquid comprises the following raw materials in percentage by mass: 18-28% of ethanol, 1-7% of PEG, 0.01-0.1% of fatty alcohol-polyoxyethylene ether, 0.035-1.05% of anticoagulant, 0.25-2% of phosphate buffer, 0.6-0.9% of sodium chloride and the balance of ultrapure water; the PEG is selected from one or more of PEG-200, PEG-400 and PEG-600. The method and the device solve the defects of unavailable blood cell treatment, positive cell stacking, cell nucleus shrinking and the like, prolong the preservation time of the cells, are beneficial to the dispersion and observation of the cells, and improve the detection accuracy.

Description

Liquid-based cell preservation treatment liquid and preparation method and application thereof

Technical Field

The application relates to a cell preservation solution, in particular to a liquid-based cell preservation treatment solution and a preparation method and application thereof.

Background

Cervical cancer is currently one of the most common malignancies in women. Early diagnosis of cervical cancer and precancerous lesions is the key to improving the cure rate of cervical cancer and the survival rate of patients, and cytological examination is the main method for screening cervical cancer which is the most widely used clinically at present. Liquid-based cytology examination is a new cytology technology invented at the end of the last 90 years, has high accuracy in diagnosing cervical cancer and precancerous lesions, and is considered as an optimal method for early diagnosis and early prevention of cervical cancer.

Liquid-based cytology examination is a new cytology technology invented at the end of the last 90 years, has high accuracy in diagnosing cervical cancer and precancerous lesions, and is considered as an optimal method for early diagnosis and early prevention of cervical cancer. The most critical reagent in the liquid-based thin-layer cytology detection technology is cell preservation solution. When analyzing cells collected from a living body, since the cells start to decompose themselves after being separated from the living body, it is necessary to appropriately store the cells from the collection of the cells to the analysis. The living cell specimen with inherent cell morphology and structure has application value in many aspects such as clinical medical diagnosis, once the cells are separated from the original living environment, the morphological structure of the cells will be changed, and correct diagnosis can be ensured only by a correct cell preservation method. The liquid-based cell preservation solution is a main consumable material of a liquid-based thin-layer cytological detection technology, overcomes the defect of the traditional pap smear, almost all collected cells are preserved in the preservation solution, the smear cells are less overlapped, have no degeneration or have slight degeneration, the background is clean and clear, and the sensitivity and the specificity of the liquid-based cytological examination are superior to those of the traditional pap smear.

At present, the clinical price of the preservation solution produced by Xinbai in the United states is high, the domestic liquid-based cell preservation solution is usually made of alcohols as a fixing preservative, such as methanol, ethanol and isopropanol, wherein the methanol and the isopropanol belong to toxic and harmful components and are not beneficial to the health of operators, and the methanol is used as the fixing preservative, which can cause cell agglomeration and caking, and protein mucus is also easy to agglomerate and deposit and is not beneficial to the dispersion and observation of cells. And when the ethanol content is high, the protein with high fluidity can be fixed, but sediment is formed immediately, the sediment cannot be transferred to a glass slide well, and cell surface deformation and cell degradation are caused, which brings trouble to cytology examination. When the concentration of the ethanol is lower, the cells can not be fixed and stored for a long time, and the liquid-based cell preservation solution can not maintain the original morphological structure and physiological function of the cells, so that the accuracy of the detection result is greatly reduced.

Disclosure of Invention

In order to overcome the defects that alcohol cell preservation solution is easy to form sediments and causes cell surface deformation and cell degeneration, the application provides a liquid-based cell preservation treatment solution and a preparation method and application thereof.

In a first aspect, the present application provides a liquid-based cell preservation treatment solution, which is implemented by adopting the following technical scheme:

a liquid-based cell preservation treatment liquid comprises the following raw materials in percentage by mass:

the PEG is selected from one or more of PEG-200, PEG-400 and PEG-600.

By adopting the technical scheme, the ethanol is used as the fixed dehydrating agent, the content of the ethanol is 18-28 wt%, the cell agglomeration and the protein mucus are not easy to agglomerate and deposit, and the cell dispersion and observation are facilitated. And the combination of ethanol and PEG solves the fixation effect of the monoethanol, has less dosage, reduces the cost, overcomes the problem of methanol, has lower toxicity, inhibits the volatilization of the ethanol, has small cell deformation, and prolongs the preservation time of the cell. PEG is used as a fixation enhancer, the molecular weight of the PEG affects the effect of the liquid-based cell preservation solution, the PEG with the molecular weight higher than 600 is in a colloid state, the production is not facilitated, the higher the molecular weight is, the lower the fixation and permeability are, the viscosity of the system can be increased, the cell agglomeration and protein mucus agglomeration and deposition are caused, and the dispersion and observation of cells are not facilitated. The fatty alcohol-polyoxyethylene ether and PEG act together, so that the solubilization and the dispersibility of the system can be increased, the softening and scattering effects of mucus are improved, the difficulty of digestion and damage of cells is effectively reduced, the deformation of the cells is small, and the storage time of the cells is prolonged.

Preferably, the liquid-based cell preservation treatment liquid comprises the following raw materials in percentage by mass:

by adopting the technical scheme, the components of the raw materials of the liquid-based cell preservation treatment liquid are regulated, so that the formation of protein into sediments is effectively avoided, the cell surface deformation and cell degeneration are reduced, the preservation time is further prolonged, the hemolysis effect is also improved, the cell nucleus structure is clear, and the background coloring is avoided.

Preferably, the HLB value of the fatty alcohol-polyoxyethylene ether is 13-14.

By adopting the technical scheme, the fatty alcohol-polyoxyethylene ether with the HLB value of 13-14 has good water solubility, good softening and scattering effect on mucus, good hemolysis effect, capability of enhancing the permeability of cell membranes, effective reduction of cell deformability, improvement of the preservation time of cells, and little cell deformation even after being preserved for 112 days.

Preferably, the mass ratio of the fatty alcohol-polyoxyethylene ether to the PEG-400 is 1: (61.5-72.7); more preferably, the mass ratio of the fatty alcohol-polyoxyethylene ether to the PEG-400 is 1: 66.7.

by adopting the technical scheme, the mass ratio of the fatty alcohol-polyoxyethylene ether to the PEG-400 is adjusted to be 1: (61.5-72.7), especially the mass ratio of 1: 66.7, the surface activity of the cell preservation treatment solution can be reduced, the solubilization of mucus and the dispersion of cells can be promoted, the softening and scattering effects of mucus are optimized, the cells are not easy to digest and damage, and the preservation time of the cells is prolonged.

Preferably, the liquid-based cell preservation treatment liquid further comprises 0.1-2% of urea and 1-3% of KOH by mass percent; more preferably, the liquid-based cell preservation treatment liquid further comprises 0.8-1.3% of urea and 1.5-2.5% of KOH by mass percent; most preferably, the liquid-based cell preservation treatment liquid further comprises 1.1% of urea and 2% of KOH by mass percentage.

By adopting the technical scheme, urea and KOH with different contents are added into the cell preservation treatment solution, the urea content can influence the hemolysis effect of the cell preservation treatment solution, and the KOH content not only influences the buffer capacity of the phosphate buffer solution, but also influences the dispersibility of the system and the cell permeability of PEG, especially the cell deformability after 112 days.

Preferably, the mass ratio of the urea to the fatty alcohol-polyoxyethylene ether is (16-17): 1.

by adopting the technical scheme, the urea and the fatty alcohol-polyoxyethylene ether are compounded to be used as the hemolytic agent, and the proportion of the urea to the fatty alcohol-polyoxyethylene ether is controlled to be (16-17): 1, not only optimizes the hemolytic effect, but also reduces the deformability of the cells when the smear is observed under a microscope without the background of bad hemolytic residues, and most importantly, improves the preservation time of the cells, so that the cells are not deformed after being placed in the cell preservation treatment solution for 56 days.

Preferably, the anticoagulant is prepared from a chelating agent and bromhexine according to the mass ratio of (10-20): 1, mixing; more preferably, the anticoagulant is prepared from a chelating agent and bromhexine according to the mass ratio of 16.7: 1 are mixed.

By adopting the technical scheme, the bromhexine and the chelating agent are compounded, so that under the action of AEO-9 and PEG400, the consumption of the bromhexine can be reduced, the curing effect of the cell preservation treatment solution and the softening and scattering effect of mucus can be improved, and the preservation time of cells can be further prolonged.

Preferably, the chelating agent is dipotassium ethylenediamine tetraacetate or ethylenediamine tetraacetic acid; more preferably, the chelating agent is dipotassium ethylenediaminetetraacetate.

By adopting the technical scheme, the compound of the dipotassium ethylenediamine tetraacetate and the bromhexine is adopted as the anticoagulant, so that the permeability of the cell preservation treatment fluid to cells is further improved, the dispersity of mucus can also be improved, and the observation of cell morphology is facilitated.

In a second aspect, the present application provides a method for preparing a liquid-based cell preservation treatment solution, which adopts the following technical scheme: a preparation method of a liquid-based cell preservation treatment liquid comprises the following steps:

adding sodium chloride, phosphate buffer solution, anticoagulant, fatty alcohol-polyoxyethylene ether and PEG into ultrapure water according to a certain proportion, dissolving, adding ethanol, and uniformly stirring to obtain the liquid-based cell preservation treatment solution.

In a third aspect, the present application provides an application of a liquid-based cell preservation treatment solution, which adopts the following technical scheme:

the application of the liquid-based cell preservation treatment liquid is applied to preservation of cells and microorganisms in a body fluid sample.

In summary, the present application has the following beneficial effects:

1. the application adopts ethanol as a fixed dehydrating agent, the content of the ethanol is 18-28 wt%, cell agglomeration is not easy to cause, protein mucus is not easy to agglomerate and deposit, and the dispersion and observation of cells are facilitated. And the combination of ethanol and PEG solves the fixation effect of the monoethanol, has less dosage, reduces the cost, overcomes the problem of methanol, has lower toxicity, inhibits the volatilization of the ethanol, has small cell deformation, and prolongs the preservation time of the cell. The fatty alcohol-polyoxyethylene ether and PEG act together, so that the solubilization and the dispersibility of the system can be increased, the mucus softening and scattering effects are improved, the difficulty of digestion and damage of cells is effectively reduced, the defects of incapability of treating blood cells, stacking of positive cells, reduction of cell nucleuses and the like are overcome, the preservation time of the cells is prolonged, the dispersion and observation of the cells are facilitated, and the detection accuracy is improved.

2. The fatty alcohol-polyoxyethylene ether with the HLB value of 13-14 is adopted, so that the water solubility is good, the softening and scattering effect on mucus is good, the permeability of cell membranes can be enhanced, the hemolysis effect is good, the cell deformability is effectively reduced, the preservation time of cells is prolonged, and even if the cells are preserved for 112 days, few cells deform.

3. The hemolytic agent is prepared by compounding urea and fatty alcohol-polyoxyethylene ether, and the proportion of the urea to the fatty alcohol-polyoxyethylene ether is controlled to be (16-17): 1, not only optimizes the hemolytic effect, but also reduces the deformability of the cells, so that the cells are not deformed after being placed in the cell preservation treatment solution for 56 days.

4. According to the application, bromhexine and a chelating agent are compounded, so that under the action of AEO-9 and PEG400, the dosage of bromhexine can be reduced, the curing effect and the mucus softening and scattering effect of the cell preservation treatment fluid can be improved, and the preservation time of cells is further prolonged.

Drawings

FIG. 1 is a schematic microscopic view of a cell smear with good mucus digestion and no overlapping accumulation of cells after staining of exfoliated cervical cells.

FIG. 2 is a schematic representation under a microscope of a cell smear without nuclear shrinkage after staining of exfoliated cervical cells.

FIG. 3 is a schematic under a microscope of a cell smear without cell deformation after staining of exfoliated cervical cells.

Detailed Description

The present application will be described in further detail with reference to examples.

The raw materials used in the present application are commercially available and, unless otherwise specified, are all purchased from chemical reagents of national drug group, ltd.

Examples

Examples 1 to 26 provide a liquid-based cell preservation treatment liquid, and the following description will be given by taking example 1 as an example.

The composition of the raw materials of the liquid-based cell preservation treatment liquid provided in example 1 is shown in table 1, and the preparation steps are as follows:

adding sodium chloride, phosphate buffer solution, disodium ethylene diamine tetraacetate, AEO-7 and PEG-200 into ultrapure water according to the mass percentage in the table 1, dissolving, adding absolute ethyl alcohol, and uniformly stirring to obtain liquid-based cell preservation treatment liquid;

wherein the ultrapure water is water with the resistivity of 18.3M omega.m (25 ℃), and is treated by an ultrapure water treatment device with the model number of HDN-B689 (purchased from environmental protection equipment Co., Ltd., Funuotai, spring State);

the phosphate buffer solution is 57.7mL of 1mol/L Na₂HPO₄And 42.3mL of 1mol/L NaH₂PO₄Preparing a solution;

the AEO-7 was purchased from Nantong Arciss chemical Co., Ltd;

the PEG-200 is purchased from the Haian petrochemical plant of Jiangsu province.

TABLE 1

Examples 2-5, like example 1, differ only in that: the liquid-based cell preservation treatment liquid is different in mass percentage of the preparation raw materials, and is specifically shown in table 1.

Example 6, like example 5, differs only in that: the PEG-200 is replaced by PEG-400, and the PEG-400 is purchased from Haian petrochemical plants in Jiangsu province.

Example 7, like example 5, differs only in that: the PEG-200 is replaced by PEG-600, and the PEG-600 is purchased from Haian petrochemical plants in Jiangsu province.

Example 8, like example 6, differs only in that: the AEO-7 was replaced with AEO-9, which was purchased from Nantong Arciss chemical Co., Ltd.

Example 9, like example 6, differs only in that: the AEO-7 was replaced with AEO-15, which was purchased from Nantong Arciss chemical Co., Ltd.

Examples 10-12, like example 8, differ only in that: the mass percentages of AEO-9 and PEG-400 are different, and are shown in Table 2.

TABLE 2

Examples	Example 8	Example 10	Example 11	Example 12
					AEO-9(wt％)	0.06	0.065	0.055	0.06
PEG-400(wt％)	4	4	4	4.5

Examples 13-17, like example 8, differ only in that: the mass percentages of the urea and the KOH are different, and are shown in Table 3.

TABLE 3

Examples	Example 8	Example 13	Example 14	Example 15	Example 16	Example 17
							Urea (wt%)	0	0.1	2	0.8	1.3	1.1
KOH(wt％)	0	1	3	1.5	2.5	2

The following describes the treatment solution for preserving liquid-based cells provided in examples 13 to 17, taking example 13 as an example.

The liquid-based cell preservation treatment liquid provided in example 13, which is prepared by the steps of:

according to the mass percentage, adding 0.78 wt% of sodium chloride, 1.1 wt% of phosphate buffer solution, 1 wt% of KOH, 0.5 wt% of disodium ethylene diamine tetraacetate, 0.1 wt% of AEO-90.06 wt% of urea and 4 wt% of PEG-4004 into ultrapure water, dissolving, adding 23 wt% of absolute ethyl alcohol, and uniformly stirring to obtain the liquid-based cell preservation treatment solution.

Examples 14 to 17 provide methods for preparing liquid-based cell preservation solutions, which are similar to example 13, except that: the mass percentages of urea and KOH were different and are shown in table 3.

Example 18, like example 17, differs only in that: the mass percentage of the urea is replaced by 0.96 wt% from 1.1 wt%.

Example 19, like example 17, differs only in that: the mass percentage of the urea is replaced by 1.02 wt% from 1.1 wt%.

Example 20, like example 18, differs only in that: and the disodium ethylene diamine tetraacetate is replaced by dipotassium ethylene diamine tetraacetate.

Example 21, like example 18, differs only in that: the disodium ethylene diamine tetraacetate is replaced by ethylene diamine tetraacetic acid.

Examples 22-25, like example 20, differ only in that: the anticoagulant has different compositions, and is specifically shown in table 4.

TABLE 4

Anticoagulant composition	Example 20	Example 22	Example 23	Example 24	Example 25
						Ethylenediaminetetraacetic acid dipotassium salt (% by weight)	0.5	0.5	0.5	0.5	0.6
Bromhexine (wt%)	0	0.01	0.05	0.03	0.03

Wherein the CAS number of the bromhexine is 3572-43-8.

Example 26, like example 24, differs only in that: the bromhexine is replaced by bromhexine hydrochloride, and the CAS number of the bromhexine hydrochloride is 611-75-6.

Comparative example

Comparative example 1, like example 2, differs only in that: the content of the absolute ethyl alcohol is changed from 28 wt% to 30 wt%.

Comparative example 2, like example 1, differs only in that: the content of the absolute ethyl alcohol is replaced by 15 wt% from 18 wt%.

Comparative example 3, like example 1, differs only in that: the AEO-7 is replaced by Tween-20, and the Tween-20 is purchased from Haian national cloud chemical Co.

Comparative example 4, like example 1, differs only in that: the AEO-7 was replaced with TX-10, and the TX-10 was purchased from Haian petrochemical plants in Jiangsu province.

Comparative example 5, like example 1, differs only in that: PEG-200 is replaced by PEG-10000, and PEG-1000 is purchased from Haian petrochemical plants in Jiangsu province.

Comparative example 6, like example 1, differs only in that: the content of PEG-200 is changed from 3 wt% to 0 wt%.

Performance test

The following performance tests were performed on the liquid-based cell preservation treatment liquids provided in examples 1 to 26 of the present application and comparative examples 1 to 6.

1. And (3) deposition: 320 samples of cervical secretions collected in multi-hospital clinical laboratory and physical laboratory in Shenzhen city during 2020, 1-12 months were prepared and processed to obtain cervical exfoliated cell specimens, 10 cervical exfoliated cell specimens were screened for each of the examples and comparative examples, the cervical exfoliated cell specimens were placed in the liquid-based cell preservation treatment solutions provided in examples 1-26 and comparative examples 1-6 for 28 days, and the number of specimens without sediment was observed, and the test results are shown in Table 5.

2. Post-cell staining state: 320 samples of cervical secretions collected from multiple hospital clinical laboratory and physical laboratory in Shenzhen, 1-12 months of 2020 were prepared and treated to obtain cervical exfoliated cell specimens, 10 cervical exfoliated cell specimens were screened for each of the examples and comparative examples, the cervical exfoliated cell specimens were placed in the liquid-based cell preservation treatment solutions provided in examples 1-26 and comparative examples 1-6 for 28 days and then removed and transferred onto glass slides to prepare 320 cell smears, and the cell smears were stained with a Papanicou EA36 (obtained from Shanghai such as Gi Biotech development Co., Ltd.), and then the number of the smears with good mucus digestion and no cell overlapping accumulation (the effect shown in FIG. 1) and no cell nucleus shrinkage (the effect shown in FIG. 2) was observed under a microscope, and the results of the tests are shown in Table 5.

3. Storage time: 960 samples of cervical secretions collected in Shenzhen, multi-family hospital clinical laboratory and physical laboratory during the period of 2020 and 1-12 months were prepared and divided into three groups, each group comprising 28 days, 56 days and 112 days, and 320 samples of cervical exfoliated cells were obtained by processing, 10 samples of cervical exfoliated cells were screened for each of the examples and comparative examples, the samples of cervical exfoliated cells were placed in the liquid-based cell preservation treatment solutions provided in examples 1-26 and comparative examples 1-6 for 28 days, 56 days and 112 days, and then taken out and transferred onto a slide glass, 320 cell smears were prepared for each group, the cell smears were stained with a Papanicolaou EA36 (available from Shanghai, such as Gibbelok Biotech development Co., Ltd.), and the number of smears without cell deformation (the effect shown in FIG. 3) was observed under a microscope, and the results of the tests are shown in Table 5.

TABLE 5

The present application is described in detail below with reference to the test data provided in table 5.

It can be seen from example 1 and comparative example 2 that, when the ethanol content is less than 18 wt%, the fixation effect is poor, the morphological integrity of the cells is greatly reduced, the remaining properties are also reduced, the cell surface deformation and cell degeneration are easily caused, and the storage time is reduced.

From the application example 2 and the comparative example 1, it is known that when the ethanol content is higher than 28 wt%, the fixing effect is good, the protein with high fluidity can be fixed, but the protein forms deposits, the protein cannot be well transferred to a glass slide, the cell surface deformation and cell degradation are easily caused, and the storage time is short.

As can be seen from the application example 1 and the comparative example 6, PEG can be used as a fixation enhancer to increase the cell permeability, the combination of ethanol and PEG solves the problem of poor fixation effect of low-dosage ethanol, and the addition of PEG can improve the dispersibility of the system, so that the cells are not easy to digest and damage, and the storage time of the cells is prolonged.

From the application example 1 and the comparative example 5, it is known that the molecular weight of PEG has a large influence on the liquid-based cell preservation solution, not only the fixing and the permeability are weakened, but also the viscosity of PEG-10000 is too high, the softening and scattering effect of mucus is poor, and on the contrary, protein is caused to form sediment, so that the cells are easily digested and damaged, the deformation of the cells is large, and the preservation time of the cells is shortened.

As can be seen from the example 1 and the comparative examples 3 to 4, under the action of PEG, the fatty alcohol-polyoxyethylene ether has better solubilization and dispersibility than polyoxyethylene sorbitan monolaurate and nonylphenol polyoxyethylene ether, the mucus softening and scattering effect is good, the cells are effectively prevented from being digested and damaged, the deformation of the cells is small, and the storage time of the cells is prolonged.

From examples 1 to 5 of the present application, it can be seen that by regulating and controlling the components of each raw material of the liquid-based cell preservation treatment solution, the formation of protein deposits is effectively avoided, the cell surface deformation and cell degeneration are reduced, the preservation time of cells is further improved, and the hemolysis effect is also improved, so that the cell nucleus structure is clear and has no background coloration, wherein example 5 is relatively preferred.

From examples 5 to 7 of the present application, it is understood that PEG-200, PEG-400 and PEG-600 all have the function of increasing cell permeability by the fixation enhancer, wherein the hydrophilicity and viscosity of PEG-400 are more suitable for suppressing the volatilization of ethanol, the fixation effect on cells is better, the preservation time of cells can be prolonged, and the cells are less deformed and less degenerated in 56 days or 112 days.

As is clear from examples 6 and 8 to 9 of the present application, AEO-9 has an ethylene oxide addition number of 9 in AEO-9, and water solubility of AEO-9 is better than that of AEO-7, and has a better softening and scattering effect on mucus, compared with AEO-15 and AEO-7; the HLB value of AEO-9 is 13-14, the permeability enhancing effect of AEO-9 on cell membranes is stronger than that of AEO-15, the hemolytic effect of AEO-15 with the HLB value of 15-16 is poorer, the AEO-9 effectively reduces the deformability of cells, improves the preservation time of the cells and has little cell deformation even after being preserved for 112 days.

From examples 8 and 10 to 12 of the present application, it is understood that the mass ratio of AEO-9 to PEG-400 has a large influence on the dispersibility and the compatibility of the system, and the mass ratio of AEO-9 to PEG-400 in examples 8, 10, 11 and 12 is 1: 66.7, 1: 61.53, 1: 72.73, 1: 75, the experimental data of examples 8 and 10-12 show that example 8 has a superior effect of preserving cells. Example 8 corresponds to a deposit-free number of 10, good mucus digestion, a cell non-overlap stacking number of 10, and a cell nucleus-free reduction of 10, and 10 cell-free deformation was also achieved after 56 days of storage in the cell storage treatment solution provided in example 8.

As can be seen from examples 8 and 13 to 17, the effect of different contents of urea and KOH on the cell preservation time of the system is large, and the effect of 1.1 wt% urea and 2 wt% KOH is excellent.

From examples 17 to 19 of the present application, urea and fatty alcohol-polyoxyethylene ether are compounded as a hemolytic agent, and the ratio of the urea to the fatty alcohol-polyoxyethylene ether is controlled to be (16-17): 1, not only optimizes the hemolytic effect, but also reduces the deformability of cells when the smear is observed under a microscope without the background of hemolytic poor residues, and most importantly, improves the preservation time of the cells, and the number of the deformation of the cells is 10 after the cells are placed in the cell preservation treatment solution for 56 days.

In practical application, besides cervical secretion cast-off cells, the liquid-based cell preservation treatment liquid can also be used for staining cast-off cells such as body fluid, body cavities and body surfaces, assisting clinical pathological diagnosis and assisting development and progress of clinical medicine.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims (4)

1. The liquid-based cell preservation treatment liquid is characterized by comprising the following raw materials in percentage by mass:

22 to 25 percent of ethanol

PEG 3-5%

0.05 to 0.07 percent of fatty alcohol-polyoxyethylene ether

Anticoagulant 0.4-0.6%

0.9-1.3% of phosphate buffer

Sodium chloride 0.75-0.8%

0.8 to 1.3 percent of urea

KOH 1.5-2.5%

The balance of ultrapure water;

the PEG is PEG-400;

the fatty alcohol-polyoxyethylene ether is AEO-9 or AEO-7;

the mass ratio of the fatty alcohol-polyoxyethylene ether to the PEG-400 is 1: 66.7;

the anticoagulant is prepared from a chelating agent and bromhexine according to the mass ratio of (10-20): 1, mixing; the chelating agent is ethylene diamine tetraacetic acid dipotassium or ethylene diamine tetraacetic acid.

2. The liquid-based cell preservation treatment liquid as claimed in claim 1, wherein the mass ratio of urea to fatty alcohol-polyoxyethylene ether is (16-17): 1.

3. the method of preparing a liquid-based cell preservation solution according to claim 1 or 2, comprising the steps of:

adding sodium chloride, phosphate buffer solution, anticoagulant, fatty alcohol-polyoxyethylene ether and PEG into ultrapure water according to a certain proportion, dissolving, adding ethanol, and uniformly stirring to obtain the liquid-based cell preservation treatment solution.

4. Use of the liquid-based cell preservation treatment liquid according to claim 1 or 2 for preserving cells and microorganisms in a body fluid sample.

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