Serosa effusion cell preserving fluid

Abstract

The invention relates to the technical field of liquid-based cytology, and particularly discloses serosa effusion cell preserving fluid. Comprising a preservation solution, an anticoagulant, a red blood cell treatment solution, a cell membrane stabilizer, an antioxidant and a metal ion chelating agent, the preservation solution contains a buffer system, an energy supply component, an antibiotic and a surfactant, and the red blood cell treatment solution contains a hemolysis active component and a cell immobilization component; the preparation method comprises the following steps: firstly, preparing the preserving fluid, adjusting the pH value, adding the active substances, mixing the antibiotics, and then completing constant volume and sterile filtration; the anticoagulant solution and the red blood cell treating fluid are independently prepared, and sterilization is achieved through filtration; and finally, packaging all the components in a sterilized storage bottle in a matched manner. The composition provided by the invention can be used for preserving cells in a clinical serosal effusion sample, and can provide a high-quality sample for subsequent cell morphology observation or detection; in addition, according to the preparation method, the controllability is high when all the components are independently prepared, and the preserving fluid with the consistent performance can be stably prepared.

Description

Technical Field

[0001] This application relates to the field of liquid-based cytology technology, and more specifically, to a serous effusion cell preservation solution. Background Technology

[0002] In the field of clinical testing, the analysis of cellular components in serous effusions is an important basis for disease diagnosis and condition assessment. For example, by observing the morphology of mesothelial cells, inflammatory cells, or tumor cells in the effusion, the type of disease such as infection or tumor can be determined. To ensure the accuracy of test results, serous effusion cell preservation solution is required to process and preserve the collected samples to avoid problems such as changes in cell morphology, decreased activity, or interference by red blood cells during the transportation and storage of the samples. This provides a qualified sample basis for subsequent microscopic observation, cell counting, and other testing steps.

[0003] However, existing cell preservation solutions for serous effusions have significant shortcomings, making it difficult to simultaneously meet multiple needs. Some preservation solutions use buffer systems with poor pH stability under low-temperature preservation conditions, which can easily lead to fluctuations in the pH of the cell environment and cause cell membrane damage. At the same time, some preservation solutions lack effective surfactants, resulting in uneven dispersion of components such as antibiotics and cell protectants, and excessively high local concentrations can irritate the cells. Furthermore, some preservation solutions are not effective in treating red blood cells, failing to completely lyse them or leaving residues that interfere with the observation of nucleated cells. These problems collectively make it difficult to guarantee the morphological integrity and detection accuracy of the preserved cells. Summary of the Invention

[0004] To address the problem that existing cell preservation solutions for serous effusions cannot simultaneously maintain the stability of the cellular physiological environment, ensure the uniform dispersion of various functional components, and effectively process erythrocytes, thereby affecting cell morphological integrity and the accuracy of subsequent detection, this application provides a cell preservation solution for serous effusions.

[0005] In a first aspect, this application provides a cell preservation solution for serous effusion, employing the following technical solution:

[0006] A cell preservation solution for serous effusion, comprising the following components: 800-1000 parts preservation solution; 15-35 parts anticoagulant; 50-150 parts red blood cell treatment solution; 5-15 parts cell membrane stabilizer; 2-8 parts antioxidant; and 1-5 parts metal ion chelating agent.

[0007] By employing the above technical solutions, the preservation solution provides the basic environment necessary for cell survival, maintaining the normal physiological state of cells and laying the foundation for the subsequent functioning of various components. The anticoagulant acts on coagulation-related components in the sample, inhibiting sample coagulation and preventing sample clumping from affecting cell separation and observation. The red blood cell treatment solution targets red blood cells in the sample, reducing interference from red blood cells in the observation of nucleated cells and improving the clarity of nucleated cell observation. The cell membrane stabilizer acts on the cell membrane structure, maintaining cell membrane integrity and reducing morphological abnormalities caused by membrane damage. The antioxidant counteracts oxidative factors during sample preservation, mitigating the effects of oxidation on cells and protecting them from oxidative damage. The metal ion chelating agent binds to metal ions in the sample, reducing the adverse effects of metal ions on cells and maintaining normal cell structure and function.

[0008] Preferably, the preservation solution comprises the following components at the following concentrations: 4-hydroxyethylpiperazine ethanesulfonic acid buffer system, concentration 15-35 mmol / L; glutathione, concentration 0.5-2.5 mmol / L; glucose, concentration 10-30 mmol / L; adenosine, concentration 1-5 mmol / L; sodium chloride, concentration 80-120 mmol / L; gentamicin, concentration 50-150 μg / mL; amphotericin B, concentration 2-8 μg / mL; surfactant polysorbate 80, concentration 0.01%-0.05%; and the balance being deionized water.

[0009] By employing the above technical solutions, the pH of the solution is adjusted and stabilized using a 4-hydroxyethylpiperazine ethanesulfonic acid buffer system, thus adapting to the physiological pH requirements of serous effusion cells and preventing cell morphological changes or functional impairment caused by pH fluctuations. Glutathione scavenge free radicals in the solution, reducing oxidative stress damage to cells and protecting the integrity of intracellular macromolecules. Glucose provides the cells with the energy required for metabolism, maintaining basic cellular physiological activities and preventing metabolic arrest due to insufficient energy. Adenosine regulates cellular metabolic rhythm and protects organelle structures, reducing cell erosion. During preservation, the stress response helps maintain normal cellular physiological functions; sodium chloride regulates the osmotic pressure of the solution, maintaining a balance with the intracellular osmotic pressure and preventing cell swelling or shrinkage due to osmotic imbalance; gentamicin and amphotericin B inhibit bacterial and fungal growth respectively, preventing microbial contamination and thus avoiding damage to cells by microbial metabolites; and the surfactant polysorbate 80 promotes uniform dispersion of functional components in the solution, preventing excessively high local concentrations of components from irritating cells, thereby ensuring the stable protective effect of each active ingredient on cells.

[0010] Preferably, the anticoagulant is one or more of sodium heparin, sodium citrate, or dipotassium ethylenediaminetetraacetate.

[0011] By employing the above-mentioned technical solutions, heparin sodium inhibits thrombin activity and prevents fibrinogen from converting into fibrin, thereby blocking the coagulation cascade reaction and preventing the coagulation of blood components in serous effusion samples and preventing cells from being encapsulated by coagulation clots. Sodium citrate chelates calcium ions in the sample, thereby depriving the sample of essential metal ions for the coagulation process, thus inhibiting the activation of coagulation factors and maintaining the fluidity of the serous effusion sample. Dipotassium ethylenediaminetetraacetate chelates calcium ions and binds to coagulation factors, thus providing a dual inhibition of the coagulation reaction, thereby maintaining the sample's non-coagulation under low-temperature storage conditions and ensuring the free state of cells. By selecting one or more of these technologies in combination, the anticoagulation strength can be flexibly adjusted to suit different coagulation levels in the sample, thus achieving effective anticoagulation for serous effusions with varying degrees of bloodiness.

[0012] Preferably, the red blood cell treatment solution comprises the following components at the following concentrations: saponins at a concentration of 0.05-0.2 g / L; glutaraldehyde at a concentration of 0.5-2.0 mL / L; a Tris-HCl buffer system at a concentration of 20-50 mmol / L and a pH value of 7.0-7.6; and the balance being deionized water.

[0013] By employing the above technical solutions, saponins act on the erythrocyte membrane, specifically disrupting its structure and promoting erythrocyte lysis, thereby reducing erythrocyte residue and preventing it from obscuring or interfering with the observation of nucleated cells. Glutaraldehyde binds to surface proteins of nucleated cells, fixing their morphology and preventing structural damage during hemolysis, thus protecting the integrity of nucleated cells and maintaining their original morphological characteristics. The Tris-HCl buffer system regulates the pH of the treatment solution, maintaining environmental stability during erythrocyte treatment, ensuring efficient hemolysis by saponins and stable cell fixation by glutaraldehyde, avoiding the impact of pH fluctuations on the effectiveness of both. These three components work synergistically to effectively treat erythrocytes while protecting nucleated cells, achieving a balance between erythrocyte clearance and nucleated cell morphology preservation.

[0014] Preferably, the cell membrane stabilizer is one or both of phosphatidylcholine and cholesterol, with a concentration of 0.1-0.5 mmol / L.

[0015] By employing the above technical solutions, phosphatidylcholine is used to supplement the core components of the cell membrane phospholipid bilayer, repairing minor damage to the cell membrane caused by low temperatures and component contact during preservation, and maintaining the continuity of the membrane structure. This prevents cytoplasmic leakage and nucleus exposure due to membrane damage, ensuring the integrity of cell morphology. Cholesterol is embedded in the cell membrane phospholipid bilayer to regulate membrane fluidity and rigidity, preventing cell membrane rupture due to excessive stiffness during low-temperature preservation or membrane dysfunction due to excessive fluidity. This maintains normal cell membrane function under low-temperature preservation conditions and reduces the impact on cell activity caused by abnormal membrane conditions. When phosphatidylcholine and cholesterol are used in combination, they synergistically maintain the cell membrane state from two dimensions: membrane structure supplementation and membrane fluidity regulation. This adapts to the needs of cell membranes under different preservation durations, avoiding the problem of insufficient effect of a single component at a specific preservation stage.

[0016] Preferably, the antioxidant is one or both of vitamin E and vitamin C, with a concentration of 0.05-0.2 mmol / L.

[0017] By employing the above technical solution, vitamin E, a lipid-soluble antioxidant, acts on the cell membrane surface and intracellular lipid structure, scavenging free radicals on the membrane and inhibiting lipid peroxidation. This prevents the cell membrane from becoming structurally loose and permeable due to lipid oxidation, thus ensuring cell membrane integrity. Vitamin C, a water-soluble antioxidant, diffuses into the intracellular and extracellular fluid environments, capturing free radicals and reducing oxidized biomolecules. This reduces damage to cytoplasmic enzymes and nucleic acids by free radicals, maintaining normal cellular metabolic function. When vitamin E and vitamin C are used together, they respectively cover the antioxidant regions of the lipid and aqueous phases, forming a synergistic antioxidant network. This adapts to the oxidative stress requirements of serous effusion cells at different preservation stages, avoiding the problem of incomplete antioxidant effects due to the limited area of ​​action of a single antioxidant.

[0018] Preferably, the metal ion chelating agent is one or both of ethylenediaminetetraacetic acid or citrate, with a concentration of 0.5-2.0 mmol / L.

[0019] By employing the above technical solutions, ethylenediaminetetraacetic acid (EDTA) forms stable chelates with calcium and magnesium ions in serous effusions, thereby depriving metal ions of their role as enzyme activators. This inhibits the activation of autolytic enzymes and oxidases due to the presence of metal ions, reducing enzymatic damage to cell structures. Citrate binds to free metal ions in the sample, reducing their concentration and mitigating their damaging effects on cell membranes. This prevents metal ion-mediated lipid peroxidation of cell membranes and maintains stable cell morphology. When EDTA and citrate are used in combination, their differing affinities for different metal ions create a synergistic chelating effect, resulting in a more comprehensive removal of various metal ions from the sample, avoiding the insufficient chelation of specific ions by a single chelating agent.

[0020] Secondly, this application provides a method for preparing a cell preservation solution for serous effusion, which adopts the following technical solution:

[0021] A method for preparing a cell preservation solution for serous effusion includes the following steps:

[0022] S1. Preparation of preservation solution: In the first reaction vessel, add 70%-80% of the total amount of deionized water at a temperature of 4-10℃. Under continuous stirring, slowly add the measured amount of 4-hydroxyethylpiperazine ethanesulfonic acid, sodium chloride and glucose at 4-10℃. Stir at a rate of 200-400 rpm until completely dissolved. The stirring time is 15-30 minutes.

[0023] S2, First pH adjustment: Add a 1 mol / L sodium hydroxide solution to the solution obtained in S1, and adjust the pH value to 7.0-7.2 under constant temperature conditions of 4-10℃;

[0024] S3. Add active ingredients: Keep the temperature at 4-10℃ and the stirring state, add glutathione and adenosine to the solution obtained in S2 in sequence. Add the next ingredient only after each ingredient is completely dissolved, and control the interval between additions to 5-10 minutes.

[0025] S4. Add surfactant: Add polysorbate 80 to the solution and continue stirring at a speed of 200-400 rpm for 10-20 minutes;

[0026] S5. Second pH adjustment: Detect the pH value of the solution again, and adjust the pH value to 6.8-7.4 using a 0.1mol / L hydrochloric acid or sodium hydroxide solution at a temperature of 4-10℃.

[0027] S6. Add antibiotics: Add gentamicin and amphotericin B to the solution obtained in S5, and stir at a speed of 200-300 rpm for 5-10 minutes.

[0028] S7. Volume Adjustment and Filtration: Transfer the solution to a volume adjustment container, wash the reaction container with the remaining deionized water and combine the washings, and finally adjust the volume to the required volume; then perform aseptic filtration using a 0.22μm microporous membrane, dispense the solution into aliquots and seal them for storage in the dark after filtration;

[0029] S8. Preparation of anticoagulant solution: Dissolve the anticoagulant components in deionized water at 18-25℃ to prepare an aqueous solution with a concentration of 5-15%. After filtration through a 0.22μm microporous membrane for sterilization, dispense and seal.

[0030] S9. Preparation of red blood cell treatment solution: Dissolve Tris base in deionized water at 18-25℃ and adjust the pH to 7.0-7.6 with hydrochloric acid; then add saponins and glutaraldehyde sequentially at 20-25℃. After adding saponins, stir at 300-500 rpm for 15-30 minutes, then add glutaraldehyde and continue stirring for 10-15 minutes; finally, bring the volume to a final volume and filter through a 0.22μm microporous membrane for sterilization.

[0031] S10. Matching Packaging: The preservation solution, anticoagulant solution, red blood cell treatment solution, cell membrane stabilizer, antioxidant, and metal ion chelating agent are packaged together with the sterilized preservation bottle.

[0032] By adopting the above technical solutions, a systematic process has been formed, from the precise preparation of each component to the matching packaging. This ensures both the stability of the activity of each component and that the final preservation solution is suitable for the preservation needs of serous effusion cells. In the preservation solution preparation stage, by controlling the low temperature environment of 4-10℃ and the stirring speed of 200-400rpm, the degradation of active components such as 4-hydroxyethylpiperazine ethanesulfonic acid, glutathione, and adenosine due to high temperature can be avoided, while also promoting the full dissolution of each component and preventing local aggregation, thus preventing cell irritation due to uneven component distribution. Through two pH adjustments, combined with acid and alkaline solutions of different concentrations, the pH of the preservation solution can be precisely controlled, avoiding excessive pH fluctuations caused by a single adjustment, and ensuring that the environment is always suitable for the physiological needs of cells. Adding gentamicin and amphotericin B in the later stage and controlling the stirring speed of 200-300rpm can reduce the contact reaction time between the antibiotics and other active ingredients, maintaining their antibacterial and antifungal effects. Meanwhile, the anticoagulant is prepared separately at 18-25℃ and formulated into a 5-15% aqueous solution to avoid the impact of low temperature on its anticoagulant activity. Strict control of the pH of the Tris-HCl buffer system and the stirring parameters of the saponins during the preparation of the red blood cell treatment solution ensures that the saponins and glutaraldehyde work synergistically to achieve hemolysis and cell fixation. Furthermore, sterilization is achieved throughout the process using a 0.22μm microporous membrane, effectively removing microbial impurities from each component and preventing microbial metabolic damage to cells during storage. The final packaging prevents cross-contamination or loss of activity of the components during storage and transportation, ensuring that all components are in optimal condition before use, thus providing a stable and safe storage environment for serous effusion cells.

[0033] Preferably, in step S7, the preservative solution before filtration is first cooled to 2-8°C and allowed to stand for 20-40 minutes, and the filtration process is carried out at 15-25°C.

[0034] By employing the above technical solution, the preservation solution is first cooled to 2-8℃ and allowed to stand for 20-40 minutes before filtration. This promotes the full sedimentation of any small, insoluble impurities that may remain in the solution, thereby reducing the clogging of the 0.22μm microporous membrane during subsequent filtration and improving filtration efficiency. Simultaneously, it prevents small impurities from entering subsequent stages with the preservation solution, preventing them from adhering to the surface of serous effusion cells or interfering with the physiological environment of the cells, thus ensuring the purity of the environment during cell preservation. Controlling the filtration process at 15-25℃ avoids the microporous membrane material becoming brittle due to low temperatures, reducing the risk of membrane breakage. It also maintains the stable dissolution of active components such as glutathione, adenosine, and antibiotics in the preservation solution, preventing the leaching of these components at low temperatures and their removal with the filter residue. This ensures smooth filtration, prevents the loss of concentrations of various functional components in the preservation solution, and maintains their original cell-protective activity. Ultimately, the filtered preservation solution meets sterility requirements and stably exerts its protective effect on serous effusion cells.

[0035] Preferably, in step S9, the saponin is added at 20-25°C, and then stirred at 300-500 rpm for 15-30 minutes.

[0036] By adopting the above technical solution, adding saponins at 20-25℃ can adapt to the solubility characteristics of saponins, promote their rapid dissolution, and maintain hemolytic activity. This avoids the slow dissolution of saponins at low temperatures, which can lead to the formation of undissolved particles, or the denaturation of saponin structures at high temperatures. This achieves the effect of uniformly dissolving saponins in the buffer system and ensuring the stable initiation of subsequent hemolytic activity. Stirring at 300-500 rpm for 15-30 minutes after adding saponins enhances the uniformity of saponin dispersion in the Tris-HCl buffer system and avoids excessively high or low local saponin concentrations. This achieves the effect of creating a uniform concentration environment for saponins in the system, allowing them to act precisely on the erythrocyte membrane without affecting the subsequent glutaraldehyde fixation effect.

[0037] In summary, this application has the following beneficial effects:

[0038] 1. Because this application uses a specific combination of preservation solution, anticoagulant, red blood cell treatment solution, cell membrane stabilizer, antioxidant and metal ion chelating agent, the 4-hydroxyethylpiperazine ethanesulfonic acid buffer system in the preservation solution can maintain the physiological environment required by the cells, polysorbate 80 helps to evenly disperse each functional component, and the saponins and glutaraldehyde in the red blood cell treatment solution work synergistically to achieve the effect of providing protection for serous effusion cells from multiple dimensions such as environmental stability, component dispersion and red blood cell treatment.

[0039] 2. In this application, phosphatidylcholine or cholesterol is preferred as a cell membrane stabilizer. Since these substances are important components of the cell membrane, they can replenish cell membrane loss and repair minor damage during preservation, prevent cytoplasmic outflow and cell nucleus exposure, and avoid cell morphological changes or death due to membrane structure damage. This achieves the effect of helping to maintain the integrity of the morphology and normal physiological function of serous effusion cells.

[0040] 3. The method of this application controls the entire preparation of the preservation solution at a low temperature of 4-10℃ and adjusts the pH value twice. The addition temperature and stirring rate of saponins are strictly controlled during the preparation of the red blood cell treatment solution. Furthermore, each component is sterilely filtered through a 0.22μm microporous membrane after preparation. Therefore, the method achieves the effects of ensuring the stability of each active component, avoiding pH fluctuations that stimulate cells, and ensuring the sterility of the preservation solution, thus providing reliable quality support for the preservation of serous effusion cells. Attached Figure Description

[0041] Figure 1 This is a flowchart of a method for preparing a cell preservation solution for serous effusion provided in this application. Detailed Implementation

[0042] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0043] Technical concept:

[0044] The core problem with existing technologies for cell preservation solutions for serous effusions lies in the difficulty of simultaneously meeting the requirements of maintaining a stable cellular physiological environment, ensuring uniform dispersion of functional components, and effectively processing erythrocytes, resulting in poor cell preservation outcomes. The root causes are: first, the choice of buffer system is inappropriate; most use phosphate buffers, which have poor pH stability under low-temperature storage conditions, easily leading to acid-base fluctuations that stimulate cell membrane damage and nuclear condensation; second, there is a lack of targeted surfactants, causing functional components such as antibiotics and cell protectants to easily aggregate, with excessively high local concentrations damaging cells and insufficient concentrations rendering them ineffective; third, the synergistic effect of erythrocyte processing components is insufficient, either due to insufficient saponin dosage leading to incomplete hemolysis and residual erythrocytes interfering with nucleated cell observation, or the lack of glutaraldehyde-assisted fixation, which easily damages the structure of nucleated cells during hemolysis.

[0045] To address the aforementioned issues, this technical solution employs multi-dimensional component optimization and synergistic design: A 4-hydroxyethylpiperazine ethanesulfonic acid buffer system is selected in the preservation solution, leveraging its low-temperature pH stability to provide a durable and stable physiological environment for cells; polysorbate 80 is added as a surfactant to promote uniform dispersion of components such as antibiotics and cell membrane stabilizers, preventing localized concentration anomalies; specific concentration ranges and synergistic usage methods for saponins and glutaraldehyde are defined in the red blood cell treatment solution to ensure effective red blood cell lysis while minimizing damage to nucleated cells; furthermore, a specific ratio of anticoagulants, cell membrane stabilizers, antioxidants, and metal ion chelators is used to achieve synergistic effects in terms of environmental stability, component dispersion, red blood cell treatment, and cell structure protection, thoroughly resolving the core deficiencies of existing technologies.

[0046] Preparation Example 1

[0047] Taking the preparation of a 500 mL Tris-HCl buffer system with a concentration of 30 mmol / L and a pH of 7.4 suitable for red blood cell processing as an example;

[0048] First, prepare analytical grade Tris powder, 1 mol / L hydrochloric acid solution, ultrapure water, a pH meter calibrated at 25°C, a 500 mL volumetric flask, a magnetic stirrer, a 0.22 μm sterile filter membrane, a clean beaker, a pipette, and sterile containers. Weigh 1.817 g of Tris powder and place it in a clean beaker. Add approximately 400 mL of ultrapure water to the beaker. Turn on the magnetic stirrer and adjust the speed to 200 rpm, stirring continuously until the Tris powder is completely dissolved, maintaining a stable solution temperature of 25°C throughout the process. Then, use a pipette to slowly add 1 mol / L hydrochloric acid solution dropwise to the above solution, maintaining stirring during the addition and monitoring the pH value of the solution with the pH meter in real time. When the pH value approaches 7.4, slow down the adding rate until the pH meter reading stabilizes at 7.4 with an error controlled within ±0.05.

[0049] Transfer the pH-adjusted solution to a 500 mL volumetric flask. Rinse the inner wall of the beaker several times with a small amount of ultrapure water, adding all the washings to the volumetric flask. Finally, add ultrapure water to the mark, tighten the stopper, and invert to mix. Filter the mixed solution through a 0.22 μm sterile filter membrane, collect the filtrate into a sterile container, seal the container, label it with the preparation date, and store it at 4°C protected from light. This buffer system is stable for 1-2 weeks. Before use, allow it to reach room temperature and retest the pH using a pH meter. Once the pH is confirmed to be within the range of 7.3-7.5, it is ready for use.

[0050] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products:

[0051] 1. 4-Hydroxyethylpiperazine ethanesulfonic acid was purchased from Wuhan Dingxintong Pharmaceutical Co., Ltd., product number DXT-SH016;

[0052] 2. Glutathione was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S20186;

[0053] 3. Adenosine was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S18049;

[0054] 4. Polysorbate 80 was purchased from Wuhan Jiyesheng Chemical Co., Ltd., product number A00288;

[0055] 5. Gentamicin was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number V30125;

[0056] 6. Amphotericin B was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S17017;

[0057] 7. Sodium heparin was purchased from Hubei Xindesheng Materials Technology Co., Ltd., product number DS-002;

[0058] 8. Dipotassium ethylenediaminetetraacetate was purchased from Hubei Xindesheng Materials Technology Co., Ltd., item number DS-003;

[0059] 9. Saponins were purchased from Shanghai Yimiao Chemical Technology Co., Ltd., product number VWRC0163;

[0060] 10. Glutaraldehyde was purchased from Wuhan Jiyesheng Chemical Co., Ltd., product number 38;

[0061] 11. Tris base was purchased from Kingclone (Beijing) Biotechnology Co., Ltd., product number CS7715;

[0062] 12. Phosphatidylcholine was purchased from Hubei Yikangyuan Chemical Co., Ltd., product number 8002-43-5;

[0063] 13. Cholesterol was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S11040;

[0064] 14. Vitamin E was purchased from Hubei Hengjingrui Chemical Co., Ltd., product number 59-02-9;

[0065] 15. Vitamin C was purchased from Nanjing Yaoshi Technology Co., Ltd., product number PB93904;

[0066] 16. Ethylenediaminetetraacetic acid (EDTA) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S30020.

[0067] Example 1

[0068] This application provides a serous effusion cell preservation solution, which is composed of the following components:

[0069] 900 parts of preservation solution; 25 parts of anticoagulant; 100 parts of red blood cell treatment solution; 10 parts of cell membrane stabilizer; 5 parts of antioxidant; 3 parts of metal ion chelating agent.

[0070] The preservation solution comprises the following components at the following concentrations: 4-hydroxyethylpiperazine ethanesulfonic acid buffer system, concentration 25 mmol / L; glutathione, concentration 1.5 mmol / L; glucose, concentration 20 mmol / L; adenosine, concentration 3 mmol / L; sodium chloride, concentration 100 mmol / L; gentamicin, concentration 100 μg / mL; amphotericin B, concentration 5 μg / mL; surfactant polysorbate 80, concentration 0.03%; and the balance being deionized water.

[0071] The red blood cell treatment solution comprises the following components at the following concentrations: saponins, 0.125 g / L; glutaraldehyde, 1.25 mL / L; a Tris-HCl buffer system, 35 mmol / L, pH 7.3; and the remainder is deionized water.

[0072] The anticoagulants were sodium heparin and sodium citrate; the cell membrane stabilizer was phosphatidylcholine at a concentration of 0.3 mmol / L; the antioxidant was vitamin E at a concentration of 0.125 mmol / L; and the metal ion chelating agent was ethylenediaminetetraacetic acid at a concentration of 1.25 mmol / L.

[0073] The preparation method of the above-mentioned serous effusion cell preservation solution includes the following steps:

[0074] S1. Preparation of preservation solution: In the first reaction vessel, add 75% of the total amount of deionized water at a temperature of 7°C. Under continuous stirring, slowly add the measured amount of 4-hydroxyethylpiperazine ethanesulfonic acid, sodium chloride and glucose at 7°C. Stir at a rate of 300 rpm until completely dissolved for 22.5 minutes.

[0075] S2, First pH adjustment: Add a 1 mol / L sodium hydroxide solution to the solution obtained in S1, and adjust the pH value to 7.1 under constant temperature of 7℃;

[0076] S3. Add active ingredients: While maintaining a temperature of 7°C and stirring, add glutathione and adenosine to the solution obtained in S2 in sequence. Add the next ingredient only after each ingredient has completely dissolved, with an interval of 7.5 minutes between each addition.

[0077] S4. Add surfactant: Add polysorbate 80 to the solution and continue stirring at 300 rpm for 15 minutes.

[0078] S5. Second pH adjustment: Detect the pH value of the solution again, and adjust the pH value to 7.1 at 7℃ using a 0.1mol / L hydrochloric acid or sodium hydroxide solution;

[0079] S6. Add antibiotics: Add gentamicin and amphotericin B to the solution obtained in S5, and stir at 250 rpm for 7.5 minutes.

[0080] S7. Volume Adjustment and Filtration: Transfer the solution to a volume adjustment container, wash the reaction container with the remaining deionized water and combine the washings, and finally adjust the volume to the required volume; then perform aseptic filtration using a 0.22μm microporous membrane, dispense the solution into aliquots and seal them for storage in the dark after filtration;

[0081] The preservative solution before filtration is first cooled to 5°C and allowed to stand for 30 minutes, while the filtration process is carried out at 20°C.

[0082] S8. Preparation of anticoagulant solution: Dissolve the anticoagulant components in deionized water at 21.5℃ to prepare a 10% aqueous solution. After sterilization by filtration through a 0.22μm microporous membrane, dispense and seal the solution.

[0083] S9. Preparation of red blood cell treatment solution: Dissolve Tris base in deionized water at 21.5℃ and adjust the pH to 7.3 with hydrochloric acid; then add saponin and glutaraldehyde sequentially at 22.5℃. After adding saponin, stir at 400 rpm for 22.5 minutes, then add glutaraldehyde and continue stirring for 12.5 minutes; finally, bring the volume to a final volume and filter through a 0.22μm microporous membrane for sterilization.

[0084] The addition of saponins was carried out at 22.5°C, and the mixture was stirred at 400 rpm for 22.5 minutes after addition.

[0085] S10. Matching Packaging: The preservation solution, anticoagulant solution, red blood cell treatment solution, cell membrane stabilizer, antioxidant, and metal ion chelating agent are packaged together with the sterilized preservation bottle.

[0086] Example 2

[0087] This application provides a serous effusion cell preservation solution, which is composed of the following components:

[0088] 800 parts of preservation solution; 15 parts of anticoagulant; 50 parts of red blood cell treatment solution; 5 parts of cell membrane stabilizer; 2 parts of antioxidant; 1 part of metal ion chelating agent.

[0089] The preservation solution comprises the following components at the following concentrations: 4-hydroxyethylpiperazine ethanesulfonic acid buffer system, concentration 15 mmol / L; glutathione, concentration 0.5 mmol / L; glucose, concentration 10 mmol / L; adenosine, concentration 1 mmol / L; sodium chloride, concentration 80 mmol / L; gentamicin, concentration 50 μg / mL; amphotericin B, concentration 2 μg / mL; surfactant polysorbate 80, concentration 0.01%; and the balance being deionized water.

[0090] The red blood cell treatment solution comprises the following components at the following concentrations: saponins, 0.05 g / L; glutaraldehyde, 0.5 mL / L; a Tris-HCl buffer system, 20 mmol / L, pH 7.0; and the remainder is deionized water.

[0091] The anticoagulants were sodium heparin and dipotassium ethylenediaminetetraacetate; the cell membrane stabilizer was cholesterol at a concentration of 0.1 mmol / L; the antioxidant was vitamin C at a concentration of 0.05 mmol / L; and the metal ion chelating agent was citrate at a concentration of 0.5 mmol / L.

[0092] The preparation method of the above-mentioned serous effusion cell preservation solution includes the following steps:

[0093] S1. Preparation of preservation solution: In the first reaction vessel, add 70% of the total amount of deionized water at a temperature of 4°C. Under continuous stirring, slowly add the measured amount of 4-hydroxyethylpiperazine ethanesulfonic acid, sodium chloride and glucose at 4°C. Stir at a rate of 200 rpm until completely dissolved for 15 minutes.

[0094] S2, First pH adjustment: Add a 1 mol / L sodium hydroxide solution to the solution obtained in S1, and adjust the pH value to 7.0 under constant temperature of 4℃;

[0095] S3. Add active ingredients: While maintaining a temperature of 4°C and stirring, add glutathione and adenosine to the solution obtained in S2 in sequence. Add the next ingredient only after each ingredient has completely dissolved, with an interval of 5 minutes between each addition.

[0096] S4. Add surfactant: Add polysorbate 80 to the solution and continue stirring at 200 rpm for 10 minutes.

[0097] S5. Second pH adjustment: Detect the pH value of the solution again, and adjust the pH value to 6.8 at 4℃ using a 0.1mol / L hydrochloric acid or sodium hydroxide solution.

[0098] S6. Add antibiotics: Add gentamicin and amphotericin B to the solution obtained in S5, and stir at 200 rpm for 5 minutes.

[0099] S7. Volume Adjustment and Filtration: Transfer the solution to a volume adjustment container, wash the reaction container with the remaining deionized water and combine the washings, and finally adjust the volume to the required volume; then perform aseptic filtration using a 0.22μm microporous membrane, dispense the solution into aliquots and seal them for storage in the dark after filtration;

[0100] The pre-filtration solution was first cooled to 2°C and allowed to stand for 20 minutes, while the filtration process was carried out at 15°C.

[0101] S8. Preparation of anticoagulant solution: Dissolve the anticoagulant components in deionized water at 18℃ to prepare a 5% aqueous solution, filter it through a 0.22μm microporous membrane for sterilization, and then dispense and seal it.

[0102] S9. Preparation of red blood cell treatment solution: Dissolve Tris base in deionized water at 18℃ and adjust the pH to 7.0 with hydrochloric acid; then add saponin and glutaraldehyde sequentially at 20℃. After adding saponin, stir at 300 rpm for 15 minutes, then add glutaraldehyde and continue stirring for 10 minutes; finally, bring the volume to a final volume and filter through a 0.22 μm microporous membrane for sterilization.

[0103] The addition of saponins was carried out at 20°C, and the mixture was stirred at 300 rpm for 15 minutes after addition.

[0104] S10. Matching Packaging: The preservation solution, anticoagulant solution, red blood cell treatment solution, cell membrane stabilizer, antioxidant, and metal ion chelating agent are packaged together with the sterilized preservation bottle.

[0105] Example 3

[0106] This application provides a serous effusion cell preservation solution, which is composed of the following components:

[0107] 1000 parts of preservation solution; 35 parts of anticoagulant; 150 parts of red blood cell treatment solution; 15 parts of cell membrane stabilizer; 8 parts of antioxidant; 5 parts of metal ion chelating agent.

[0108] The preservation solution comprises the following components at the following concentrations: 4-hydroxyethylpiperazine ethanesulfonic acid buffer system, concentration 35 mmol / L; glutathione, concentration 2.5 mmol / L; glucose, concentration 30 mmol / L; adenosine, concentration 5 mmol / L; sodium chloride, concentration 120 mmol / L; gentamicin, concentration 150 μg / mL; amphotericin B, concentration 8 μg / mL; surfactant polysorbate 80, concentration 0.05%; and the balance being deionized water.

[0109] The red blood cell treatment solution comprises the following components at the following concentrations: saponins, 0.2 g / L; glutaraldehyde, 2.0 mL / L; a Tris-HCl buffer system, 50 mmol / L, pH 7.6; and the remainder is deionized water.

[0110] The anticoagulants were sodium citrate and dipotassium EDTA; the cell membrane stabilizers were phosphatidylcholine and cholesterol, both at a concentration of 0.5 mmol / L; the antioxidants were vitamin E and vitamin C, both at a concentration of 0.2 mmol / L; and the metal ion chelating agents were EDTA and citrate, both at a concentration of 2.0 mmol / L.

[0111] The preparation method of the above-mentioned serous effusion cell preservation solution includes the following steps:

[0112] S1. Preparation of preservation solution: In the first reaction vessel, add 80% of the total amount of deionized water at a temperature of 10°C. Under continuous stirring, slowly add the measured amount of 4-hydroxyethylpiperazine ethanesulfonic acid, sodium chloride and glucose at 10°C. Stir at a rate of 400 rpm until completely dissolved for 30 minutes.

[0113] S2, First pH adjustment: Add a 1 mol / L sodium hydroxide solution to the solution obtained in S1, and adjust the pH value to 7.2 under constant temperature of 10℃;

[0114] S3. Add active ingredients: Keep the temperature at 10℃ and stir. Add glutathione and adenosine to the solution obtained in S2 in sequence. Add the next ingredient only after each ingredient has completely dissolved. The interval between additions should be controlled at 10 minutes.

[0115] S4. Add surfactant: Add polysorbate 80 to the solution and continue stirring at 400 rpm for 20 minutes.

[0116] S5. Second pH adjustment: Detect the pH value of the solution again, and adjust the pH value to 7.4 at 10℃ using a 0.1mol / L hydrochloric acid or sodium hydroxide solution.

[0117] S6. Add antibiotics: Add gentamicin and amphotericin B to the solution obtained in S5, and stir at 300 rpm for 10 minutes.

[0118] S7. Volume Adjustment and Filtration: Transfer the solution to a volume adjustment container, wash the reaction container with the remaining deionized water and combine the washings, and finally adjust the volume to the required volume; then perform aseptic filtration using a 0.22μm microporous membrane, dispense the solution into aliquots and seal them for storage in the dark after filtration;

[0119] The pre-filtration solution was first cooled to 8°C and allowed to stand for 40 minutes, while the filtration process was carried out at 25°C.

[0120] S8. Preparation of anticoagulant solution: Dissolve the anticoagulant components in deionized water at 25℃ to prepare an aqueous solution with a concentration of 15%. After filtration through a 0.22μm microporous membrane for sterilization, dispense and seal.

[0121] S9. Preparation of red blood cell treatment solution: Dissolve Tris base in deionized water at 25°C and adjust the pH to 7.6 with hydrochloric acid; then add saponin and glutaraldehyde sequentially at 25°C. After adding saponin, stir at 500 rpm for 30 minutes, then add glutaraldehyde and continue stirring for 15 minutes; finally, bring the volume to a final volume and filter through a 0.22 μm microporous membrane for sterilization.

[0122] The addition of saponins was carried out at 25°C, and the mixture was stirred at 500 rpm for 30 minutes after addition.

[0123] S10. Matching Packaging: The preservation solution, anticoagulant solution, red blood cell treatment solution, cell membrane stabilizer, antioxidant, and metal ion chelating agent are packaged together with the sterilized preservation bottle.

[0124] Comparative Example 1

[0125] The only difference between this comparative example and Example 1 is that the surfactant polysorbate 80 was not added to the preservation solution, and its composition and preparation steps are exactly the same as those in Example 1.

[0126] Comparative Example 2

[0127] The only difference between this comparative example and Example 1 is that the 4-hydroxyethylpiperazine ethanesulfonic acid buffer system in the preservation solution is replaced with an equal concentration of phosphate buffer system (PBS), while the components and preparation steps are exactly the same as in Example 1.

[0128] Comparative Example 3

[0129] The only difference between this comparative example and Example 1 is that the metal ion chelating agent ethylenediaminetetraacetic acid is replaced with citrate of the same concentration, while the composition and preparation steps are exactly the same as in Example 1.

[0130] Comparative Example 4

[0131] The only difference between this comparative example and Example 1 is that saponins are removed from the red blood cell treatment solution, and only glutaraldehyde and the Tris-HCl buffer system are retained. The components and preparation steps are exactly the same as in Example 1.

[0132] Comparative Example 5

[0133] The only difference between this comparative example and Example 1 is that the cell membrane stabilizer was omitted, while the components and preparation steps are exactly the same as in Example 1.

[0134] Comparative Example 6

[0135] The only difference between this comparative example and Example 1 is that the first pH adjustment in step S2 is omitted. After adding the active component in step S3, the pH is adjusted to 7.1 in step S5. The components and preparation steps are exactly the same as in Example 1.

[0136] 1. Determination of cell morphological integrity rate in serous effusion

[0137] Fresh serous effusion samples from the same batch, excluding those with severe contamination and bloodiness differences >10%, were selected from clinical collection. The samples were divided into 10 equal parts and mixed with the preservation solutions of Examples 1-3, Comparative Examples 1-6, and physiological saline of the blank control group at a volume ratio of 1:1. After mixing, the samples were immediately stored at 4°C, and samples were taken at four time points: 0h, 24h, 72h, and 168h.

[0138] At each time point, 100 μL of the mixture was taken and fixed with an equal volume of 4% paraformaldehyde solution for 15 minutes. Then, 20 μL of the fixative was dropped onto a glass slide to prepare a smear. After air drying, it was stained with Wright-Gymsa stain for 10 minutes, rinsed with running water, and air-dried. The smears were then observed under a 10×40x optical microscope. Five fields of view were randomly selected from each smear, and 200 nucleated cells were counted in each field. The number of morphologically intact cells was recorded. Morphologically intact cells were defined as cells with intact cell membranes, homogeneous cytoplasm, and clear nuclei without condensation or fragmentation. The cell morphology integrity rate was calculated using the following formula:

[0139]

[0140] 2. Determination of nucleated cell viability in serous effusion

[0141] Using the same sample grouping, preservation conditions and time points as the determination of cell morphology integrity rate in serous effusion, 100 μL of the mixture of preservation solutions of Examples 1-3 and Comparative Examples 1-6 was taken at each time point, and 10 μL of trypan blue staining solution with a mass concentration of 0.4% was added. After gently pipetting and mixing, the mixture was allowed to stand at room temperature for 5 minutes.

[0142] Take 20 μL of the stained mixture and add it to a hemocytometer. Observe under a 10×10x optical microscope and count all nucleated cells. Since trypan blue cannot enter living cells, cells not stained with trypan blue are considered live cells, and cells stained blue are considered dead cells. Calculate the nucleated cell viability using the following formula:

[0143]

[0144] Each sample was counted three times and the average was taken.

[0145] 3. Evaluation of erythrocyte lysis effect

[0146] Fresh serous effusion samples from the same batch were used again, divided into 10 equal parts, and then mixed with the preservation solutions of Examples 1-3, Comparative Examples 1-6, and physiological saline of the blank control group at a volume ratio of 1:1. The mixtures were stored at 4°C, and samples were taken at 0h and 24h. At each time point, 1mL of the mixture was taken and centrifuged at 3000rpm for 5 minutes in a centrifuge tube. The clarity of the supernatant was observed and recorded after centrifugation, and was divided into four grades: clear and transparent, slightly turbid, obviously turbid, and visible red blood cell precipitation. The absorbance of the supernatant was then measured at 540nm using a UV-Vis spectrophotometer. A blank control group was set up. The blank control group was treated as follows: fresh bloody serous effusion samples from the same batch as the experimental group were mixed with an equal volume of physiological saline at a 1:1 volume ratio. The remaining treatments were completely consistent with Examples 1-3 and Comparative Examples 1-6. The completely hemolyzed group in the original design was also retained. The same serous effusion samples were diluted 10 times with deionized water and subjected to three freeze-thaw cycles to completely lyse the red blood cells. The lysis rate was calculated to calibrate the lysis rate. The formula for calculating the red blood cell lysis rate is as follows:

[0147]

[0148] The results of the determination of cell morphological integrity rate in serous effusion are shown in Table 1.

[0149] Table 1:

[0150]

[0151]

[0152] The results of the determination of the survival rate of nucleated cells in serous effusion are shown in Table 2.

[0153] Table 2:

[0154] Group Saved for 0 hours Store for 24 hours Store for 72 hours Save for 168 hours Example 1 97.8％ 94.5％ 89.2％ 80.6％ Example 2 97.5％ 91.3％ 84.1％ 74.5％ Example 3 98.2％ 95.9％ 92.1％ 85.3％ Comparative Example 1 97.6％ 90.4％ 82.1％ 71.3％ Comparative Example 2 97.4％ 87.2％ 78.3％ 66.5％ Comparative Example 3 97.5％ 92.0％ 86.5％ 76.8％ Comparative Example 4 97.3％ 84.5％ 75.1％ 64.2％ Comparative Example 5 97.7％ 88.3％ 79.2％ 67.8％ Comparative Example 6 97.5％ 89.4％ 81.3％ 70.2％ Blank control group 97.9％ 81.2％ 69.3％ 53.7％

[0155] The evaluation results of the erythrocyte lysis effect are shown in Table 3.

[0156] Table 3:

[0157]

[0158]

[0159] Summarize:

[0160] Combining Examples 1-3 and Comparative Example 1 with Tables 1, 2, and 3, it can be seen that the addition of the surfactant polysorbate 80 to the preservation solution has a significant impact on product performance. Examples 1-3, due to the addition of polysorbate 80, showed higher cell morphology integrity and nucleated cell viability at different preservation times compared to Comparative Example 1, and also exhibited better supernatant clarity. In contrast, Comparative Example 1, without the addition of polysorbate 80, showed a more significant decrease in cell morphology integrity and viability with preservation time, and the supernatant after hemolysis was also noticeably turbid at 0 h. This is because polysorbate 80 helps to evenly disperse antibiotics and active components in the preservation solution, preventing excessively high local concentrations from stimulating cells. It also helps to improve the dissolution state of hemoglobin after erythrocyte lysis, reducing supernatant turbidity. The absence of polysorbate leads to uneven component dispersion, affecting cell morphology maintenance and viability, and also reducing the clarity of the supernatant after hemolysis.

[0161] Combining Examples 1-3 and Comparative Example 2 with Tables 1 and 2, it can be seen that the type of buffer system in the preservation solution significantly affects the cell preservation effect. Examples 1-3, using the 4-hydroxyethylpiperazine ethanesulfonic acid buffer system, showed higher cell morphology integrity and nucleated cell viability after 24h, 72h, and 168h of preservation compared to Comparative Example 2, which used the phosphate buffer system. The difference became more pronounced with prolonged preservation time. This is because 4-hydroxyethylpiperazine ethanesulfonic acid exhibits stronger pH stability under 4°C low-temperature preservation conditions, maintaining the physiological pH environment required by serous effusion cells for a longer period. In contrast, the phosphate buffer system is prone to pH fluctuations during low-temperature preservation. These fluctuations can stimulate cells, leading to cell membrane damage, nuclear condensation, and other problems, thereby reducing cell morphology integrity and viability.

[0162] Combining Examples 1-3 and Comparative Example 3 with Tables 1, 2, and 3, it can be seen that the type of metal ion chelating agent affects the cell preservation performance. In Example 1, ethylenediaminetetraacetic acid (EDTA) was used as the chelating agent. In Comparative Example 3, after replacing it with an equal concentration of citrate, although the difference in erythrocyte lysis was not significant, the cell morphology integrity and nucleated cell viability in Comparative Example 3 were slightly lower than in Example 1 at all preservation time points. This is because EDTA has a stronger chelating ability for metal ions than citrate, and can more effectively chelate calcium, magnesium, and other metal ions that may be present in serous effusions, reducing the probability of these ions activating cell autolysins and oxidases, thereby better maintaining cell morphology and viability. Citrate, on the other hand, has a weaker chelating ability and cannot completely inhibit metal ion-mediated enzymatic reactions, resulting in slightly poorer cell morphology and viability.

[0163] Combining Examples 1-3 and Comparative Example 4 with Tables 1, 2, and 3, it can be seen that the addition or absence of saponins in the red blood cell treatment solution is crucial to the red blood cell lysis effect and subsequent cell preservation. In Examples 1-3, due to the addition of saponins, the red blood cell lysis rate reached over 72.3% at 0 h, and further increased at 24 h, with no obvious precipitation in the supernatant. In contrast, after removing saponins in Comparative Example 4, the red blood cell lysis rate was extremely low, with visible red blood cell precipitation at both 0 h and 24 h, and the cell morphology integrity rate and nucleated cell survival rate were also far lower than in Examples 1-3. This is because saponins can specifically disrupt the red blood cell membrane, achieving effective red blood cell lysis and preventing residual red blood cells from interfering with the observation and preservation environment of nucleated cells. Without saponins, red blood cells cannot be effectively lysed, and a large number of residual red blood cells will occupy cell preservation space and may also release harmful substances that affect nucleated cells, leading to morphological damage and decreased survival capacity of nucleated cells.

[0164] Combining Examples 1-3 and Comparative Example 5 with Tables 1 and 2, it can be seen that cell membrane stabilizers are crucial for maintaining the morphology and survival of cells in serous effusion. Examples 1-3, which included cell membrane stabilizers, showed higher cell morphology integrity and nucleated cell survival rates at each storage time point compared to Comparative Example 5, which did not include stabilizers. This is because cell membrane stabilizers replenish the lipid components required by the cell membrane, repair minor cell membrane damage caused by environmental stimuli during storage, and prevent cytoplasmic leakage and nucleus exposure, thereby maintaining the cell's intact morphology and normal physiological function. Without cell membrane stabilizers, the cell membrane is easily damaged during low-temperature storage and contact with the storage solution, leading to a decrease in morphological integrity and reduced survival rate. However, it has a relatively small impact on erythrocyte lysis; the clarity and lysis rate of the supernatant in Comparative Example 5 are similar to those in Example 1.

[0165] Combining Examples 1-3 and Comparative Example 6 with Tables 1, 2, and 3, it can be seen that whether or not a first pH adjustment is performed during the preparation of the preservation solution affects the cell preservation effect. Examples 1-3, with two pH adjustments, showed higher cell morphology integrity and nucleated cell viability than Comparative Example 6, which only underwent one pH adjustment. Furthermore, the supernatant of erythrocytes in Comparative Example 6 showed significant turbidity at 0 h after lysis. This is because the first pH adjustment coarsely adjusts the solution pH to near the optimal buffer range of 4-hydroxyethylpiperazine ethanesulfonic acid, providing a stable pH environment for the subsequent addition of active components such as glutathione and adenosine, preventing these components from degrading or denaturing at extreme pH levels. While skipping the first pH adjustment and relying solely on the second adjustment can ultimately reach the target pH, pH fluctuations during the addition of active components can cause some active components to become ineffective. This affects cell energy supply and protection, and also slightly impacts the hemolytic activity of saponins, leading to decreased cell morphology and viability, and poorer supernatant clarity.

[0166] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A serosal fluid cell preservation solution, characterized by: Consists of the following components: preservative solution 800-1000 parts; anticoagulant 15-35 parts; red blood cell processing solution 50-150 parts; cell membrane stabilizer 5-15 parts; antioxidant 2-8 parts; metal ion chelator 1-5 parts.

2. The cell preservation solution for serous membrane effusion according to claim 1, wherein: The preservative solution comprises the following components at the following concentrations: 4-hydroxyethyl piperazine ethanesulfonic acid buffer system, at a concentration of 15-35 mmol / L; glutathione, at a concentration of 0.5-2.5 mmol / L; glucose, at a concentration of 10-30 mmol / L; adenosine, at a concentration of 1-5 mmol / L; sodium chloride, at a concentration of 80-120 mmol / L; gentamicin, at a concentration of 50-150 μg / mL; amphotericin B, at a concentration of 2-8 μg / mL; surfactant polysorbate 80, at a concentration of 0.01%-0.05%; and the balance is deionized water.

3. The cell preservation solution for serosal fluid accumulation according to claim 1, wherein: The anticoagulant is one or more of sodium heparin, sodium citrate or dipotassium ethylenediaminetetraacetate.

4. The cell preservation solution for serous membrane fluid accumulation according to claim 1, wherein: The red blood cell processing solution comprises the following components at the following concentrations: saponin, at a concentration of 0.05-0.2 g / L; glutaraldehyde, at a concentration of 0.5-2.0 mL / L; Tris-HCl buffer system, at a concentration of 20-50 mmol / L, pH 7.0-7.6; and the balance is deionized water.

5. The cell preservation solution for serosal fluid accumulation according to claim 1, wherein: The cell membrane stabilizer is one or both of phosphatidylcholine or cholesterol, at a concentration of 0.1-0.5 mmol / L.

6. The cell preservation solution for serous membrane fluid accumulation according to claim 1, wherein: The antioxidant is one or both of vitamin E or vitamin C, at a concentration of 0.05-0.2 mmol / L.

7. The cell preservation solution for serosal fluid accumulation according to claim 1, wherein: The metal ion chelator is one or both of ethylenediaminetetraacetic acid or citrate, at a concentration of 0.5-2.0 mmol / L.

8. A method for preparing a serosal fluid cell preservation solution, characterized by, A serous effusion cell preservative solution for use in any one of claims 1-7, comprising the following steps: S1, preservative solution preparation: in a first reaction vessel, add deionized water accounting for 70%-80% of the total amount, at a temperature of 4-10°C, under continuous stirring, slowly add a measured amount of 4-hydroxyethyl piperazine ethanesulfonic acid, sodium chloride and glucose at a temperature of 4-10°C, stir at a rate of 200-400 rpm until completely dissolved, and the stirring time is 15-30 minutes; S2, first pH adjustment: add a 1 mol / L sodium hydroxide solution to the solution obtained in S1, and adjust the pH to 7.0-7.2 under constant temperature conditions of 4-10°C; S3, addition of active components: maintain a temperature of 4-10°C and stirring, and sequentially add glutathione and adenosine to the solution obtained in S2, after each component is completely dissolved, add the next one, and the interval time is controlled to be 5-10 minutes; S4, addition of surfactant: add polysorbate 80 to the solution, and continue stirring at a rate of 200-400 rpm for 10-20 minutes; S5, second pH adjustment: detect the pH of the solution again, and adjust the pH to 6.8-7.4 using a 0.1 mol / L hydrochloric acid or sodium hydroxide solution at a temperature of 4-10°C; S6, adding antibiotics: adding gentamicin and amphotericin B to the solution obtained in S5, stirring at a speed of 200-300 rpm for 5-10 minutes; S7, volume setting and filtration: transferring the solution to a volume setting container, washing the reaction container with the remaining deionized water and combining the washings, finally setting the volume to the required volume; then using a 0.22 μm microporous filter for sterile filtration, after filtration is completed, aliquot and seal to protect from light; S8, preparation of anticoagulant solution: dissolving the anticoagulant ingredients in deionized water at 18-25℃ to prepare a 5-15% aqueous solution, filtering and sterilizing with a 0.22 μm microporous filter, then aliquot and seal; S9, preparation of red blood cell treatment solution: dissolving Tris base in deionized water at 18-25℃, adjusting the pH to 7.0-7.6 with hydrochloric acid; then adding saponin and glutaraldehyde successively at 20-25℃, stirring at a speed of 300-500 rpm for 15-30 minutes after adding saponin, and then adding glutaraldehyde and continuing to stir for 10-15 minutes; finally, volume setting and filtering with a 0.22 μm microporous filter to remove bacteria; S10, matching packaging: matching packaging of the preservation solution, anticoagulant solution, red blood cell treatment solution, and cell membrane stabilizer, antioxidant, metal ion chelating agent components with sterilized preservation bottles.

9. The method for preparing a serous effusion cell preservation solution according to claim 8, characterized in that: In step S7, the preservation solution before filtration is cooled to 2-8℃ and left standing for 20-40 minutes, and the filtration process is carried out in an environment of 15-25℃.

10. The method for preparing a serous effusion cell preservation solution according to claim 8, characterized in that: In step S9, the addition of saponin is carried out at 20-25℃, and stirring is carried out at a speed of 300-500 rpm for 15-30 minutes after addition.

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