Exfoliated cell preserving fluid as well as preparation method and application thereof

Abstract

The invention relates to the technical field of sample preservation. The invention provides an exfoliated cell preserving fluid and a preparation method and application thereof, the preserving fluid comprises a cracking agent, a buffering agent, a chelating agent, a mucus dissolving agent and a preservative, the cracking agent and the buffering agent comprise ammonium salt, and the ammonium salt comprises at least one selected from the group consisting of ammonium chloride, ammonium acetate and ammonium bicarbonate. The preserving fluid can selectively lyse red blood cells to reduce blood background interference, and strongly disperse and dissolve mucus to effectively release wrapped target cells; the morphological integrity and nucleic acid integrity of nucleated cells are protected, and morphological abnormalities such as solid shrinkage, dissolution or expansion of the cells are avoided. The biological stability of the sample in the long-term storage and transportation process is ensured due to strong anti-corrosion and antibacterial capabilities; the preservation solution can meet the requirements of cell morphology examination and molecular detection at the same time, double-detection compatibility is achieved, the preparation technology is simple and convenient, and large-scale production is easy to achieve.

Description

Technical Field

[0001] This invention relates to the field of sample preservation technology, specifically to a cell preservation solution, its preparation method, and its application. Background Technology

[0002] Exfoliative cytology uses cells that are naturally shed from the body or obtained through non-invasive / minimally invasive methods (such as collecting sputum, secretions, and serous cavity effusions) for diagnosis. With its core advantages of being non-invasive / minimally invasive, simple, cost-effective, efficient, and highly reproducible, it plays a key role in early disease screening, preliminary diagnosis, and long-term follow-up monitoring. It is widely used in clinical scenarios such as cervical cancer screening (e.g., TCT, HPV), lung cancer diagnosis (e.g., sputum, lavage fluid), and determination of the nature of serous cavity effusions (e.g., cytological examination of pleural effusion, ascites, and pericardial effusion).

[0003] Obtaining high-quality exfoliated cell samples is a prerequisite for accurate interpretation of cell morphology examinations and reliable molecular detection results. This requires the sample preservation solution to simultaneously meet multiple stringent standards: maintain cell morphological integrity for microscopic observation; protect the integrity of intracellular nucleic acids (DNA / RNA) to ensure molecular detection sensitivity; efficiently remove complex interfering substances such as mucus and blood (especially selectively lysing red blood cells and protecting the morphology and nucleic acids of nucleated cells); and be compatible with both morphological examinations and molecular detection requirements. In addition, the sample solution must have long-term stable preservation capabilities to adapt to clinical circulation and ensure that the operation process is safe and non-toxic.

[0004] Currently, widely used alcohol / aldehyde-based exfoliated cell preservation solutions still face challenges in the following aspects: balancing fixation effectiveness and preservation time. High concentrations of alcohol / aldehyde can fix cells for a short period, but cell shrinkage and deformation can impair the accuracy of subsequent cell morphology analysis. Conversely, low concentrations can lead to insufficient fixation and inadequate stability, making cells prone to degradation and significantly shortening the effective preservation time of samples, which is insufficient to meet the actual clinical needs for sample transportation, batch processing, or long-term preservation. Furthermore, certain components in the formulation can damage intracellular DNA / RNA, reduce nucleic acid integrity, affect the sensitivity and reliability of molecular detection (e.g., PCR, FISH), and increase the risk of false negatives.

[0005] Furthermore, these formulations require further improvement in removing complex interfering substances from samples. Their insufficient removal of mucus leads to cell obscuring and affects morphological observation. The processing of blood needs improvement to enable lysis of red blood cells to reduce background interference, while specifically protecting the morphology and nucleic acid integrity of nucleated cells (e.g., leukocytes, tumor cells). This requirement is particularly important in the testing of samples rich in blood components, such as cerebrospinal fluid and serous cavity effusion, directly affecting the identification, counting, and molecular analysis of nucleated cells. A single formulation also struggles to simultaneously meet the dual requirements of intact cell morphology preservation (for morphological examination) and high-quality nucleic acid extraction (for molecular detection), limiting the potential for multi-methodological applications of the same sample. Moreover, most alcohol / aldehyde components are volatile and toxic, posing health risks to operators, and their instability further restricts the long-term preservation of samples.

[0006] Therefore, further research and development is still needed to obtain sample preservation solutions that simultaneously achieve fixation and morphological balance, long-term preservation capability, nucleic acid protection, removal of complex interfering substances (especially for blood components), and compatibility of dual detection of cell morphology and molecular detection, so as to meet the high standards and diverse needs of various exfoliated cell samples in transportation, storage and clinical testing. Summary of the Invention

[0007] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, this invention provides an exfoliated cell preservation solution, its preparation method, and its applications, aiming to overcome the systematic shortcomings of traditional alcohol / aldehyde-based preservation solutions in the preservation of exfoliated cell samples. The preservation solution proposed in this invention, through innovative formulation design, effectively maintains the integrity of cell morphology, avoiding shrinkage, dissolution, or swelling, providing a clear basis for interpretation of cell morphology examinations; simultaneously, it strongly protects the integrity of intracellular DNA / RNA, ensuring high sensitivity and accuracy of molecular detection (e.g., PCR, FISH); and it optimizes its ability to clear complex interfering substances—efficiently dispersing mucus, selectively lysing red blood cells, and protecting the morphology and nucleic acids of nucleated cells (such as white blood cells and tumor cells). A single formulation can simultaneously meet the high-quality slide preparation requirements of cell morphology examination and the high-quality nucleic acid extraction requirements of molecular detection, achieving compatibility for both cell morphology examination and molecular detection, and supporting long-term stable preservation of samples. Combining its safety (non-toxic and environmentally friendly) and efficiency, this preservation solution lays a reliable high-quality sample foundation for clinical testing of various exfoliated cell samples (especially blood samples), significantly improving the accuracy and reliability of test results.

[0008] Therefore, a first aspect of the present invention provides a preservation solution. According to an embodiment of the present invention, the preservation solution comprises a pyrolysis agent, a buffer, a chelating agent, a mucin dissolving agent, and a preservative, wherein the pyrolysis agent and the buffer comprise ammonium salts, the ammonium salts comprising at least one selected from the group consisting of ammonium chloride, ammonium acetate, and ammonium bicarbonate.

[0009] According to embodiments of the present invention, the preservation solution achieves efficient processing and long-term preservation of complex exfoliated cell samples through the specific ratio and synergistic effect of key components. The preservation solution selectively lyses red blood cells to significantly reduce blood background interference, while powerfully dispersing and dissolving mucus to effectively release encapsulated target cells and prevent blood coagulation in the sample. While eliminating interference, the formulation protects the morphological integrity and nucleic acid (DNA / RNA) integrity of nucleated cells (such as leukocytes and tumor cells), preventing morphological abnormalities such as cell shrinkage, dissolution, or swelling, laying the foundation for subsequent detection. Its highly efficient antiseptic and antibacterial capabilities ensure the biological stability of the sample during preservation and transportation, supporting long-term storage. This single formulation can simultaneously meet the requirements of high-quality cell morphology for cell morphology examination and high-quality nucleic acid extraction for molecular detection (such as PCR and FISH), achieving "dual-detection compatibility." Furthermore, the preparation process of this preservation solution is simple and easy to scale up.

[0010] The aforementioned lysing agent and buffer work synergistically to generate an osmotic pressure effect, selectively lysing red blood cells and effectively reducing background interference from the blood. At the same time, their weakly alkaline environment helps to disperse mucus and release the encapsulated target cells. The lysing agent needs to work synergistically with the buffer to generate a suitable osmotic pressure for selective lysis of red blood cells. Experimental verification of this invention has shown that it has a better effect than using either of the two alone.

[0011] According to embodiments of the present invention, the preservation solution may further include at least one of the following additional technical features: According to an embodiment of the present invention, the pyrolysis agent includes at least one selected from the following: ammonium chloride, ammonium acetate.

[0012] According to an embodiment of the present invention, the buffer comprises ammonium bicarbonate.

[0013] According to an embodiment of the present invention, the mass-to-volume ratio of the pyrolysis agent is 0.5% to 0.9%. Exemplarily, the mass-to-volume ratio (w / v) of the pyrolysis agent is 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, or 0.9%, or a range between any two of the above values.

[0014] According to an embodiment of the present invention, the mass-to-volume ratio of the buffer is 0.05% to 0.09%. Exemplarily, the mass-to-volume ratio of the buffer is 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, or 0.09%, or a range between any two of the above values.

[0015] According to an embodiment of the present invention, the chelating agent comprises at least one selected from the following: ethylenediaminetetraacetic acid, citrate, and EGTA.

[0016] According to an embodiment of the present invention, the mucus solvent comprises a thiol compound.

[0017] According to an embodiment of the present invention, the thiol compound is selected from at least one of the following: cysteine, dithiothreitol (DTT), and N-acetylcysteine ​​(NAC).

[0018] According to an embodiment of the present invention, the mucus dissolving agent is selected from cysteine.

[0019] According to an embodiment of the present invention, the preservative includes an azide and an oxidant.

[0020] According to an embodiment of the present invention, the azide comprises sodium azide.

[0021] According to an embodiment of the present invention, the oxidant includes sodium nitrite.

[0022] According to an embodiment of the present invention, the mass-to-volume ratio of the chelating agent is 0.001% to 0.005%. Exemplarily, the mass-to-volume ratio of the chelating agent is 0.001%, 0.002%, 0.003%, 0.004%, or 0.005%, or any range of two of the above values. Preferably, the mass-to-volume ratio of the chelating agent is 0.002% to 0.005%, and more preferably, the mass-to-volume ratio of the chelating agent is 0.002% to 0.003%.

[0023] According to an embodiment of the present invention, the mass-to-volume ratio of the mucus solvent is 0.06% to 0.14%. Exemplarily, the mass-to-volume ratio of the mucus solvent is 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, or 0.14%, or a range between any two of the above values. Preferably, the mass-to-volume ratio of the mucus solvent is 0.06% to 0.10%.

[0024] According to an embodiment of the present invention, the preservative has a mass-to-volume ratio of 0.1% to 0.3%. Exemplarily, the mass-to-volume ratio of the preservative is 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, or 0.3%, or a range between any two of the above values.

[0025] According to an embodiment of the present invention, the mass ratio (w / w) of the pyrolysis agent to the buffer is (8~12):(1~2). Exemplarily, the mass ratio of the pyrolysis agent to the buffer is 4:1, 9:2, 5:1, 8:1, 9:1, or 10:1, or a range between any two of the above mass ratios, with a preferred mass ratio of 10:1. Therefore, at this mass ratio, the synergistic effect between the pyrolysis agent and the buffer is stronger, resulting in a stronger preservation effect and stability of the preservation solution.

[0026] According to an embodiment of the present invention, the pyrolysis agent is ammonium chloride, and the mass-volume ratio of the ammonium chloride is 0.5% to 0.9%. Exemplarily, the mass-volume ratio of the ammonium chloride is 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, or 0.9%, or a range between any two of the above values.

[0027] According to an embodiment of the present invention, the buffer is ammonium bicarbonate, and the mass-to-volume ratio of the ammonium bicarbonate is 0.05% to 0.09%. Exemplarily, the mass-to-volume ratio of the ammonium bicarbonate is 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, or 0.09%, or a range between any two of the above values.

[0028] According to a specific embodiment of the present invention, ammonium chloride and ammonium bicarbonate serve as key lysing agents and buffers, respectively. The two work synergistically to generate an osmotic pressure effect, selectively lysing red blood cells and effectively reducing background interference from blood. At the same time, their weakly alkaline environment helps to disperse mucus and release the encapsulated target cells.

[0029] According to an embodiment of the present invention, the chelating agent is ethylenediaminetetraacetic acid (EDTA), and the mass-to-volume ratio of EDTA is 0.001% to 0.005%. In the preservation solution of a specific embodiment of the present invention, EDTA mainly acts as a chelating agent, exerting a strong anticoagulant effect by chelating divalent metal ions (such as calcium and magnesium ions), preventing blood coagulation in the sample, protecting the integrity of nucleic acids (DNA / RNA), inhibiting nuclease activity, and laying the foundation for subsequent molecular detection.

[0030] According to an embodiment of the present invention, the mucus dissolving agent is cysteine, and the mass-to-volume ratio of cysteine ​​is 0.06% to 0.14%. Exemplarily, the mass-to-volume ratio of the mucus dissolving agent is 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, or 0.14%, or a range between any two of the above values. Preferably, the mass-to-volume ratio of cysteine ​​is 0.06% to 0.10%.

[0031] According to a specific embodiment of the present invention, the preservation solution uses cysteine ​​as a reducing agent and mucus dissolving agent. Its thiol group (-SH) can effectively cleave the disulfide bonds in the mucus protein, strongly disperse and dissolve the mucus, significantly improve sample homogeneity and expose the target cells.

[0032] According to an embodiment of the present invention, the preservative is sodium nitrite and sodium azide, wherein the mass-to-volume ratio of sodium nitrite is 0.05%~0.15%, preferably 0.05%~0.10%, and the mass-to-volume ratio of sodium azide is 0.05%~0.15%, preferably 0.05%~0.10%. The preservation solution according to a specific embodiment of the present invention provides highly efficient antiseptic and antibacterial capabilities through the synergistic effect of sodium nitrite and sodium azide, maintaining the biological stability of samples and supporting long-term preservation. Experimental verification by the present invention has shown that using sodium nitrite and sodium azide together is more effective than using sodium nitrite and sodium azide alone. Exemplarily, the mass-to-volume ratio of sodium nitrite is 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, or 0.15%, or a range between any two of the above values. For example, the mass-volume ratio of the sodium azide is 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, or 0.15%, or a range between any two of the above values.

[0033] According to an embodiment of the present invention, in the preservative, the mass ratio of sodium nitrite to sodium azide is (1~2):(1~2). Exemplarily, the mass ratio of sodium nitrite to sodium azide is 1:2, 1:1, or 2:1, or a range between any two of the above mass ratios, preferably 1:1.

[0034] According to an embodiment of the present invention, the preservation solution further comprises purified water.

[0035] According to an embodiment of the present invention, the pH value of the preservation solution is 5.0 to 7.2. Exemplarily, the pH value of the preservation solution is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, or 7.2, or a range between any two of the above values. Therefore, a preservation solution with suitable acidity and alkalinity can be prepared within this range, and the pH value of the preservation solution can be closer to the body's pH.

[0036] According to an embodiment of the present invention, the pH value of the preservation solution is 5.0 to 6.5. Exemplarily, the pH value of the preservation solution is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, or a range between any two of the above values. In some specific embodiments, this pH value simulates the in vivo environment pH value of cervical exfoliated cells, and cervical exfoliated cells exhibit high stability when preserved in a preservation solution with this pH value.

[0037] According to embodiments of the present invention, the pH value of the preservation solution is 6.7 to 7.2. Exemplarily, the pH value of the preservation solution is 6.7, 6.8, 6.9, 7.0, 7.1, or 7.2, or a range between any two of the above values. In some specific embodiments, cells other than cervical exfoliated cells exhibit high stability when preserved in a preservation solution at this pH value, such as exfoliated cells from sputum, pleural effusion, or ascites.

[0038] A second aspect of the present invention provides a method for preparing the preservation solution described in the first aspect. According to an embodiment of the present invention, the method includes: (1) The solvent is first mixed with the pyrolysis agent, buffer, and chelating agent to obtain solution A. (2) The solvent is mixed with the mucin solvent and preservative in a second mixing process to obtain solution B. (3) After mixing solutions A and B, a preservative is added for a third mixing treatment to obtain the preservation solution. The pyrolysis agent and the buffer include ammonium salts, wherein the ammonium salts include at least one selected from the following: ammonium chloride, ammonium acetate, and ammonium bicarbonate.

[0039] According to embodiments of the present invention, the method for preparing the above-mentioned preservation solution may further include at least one of the following additional technical features: According to an embodiment of the present invention, the pyrolysis agent includes at least one selected from the following: ammonium chloride, ammonium acetate.

[0040] According to an embodiment of the present invention, the buffer comprises ammonium bicarbonate.

[0041] According to an embodiment of the present invention, the mass-to-volume ratio of the pyrolysis agent is 0.5% to 0.9%. Exemplarily, the mass-to-volume ratio of the pyrolysis agent is 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, or 0.9%, or a range between any two of the above values.

[0042] According to an embodiment of the present invention, the mass-to-volume ratio of the buffer is 0.05% to 0.09%. Exemplarily, the mass-to-volume ratio of the buffer is 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, or 0.09%, or a range between any two of the above values.

[0043] According to an embodiment of the present invention, the chelating agent comprises at least one selected from the following: ethylenediaminetetraacetic acid, citrate, and EGTA.

[0044] According to an embodiment of the present invention, the mucus solvent comprises a thiol compound.

[0045] According to an embodiment of the present invention, the thiol compound is selected from at least one of the following: cysteine, dithiothreitol, and N-acetylcysteine.

[0046] According to an embodiment of the present invention, the mucus dissolving agent is selected from cysteine.

[0047] According to an embodiment of the present invention, the preservative includes an azide and an oxidant.

[0048] According to an embodiment of the present invention, the azide comprises sodium azide.

[0049] According to an embodiment of the present invention, the oxidant includes sodium nitrite.

[0050] According to an embodiment of the present invention, the chelating agent has a mass-to-volume ratio of 0.001% to 0.005%. Exemplarily, the mass-to-volume ratio of the chelating agent is 0.001%, 0.002%, 0.003%, 0.004%, or 0.005%, or a range of any two of the above values.

[0051] According to an embodiment of the present invention, the mass-to-volume ratio of the mucus solvent is 0.06% to 0.14%. Exemplarily, the mass-to-volume ratio of the mucus solvent is 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, or 0.14%, or a range between any two of the above values.

[0052] According to an embodiment of the present invention, the preservative has a mass-to-volume ratio of 0.1% to 0.3%. Exemplarily, the mass-to-volume ratio of the preservative is 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, or 0.3%, or a range between any two of the above values.

[0053] According to an embodiment of the present invention, the mass ratio of the pyrolysis agent to the buffer is (8~12):(1~2). Exemplarily, the mass ratio of the pyrolysis agent to the buffer is 4:1, 9:2, 5:1, 8:1, 9:1, or 10:1, or a range between any two of the above mass ratios, with a preferred mass ratio of 10:1. Therefore, at this mass ratio, the synergistic effect between the pyrolysis agent and the buffer is stronger, resulting in a stronger preservation effect and stability of the preservation solution.

[0054] According to an embodiment of the present invention, the pyrolysis agent is ammonium chloride, and the mass-volume ratio of the ammonium chloride is 0.5% to 0.9%. Exemplarily, the mass-volume ratio of the ammonium chloride is 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, or 0.9%, or a range between any two of the above values.

[0055] According to an embodiment of the present invention, the buffer is ammonium bicarbonate, and the mass-to-volume ratio of the ammonium bicarbonate is 0.05% to 0.09%. Exemplarily, the mass-to-volume ratio of the ammonium bicarbonate is 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, or 0.09%, or a range between any two of the above values.

[0056] According to a specific embodiment of the present invention, ammonium chloride and ammonium bicarbonate serve as key lysing agents and buffers, respectively. The two work synergistically to generate an osmotic pressure effect, selectively lysing red blood cells and effectively reducing background interference from blood. At the same time, their weakly alkaline environment helps to disperse mucus and release the encapsulated target cells.

[0057] According to an embodiment of the present invention, the chelating agent is ethylenediaminetetraacetic acid (EDTA), and the mass-to-volume ratio of EDTA is 0.001% to 0.005%. In the preservation solution of a specific embodiment of the present invention, EDTA mainly acts as a chelating agent, exerting a strong anticoagulant effect by chelating divalent metal ions (such as calcium and magnesium ions), preventing blood coagulation in the sample, protecting the integrity of nucleic acids (DNA / RNA), inhibiting nuclease activity, and laying the foundation for subsequent molecular detection.

[0058] According to an embodiment of the present invention, the mucus dissolving agent is cysteine, and the mass-volume ratio of cysteine ​​is 0.06%~0.14%. In the preservation solution of a specific embodiment of the present invention, cysteine ​​is used as a reducing agent and a mucus dissolving agent. Its thiol group (-SH) can effectively cleave the disulfide bonds in mucus proteins, strongly disperse and dissolve mucus, significantly improve sample homogeneity, and expose target cells.

[0059] According to an embodiment of the present invention, the preservative is sodium nitrite and sodium azide, wherein the mass-to-volume ratio of sodium nitrite is 0.05%~0.15%, preferably 0.05%~0.10%, and the mass-to-volume ratio of sodium azide is 0.05%~0.15%, preferably 0.05%~0.10%. The preservation solution according to a specific embodiment of the present invention provides highly efficient preservative and antibacterial capabilities through the synergistic effect of sodium nitrite and sodium azide, maintaining the biological stability of the sample and supporting long-term preservation. Exemplarily, the mass-to-volume ratio of sodium nitrite is 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, or 0.15%, or a range between any two of the above values. For example, the mass-volume ratio of the sodium azide is 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, or 0.15%, or a range between any two of the above values.

[0060] According to an embodiment of the present invention, in the preservative, the mass ratio of sodium nitrite to sodium azide is (1~2):(1~2). Exemplarily, the mass ratio of sodium nitrite to sodium azide is 1:2, 1:1, or 2:1, or a range between any two of the above mass ratios, preferably 1:1.

[0061] According to an embodiment of the present invention, the preservation solution further comprises purified water.

[0062] According to an embodiment of the present invention, the pH value of the preservation solution is 5.0 to 7.2. Exemplarily, the pH value of the preservation solution is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, or 7.2, or a range between any two of the above values. Thus, a preservation solution with suitable acidity or alkalinity can be prepared within this range.

[0063] According to embodiments of the present invention, the pH value of the preservation solution is 5.0 to 6.5. Exemplarily, the pH value of the preservation solution is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5, or a range between any two of the above values. In some specific embodiments, cervical exfoliated cells exhibit high stability when preserved in a preservation solution with this pH value.

[0064] According to embodiments of the present invention, the pH value of the preservation solution is 6.7 to 7.2. Exemplarily, the pH value of the preservation solution is 6.7, 6.8, 6.9, 7.0, 7.1, or 7.2, or a range between any two of the above values. In some specific embodiments, cells other than cervical exfoliated cells exhibit high stability when preserved in a preservation solution at this pH value, such as exfoliated cells derived from sputum, saliva, upper respiratory tract mucus, lavage fluid, pleural effusion, ascites, or pericardial effusion.

[0065] According to an embodiment of the present invention, the first mixing process is carried out by stirring at 22°C to 27°C for 15 to 25 minutes.

[0066] According to an embodiment of the present invention, the second mixing process is carried out by stirring at 22°C to 27°C for 5 to 10 minutes.

[0067] According to an embodiment of the present invention, the third mixing process is carried out by stirring at 22°C to 27°C for 3 to 10 minutes.

[0068] A third aspect of this invention provides the use of the preservation solution described in the first aspect in the preparation of a reagent kit. According to an embodiment of the invention, the reagent kit is used to preserve samples. As previously described, the preservation solution described in the first aspect can selectively lyse red blood cells to significantly reduce blood background interference, while strongly dispersing and dissolving mucus to effectively release encapsulated target cells and prevent blood coagulation in the sample. While eliminating interference, this formulation protects the morphological integrity and nucleic acid (DNA / RNA) integrity of nucleated cells (such as leukocytes and tumor cells), preventing morphological abnormalities such as cell shrinkage, dissolution, or swelling, laying the foundation for subsequent detection. Its highly efficient antiseptic and antibacterial capabilities ensure the biological stability of the sample during preservation and transportation, supporting long-term storage. This single formulation can simultaneously meet the requirements of high-quality cell morphology for cell morphology examination and high-quality nucleic acid extraction for molecular detection (such as PCR, FISH), achieving "dual-detection compatibility." Furthermore, the preparation process of this preservation solution is simple and easy to scale up. The reagent kit prepared with this preservation solution also possesses the above-mentioned technical effects.

[0069] According to embodiments of the present invention, the samples include, but are not limited to, tissues, cells, cervical mucus, sputum, saliva, upper respiratory tract mucus, lavage fluid, pleural effusion, ascites, or pericardial effusion.

[0070] According to an embodiment of the present invention, the tissue includes the cervical canal, the external cervical os, the bronchus, the pleura, and the pericardium.

[0071] According to an embodiment of the present invention, the cells include exfoliated epithelial cells of the cervical canal and external cervical os, exfoliated epithelial cells of the bronchi and alveoli, exfoliated cells of the pleural surface, exfoliated cells of the peritoneal surface, and exfoliated cells of the pericardial surface.

[0072] A third aspect of the present invention provides a reagent kit. According to an embodiment of the present invention, the reagent kit comprises the preservation solution described in the first aspect.

[0073] A fourth aspect of the present invention provides a method for preserving samples. According to an embodiment of the present invention, the method includes: placing the cells to be preserved in the preservation solution described in the first aspect or the kit described in the third aspect for preservation. In a specific method according to an embodiment of the present invention, the temperature during the preservation process is 2°C to 40°C, and the preservation time is at least 30 days.

[0074] According to embodiments of the present invention, the samples include, but are not limited to, tissues, cells, cervical mucus, sputum, saliva, upper respiratory tract mucus, lavage fluid, pleural effusion, ascites, or pericardial effusion.

[0075] According to an embodiment of the present invention, the tissue includes the cervical canal, the external cervical os, the bronchus, the pleura, and the pericardium.

[0076] According to an embodiment of the present invention, the cells include exfoliated epithelial cells of the cervical canal and external cervical os, exfoliated epithelial cells of the bronchi and alveoli, exfoliated cells of the pleural surface, exfoliated cells of the peritoneal surface, and exfoliated cells of the pericardial surface.

[0077] Compared with the prior art, the beneficial effects of the present invention include at least the following: (1) The exfoliated cell preservation solution prepared by the method and formulation of the present invention can efficiently handle complex sample interference, specifically in that selective lysis of red blood cells significantly reduces blood background interference; strongly disperses and dissolves mucus, effectively releasing the encapsulated target cells; prevents blood coagulation in the sample and maintains sample fluidity; (2) The exfoliated cell preservation solution prepared by the method and formulation of the present invention can maintain the optimal state of cells and nucleic acids, specifically by protecting the morphological integrity of nucleated cells (such as leukocytes and tumor cells) and the integrity of nucleic acids (DNA / RNA), laying the foundation for subsequent detection; providing a suitable microenvironment to avoid abnormal cell morphology such as shrinkage, dissolution or swelling; (3) The exfoliated cell preservation solution prepared by the method and formulation of the present invention has high stability and can ensure long-term stability of the sample. Specifically, it has high antiseptic and antibacterial ability to maintain the biological stability of the sample during preservation and transportation and support long-term storage. (4) The exfoliated cell preservation solution prepared by the method and formula of the present invention achieves "dual detection compatibility" of cell morphology examination and molecular detection through a single formula.

[0078] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0079] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0080] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0081] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0082] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0083] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0084] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0085] In this document, the term "mass-volume ratio" can be expressed as "w / v" and refers to the ratio between the mass and volume of a substance, specifically the mass (g) of a substance contained in every 100 mL. It is usually used to express the concentration of a solution. For example, in the preservation solution, the mass-volume ratio of the lysis agent is 0.4% to 0.8%, which means that the mass of the lysis agent in every 100 mL of preservation solution is 0.4 to 0.8 g.

[0086] In this article, the term "mass ratio" can be expressed as "w / w", which refers to the ratio between the mass of a substance and its mass.

[0087] In this article, the term "ammonium salt" refers to an ionic compound composed of ammonium ions and acid radicals, which are generally obtained by the reaction of ammonia with an acid. They are usually colorless crystals that are easily soluble in water, such as ammonium chloride, ammonium acetate, ammonium bicarbonate, ammonium carbonate, ammonium sulfate, ammonium nitrate, ammonium fluoride, ammonium iodide, and ammonium bromide.

[0088] In this article, the term "thiol compound" refers to a compound containing a thiol group, also known as a thiol group or thiol group. The thiol group is a negatively charged functional group consisting of a sulfur atom and a hydrogen atom bonded together, with the chemical formula -SH.

[0089] In this document, the term "azide" refers to a class of compounds containing a triple-nitrogen structure, generally denoted by RN3. Azide compounds are inhibitors of electron transport systems, forming coordination compounds with cytochromes and preventing the reduction of the oxidized a3 component of cytochrome oxidase. Sodium azide used in this application is an antimicrobial agent used as a preservative.

[0090] The formulation and efficacy of the exfoliated cell preservation solution described in this invention are further illustrated by the following first-stage grouped experiments in Example 1. All experimental groups used purified water as the solvent and were prepared at room temperature and pressure. The group allocations are shown in Table 1. The second-stage temperature-time variable experimental group in Example 2 verified the long-term preservation ability of the formulation at common clinical temperatures (control group: commercially available sample preservation solution containing 50% ethanol).

[0091] In some specific embodiments of the present invention, the present invention provides a preservation solution comprising: Ammonium chloride, wherein the mass-volume ratio of ammonium chloride is 0.5%~0.9%. Ammonium bicarbonate, wherein the mass-volume ratio of ammonium bicarbonate is 0.05%~0.09%. Ethylenediaminetetraacetic acid, wherein the mass-to-volume ratio of ethylenediaminetetraacetic acid is 0.001%~0.005%. Sodium nitrite, wherein the mass-to-volume ratio of sodium nitrite is 0.05%~0.15%. Cysteine, wherein the mass-to-volume ratio of cysteine ​​is 0.06% to 0.14%. Sodium azide, wherein the mass-volume ratio of sodium azide is 0.05%~0.15%. The remainder is purified water.

[0092] In other specific embodiments of the present invention, a method for preparing a preservation solution is provided, the method comprising: (1) Mix water with ammonium chloride, ammonium bicarbonate and ethylenediaminetetraacetic acid, and stir at 22℃~27℃ for 15~25min to obtain solution A; (2) Mix water with sodium nitrate and cysteine, stir for 5-10 minutes to obtain solution B; (3) Mix solution A and solution B, add sodium azide and stir for 5 minutes, then add water to make up to volume.

[0093] (4) Adjust the pH of the product from step (4) to 5.0~7.2 to obtain the preservation solution.

[0094] It should be noted that the water is purified water. Those skilled in the art can select a suitable pH value according to the type of sample. In a specific embodiment of the present invention, the suitable pH value for cervical exfoliated cells is 5.0-6.5, and the suitable pH value for other exfoliated cells is 6.7-7.2, such as exfoliated cells from sputum, saliva, upper respiratory tract mucus, lavage fluid, pleural fluid, ascites, or pericardial effusion.

[0095] In the preservation solution, the mass-volume ratio of ammonium chloride is 0.5%~0.9%, the mass-volume ratio of ammonium bicarbonate is 0.05%~0.09%, the mass-volume ratio of ethylenediaminetetraacetic acid is 0.001%~0.005%, the mass-volume ratio of cysteine ​​is 0.06%~0.14%, the mass-volume ratio of sodium nitrite is 0.05%~0.15%, and the mass-volume ratio of sodium azide is 0.05%~0.15%.

[0096] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0097] Example 1: Optimization of Exfoliated Cell Preservation Solution This embodiment first screens the components of the exfoliated cell preservation solution and then optimizes its concentration. It should be noted that the specific detection indicators in sections 1.1, 1.2, and 1.3 refer to section 1.4 of this embodiment and Example 2, such as detection indicators for cell lysis efficiency, nucleated cell membrane detection, nuclease activity, nucleic acid protection, mucus dissolution effect, and preservative effect. The specific experimental details are as follows: 1.1 Component Screening This invention is based on the analysis of the shortcomings of traditional alcohol / aldehyde preservation solutions. Through functional modular screening, a novel exfoliated cell preservation solution formulation was designed. The following are the screening ideas and conclusions for each module component, providing a basis for subsequent concentration optimization.

[0098] (1) Screening principles and objectives The aim is to avoid the problems of cell shrinkage, nucleic acid degradation, and toxicity caused by traditional fixatives, and to achieve multiple functions such as cell morphology protection, maintenance of nucleic acid integrity, removal of interfering substances, and preservation. The initial screening adopted functional module division (lysis system, nucleic acid protectant, mucin dissolving agent, and preservation system). The efficacy and compatibility of various compounds were tested using simulated samples. The specific experimental design and conclusions are shown in Table 1. It should be noted that no other additives were added during the screening of components in each functional module; only specific candidate substances in the following single modules were screened. For example, in the lysis system, only ammonium chloride or ammonium acetate combined with buffers such as ammonium bicarbonate, methanol, and ethanol from the candidate categories were used to preserve exfoliated cells, and the screening conclusions for each component were obtained through conventional methods.

[0099] Table 1

[0100] 1.2 Concentration Screening Group Design This experiment, based on section 1.1, conducted preliminary screening of the concentrations of various substances. The specific experimental setup and results are shown in Table 2. It should be noted that, apart from the screening indicators listed in the table below, no other additives were added during this preliminary screening. Only the following single indicators were screened. For example, the preliminary screening of the lysis agent and buffer concentrations involved preserving the samples (containing exfoliated cells) using a series of concentrations of a mixture of ammonium chloride and ammonium bicarbonate (pH 6.0). The preliminary screening results were obtained after testing using the following standard experimental methods. These results were used to guide subsequent concentration optimization. The designs for other groups similarly involved preserving the samples using only the components to be screened and testing the relevant indicators using standard methods to obtain preliminary screening results. Unless otherwise specified, the detection methods used in this experiment are standard methods in the field and will not be elaborated upon here.

[0101] Table 2

[0102] 1.3 Concentration-Optimized Group Design This experiment was conducted based on sections 1.1 and 1.2 above. The preparation methods for the preservation solutions of each group were standardized as follows, and the variables of each component are shown in Table 3: (1) Preparation of solution A: Mix 60% of the solvent (purified water) with ammonium chloride, ammonium bicarbonate and EDTA, and stir at 37°C for 20 minutes; (2) Preparation of solution B: Mix the remaining purified water according to the formula with sodium nitrite and cysteine, and stir for 10 minutes; (3) Mixing and adjusting the volume: Mix solution A and solution B, add sodium azide and stir for 5 minutes, then adjust the volume to 1L; (4) pH adjustment: In this application, the pH of the cervical cancer exfoliated cell preservation solution is adjusted to 5.0~6.5 with dilute hydrochloric acid or sodium hydroxide, while the pH of other types of exfoliated cell preservation solutions is 6.8~7.2.

[0103] Unless otherwise specified, all component concentrations are calculated as w / v%, the basic preparation method is as described above, and the core variable groups are as follows: Table 3: Key Component Core Variable Group Table

[0104] 1.4 Core performance testing of each group of preservation solutions This experiment used cervical exfoliated cells (20 cases), sputum (15 cases), and pleural and peritoneal fluid (15 cases) to evaluate the effectiveness of the preservation solution prepared in section 1.1 above. The evaluation indicators and standards are shown in Table 4. The preservation conditions for each group in this experiment were the same, which was 25℃ for 30 days. Among the following detection methods, PCR was used to detect the Ct value of the internal reference gene (ACTB). Since all detection methods are routine methods in this field, the specific operation steps will not be described in detail in this experiment.

[0105] The evaluation results of each group of preservation solutions are shown in Table 5. The data are analyzed as follows: (1) Experimental groups 1-3 showed that the concentration of the pyrolysis agent played a decisive role: Experimental group 1 (0.8 w / v% ammonium chloride + 0.08% w / v% ammonium bicarbonate): erythrocyte lysis rate 99.3%, nucleated cell integrity rate 98.7% (optimal balance); Experimental group 2 (1.0 w / v% high concentration of ammonium chloride + 0.1 w / v% ammonium bicarbonate): nucleated cell damage (excessive osmotic pressure, nucleated cell integrity rate 86.2%). Experimental group 3 (0.4 w / v% low concentration ammonium chloride + 0.04 w / v% ammonium bicarbonate): Insufficient erythrocyte lysis (erythrocyte lysis rate 82.1%).

[0106] (2) Validation of the minimum effective concentration of EDTA: Experimental group 4: A concentration of 0.003 w / v% achieved complete nucleic acid (OD260 / 280=1.85) and 100% PCR success rate; Experimental group 5: In the absence of EDTA, nucleic acids were completely degraded (OD260 / 280=1.51), and nucleated cells morphologically disintegrated (nucleated cell integrity rate was 75.4%).

[0107] (3) The advantage of low-dose cysteine: Experimental group 6: At a concentration of 0.05 w / v%, it was sufficient to maintain a mucus clearance rate of 96.5% and the cell integrity rate was the highest (nucleated cell integrity rate of 99.1%). Experimental group 7: At a concentration of 0.15 w / v%, mucus clearance was better (99.0%), but due to the excessive reducing environment, the rate of nucleated cell damage increased.

[0108] (4) Preservative synergistic threshold: Experimental group 8: When the concentration of sodium nitrite is 0.05 w / v% and the concentration of sodium azide is 0.05 w / v%, it provides 100% long-term stability; Experimental group 9: When the sodium nitrite concentration was 0.01 w / v% and the sodium azide concentration was 0.01 w / v%, the PCR failure rate reached 62% when stored for 30 days due to microbial contamination.

[0109] Therefore, the optimal concentration formula of each component in the exfoliated cell preservation solution is: ammonium chloride 0.8 w / v, ammonium bicarbonate 0.08 w / v, ethylenediaminetetraacetic acid (EDTA) 0.003 w / v, sodium nitrite 0.05 w / v, cysteine ​​0.05 w / v, sodium azide 0.05 w / v, with the balance being water.

[0110] Table 4: Evaluation Indicators and Standards

[0111] Table 5: Evaluation results of preservation solutions for each group

[0112] Example 2: Stability Testing of Preservative Solution This embodiment is used to verify the preservation capabilities of the optimal formulation preservation solution obtained in Example 1 and the commercially available sample preservation solution containing 50% ethanol at common clinical temperatures.

[0113] 2.1 Experimental Design: (1) Sample types: cervical exfoliated cells (15 cases) and sputum (15 cases) obtained clinically.

[0114] (2) Grouping of preservation solutions: Experimental group: Preservative solution of the present invention (prepared using the preferred formulation and method of Example 1). Control group: conventional alcohol / aldehyde preservation solution (i.e., 50% ethanol).

[0115] (3) The detection indicators and standards are shown in Table 6, and the detection methods for each indicator are as described in Example 1.

[0116] The evaluation results are shown in Table 7. The data are analyzed as follows: (1) Low temperature stability (4℃ / 30 days): The cell integrity rate of the present invention (98.2%) is significantly higher than that of the traditional formula (89.5%), and there is no microbial contamination (the contamination rate of the traditional liquid is 8%). (2) Long-lasting effect at room temperature (25℃ / 30 days): The Ct value of the nucleic acid of the present invention only shifts by 1.2 (Ct=27.3), which meets the detection requirements; the nucleic acid of the traditional formula is completely degraded; (3) High temperature tolerance (37℃): ① Day 7: The cell morphology integrity rate of the present invention is 97.0%, while that of the traditional solution has dropped to 78.6%; ② Day 30: The Ct value of the present invention is still 28.9 (offset <2.8), while the traditional solution is completely ineffective.

[0117] Therefore, the preservation solution obtained by this invention has wide temperature range adaptability, maintaining cell morphology and nucleic acid integrity for at least 30 days at temperatures ranging from 4℃ to 37℃, overcoming the limitations of traditional alcohol / aldehyde formulations that are dependent on low temperatures and prone to high-temperature inactivation. The preservation solution obtained by this invention exhibits extreme condition tolerance; after 30 days of storage at 37℃, the cell morphology integrity rate is >93%, and the nucleic acid Ct value remains ≤28.9 (offset <2.8), meeting the requirements for molecular detection, while traditional formulations become ineffective after 7 days. The preservation solution obtained by this invention demonstrates reliable preservative protection: the microbial contamination rate is 0% within 30 days under all temperature conditions, significantly better than the minimum 8% contamination rate of traditional formulations (25℃ / 30 days).

[0118] Table 6: Testing Indicators and Standards

[0119] Table 7: Comparison of Experimental Results for Temperature-Time Variables

[0120] In summary, this invention achieves long-term stable preservation of exfoliated cell samples in diverse clinical environments (refrigeration, room temperature, and body temperature simulation) by optimizing the synergistic effect of components, ensuring "dual-test compatibility".

[0121] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0122] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A preservation solution, characterized in that, It comprises a pyrolysis agent, a buffer, a chelating agent, a mucin dissolving agent, and a preservative, wherein the pyrolysis agent and the buffer comprise an ammonium salt, the ammonium salt comprising at least one selected from the following: ammonium chloride, ammonium acetate, and ammonium bicarbonate.

2. The preservation solution according to claim 1, characterized in that, The pyrolysis agent includes at least one of the following: ammonium chloride, ammonium acetate; Optionally, the buffer includes ammonium bicarbonate; Optionally, the mass-to-volume ratio of the pyrolysis agent is 0.5% to 0.9%; Optionally, the mass-to-volume ratio of the buffer is 0.05% to 0.09%.

3. The preservation solution according to claim 1, characterized in that, The chelating agent includes at least one selected from the following: ethylenediaminetetraacetic acid, citrate, and EGTA; Optionally, the mucus solvent includes a thiol compound; Optionally, the thiol compound is selected from at least one of the following: cysteine, dithiothreitol, and N-acetylcysteine; Optionally, the mucus solvent is selected from: cysteine; Optionally, the preservative includes azide and oxidant; Optionally, the azide comprises sodium azide; Optionally, the oxidizing agent includes sodium nitrite.

4. The preservation solution according to claim 1, characterized in that, The chelating agent has a mass-to-volume ratio of 0.001% to 0.005%; Optionally, the mucus solvent has a mass-to-volume ratio of 0.06% to 0.14%. Optionally, the preservative has a mass-to-volume ratio of 0.1% to 0.3%.

5. The preservation solution according to claim 1, characterized in that, The mass ratio of the pyrolytic agent to the buffer is (8~12):(1~2), preferably 10:1; Optionally, the pyrolysis agent is ammonium chloride, and the mass-volume ratio of the ammonium chloride is 0.5% to 0.9%; Optionally, the buffer is ammonium bicarbonate, and the mass-volume ratio of the ammonium bicarbonate is 0.05% to 0.09%. Optionally, the chelating agent is ethylenediaminetetraacetic acid (EDTA), and the mass-to-volume ratio of EDTA is 0.001% to 0.005%. Optionally, the mucus dissolving agent is cysteine, and the mass-volume ratio of cysteine ​​is 0.06%~0.14%; Optionally, the preservative is sodium nitrite and sodium azide, wherein the mass-volume ratio of sodium nitrite is 0.05%~0.15%, and the mass-volume ratio of sodium azide is 0.05%~0.15%. Optionally, in the preservative, the mass ratio of sodium nitrite to sodium azide is (1~2):(1~2), preferably 1:

1.

6. The preservation solution according to claim 1, characterized in that, The preservation solution also contains purified water; Optionally, the pH value of the preservation solution is 5.0 to 7.2; Optionally, the pH value of the preservation solution is 5.0~6.5 or 6.7~7.

2.

7. A method for preparing the preservation solution according to any one of claims 1 to 6, characterized in that, include: (1) The solvent is first mixed with the pyrolysis agent, buffer, and chelating agent to obtain solution A. (2) The solvent is mixed with the mucin solvent and preservative in a second mixing process to obtain solution B. (3) After mixing solutions A and B, a preservative is added for a third mixing treatment to obtain the preservation solution. The pyrolysis agent and the buffer include ammonium salts, wherein the ammonium salts include at least one selected from the following: ammonium chloride, ammonium acetate, and ammonium bicarbonate.

8. The method according to claim 7, characterized in that, The first mixing process is carried out by stirring at 22℃~27℃ for 15~25 min; Optionally, the second mixing process is carried out by stirring at 22°C to 27°C for 5 to 10 minutes; Optionally, the third mixing process is carried out by stirring at 22°C to 27°C for 3 to 10 minutes.

9. A reagent kit, characterized in that, It includes the preservation solution according to any one of claims 1 to 6.

10. A method for preserving samples, characterized in that, include: The sample to be preserved is placed in the preservation solution according to any one of claims 1 to 6 or the kit according to claim 9.

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