Preserving fluid of bronchoalveolar lavage fluid, kit and cytological test method

Abstract

The invention belongs to the technical field of biology, and relates to a bronchoalveolar lavage fluid (BALF) preserving fluid, a kit and a cytological test method. The preservation solution is composed of TCEP, N-acetylcysteine, EDTA.2Na, trehalose, Proclin300, methanol, sodium chloride and HEPES, and all the components have a synergistic effect to achieve sample viscosity removal, oxidation resistance, corrosion prevention, cell protection and pH stabilization. The preserving fluid is suitable for morphological observation and nucleic acid detection of BALF cells, and can stably preserve samples at room temperature and maintain completeness of cell membranes and clear morphological structure. The kit comprises two preparations which are mixed to form a working solution, and the working solution can be directly used for sample preservation on a sampling site. The cytological test method comprises the steps of sample collection, preservation, centrifugal slide preparation, Wright-Giemsa staining and microscopic observation. Experimental results show that preservation solutions of different formulas are compatible with a chromosome system, the cell morphology is kept stable within 3 days, the dyeing effect is good, and it is proved that the preservation system can remarkably improve the preservation stability and detection reliability of BALF samples and is suitable for cytology and molecular diagnosis application of lower respiratory diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a preservation solution, kit, and method for cytological examination of bronchoalveolar lavage fluid. Background Technology

[0002] Bronchoalveolar lavage fluid (BALF) is a sample of alveolar lining fluid collected by injecting a suitable amount of sterile saline into the lower bronchial or subsegmental lungs via fiberoptic bronchoscopy. BALF specimens can be comprehensively analyzed using methods such as cytology, etiology, immunology, and molecular biology. It is of great significance in the diagnosis, treatment monitoring, prognostic assessment, and pathogenesis research of respiratory diseases, especially lower respiratory tract diseases (including pulmonary infections, interstitial lung disease, and lung cancer).

[0003] According to the recommendations of the "Chinese Expert Consensus on the Examination of Cell Morphology in Bronchoalveolar Lavage Fluid (2023)," BALF specimens should be submitted for testing within 2 hours of collection at room temperature. Before testing, specimens can be stored at room temperature for 4 hours, and after analysis, they can be stored at 2–8°C for 24 hours. Before the experiment, the characteristics of the lavage fluid should be observed. If it contains a large amount of mucus, add 2 times the volume of dithiothreitol (DTT) reagent at a concentration of 0.1 g / dl, and treat it on a constant-temperature shaker at 300 r / min and 37°C for 30–60 minutes. If a constant-temperature shaker is unavailable, the mixture can be placed in a 37°C water bath and manually inverted every 10 minutes for 30–60 minutes to reduce the influence of mucus components on subsequent testing.

[0004] However, the following technical problems still exist in the current BALF sample processing and analysis process: 1. Poor sample stability and stringent preservation conditions Cells and soluble biomarkers in BALF are highly susceptible to the effects of time, temperature and storage medium, resulting in problems such as cell structure degeneration, cytokine degradation and RNA damage. In particular, in multi-center studies or delayed detection, the consistency of results is difficult to guarantee.

[0005] 2. Detection interference from impurities and mucus components in the sample BALF often contains a large amount of mucus, blood components, and debris, which affects subsequent centrifugation, staining, cell counting, and molecular detection. The commonly used desticator (dithiothreitol, DTT) varies greatly in concentration, temperature, and time among different laboratories, indicating a lack of standardization. Furthermore, dithiothreitol (DTT) has a strong odor, is easily oxidized, and its effective reducing power decreases as the pH value decreases.

[0006] 3. Currently available treatment agents have obvious limitations. Currently, commonly used clinical processing or preservation reagents are mostly sputum preparation agents or sputum nucleic acid preservation solutions. These preparations are specifically designed for sputum samples with high viscosity and high protein content, and their formulations usually contain cationic surfactants, DTT, and other strong liquefying and membrane-breaking components. When these components are used in bronchoalveolar lavage fluid, they can easily lead to cell membrane rupture, loss of cell morphology, and increased risk of nucleic acid degradation, failing to meet the compatibility requirements of BALF samples for cytological observation and nucleic acid detection. Furthermore, these reagents often require an alkaline or strongly reducing environment to function, making them unsuitable for the weakly acidic staining systems commonly used in BALF (such as Wright-Gymsa staining).

[0007] Therefore, it is essential to develop a dedicated processing and preservation reagent suitable for the characteristics of bronchoalveolar lavage fluid (BALF) samples. This reagent should be able to be added to the sample immediately after sampling, stably preserve cell structure at room temperature, inhibit nuclease activity, prevent microbial proliferation, and be compatible with routine staining and molecular detection procedures. Unlike traditional sputum processing reagents, this reagent is not designed for strong liquefaction and membrane permeation, but rather for cell-based... With protection and nucleic acid stability as its core features, it can maintain the integrity of alveolar cell membranes, clear nuclei, and uniform cytoplasm distribution in weakly acidic to neutral environments, ensuring that BALF samples still have good morphological recognizability and nucleic acid detection integrity even in the case of delayed detection or cross-center transportation. Summary of the Invention

[0008] This invention aims to establish a chemically mild, conditionally flexible, and highly stable bronchoalveolar lavage fluid (BALF) sample preservation system to address key issues in existing technologies, such as short BALF sample preservation time, cell structure damage, rapid nucleic acid degradation, and poor staining adaptability. This provides a reliable and standardized sample pretreatment protocol for clinical and research purposes, improving the accuracy and comparability of lower respiratory tract disease test results. The solution to the above technical problems is as follows: A preservation solution for bronchoalveolar lavage fluid, comprising the following components: TCEP 0.5-1 g / L; N-acetylcysteine ​​0.1-0.5 g / L, EDTA·2Na 0.08-0.12 g / L, trehalose 4-10 g / L, Proclin 300 0.8-1.3 mL / L, methanol 130-230 mL / L, sodium chloride 0.9 g / L, pH buffer HEPES 2.5-4 g / L.

[0009] Furthermore, a preservation solution for bronchoalveolar lavage fluid includes the following components: TCEP 0.8-1 g / L; N-acetylcysteine ​​0.2-0.5 g / L, EDTA·2Na 0.08-0.1 g / L, trehalose 5-10 g / L, Proclin 300 0.8-1 mL / L, methanol 150-200 mL / L, sodium chloride 0.9 g / L, pH buffer HEPES 2.5-3 g / L.

[0010] Furthermore, a preservation solution for bronchoalveolar lavage fluid includes the following components: TCEP 1g / L; N-acetylcysteine ​​0.5g / L, EDTA·2Na 0.1g / L, trehalose 8g / L, Proclin 300 1mL / L, methanol 150mL / L, sodium chloride 0.9g / L, pH buffer HEPES 3g / L.

[0011] Furthermore, a preservation solution for bronchoalveolar lavage fluid includes the following components: TCEP 1g / L; N-acetylcysteine ​​0.3g / L, EDTA·2Na 0.12g / L, trehalose 10g / L, Proclin 300 1.3mL / L, methanol 200mL / L, sodium chloride 0.9g / L, pH buffer HEPES 4g / L.

[0012] Furthermore, a preservation solution for bronchoalveolar lavage fluid includes the following components: TCEP 0.8 g / L; N-acetylcysteine ​​0.2 g / L, EDTA·2Na 0.08 g / L, trehalose 4 g / L, Proclin 300 0.8 mL / L, methanol 230 mL / L, sodium chloride 0.9 g / L, pH buffer HEPES 4 g / L.

[0013] Furthermore, a preservation solution for bronchoalveolar lavage fluid includes the following components: TCEP 0.5 g / L; N-acetylcysteine ​​0.1 g / L, EDTA·2Na 0.08 g / L, trehalose 4 g / L, Proclin 300 0.8 mL / L, methanol 180 mL / L, sodium chloride 0.9 g / L, pH buffer HEPES 4 g / L.

[0014] The inventive concept of this invention lies in providing a preservation solution for the morphological identification of cells in bronchoalveolar lavage fluid (BALF). This preservation solution, through the synergistic action of multiple components, effectively preserves BALF cells by removing mucus, fixing cell morphology, providing antioxidant protection, and maintaining osmotic pressure and pH balance. Samples treated with this preservation solution can be directly stained with Wright-Gymsa for cell morphology examination, ensuring reliable representation of cell structure and staining characteristics.

[0015] In terms of formulation design, the concept of this invention is as follows: 1. Tris(2-carboxyethyl)phosphine (TCEP) was selected as the main reducing agent for removing mucus.

[0016] TCEP is a stable thiol reducing agent, maintaining its activity across a wide acidic to alkaline range (pH 3-9), while traditional dithiothreitol (DTT) is only effective under alkaline conditions (pH > 7). Since bronchoalveolar lavage fluid samples require a weakly acidic environment for Wright-Gymsa staining, TCEP is more suitable than DTT for BALF cell preservation systems. Secondly, DTT is a typical sulfur-containing compound with a pronounced thiol odor, while TCEP is almost odorless, making it more suitable for use in closed laboratory or medical settings. DTT is easily oxidized to disulfides in air, requiring storage and use under low-temperature, anaerobic, or sealed conditions, while TCEP is less prone to oxidation and can be stored stably for extended periods.

[0017] 2. N-acetylcysteine ​​(NAC) acts as an auxiliary deviscosifying component. The thiol group in its molecule can break the disulfide bonds in the mucin molecule, further reducing the viscosity of the sample solution and improving cell dispersibility.

[0018] 3. Regarding stability and anticoagulation, disodium ethylenediaminetetraacetate (EDTA·2Na) is added as a chelating agent to maintain the stability of the chemical composition of the preservation solution. Simultaneously, since BALF samples often contain a small amount of blood, EDTA·2Na can prevent coagulation by binding with Ca²⁺ in the blood, thus avoiding the formation of blood clots in the sample that could affect observation.

[0019] 4. To prevent microbial contamination, Proclin 300 is selected as a preservative in this invention. This ingredient has broad-spectrum antibacterial properties and is effective against Gram-positive bacteria, Gram-negative bacteria, and fungi. It can rapidly inhibit microbial activity and prevent biofilm formation, thereby ensuring the biosafety and stability of samples during storage.

[0020] 5. In terms of cell protection, trehalose is added to stabilize the cell membrane structure and prevent cell membrane damage or permeability changes during preservation, thereby improving the overall stability and survival morphology of cells.

[0021] 6. To maintain the osmotic balance of cells, sodium chloride (NaCl) is added in this invention to regulate osmotic pressure, prevent cells from dehydrating, shrinking or rupturing, and maintain the normal shape of cells.

[0022] 7. Use HEPES as a buffer component to ensure that the solution pH is stable between 6.8 and 7.2 to meet the weakly acidic requirements of Wright-Gymsa staining and to prevent pH fluctuations from affecting staining quality and cell morphology.

[0023] In summary, the present invention takes into account de-adhesion, anti-oxidation, anti-corrosion, cell protection and staining compatibility in its formulation design. Through the synergistic effect of multiple components, it achieves comprehensive and stable preservation of cells in bronchoalveolar lavage fluid, ensuring the accuracy and reproducibility of morphological observation results.

[0024] A bronchoalveolar lavage fluid kit comprises two formulations. The first formulation includes TCEP 0.5-1 g / L; N-acetylcysteine ​​0.1-0.5 g / L; EDTA·2Na 0.08-0.12 g / L; trehalose 4-10 g / L; sodium chloride 0.9 g / L; and pH buffer HEPES 2.5-4 g / L. The second formulation is a solvent comprising Proclin 300 0.8-1.3 mL / L and methanol 130-230 mL / L. The kit is prepared by mixing the first and second formulations to form a solution. This solution can be stored at room temperature for extended periods.

[0025] A method for cytological examination of bronchoalveolar lavage fluid includes the following steps: Step 1: Sample collection: First, local anesthesia is performed on the target lung segment. Then, the tip of the fiberoptic bronchoscope is wedged into the bronchial opening, and a total volume of 100 to 250 ml of 37°C sterile saline is injected. Finally, the lavage fluid is aspirated and recovered with a negative pressure of 50 to 100 mmHg. Step 2: Adding preservation solution to the sample: Within 1 hour of receiving the collected irrigation fluid sample, add the preservation solution described in claim 1, with a sample to preservation solution mixing ratio of 1:4, then seal and store at room temperature in a fume hood; Step 3: Sample preparation and staining: Centrifuge the sample with preservation solution added in Step 2, discard the supernatant, retain the cell precipitate at the bottom, take an appropriate amount of precipitate and spread it on a glass slide, let it stand and fix, then stain it using the Wright-Gymsa method, and after the stained slide dries, mount it for observation. Step 4: Observe the stained slides under a microscope to assess changes in the proportions of macrophages, lymphocytes, neutrophils, and eosinophils, and make a judgment based on the abnormality of the cells.

[0026] Furthermore, the abnormal cell condition refers to the presence of tumor cells or pathogens. Attached Figure Description

[0027] Figure 1 These are the macrophage morphologies of Example 1, where a is the morphology on day 1, b is the morphology on day 2, and c is the morphology on day 3.

[0028] Figure 2 This is the field of view after treatment with the preservation solution in Example 1 and staining with Wright-Gymsa.

[0029] Figure 3 These are the macrophage morphologies of Example 2, where a is the morphology of day 1, b is the morphology of day 2, and c is the morphology of day 3.

[0030] Figure 4 This is the field of view after treatment with the preservation solution in Example 2 and staining with Wright-Gymsa.

[0031] Figure 5 These are the neutrophil morphologies from Example 3, where a represents the morphology on day 1, b represents the morphology on day 2, and c represents the morphology on day 3.

[0032] Figure 6 This is the field of view after treatment with the preservation solution in Example 3 and staining with Wright-Gymsa.

[0033] Figure 7 The morphology of ciliated columnar epithelial cells in Example 4 is shown, where a represents the morphology on day 1, b represents the morphology on day 2, and c represents the morphology on day 3.

[0034] Figure 8 This is the field of view after treatment with the preservation solution in Example 4 and staining with Wright-Gymsa.

[0035] Figure 9 The morphology of squamous epithelial cells in Example 5 is shown, where a is the morphology on day 1, b is the morphology on day 2, and c is the morphology on day 3.

[0036] Figure 10 This is the field of view after treatment with the preservation solution in Example 5 and staining with Wright-Gymsa.

[0037] Figure 11 The field of view after treatment with the preservation solution of Comparative Example 1 and staining with Wright-Gymsa.

[0038] Figure 12 The field of view after treatment with the preservation solution of Comparative Example 2 and staining with Wright-Gymsa.

[0039] Figure 13 The field of view after treatment with the preservation solution of Comparative Example 3 and staining with Wright-Gymsa.

[0040] Figure 14 The field of view after treatment with the preservation solution of Comparative Example 4 and staining with Wright-Gymsa.

[0041] Figure 15 The field of view after treatment with the preservation solution of Comparative Example 5 and staining with Wright-Gymsa.

[0042] Figure 16 The field of view after treatment with the preservation solution of Comparative Example 6 and staining with Wright-Gymsa. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0044] Example 1: The bronchoalveolar lavage fluid preservation solution of this embodiment includes the following components: Mucus treatment agent: TCEP 0.5g / L; N-acetylcysteine ​​0.5g / L Chelating agent: EDTA·2Na 0.08 g / L Cell membrane protectant: Trehalose 5g / L Preservative: Proclin 300 0.8 mL / L Fixative: Methanol 130ml / L (v / v) Maintain osmotic pressure: Sodium chloride 0.9% pH buffer: HEPES 2.5g / L The cells observed in the slides were macrophages.

[0045] Example 2: The bronchoalveolar lavage fluid preservation solution of this embodiment includes the following components: Mucus treatment agent: TCEP 1g / L; N-acetylcysteine ​​0.5g / L Chelating agent: EDTA·2Na 0.1g / L Cell membrane protectant: Trehalose 8g / L Preservative: Proclin 300 1mL / L Fixative: Methanol 150ml / L (v / v) Maintain osmotic pressure: Sodium chloride 0.9% pH buffer: HEPES 3g / L The cells observed in the slides were macrophages.

[0046] Example 3: The bronchoalveolar lavage fluid preservation solution of this embodiment includes the following components: Mucus treatment agent: TCEP 1g / L; N-acetylcysteine ​​0.3g / L Chelating agent: EDTA·2Na 0.12g / L Cell membrane protectant: Trehalose 10g / L Preservative: Proclin 300 1.3 mL / L Fixative: Methanol 200ml / L (v / v) Maintain osmotic pressure: Sodium chloride 0.9% pH buffer: HEPES 4g / L The cells observed on the slide were neutrophils.

[0047] Example 4: The bronchoalveolar lavage fluid preservation solution of this embodiment includes the following components: Mucus treatment agent: TCEP 0.8 g / L; N-acetylcysteine ​​0.2 g / L Chelating agent: EDTA·2Na 0.08 g / L Cell membrane protectant: Trehalose 4g / L Preservative: Proclin 300 0.8 mL / L Fixative: Methanol 230ml / L (v / v) Maintain osmotic pressure: Sodium chloride 0.9% pH buffer: HEPES 4g / L The cells observed on the slide were ciliated columnar epithelial cells.

[0048] Example 5: The bronchoalveolar lavage fluid preservation solution of this embodiment includes the following components: Mucus treatment agent: TCEP 0.5g / L; N-acetylcysteine ​​0.1g / L Chelating agent: EDTA·2Na 0.08 g / L Cell membrane protectant: Trehalose 4g / L Preservative: Proclin 300 0.8 mL / L Fixative: Methanol 180ml / L (v / v) Maintain osmotic pressure: Sodium chloride 0.9% pH buffer: HEPES 4g / L The cells observed in the slide were squamous epithelial cells.

[0049] Example 6: Performance Test Experiment: Different bronchoalveolar lavage fluid (BALF) preservation solutions were prepared according to the formulations described in Examples 1-5 to preserve BALF cell samples from different sources. Although the preservation solution formulations and sample sources in Examples 1-5 differed slightly, a unified cytological testing method and evaluation procedure were used. The specific steps are as follows: Sample collection: First, local anesthesia is performed on the target lung segment. Then, the tip of the fiberoptic bronchoscope is wedged into the bronchial opening, and a total volume of 100 to 250 ml of 37°C sterile saline is injected. Finally, the lavage fluid is collected by suction with a negative pressure of 50 to 100 mmHg. Bronchoalveolar lavage fluid (BALF) samples were immediately sealed and mixed with 40 ml of preservation solution per 10 ml of sample upon receipt, and stored at room temperature in a fume hood. Upon sampling, the samples were centrifuged (400 g relative centrifugation force, 10 min), and the supernatant was removed using a disposable pipette, retaining the bottom precipitate for slide preparation. The precipitate was mixed thoroughly, and 5–10 μl was placed on the right side of a glass slide, spreading it from right to left. After spreading, the slide was allowed to stand for fixation and then stained with Wright-Giemsa stain.

[0050] For three consecutive days, the preserved samples were prepared into slides, stained with Wright-Giemsa, and observed under 100x oil immersion to compare changes in cell morphology. The preservation solution was evaluated for its effect on the preservation of cell morphology in BALF samples.

[0051] like Figures 1 to 10 As shown, the evaluation results are as follows: the preservation solution did not interfere with Wright-Giemsa staining of the cells, and the intracellular and extracellular morphology could be stained normally with clear structure. After adding the preservation solution, the cell morphology remained stable for 3 days, the cell membrane remained intact, and the morphology of the whole cell and the granules inside the cell could still be clearly identified on the 3rd day.

[0052] Comparative Example 1: This embodiment provides a preservation solution for bronchoalveolar lavage fluid, which differs from Example 1 in that it does not contain the cell membrane protectant trehalose 5g / L. Everything else is the same as in Example 1.

[0053] Comparative Example 2: Unlike Example 1, the pH buffer is Tris(hydroxymethyl)aminomethane 2.5 g / L, with a pH range of 7.0 to 9.0, replacing the original HEPES buffer. Otherwise, it is the same as in Example 1.

[0054] Comparative Example 3: Unlike Example 1, the mucus treatment agent was: dithiothreitol (DTT) 1.0 g / L, replacing the original TCEP 0.5 g / L; N-acetylcysteine ​​0.5 g / L, and the rest was the same as in Example 1.

[0055] Comparative Example 4: Unlike Example 1, this example added a cell protectant: albumin (BSA) 1.5 g / L, otherwise it was the same as Example 1.

[0056] Comparative Example 5: Patent CN202411580236.7 describes a sputum sample microbial community nucleic acid (DNA) preservation solution. The solution comprises: weighing 1.5g of tris(hydroxymethyl)aminomethane, 0.6g of TCEP, 1.1g of sodium chloride, 1.2g of sodium citrate, 1.3g of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 0.3g of EDTA, 4g of N-octylpyridine bromide, and 40g of anhydrous ethanol. These are then dissolved in sterile deionized water and brought to a final volume of 100mL with sterile deionized water. The solution is then filtered through a 0.22μm sterile filter membrane to obtain the sputum preservation solution.

[0057] Comparative Example 6: CN111020001A A novel saliva preservation solution, comprising the following components and contents: EDTA 10 mmol / L, Tris-HCl 20 mmol / L, sodium chloride 100 mmol / L, sodium diacetate 1% (w / v), proclin 300 0.01% (v / v), TWEEN 20 1% (w / v), SDS 1% (w / v), with a system pH of 8.

[0058] Performance testing experiments: Different bronchoalveolar lavage fluid (BALF) preservation solutions were prepared according to the formulations described in Comparative Examples 1–5 to preserve BALF cell samples from different sources. Although the preservation solution formulations and sample sources of Comparative Examples 1–6 differed slightly, a unified cytological testing method and evaluation procedure were used for all of them. The specific steps are as follows: Sample collection: First, local anesthesia is performed on the target lung segment. Then, the tip of the fiberoptic bronchoscope is wedged into the bronchial opening, and a total volume of 100 to 250 ml of 37°C sterile saline is injected. Finally, the lavage fluid is collected by suction with a negative pressure of 50 to 100 mmHg. Bronchoalveolar lavage fluid (BALF) samples were immediately sealed and mixed with 40 ml of preservation solution per 10 ml of sample upon receipt, and stored at room temperature in a fume hood. Upon sampling, the samples were centrifuged (400 g relative centrifugation force, 10 min), and the supernatant was removed using a disposable pipette, retaining the bottom precipitate for slide preparation. The precipitate was mixed thoroughly, and 5–10 μl was placed on the right side of a glass slide, spreading it from right to left. After spreading, the slide was allowed to stand for fixation and then stained with Wright-Giemsa stain.

[0059] For three consecutive days, the preserved samples were prepared into slides, stained with Wright-Giemsa, and observed under 100x oil immersion to compare changes in cell morphology. The preservation solution was evaluated for its effect on the preservation of cell morphology in BALF samples.

[0060] like Figures 11 to 16The images shown are all Wright-Gymsa staining fields after treatment with preservation solution on day 1. The evaluation results are as follows: (The images were obtained through...) Figure 11 It can be observed that in Comparative Example 1 without the addition of trehalose, the morphology and structure of the cell membrane are significantly different from those of cells under normal conditions, and the cell membrane structure is not obvious. Figure 12 In Comparative Example 2, cells were observed to be affected by osmotic pressure, with some cells showing indistinct distinction between cytoplasm and nucleus. Comparative Example 3... Figure 13 Mucus was not completely removed from the sample, and mucus residue was clearly visible in the background. (Comparative Example 4) Figure 14 The field of view was observed to have an overall reddish tint. Preliminary assessment suggests this is due to the reaction between albumin in the background and the staining solution, resulting in a reddish undertone throughout the field of view. This affected the normal staining of the field of view. (Comparative Example 5) Figure 15 It can be seen that some cell membranes have dissolved and ruptured, and the cell morphology cannot be maintained. (Comparative Example 6) Figure 16 Due to the alkaline pH of the preservation solution, the overall staining was bluish, and the staining of acidophilic particles was significantly affected.

[0061] In Comparative Example 5, during sputum sample preservation, due to the highly viscous nature and complex mucin network structure of sputum, de-liquidation is necessary to release the encapsulated microorganisms and cellular components. Therefore, traditional sputum preservation solutions typically require the addition of chemical components with strong reducing or surface-active properties, such as DTT, TCEP, N-acetylcysteine, or the cationic surfactant N-octylpyridine bromide, to break disulfide bonds in the mucin and disrupt the mucin polymer structure, thus achieving sample liquefaction. However, during sputum liquefaction, the sample often contains a large number of nucleases derived from cells and bacteria. Without effective enzyme inhibition or chemical fixation, the released DNA is easily degraded, leading to damage to the sample's nucleic acid integrity. Therefore, traditional sputum preservation systems need to simultaneously introduce chelating agents (such as EDTA and DOTA) and chemical fixatives (such as paraformaldehyde and alcohols) to inhibit nuclease activity during liquefaction, thereby balancing the conflicting needs of liquefaction and DNA stability.

[0062] In contrast, bronchoalveolar lavage fluid (BALF) samples are characterized by low cell count, low viscosity, and thin fluid volume. They do not inherently require intensive demucinization treatment, nor are surfactants suitable for use to avoid damaging cell membranes. The key to BALF sample preservation lies in protecting the limited cellular components and stabilizing their nucleic acid structure for subsequent cell morphology observation (such as Wright-Gissam staining) and molecular detection (including nucleic acid extraction and gene sequencing).

[0063] In Comparative Example 6, the preservation solution was generally weakly alkaline. Morphological examination of cells in bronchoalveolar lavage fluid required Wright-Gymsa staining, which is sensitive to pH and needs to be performed in a weakly acidic environment, resulting in poor performance.

[0064] In summary, this invention aims to provide a reliable and efficient pretreatment solution for clinical and research purposes by establishing a chemically mild, conditionally flexible, and functionally integrated bronchoalveolar lavage fluid sample preservation system, thereby improving the accuracy, comparability, and ease of operation of lower respiratory tract disease test results.

[0065] The above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above content. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A preservation solution for bronchoalveolar lavage fluid, characterized in that: Includes the following components: TCEP 0.5-1 g / L; N-acetylcysteine ​​0.1-0.5 g / L, EDTA·2Na 0.08-0.12 g / L, trehalose 4-10 g / L, Proclin 300 0.8-1.3 mL / L, methanol 130-230 mL / L, sodium chloride 0.9 g / L, pH buffer HEPES 2.5-4 g / L.

2. The bronchoalveolar lavage fluid preservation solution according to claim 1, characterized in that: Includes the following components: TCEP 0.8-1 g / L; N-acetylcysteine ​​0.2-0.5 g / L, EDTA·2Na 0.08-0.1 g / L, trehalose 5-10 g / L, Proclin 300 0.8-1 mL / L, methanol 150-200 mL / L, sodium chloride 0.9 g / L, pH buffer HEPES 2.5-3 g / L.

3. The preservation solution for bronchoalveolar lavage fluid according to claim 1, characterized in that: Includes the following components: TCEP 1g / L; N-acetylcysteine ​​0.5g / L, EDTA·2Na 0.1g / L, trehalose 8g / L, Proclin 300 1mL / L, methanol 150ml / L, sodium chloride 0.9g / L, pH buffer HEPES 3g / L.

4. The preservation solution for bronchoalveolar lavage fluid according to claim 1, characterized in that: Includes the following components: TCEP 1g / L; N-acetylcysteine ​​0.3g / L, EDTA·2Na 0.12g / L, trehalose 10g / L, Proclin 300 1.3mL / L, methanol 200mL / L, sodium chloride 0.9g / L, pH buffer HEPES 4g / L.

5. The preservation solution for bronchoalveolar lavage fluid according to claim 1, characterized in that: Includes the following components: TCEP 0.8 g / L; N-acetylcysteine ​​0.2 g / L, EDTA·2Na 0.08 g / L, trehalose 4 g / L, Proclin 300 0.8 mL / L, methanol 230 mL / L, sodium chloride 0.9 g / L, pH buffer HEPES 4 g / L.

6. The preservation solution for bronchoalveolar lavage fluid according to claim 1, characterized in that: Includes the following components: TCEP 0.5 g / L; N-acetylcysteine ​​0.1 g / L, EDTA·2Na 0.08 g / L, trehalose 4 g / L, Proclin 300 0.8 mL / L, methanol 180 mL / L, sodium chloride 0.9 g / L, pH buffer HEPES 4 g / L.

7. A bronchoalveolar lavage fluid kit, characterized in that: The kit contains two formulations. The first formulation includes TCEP 0.5-1 g / L; N-acetylcysteine ​​0.1-0.5 g / L; EDTA·2Na 0.08-0.12 g / L; trehalose 4-10 g / L; sodium chloride 0.9 g / L; and pH buffer HEPES 2.5-4 g / L. The second formulation is a solvent, including Proclin 300 0.8-1.3 mL / L and methanol 130-230 mL / L. The kit is prepared as a solution when the first and second formulations are mixed.

8. A method for cytological examination of bronchoalveolar lavage fluid, characterized in that: Includes the following steps: Step 1: Sample collection: First, local anesthesia is performed on the target lung segment. Then, the tip of the fiberoptic bronchoscope is wedged into the bronchial opening, and a total volume of 100 to 250 ml of 37°C sterile saline is injected. Finally, the lavage fluid is aspirated and recovered with a negative pressure of 50 to 100 mmHg. Step 2: Adding preservation solution to the sample: Within 1 hour of receiving the collected irrigation fluid sample, add the preservation solution described in claim 1, with a sample to preservation solution mixing ratio of 1:4, then seal and store at room temperature in a fume hood; Step 3: Sample preparation and staining: Centrifuge the sample with preservation solution added in Step 2, discard the supernatant, retain the cell precipitate at the bottom, take an appropriate amount of precipitate and spread it on a glass slide, let it stand and fix, then stain it using the Wright-Gymsa method, and after the stained slide dries, mount it for observation. Step 4: Observe and evaluate the stained slides under a microscope, and make a judgment based on the presence of abnormal cells.

9. The method for cytological examination of bronchoalveolar lavage fluid according to claim 8, characterized in that: The abnormal cell condition refers to the presence of tumor cells or pathogens.

10. The method for bronchoalveolar lavage fluid cytological examination according to claim 8, characterized in that: The evaluation targets were macrophages, lymphocytes, neutrophils, and eosinophils.

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