Cell cleaning fluid, reagent combination for serous cavity liquid-based cell treatment, serous cavity liquid-based cell staining kit and staining method

Abstract

The invention relates to a cell cleaning solution, a reagent combination for serosal cavity liquid-based cell treatment, a serosal cavity liquid-based cell staining kit and a staining method, and belongs to the technical field of biological detection. In order to consider the morphology and the ICC dyeing effect, the invention provides a formula of the cell cleaning fluid based on a large number of experimental studies in the early stage, and subsequent experiments prove that when the cell cleaning fluid is used for treating a bloody specimen, the effects of rapid hemolysis, mucus and protein fluid removal and the like can be realized, and meanwhile, other cells are effectively fixed; the morphological integrity and good antigenicity of other cells are ensured. And a thin and uniform cell layer is prepared through natural sedimentation, is suitable for cytological detection, has a clear background, and can completely show the morphology and structure of cells. The cell cleaning fluid is a medium for cleaning and dissolving red blood cells, can generate a destructive effect on the red blood cells, and is an effective reagent capable of ensuring the morphological integrity and good antigenicity of other cells.

Description

Technical Field

[0001] The invention relates to a cell cleaning solution, a reagent combination for treating serous cavity liquid-based cells, a serous cavity liquid-based cell staining kit and a staining method, and belongs to the technical field of biological detection. Background Art

[0002] Serous effusion refers to the excessive fluid accumulated in body cavities such as the pleural cavity, pericardial cavity and abdominal cavity. It is a common clinical manifestation of tumors and non-tumor diseases, and sometimes even the only symptom of patients. All kinds of benign and malignant diseases can cause serous effusion. It is the earliest clinical manifestation of some malignant tumors, especially malignant mesothelioma and some lymphohematopoietic system tumors. For malignant tumors, in addition to detached free tumor cells, serous effusion also includes immune cells, mesothelial cells, red blood cells, mesenchymal stem cells, and a large amount of proteins, amino acids, lipids, sugars, electrolytes, etc. These components are closely related to the tumor microenvironment and provide an important source of clinical materials for studying the occurrence and development of tumors. Therefore, the judgment of its nature is helpful for the diagnosis of malignant tumors, and tracing its source, category, etc., provides a basis for clinical diagnosis and treatment.

[0003] At present, conventional cytology is mainly used to distinguish benign and malignant effusions. However, this method has the limitations of low sensitivity and susceptibility to subjective factors of pathologists. Therefore, effusion cytology evaluation has gradually become one of the most challenging areas in cytopathological diagnosis. In many cases, a clear diagnosis cannot be made based on cell morphology alone. On the one hand, during the preparation of cytological specimens for bloody pleural and ascites, the specimens contain a large number of red blood cells, especially when some specimens themselves contain more blood cells and fewer cancer cells. The red blood cells will cover up the few cancer cells, resulting in missed cytological diagnosis; on the other hand, due to certain limitations in the location and quantity of serous cavity effusions, it is not easy to find cancer cells. Therefore, when the cytological test results are negative, the possibility of malignant tumors cannot be completely ruled out, and there is still a problem of missed diagnosis. In addition, benign mesothelial cells undergo countless structural and cellular changes under numerous stimuli, and well-differentiated or borderline (marginal) malignant cells can disguise themselves as benign cells, which easily leads to overlapping of cell morphological characteristics, making it impossible to distinguish free malignant cells (especially metastatic adenocarcinoma and reactive mesothelial cells). Therefore, sometimes a clear cytological diagnosis of serous effusion cannot be made based on cell morphology alone.

[0004] With the advancement of medical science, simple cell morphology diagnosis can no longer meet the needs of clinical diagnosis and treatment, and immunohistochemistry and molecular detection have become necessary auxiliary means. In the past decade, a large amount of literature data has shown that among all available methods, immunocytochemistry (ICC) staining has significant superiority and irreplaceable significance in the judgment of the nature of effusion in clinical diagnosis and treatment. It is a relatively objective and most commonly used staining technique. Immunocytochemistry has been used in many laboratories to judge the benign and malignant nature of effusions, and then provide a reference for the judgment of the benign and malignant nature of tumors, but most of the tests use cell wax blocks. However, cell wax blocks have the problems of long preparation cycle, cumbersome steps, relatively time-consuming inspection process, and easy loss when there are few tumor cells.

[0005] The liquid-based thin-layer cell preparation method is an internationally advanced cytological detection technology developed in recent years. It is to place the collector for collecting exfoliated samples in a device of cell preservation treatment fluid, rinse the exfoliated cell samples with the cell preservation treatment fluid, and then use manual, semi-automatic or fully automatic methods to disperse and filter the samples for preparation, and finally perform fiber detection and diagnosis. The liquid-based thin-layer cell preparation method is particularly suitable for cytological classification detection of serous cavity effusions. Therefore, the exfoliated cell preservation fluid that prevents the morphological structure of exfoliated cells from changing is crucial for liquid-based thin-layer cell preparation and subsequent cytological detection. At present, most of the exfoliated cell preservation fluids mainly play the role of maintaining and fixing the original morphological structure of exfoliated cells, but cannot remove more red blood cells, so that more red blood cells interfere with the clinical diagnosis of exfoliated cells.

[0006] Therefore, in clinical practice, serous cavity effusions are generally pre-treated to remove red blood cells. Pathology departments use potassium chloride hypotonicity, glacial acetic acid or other red blood cell removal agents, but the operation is cumbersome, the use is not convenient, and the treatment effect is not satisfactory. At present, most of the cell cleaning fluids available on the market are imported brands with high costs. They are mainly used to meet the requirements of Papanicolaou morphological staining, and there is no guarantee for the results of immune cell staining. There may even be false negative staining results, which seriously affect the judgment of the results.

[0007] Therefore, developing a cell washing solution or preservation solution that can effectively remove red blood cells is of great significance for improving the quality of liquid-based thin-layer cell preparation and preventing excessive red blood cells in bloody serous effusion from interfering with the clinical diagnosis of exfoliated cells. Summary of the invention

[0008] The first object of the present invention is to provide a cell washing solution, which provides a cell washing solution for the prior art that can maintain the integrity of the morphology of the detected cells and good antigenicity while effectively removing red blood cells.

[0009] The second object of the present invention is to provide a reagent combination for treating serous cavity liquid-based cells, which provides a reagent combination for treating serous cavity liquid-based cells that can improve the subsequent staining effect for the prior art.

[0010] The third object of the present invention is to provide a serous cavity liquid-based cell staining kit, which provides a serous cavity liquid-based cell staining kit with high sensitivity and taking into account both morphological and immunocytochemical staining effects for the prior art.

[0011] The fourth object of the present invention is to provide a method for staining serous cavity fluid-based cells, which provides a method for staining serous cavity fluid-based cells with high specificity and accuracy for the prior art.

[0012] In order to achieve the above object, a technical solution of a cell washing solution of the present invention is:

[0013] A cell cleaning solution, the cell cleaning solution consists of component A and component B: the component A is an ethanol solution with a volume fraction of 40-50%; the component B consists of the following ingredients: 10 vol% formalin, 0.8-0.9 wt% ion maintainer, 0.095-0.190 wt% buffer, and water as the balance; the pH of the component B is 7.20-7.40; when used, the components A and B are mixed at a volume ratio of 1:1.

[0014] The beneficial effect of the above scheme is that the cell cleaning solution provided by the present invention is a pioneering invention. In order to take into account the morphological and ICC staining effects, the present invention provides a formula of a cell cleaning solution through a large number of experimental discoveries in the early stage. Subsequent experiments have proved that the cell cleaning solution can achieve rapid hemolysis, mucus and protein liquid removal and other effects when processing blood specimens, while effectively fixing other cells, ensuring the morphological integrity and good antigenicity of other cells. Moreover, a thin and uniform cell layer is prepared by natural sedimentation, which is suitable for cytological detection, with a clear background, and can fully show the cell morphology and structure. The cell cleaning solution of the present invention is a medium for cleaning and dissolving red blood cells, which can have a destructive effect on red blood cells, and is also an effective reagent that can ensure the morphological integrity and good antigenicity of other cells.

[0015] Furthermore, the cell washing solution provided by the present invention can process red blood cells faster and more effectively, taking into account the morphological and immunocytochemical staining effects, which is conducive to breaking the state of cell washing solution dependence on imported brands, promoting the development of domestic original research products, and reducing costs.

[0016] Specifically, ethanol is an organic solvent. After adding an appropriate concentration of ethanol to a sample rich in red blood cells, a large number of ethanol molecules enter the red blood cells along the concentration difference, making the osmotic pressure inside the red blood cells higher than the osmotic pressure outside the red blood cells, and the water channels are open, so that a large number of water molecules enter the red blood cells, burst the red blood cells, and achieve the purpose of breaking red blood cells. Experiments have found that excessively high concentrations of ethanol can cause the surface proteins of red blood cells to agglutinate rapidly and prevent the infiltration of ethanol, making the effect of breaking red blood cells sharply worse. At the same time, ethanol can also act as a fixative. Formaldehyde solution has the characteristics of strong permeability, uniform fixation, and small cell shrinkage. It can effectively fix cells with nuclei and fix the precipitates (i.e. other cells) after breaking. The two work together to quickly dissolve the red blood cells in the sample and quickly and effectively fix the nucleated cells, retaining the morphological structure of the cells, so as to better perform cell detection. Buffers can effectively alleviate the pH changes caused by the release of autolysis byproducts by cells and maintain cell morphological characteristics. Ionic strength maintainers can adjust the osmotic pressure of the solution to maintain the original morphology of the cells.

[0017] Specifically, formalin is a formaldehyde solution with a concentration of 37-40%.

[0018] As a further improvement, the buffer includes one or two of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate; the ion maintainer includes one, two, or three of sodium chloride, potassium chloride, and ammonium chloride.

[0019] In order to achieve the above-mentioned object, the technical scheme of a reagent combination for treating serous cavity liquid-based cells of the present invention is:

[0020] A reagent combination for treating serous cavity liquid-based cells, the reagent combination comprising the cell cleaning solution and the cell preservation solution; the cell preservation solution is composed of the following components: 18-22 vol% ethanol, 4-8 vol% ethylene glycol, 6-12 vol% isopropanol, 1-2 mM / L chelating agent, 1.0-1.2 wt% osmotic pressure maintaining agent, 0.05 vol% surfactant, and the solvent is a buffer solution; the pH of the cell preservation solution is 6.80-7.20.

[0021] The beneficial effect of the above scheme is that the present invention proves through experiments that after the blood cell sample is treated with the cell washing solution, the red blood cells are quickly and effectively lysed, while other cells (such as mesothelial cells, lymphocytes, etc.) have intact structures, no deformation, distinct nucleus and cytoplasm, and clear nucleoli. The treated cell sample is stored in the cell preservation solution provided by the present invention, which can effectively preserve its structural morphology. The combined use of the two is helpful to improve the quality of film preparation and improve the accuracy and reliability of subsequent cytological analysis.

[0022] As a further improvement, the buffer is one of PBS buffer, Tris-HCl buffer, PIPES buffer, acetic acid buffer, sodium acetate buffer, phosphate buffer, citric acid buffer, imidazole buffer, and triethanolamine hydrochloride buffer.

[0023] As a further improvement, the chelating agent is selected from one or two of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, sodium citrate, heparin, hepatokinase, streptokinase, and sodium citrate.

[0024] As a further improvement, the osmotic pressure maintaining agent is selected from one or two of sodium chloride, potassium chloride, and ammonium chloride; the surfactant is selected from one or two of Tween-20, Triton X-100, and ethylphenyl polyethylene glycol.

[0025] In order to achieve the above object, the technical solution of a serous cavity liquid-based cell staining kit of the present invention is:

[0026] A serous cavity liquid-based cell staining kit comprises the serous cavity liquid-based cell processing reagent combination.

[0027] The beneficial effect of the above scheme is that the serous cavity liquid-based cell staining kit of the present invention includes the above-mentioned cell washing solution, which can quickly and effectively dissolve red blood cells when facing bloody serous cavity effusion, preserve the cell morphology and good antigenicity of the specimen intact, improve the preparation quality, make the cells in the prepared liquid-based cell slice more evenly distributed, the background clearer, the cell structure easier to observe, and improve the accuracy of diagnosis.

[0028] Furthermore, the serous cavity liquid-based cell staining kit provided by the present invention is adapted to a fully automatic immunocytochemical staining machine, which can realize fully automatic liquid-based slide preparation, improve work efficiency, reduce errors, improve slide preparation quality, reduce pollution, and improve slide preparation safety. In addition, the sedimentation chamber delimits the cell area, and the reagent is added separately to avoid cross contamination, avoid reagent loss, improve the positive detection rate, and avoid false negatives.

[0029] As a further improvement, the serous cavity liquid-based cell staining kit includes a peroxidase blocking agent, a primary antibody post-reagent, a polymer, a DAB substrate, a DAB buffer, and hematoxylin.

[0030] The beneficial effect of the above scheme is that the serous cavity liquid-based cell staining kit provided by the present invention adopts a non-biotin secondary antibody detection system based on horseradish peroxidase-labeled micropolymer-labeled antibodies, adopts the independently developed "MicroStracker" micropolymer stacking technology, eliminates the Fc fragment, has better specificity, a micropolymer skeleton, a faster penetration rate, and a higher sensitivity.

[0031] In order to achieve the above object, the technical solution of a serous cavity liquid-based cell staining method of the present invention is:

[0032] A method for staining serous cavity liquid-based cells, comprising the following steps:

[0033] (1) centrifuging the serous cavity effusion sample to be sliced, adding the cell washing solution in the serous cavity fluid-based cell treatment reagent combination to the precipitate, shaking and mixing, centrifuging, and retaining the precipitate in the cell preservation solution in the serous cavity fluid-based cell treatment reagent combination;

[0034] (2) The sample stored in the cell preservation solution in step (1) is subjected to liquid-based slide preparation and subsequent staining.

[0035] The beneficial effect of the above scheme is that in the serous cavity liquid-based cell staining method of the present invention, the serous cavity effusion cell sample is treated with a cell washing solution and preserved in a cell preservation solution before liquid-based slide preparation. In the subsequent staining process, it effectively solves the problem that some trace cytological specimens, such as fine needle aspiration cytological specimens, cannot be embedded in paraffin, and provides patients with a rapid and accurate basis for diagnosis and treatment. Moreover, after treatment, the cell morphology and good antigenicity of the specimen are well preserved. The cells in the prepared liquid-based cell sheet are more evenly distributed, the background is clearer, and the cell structure is easier to observe, thereby improving the accuracy of diagnosis.

[0036] As a further improvement, the staining includes Papanicolaou staining and / or immunocytochemical staining.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] First, the present invention discloses a cell cleaning solution, which can clean the serous cavity effusion sample, dissolve red blood cells, prevent protein precipitation, dissolve mucus and other pretreatments, and preserve the cell morphology and good antigenicity of the specimen. The cell distribution in the finished liquid-based cell sheet is more uniform, the background is clearer, the cell structure is easier to observe, and the diagnostic accuracy is improved.

[0039] Second, a serous cavity liquid-based cell detection kit disclosed in the present invention can be equipped with a fully automatic immunocytochemical staining machine (such as: CNT480). Fully automatic liquid-based slide making improves work efficiency and reduces errors; improves slide making quality and reduces pollution; and improves slide making safety. It supports continuous machine loading, three independent reaction chambers, and 48 slides are loaded at the same time. The slides can be replaced continuously. After one batch is completed, the next batch of experiments can be executed immediately, and various immunocytochemical staining programs can be edited at will. In addition, the sedimentation chamber delimits the cell area, and the reagents are added separately to avoid cross contamination, avoid reagent loss, improve the positive detection rate, and avoid false negatives. The specific advantages are as follows: ① Instant detection: Liquid-based preparation can be directly used for immunocytochemical staining, without the need for cell wax block embedding process, which improves the diagnosis speed; ② Wide range of applicable sample types: Suitable for trace cell specimens and samples that are difficult to embed in wax blocks; ③ Clean background: Clear staining can effectively improve the false positive effect of bloody serous cavity effusion samples; ④ Accurate and reliable staining results: Natural sedimentation method preparation, preferential capture of diseased cells, and effective enrichment of more cells; ⑤ Replicate films: The same sample can be used to prepare multiple cell white films for multi-index staining at the same time; ⑥ Multi-functional free choice: Fully automatic liquid-based preparation + Papanicolaou staining + immunocytochemical staining; ⑦ Flexible design: Single chamber / double chamber, doubled throughput, free combination of staining indicators. In summary, the serous cavity liquid-based cell detection kit of the present invention can realize full automation of the entire process of preparation and staining, without manual intervention, simple and convenient operation, and greatly improves the staining efficiency and laboratory standardization level.

[0040] Third, after pre-treatment, the serous cavity effusion cell sample of the present invention can be equipped with a fully automatic immunocytochemical staining machine to achieve both Papanicolaou staining and immunocytochemical staining. When conducting serous cavity fluid-based cell detection, the combined use of cell morphology and immunocytochemical staining can greatly improve the sensitivity, specificity and accuracy of diagnosis, provide a reliable basis for clinical treatment, and serve as an important supplement to accompanying diagnosis when tissue samples are lacking. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the result of Papanicolaou staining after preparation of liquid-based cells of bloody pleural effusion sample in Experimental Example 2 of the present invention (40×);

[0042] Figure 2 This is a Synaptophsin staining result of the liquid-based cell preparation of the human small cell lung cancer cell line NCI-H146 in Experimental Example 3 of the present invention (40×);

[0043] Figure 3 This is the CK7 staining result of the bloody pleural effusion sample liquid-based cell preparation in Experimental Example 4 of the present invention (40×);

[0044] Figure 4This is the CK7 staining result of the bloody pleural effusion sample liquid-based cell preparation in Experimental Example 5 of the present invention (40×);

[0045] Figure 5 This is the CK7 staining result of the bloody pleural effusion sample liquid-based cell preparation in Experimental Example 6 of the present invention (40×);

[0046] Figure 6 This is a TTF-1 staining result of the bloody pleural effusion sample liquid-based cell preparation in Experimental Example 7 of the present invention (40×);

[0047] Figure 7 This is a picture of TTF-1 and NapsinA staining results after preparation of liquid-based cell slides of thoracic puncture liquid-based samples in Experimental Example 8 of the present invention (40×);

[0048] Figure 8 This is the Her-2 and Gata-3 staining result of the ascites sample liquid-based cell preparation in Experimental Example 9 of the present invention (40×);

[0049] Fig. 9 This is a comparison chart of the results of ICC staining of ascites liquid-based cell preparation and IHC staining of paraffin sections in Experimental Example 10 of the present invention (20×);

[0050] Fig.10 This is a Papanicolaou staining result (40×) of a bloody pleural effusion sample treated with a commercially available imported H brand cell washing solution in Experimental Example 11 of the present invention;

[0051] Fig.11 This is a Papanicolaou staining result diagram (40×) of a bloody pleural effusion sample treated with the cell washing solution of the present invention in Experimental Example 11 of the present invention;

[0052] Fig.12 This is a TTF-1 staining result diagram (40×) of a concentrated bloody pleural effusion sample treated with a commercially available imported H brand cell washing solution in Experimental Example 12 of the present invention;

[0053] Fig.13 This is a TTF-1 staining result diagram (40×) of a bloody pleural effusion sample treated with the cell washing solution of the present invention in Experimental Example 12 of the present invention;

[0054] Fig.14 The figures are the effects of treating purulent pleural effusion samples with the commercially available imported H brand cell washing solution and the cell washing solution of the present invention in Experimental Examples 11 and 12 of the present invention. DETAILED DESCRIPTION

[0055] In existing clinical tests, bloody pleural and abdominal effusion samples are often encountered. In the process of cytological specimen preparation, such samples contain a large number of red blood cells. If they are not treated or the treatment effect is not good, it is easy to cause the background color of Papanicolaou staining or immunohistochemical staining to be dark, and the cell morphology is not easy to distinguish, which seriously affects the diagnosis of pathologists. In particular, when there are more blood cells and fewer cancer cells, red blood cells will cover up a few cancer cells, causing cytological diagnosis to be missed. Most of the exfoliated cell preservation fluids in the prior art mainly play the role of maintaining and fixing the original morphological structure of exfoliated cells, but cannot remove more red blood cells or the effect of removing red blood cells is poor. Therefore, clinically, pretreatment is generally performed for serous cavity effusion to remove red blood cells. Pathology departments use potassium chloride hypotonic, glacial acetic acid or other red blood cell removal agents, but the operation is cumbersome, the use is not convenient enough, and the treatment effect is not satisfactory. At present, most of the cell cleaning fluids available on the market are imported brands, with high costs, and are mainly for satisfying Papanicolaou morphological staining. There is no guarantee for the results of immune cell staining, and even false negative staining results occur, which seriously affects the result judgment. Based on this, the present invention provides a cell cleaning solution.

[0056] The cell cleaning solution of the present invention is composed of component A and component B: the cell cleaning solution is composed of component A and component B: the component A is an ethanol solution with a volume fraction of 40-50%; the component B is composed of the following ingredients: 10 vol% formalin, 0.8-0.9 wt% ion maintainer, 0.095-0.190 wt% buffer, and water balance; the pH of the component B is 7.20-7.40; when used, the component A and the component B are mixed in a volume ratio of 1:1. Experiments have shown that after the blood cell sample is treated with the cell cleaning solution, the red blood cells are quickly and effectively lysed, while other cells (such as mesothelial cells, lymphocytes, etc.) have complete structures, no deformation, distinct nucleus and cytoplasm, and clear nucleoli. Moreover, the treated sample is suitable for cytological detection, the background is clearer, and the cell structure is easier to observe, which helps to improve the quality of film making and the accuracy and reliability of subsequent cytological analysis.

[0057] Taking pleural effusion exfoliated cells (a type of blood cytology sample) as an example, the steps of immunocytochemical staining using a serous cavity liquid-based cell detection kit in the following embodiment of the present invention are described:

[0058] (1) Sample pretreatment:

[0059] After the serous cavity effusion sample is treated with cell washing solution, it is stored in a cell preservation solution bottle and then prepared by a fully automatic immunocytochemical staining machine for preparation, Papanicolaou staining or immunocytochemical staining.

[0060] Before use, component A: component B = 1:1 mixed to obtain a cell washing solution that can be used to wash cells.

[0061] It is recommended that the cell washing solution obtained by mixing in the above proportions be used within one week.

[0062] The specific steps for sample cleaning are:

[0063] a) Mix the serous cavity effusion sample thoroughly, take 50 mL and place it in a disposable conical bottom centrifuge tube, centrifuge at 2500r / min for 5 minutes. Observe the stratification of the supernatant and the precipitate. When the blood content of the precipitate is greater than 1 / 2 of the precipitate, it is recommended to use cell washing solution for washing.

[0064] b) When the sample is separated by centrifugation, in actual situations, the amount of supernatant and sediment in most samples is greater than 2 mL, and can be moved at the bottom of the tube: discard the supernatant, take all the sediment at the bottom of the tube, add 20-25 mL of cell washing solution, oscillate in a micro-oscillator for 10-20 minutes, mix thoroughly, wait for the sediment at the bottom of the centrifuge tube to disperse and dissolve until the color turns light red, centrifuge at 2500 r / min for 5 minutes, and discard the supernatant. Add phosphate buffered saline (PBS) to 50 mL, centrifuge at 2500 r / min for 5 minutes, discard the supernatant, and transfer to a cell preservation solution bottle.

[0065] c) When the sample is consistent before and after centrifugation and not stratified:

[0066] For this type of purulent and bloody sample, take 10 mL of the sample into a 50 mL disposable conical bottom centrifuge tube, add 40 mL of cell washing solution, oscillate in a micro-oscillator for 10 to 20 minutes, mix thoroughly, wait for the sediment at the bottom of the centrifuge tube to disperse and dissolve until the color turns light red, centrifuge at 2500 r / min for 5 minutes, and discard the supernatant. Add phosphate buffered saline (PBS) to 50 mL, wash and centrifuge at 2500 r / min for 5 minutes, discard the supernatant, and transfer to a cell preservation solution bottle.

[0067] (2) Liquid-based thin layer cell slide preparation:

[0068] The pleural effusion samples stored in the cell preservation solution bottle were directly placed in the designated position of the fully automatic immunocytochemical staining machine, and 500 μL of cell sample was added to each slide to obtain a thin layer of cell white slides based on the staining solution.

[0069] (3) Fixation:

[0070] Fix with 95% ethanol at room temperature for 5 minutes, and rinse with cleaning solution 3 times, 2 minutes each time.

[0071] (4) Antigen retrieval:

[0072] The Tris-EDTA antigen repair solution was heated to 70-85°C and repaired for 20 minutes to effectively remove the cross-linking between proteins caused by aldehyde fixatives and fully expose the antigen epitopes in the cell sheets.

[0073] (5) Add peroxidase blocking agent

[0074] Add 150 μL of endogenous peroxidase blocking agent and incubate at room temperature for 5 min. Rinse with cleaning solution 3 times, 2 min each time.

[0075] (6) Primary antibody incubation:

[0076] Add 150 μL of primary antibody reagent and incubate at room temperature for 20 to 30 minutes to bind with antigen proteins in the cell sheet to form antigen-antibody complexes. Rinse with cleaning solution 3 times, 2 minutes each time.

[0077] (7) Post-primary antibody incubation

[0078] Add 150 μL of primary antibody reagent and incubate at room temperature for 8 to 10 minutes to allow the primary antibody to bind to the antigen-antibody complex. Rinse with cleaning solution 3 times, 2 minutes each time.

[0079] (8) Polymer incubation

[0080] Add 150 μL of horseradish peroxidase (HRP) labeled polymer and incubate at room temperature for 8-12 minutes to allow the secondary antibody to specifically bind to the primary antibody reagent. Rinse with cleaning solution 3 times, 2 minutes each time.

[0081] Note: The horseradish peroxidase (HRP) labeled polymer is: polymer HRP-GAM (goat anti-mouse IgG), the secondary antibody is able to bind to the primary antibody reagent, and the secondary antibody is labeled with horseradish peroxidase (HRP), and its main function is to detect the presence of the antibody, amplify the signal of the primary antibody, and introduce it into the color development system.

[0082] (9) DAB color development

[0083] Add 150 μL of DAB color development solution and incubate at room temperature for 2 to 5 minutes until a brown-yellow precipitate appears. Rinse with deionized water 3 times, 2 minutes each time.

[0084] Note: DAB substrate: substrate buffer = 1:20 dilution ratio.

[0085] (10) Hematoxylin counterstaining

[0086] Counterstain at room temperature for 10s to 30s to stain the cell nucleus. Rinse with purified water 5 times, 2s each time.

[0087] (11) Dehydrate, transparentize, and seal the slides, and examine them under a microscope.

[0088] The present invention is further described in detail below in conjunction with specific examples. Unless otherwise specified, the equipment and reagents used in each embodiment, experimental example and comparative example can be obtained from commercial sources.

[0089] The model of the fully automatic immunocytochemical staining machine used in the following examples of the present invention is CNT480, which was purchased from Henan Synotech Biotechnology Co., Ltd.

[0090] 1. A specific embodiment of a cell cleaning solution of the present invention:

[0091] Example 1

[0092] The cell washing solution of this embodiment is composed of component A, component B and water; the component A is an ethanol solution with a volume fraction of 50%; the component B is composed of the following ingredients: 10 vol% formalin, 0.875 wt% sodium chloride, 0.115 wt% anhydrous disodium hydrogen phosphate, 0.0272 wt% anhydrous potassium dihydrogen phosphate, and the balance water. The pH of component B is 7.21.

[0093] Before using the cell washing solution, mix component A and component B at a volume ratio of 1:1.

[0094] Example 2

[0095] The cell washing solution of this embodiment is composed of component A, component B and water; the component A is an ethanol solution with a volume fraction of 40%; the component B is composed of the following ingredients: 10 vol% formalin, 0.875 wt% sodium chloride, 0.100 wt% anhydrous disodium hydrogen phosphate, 0.0408 wt% anhydrous potassium dihydrogen phosphate, and the balance water. The pH of component B is 7.32.

[0096] Before using the cell washing solution, mix component A and component B at a volume ratio of 1:1.

[0097] Example 3

[0098] The cell washing solution of this embodiment is composed of component A, component B and water; the component A is an ethanol solution with a volume fraction of 45%; the component B is composed of the following ingredients: 10 vol% formalin, 0.819 wt% sodium chloride, 0.071 wt% anhydrous disodium hydrogen phosphate, 0.068 wt% anhydrous potassium dihydrogen phosphate, and the balance water. The pH of component B is 7.32.

[0099] Before using the cell washing solution, mix component A and component B at a volume ratio of 1:1.

[0100] 2. Specific embodiments of a reagent combination for treating serous cavity liquid-based cells according to the present invention:

[0101] Example 4

[0102] The reagent combination for treating cells with a liquid-based cell in the serous cavity of this embodiment is composed of the cell washing solution and the cell preservation solution of Example 1; the specific composition of the cell preservation solution is as follows: 20 (V / V)% ethanol, 5 (V / V)% ethylene glycol, 12 (V / V)% isopropanol, 2 mM / L disodium ethylenediaminetetraacetic acid, 1.1175wt% potassium chloride, 0.1064wt% disodium hydrogen phosphate anhydrous, 0.0899wt% sodium dihydrogen phosphate anhydrous, 0.05 (V / V)% Tween-20, and the balance water. The pH of the cell preservation solution is 7.05.

[0103] Example 5

[0104] The reagent combination for treating the serous cavity liquid-based cells of this embodiment is composed of the cell washing solution and the cell preservation solution of Example 2; the specific composition of the cell preservation solution is as follows: 18 (V / V)% ethanol, 4 (V / V)% ethylene glycol, 6 (V / V)% isopropanol, 1 mM / L disodium ethylenediaminetetraacetic acid, 1.043wt% potassium chloride, 0.1277wt% disodium hydrogen phosphate anhydrous, 0.0720wt% sodium dihydrogen phosphate anhydrous, 0.05 (V / V)% Tween-20, and the balance water. The pH of the cell preservation solution is 7.10.

[0105] Example 6

[0106] The reagent combination for treating the serous cavity liquid-based cells in this embodiment is composed of the cell washing solution and the cell preservation solution of Example 3; the specific composition of the cell preservation solution is as follows: 22 (V / V)% ethanol, 8 (V / V)% ethylene glycol, 12 (V / V)% isopropanol, 2 mM / L disodium ethylenediaminetetraacetic acid, 1.1175wt% potassium chloride, 0.1489wt% disodium hydrogen phosphate anhydrous, 0.0539wt% sodium dihydrogen phosphate anhydrous, 0.05 (V / V)% Tween-20, and the balance water. The pH of the cell preservation solution is 7.20.

[0107] 3. Specific embodiments of a serous cavity liquid-based cell staining kit of the present invention:

[0108] Example 7

[0109] The present embodiment of the serous cavity liquid-based cell staining kit includes the serous cavity liquid-based cell treatment reagent combination in Example 4, and the specific composition is as follows:

[0110] (1) Mouse anti-human cytokeratin 7 monoclonal antibody (species: mouse, clone number C1C10, catalog number: TCM0120);

[0111] (2) Immunochromogenic reagent (immunocytochemistry method, Henan Zheng Equipment No.: 20240271, Catalog No.: SD9030);

[0112] The immunochromogenic reagent consists of the following 6 components: peroxidase blocking agent, primary antibody post-reagent, polymer, DAB substrate (20×), DAB buffer, and hematoxylin;

[0113] (3) Cell preservation medium (Cat. No.: TC1001);

[0114] (4) Cell washing solution (Yu Zheng Xie Bei 20240270, catalog number: TC3002).

[0115] Example 8

[0116] The present embodiment of the serous cavity liquid-based cell staining kit includes the serous cavity liquid-based cell treatment reagent combination in Example 4, and the specific composition is as follows:

[0117] (1) Mouse anti-human thyroid transcription factor (TTF-1) monoclonal antibody (species: mouse, clone number SPT24, catalog number: TTM0010);

[0118] (2) Immunochromogenic reagent (immunocytochemistry method, Henan Zheng Equipment No.: 20240271, Catalog No.: SD9030);

[0119] The immunochromogenic reagent consists of the following 6 components: peroxidase blocking agent, primary antibody post-reagent, polymer, DAB substrate (20×), DAB buffer, and hematoxylin;

[0120] (3) Cell preservation medium (Cat. No.: TC1001);

[0121] (4) Cell washing solution (Yu Zheng Xie Bei 20240270, catalog number: TC3002).

[0122] Example 9

[0123] The present embodiment of the serous cavity liquid-based cell staining kit includes the serous cavity liquid-based cell treatment reagent combination in Example 4, and the specific composition is as follows:

[0124] (1) Mouse anti-human thyroid transcription factor (TTF-1) monoclonal antibody (species: mouse, clone number SPT24);

[0125] (2) Mouse anti-human Napsin A monoclonal antibody (species: mouse, clone number C2C2);

[0126] (3) Immunochromogenic reagent (immunocytochemistry method, Henan Zheng Equipment No.: 20240271, Catalog No.: SD9030);

[0127] The immunochromogenic reagent consists of the following 6 components: peroxidase blocking agent, primary antibody post-reagent, polymer, DAB substrate (20×), DAB buffer, and hematoxylin;

[0128] (4) Cell preservation medium (Cat. No.: TC1001);

[0129] (5) Cell washing solution (Yu Zheng Xie Bei 20240270, catalog number: TC3002).

[0130] Example 10

[0131] The present embodiment of the serous cavity liquid-based cell staining kit includes the serous cavity liquid-based cell treatment reagent combination in Example 4, and the specific composition is as follows:

[0132] (1) Mouse anti-human Her-2 monoclonal antibody (species: mouse, clone number C1F7, catalog number: CCM-0844);

[0133] (2) Mouse anti-human GATA3 monoclonal antibody (species: mouse, clone number L50-823, catalog number: TGM0010);

[0134] (3) Immunochromogenic reagent (immunocytochemistry method, Henan Zheng Equipment No.: 20240271, Catalog No.: SD9030);

[0135] The immunochromogenic reagent consists of the following 6 components: peroxidase blocking agent, primary antibody post-reagent, polymer, DAB substrate (20×), DAB buffer, and hematoxylin;

[0136] (4) Cell preservation medium (Cat. No.: TC1001);

[0137] (5) Cell washing solution (Yu Zheng Xie Bei 20240270, catalog number: TC3002).

[0138] Embodiment 11

[0139] The present embodiment of the serous cavity liquid-based cell staining kit includes the serous cavity liquid-based cell treatment reagent combination in Example 4, and the specific composition is as follows:

[0140] (1) Mouse anti-human pax-8 monoclonal antibody (species: mouse, clone number C12A32, catalog number: TPM0040);

[0141] (2) Mouse anti-human ovarian cancer antigen monoclonal antibody (species: mouse, clone number OC125, catalog number: TCM0010);

[0142] (3) rabbit anti-human nephroblastoma monoclonal antibody (species: rabbit, clone number MyR1-WT1, catalog number: TWR0010);

[0143] (4) Mouse anti-human p53 protein monoclonal antibody (species: mouse, clone number C2H10, catalog number: TPM0030);

[0144] (5) Immunochromogenic reagent (immunocytochemistry method, Henan Zheng Equipment No.: 20240271, Catalog No.: SD9030);

[0145] The immunochromogenic reagent consists of the following 6 components: peroxidase blocking agent, primary antibody post-reagent, polymer, DAB substrate (20×), DAB buffer, and hematoxylin;

[0146] (6) Cell preservation medium (Cat. No.: TC1001);

[0147] (7) Cell washing solution (Yu Zheng Xie Bei 20240270, catalog number: TC3002).

[0148] IV. Experimental Example: A method for staining cells in the serosa cavity liquid-based

[0149] Experimental Example 1

[0150] 1. Single factor analysis was used to investigate the effect of different concentrations of ethanol in component A of cell washing solution on the treatment of bloody pleural effusion samples.

[0151] The specific results are shown in Table 1. When the ethanol concentration in the cell washing solution A component is ≤50 (V / V)%, the red blood cell processing speed is accelerated and the effect is better. Too low ethanol concentration causes some nucleated cells to shrink and deform, but no cell overlap occurs. The preferred ethanol concentration is 40-50% (V / V).

[0152] Table 1 Effects of different concentrations of ethanol on red blood cells in component A of cell washing solution

[0153]

[0154] When the cell washing solution B component is used alone to treat bloody pleural effusion samples, the cell morphology fixation effect is better. However, it takes a long time to process, even two washing processes, and there are still a large number of red blood cells that are not completely lysed.

[0155] When the cell washing solution component A: 40-50% ethanol and the cell washing solution component B are mixed in a volume ratio of 1:1, the treatment effect of red blood cells in bloody pleural and peritoneal effusion samples is better. The coordinated use of the two allows the red blood cells in the sample to be quickly dissolved and the nucleated cells to be quickly and effectively fixed, retaining the morphological structure of the cells for better subsequent cytological testing. The specific results are shown in Table 2.

[0156] Table 2 Effect of cell washing solution on red blood cells

[0157] Cell washing solution Red blood cells background Cell distribution Cell morphology Example 1 disappear Clarity Uniform, single layer Cell integrity Example 2 disappear Clarity Uniform, single layer Cell integrity Example 3 disappear Clarity Uniform, single layer Cell integrity

[0158] 2. Compare the effects of currently used cell washing solutions on treating red blood cells. The specific operations are as follows:

[0159] After the red blood cells of bloody pleural effusion samples were removed using cell washing solutions of different formulas, the liquid-based slides were subjected to Papanicolaou staining and ICC staining, respectively, to compare the treatment effects of different cell washing solutions. The specific formulas and results are shown in Table 3.

[0160] Table 3 Cell cleaning solution formula and effect statistics

[0161]

[0162] Wherein, 10% NBF is 10% neutral buffered formalin solution, comprising: 10% formaldehyde (V / V%), 0.875% (wt%) sodium chloride, 0.115% (wt%) anhydrous disodium hydrogen phosphate, and 0.0275% (wt%) anhydrous sodium dihydrogen phosphate.

[0163] By examining the red blood cell removal schemes in four different pleural and abdominal effusion samples, the effects of Pap and ICC staining were summarized: I≥J>H>G. Among them, the background of scheme I is cleaner, and the cells are more and more uniformly enriched. In schemes G and H, there are flocs in Pap and ICC staining, and the number of cells is relatively small and unevenly distributed. The effect of scheme J on removing red blood cells and the subsequent Pap and ICC staining effects are not significantly different from those of scheme I. Both schemes H and J use glacial acetic acid, which is not friendly to experimental operators. In summary, scheme I is preferred for bloody pleural and abdominal effusion samples, and 50% ethanol + 10% NBF is used for treatment. After verification in more bloody pleural and abdominal effusions, it is confirmed that this method has good stability in the treatment of red blood cells, and the Pap and ICC staining effects meet clinical diagnostic needs.

[0164] Experimental Example 2

[0165] In this experimental example, the bloody pleural effusion sample was treated with the serous cavity liquid-based cell treatment reagent combination of Example 4, and the liquid-based slide was prepared. Then, the pleural effusion sample was Papanicolaou staining solution (Yu Zheng Equipment Preparation No.: 20190234) was used to perform Papanicolaou staining on the pleural effusion sample. The Papanicolaou staining solution includes: hematoxylin staining solution and EA / OG staining solution. The specific process is as follows:

[0166] 1. Sample pretreatment:

[0167] After the bloody pleural effusion sample is treated with cell washing solution, it is stored in a cell preservation solution bottle and then used for slide preparation and immunocytochemical staining by a fully automatic immunocytochemical staining machine. Before using the cell washing solution, component A and component B are mixed at a ratio of 1:1 to obtain a buffer solution that can be used to wash cells. It is recommended that the cell washing solution obtained by mixing in the above ratio be used within one week.

[0168] The specific processing steps are as follows:

[0169] a) Mix the bloody pleural effusion sample thoroughly, take 50 mL and place it in a disposable conical bottom centrifuge tube, and centrifuge at 2500 r / min for 5 min.

[0170] b) After centrifugation, the sample is separated into layers, and the supernatant and sediment are greater than 2 mL and can be moved at the bottom of the tube:

[0171] Discard the supernatant, take all the sediment at the bottom of the tube, add 25mL of cell washing solution, oscillate in a micro-oscillator for 10 minutes, mix thoroughly, wait for the sediment at the bottom of the centrifuge tube to disperse and dissolve until the color turns light red, centrifuge at 2500r / min for 5 minutes, and discard the supernatant. Add phosphate buffered saline (PBS) to 50mL, centrifuge at 2500r / min for 5 minutes, discard the supernatant, and transfer to a cell preservation solution bottle.

[0172] 2. Liquid-based thin layer cell slide preparation

[0173] The pleural effusion samples stored in the cell preservation fluid bottle were directly placed in the designated position of the fully automatic immunocytochemical staining machine, and 500 μL of cell sample was added to each slide to obtain a liquid-based thin-layer cell white slide to be stained (hereinafter referred to as a cell slide).

[0174] 3. Fixed

[0175] Place the cell slide prepared in step 2 in 95% ethanol fixative and fix for 5 min at room temperature, then rinse with cleaning solution 3 times, 2 min each time.

[0176] Note: The cleaning solution is obtained by diluting the concentrated cleaning solution (10×) (Yu Zheng Xie Bei No. 20140002) to provide a stable working environment for the dyeing system.

[0177] 4. Papanicolaou staining

[0178] The cell slides fixed in step 3 were rinsed with buffer 3 times for 1 min each time, stained with hematoxylin for 20 s, rinsed with deionized water 3 times for 1 min each time, rinsed with buffer 3 times for 1 min each time, rinsed with anhydrous ethanol 2 times for 15 s each time, stained with EAOG for 30 s, and rinsed with anhydrous ethanol 4 times for 10 s each time.

[0179] Note: Buffer solution (Yuzheng Equipment No.: 20240194) is used to maintain the reaction environment.

[0180] 5. Dehydration and transparency

[0181] Dehydrate the cell slide stained in step 4 in anhydrous ethanol twice, each time for 1 min, take the cell slide out of the anhydrous ethanol, and soak it in xylene twice, each time for 1 min.

[0182] 6. Neutral gum sealing

[0183] After the transparency is completed, the cell slide is removed from the xylene and sealed with neutral gum. The staining results are observed under a microscope and photographed.

[0184] The staining results are shown in Figure 1 The clinical diagnosis of this case was lung adenocarcinoma derived from pleural effusion. After treatment with cell washing solution, the results of Papanicolaou staining showed that red blood cells were completely removed, cells were evenly distributed, the background was clean, and the three-dimensional structure of adenocarcinoma cell clusters was clearly visible.

[0185] Experimental Example 3

[0186] In this experimental example, a liquid-based thin layer of cells was prepared for the small cell lung cancer cell line NCI-H146 liquid cell line, and then immunocytochemical staining was performed using the serous cavity liquid-based cell staining kit of Example 7. The specific process is as follows:

[0187] 1. Liquid-based thin layer cell slide preparation

[0188] The human small cell lung cancer cell line NCI-H146 liquid cell line (the NCI-H146 cell line was purchased from BNCC Beina Biotechnology, number: BNCC278933) was used, and 500 μL of cell sample was added to each slide to obtain a liquid-based thin layer cell white slide to be stained (hereinafter referred to as a cell slide).

[0189] 2. Fixation

[0190] Place the cell slide prepared in step 2 in 95% ethanol fixative and fix for 5 min at room temperature, then rinse with cleaning solution 3 times, 2 min each time.

[0191] 3. Antigen retrieval

[0192] Place the cell slide fixed in step 2 in Tris-EDTA antigen retrieval solution, heat to 75°C, and retrieval for 20 minutes.

[0193] 4. Add peroxidase blocking agent

[0194] Add 150 μL of endogenous peroxidase blocking agent and incubate at room temperature for 5 min. Rinse with cleaning solution 3 times, 2 min each time.

[0195] 5. Primary Antibody Incubation

[0196] Add 150 μL of primary antibody reagent and incubate at room temperature for 20 min to bind to the antigen protein in the cell sheet to form an antigen-antibody complex. Rinse with cleaning solution 3 times, 2 min each time.

[0197] Note: the primary antibody reagent is: mouse anti-human synaptophysin monoclonal antibody (species: mouse, clone number C9D11, catalog number: TSM0020).

[0198] 6. Post-primary antibody incubation

[0199] Add 150 μL of primary antibody reagent and incubate at room temperature for 8 minutes to allow the primary antibody to bind to the antigen-antibody complex. Rinse with cleaning solution 3 times, 2 minutes each time.

[0200] 7. Polymer incubation

[0201] Add 150 μL of horseradish peroxidase (HRP) labeled polymer and incubate at room temperature for 12 min to allow the secondary antibody to specifically bind to the primary antibody reagent. Rinse with cleaning solution 3 times, 2 min each time.

[0202] Note: The horseradish peroxidase (HRP) labeled polymer is: polymer HRP-GAM (goat anti-mouse IgG), the secondary antibody is able to bind to the primary antibody reagent, and the secondary antibody is labeled with horseradish peroxidase (HRP), and its main function is to detect the presence of the antibody, amplify the signal of the primary antibody, and introduce it into the color development system.

[0203] 8. DAB color development

[0204] Add 150 μL of DAB color development solution and incubate at room temperature for 2 min until a brown-yellow precipitate appears. Rinse with deionized water 3 times, 2 min each time.

[0205] Note: The DAB color development working solution is DAB substrate: substrate buffer = 1:20 dilution ratio.

[0206] 9. Hematoxylin counterstaining

[0207] Counterstain at room temperature for 10 seconds to stain the cell nucleus, and rinse with purified water 5 times, 2 seconds each time.

[0208] 10. Dehydrate, make transparent, seal the slides, and read the slides under a microscope.

[0209] The staining results are shown in Figure 2 As shown. It is impractical for each laboratory to maintain a separate library of quality control materials to cover every possible combination of fixation, processing, and specimen type, especially considering that immunocytochemistry is generally a low-volume test in most laboratories. For example, for some antibody projects where it is difficult to collect positive quality control samples, such as in this experimental example, Synaptophsin is used as a small cell lung cancer marker, and the human small cell lung cancer cell line NCI-H146 liquid cell line is used as a quality control sample. The cytoplasmic staining is clearly localized and the positive signal expression is moderate. It can be used as one of the quality control sample types for the serous cavity liquid-based cell detection kit.

[0210] Experimental Example 4

[0211] In this experimental example, the serous cavity fluid-based cell staining kit of Example 7 was used to perform immunocytochemical staining on the bloody pleural effusion sample. The specific process is as follows:

[0212] 1. Sample pretreatment:

[0213] After the bloody pleural effusion sample is treated with cell washing solution, it is stored in a cell preservation solution bottle and then used for slide preparation and immunocytochemical staining by a fully automatic immunocytochemical staining machine. Before using the cell washing solution, component A and component B are mixed at a ratio of 1:1 to obtain a buffer solution that can be used to wash cells. It is recommended that the cell washing solution obtained by mixing in the above ratio be used within one week.

[0214] The specific processing steps are as follows:

[0215] a) Mix the bloody pleural effusion sample thoroughly, take 50 mL and place it in a disposable conical bottom centrifuge tube, and centrifuge at 2500 r / min for 5 min.

[0216] b) After centrifugation, the sample is separated into layers, and the supernatant and sediment are greater than 2 mL and can be moved at the bottom of the tube:

[0217] Discard the supernatant, take all the sediment at the bottom of the tube, add 20mL of cell washing solution, oscillate in a micro-oscillator for 10 minutes, mix thoroughly, wait for the sediment at the bottom of the centrifuge tube to disperse and dissolve until the color turns light red, centrifuge at 2500r / min for 5 minutes, and discard the supernatant. Add phosphate buffered saline (PBS) to 50mL, centrifuge at 2500r / min for 5 minutes, discard the supernatant, and transfer to a cell preservation solution bottle.

[0218] 2. Liquid-based thin layer cell slide preparation

[0219] The pleural effusion samples stored in the cell preservation fluid bottle were directly placed in the designated position of the fully automatic immunocytochemical staining machine, and 500 μL of cell sample was added to each slide to obtain a liquid-based thin-layer cell white slide to be stained (hereinafter referred to as a cell slide).

[0220] 3. Fixed

[0221] Place the cell slide prepared in step 2 in 95% ethanol fixative and fix for 5 min at room temperature, then rinse with cleaning solution 3 times, 2 min each time.

[0222] 4. Antigen retrieval

[0223] Place the cell slide fixed in step 3 in Tris-EDTA antigen retrieval solution, heat to 80°C, and retrieval for 20 minutes.

[0224] 5. Add peroxidase blocking agent

[0225] Add 150 μL of endogenous peroxidase blocking agent and incubate at room temperature for 5 min. Rinse with cleaning solution 3 times, 2 min each time.

[0226] 6. Primary Antibody Incubation

[0227] Add 150 μL of primary antibody reagent and incubate at room temperature for 30 min to bind to the antigen protein in the cell sheet to form an antigen-antibody complex. Rinse with cleaning solution 3 times, 2 min each time.

[0228] Note: the primary antibody reagent is: mouse anti-human cytokeratin 7 monoclonal antibody (species: mouse, clone number C1C10, catalog number: TCM0120).

[0229] 7. Post-primary antibody incubation

[0230] Add 150 μL of primary antibody reagent and incubate at room temperature for 8 minutes to allow the primary antibody to bind to the antigen-antibody complex. Rinse with cleaning solution 3 times, 2 minutes each time.

[0231] 8. Polymer incubation

[0232] Add 150 μL of horseradish peroxidase (HRP) labeled polymer and incubate at room temperature for 12 min to allow the secondary antibody to specifically bind to the primary antibody reagent. Rinse with cleaning solution 3 times, 2 min each time.

[0233] Note: The horseradish peroxidase (HRP) labeled polymer is: polymer HRP-GAM (goat anti-mouse IgG), the secondary antibody is able to bind to the primary antibody reagent, and the secondary antibody is labeled with horseradish peroxidase (HRP), and its main function is to detect the presence of the antibody, amplify the signal of the primary antibody, and introduce it into the color development system.

[0234] 9. DAB color development

[0235] Add 150 μL of DAB color development solution and incubate at room temperature for 3 min until a brown-yellow precipitate appears. Rinse with deionized water 3 times, 2 min each time.

[0236] Note: The DAB color development working solution is DAB substrate: substrate buffer = 1:20 dilution ratio.

[0237] 10. Hematoxylin counterstaining

[0238] Counterstain at room temperature for 10 seconds to stain the cell nucleus, and rinse with purified water 5 times, 2 seconds each time.

[0239] 11. Dehydrate, make transparent, seal the slides, and read the slides under a microscope.

[0240] The results are as follows Figure 3 As shown in the figure, the sample is a case of lung adenocarcinoma metastasis derived from pleural effusion. Immunocytochemical staining shows cytoplasmic positive expression of CK7. The positive signal staining intensity is moderate, the background is clean, and there is no nonspecific staining.

[0241] The specific operation steps of Experimental Examples 5 to 9 are the same as those of Experimental Example 4. The processed samples and specific experimental conditions of each experimental example are shown in Table 4.

[0242] Table 4 Processed samples and specific experimental conditions for immunocytochemical staining of Experimental Examples 5 to 9

[0243]

[0244]

[0245] Specific test results such as Figures 4 to 8 The staining results of Experimental Examples 5 to 9 are summarized in Table 5.

[0246] Table 5 Summary of staining results of experimental examples 5 to 9

[0247]

[0248] Experimental Example 10

[0249] This experimental example uses ascites samples, and uses the serosal cavity liquid-based cell staining kit of Example 11 and the immunochromogenic reagent (Yu Zheng Xie Bei 20180257, item number: SD3102) after paraffin embedding and sectioning to perform staining, and compares the staining effects. The specific operation is as follows:

[0250] 1. Immunocytochemical staining of ascites samples using the serous cavity liquid-based cell staining kit

[0251] The ascites sample was subjected to immunocytochemical staining using the serous cavity liquid-based cell staining kit of Example 11, and the specific operation process was as described in Example 3. The primary antibody reagent was:

[0252] Mouse anti-human pax-8 monoclonal antibody (species: mouse, clone number C12A32, catalog number: TPM0040);

[0253] Mouse anti-human ovarian cancer antigen monoclonal antibody (species: mouse, clone number OC125, catalog number: TCM0010);

[0254] Rabbit anti-human Wilms tumor monoclonal antibody (species: rabbit, clone number MyR1-WT1, catalog number: TWR0010);

[0255] Mouse anti-human p53 protein monoclonal antibody (species: mouse, clone number C2H10, catalog number: TPM0030).

[0256] 2. Paraffin-embedded ascites samples and sections for immunohistochemical staining

[0257] First, the ascites samples were embedded in paraffin and sliced, baked, and manually stained with immunochromogenic reagent (Yu Zheng Xie Bei 20180257, item number: SD3102). The specific steps are as follows:

[0258] 1) Sample processing

[0259] After the ascites sample was treated with cell washing solution, it was stored in a cell preservation solution bottle, and the cell centrifugal sediment was embedded in paraffin to obtain a cell wax block. The cell wax block slices were 3 to 5 μm thick and adhered to the anti-detachment glass slide. After removing the water droplets in the tissue slices, they were placed in a 60°C (±5°C) incubator for 60 minutes, removed from the incubator, and cooled at room temperature.

[0260] 2) Dewaxing and hydration

[0261] The paraffin sections were placed in fresh xylene and immersed twice, 10 min each time;

[0262] After removing excess liquid, soak in anhydrous ethanol twice, 5 min each time;

[0263] After removing excess liquid, place in 95% ethanol and soak for 5 minutes;

[0264] After removing excess liquid, place in 85% ethanol and soak for 5 minutes;

[0265] After removing excess liquid, place in 70% ethanol and soak for 5 minutes;

[0266] Rinse with purified water for 5 minutes;

[0267] 3) Antigen retrieval

[0268] Add repair liquid to the pressure cooker and heat to boiling, then repair for 2.5 to 3 minutes. Cool naturally, soak in purified water twice, 5 minutes each time; soak in cleaning solution once, 5 minutes each time.

[0269] Note: During antigen retrieval, the amount of retrieval solution must ensure that the sections can always be immersed in the liquid.

[0270] 4) Add endogenous peroxidase blocking agent

[0271] Remove the cleaning solution, add 100 μL of endogenous peroxidase blocking agent to the tissue area to be tested, and incubate at room temperature for 5 minutes. Then soak in the cleaning solution twice, 5 minutes each time.

[0272] 5) Add antibody reagent

[0273] Remove the cleaning solution, add 100 μL of antibody reagent or blank control reagent, incubate at room temperature for 60 min, and soak in the cleaning solution twice, 5 min each time.

[0274] Note: The primary antibody reagent is:

[0275] Mouse anti-human pax-8 monoclonal antibody (species: mouse, clone number C12A32, catalog number: CPM-0252);

[0276] Mouse anti-human ovarian cancer antigen monoclonal antibody (species: mouse, clone number OC125, catalog number: CCM-0151);

[0277] Rabbit anti-human Wilms tumor monoclonal antibody (species: rabbit, clone number MyR1-WT1, catalog number: CWR-0012);

[0278] Mouse anti-human p53 protein monoclonal antibody (species: mouse, clone number C2H10, catalog number: CPM-0142).

[0279] 6) Enzyme-labeled goat anti-mouse / rabbit polymer

[0280] Remove the cleaning solution, add 100 μL of enzyme-labeled goat anti-mouse / rabbit polymer, incubate for 30 min, and soak in the cleaning solution twice, 5 min each time.

[0281] 7) DAB color development

[0282] Remove the cleaning solution, add 100 μL of freshly prepared DAB color developing solution, incubate for 5 to 8 minutes, and observe the staining results under an optical microscope for generally no more than 10 minutes.

[0283] 8) Hematoxylin counterstaining

[0284] Add 100 μL of hematoxylin staining solution and incubate for 1 to 3 minutes, then rinse with purified water to return to blue.

[0285] Dehydrate, make transparent, seal the slides, and read the slides under a microscope.

[0286] The specific staining results of the two staining methods are as follows Fig. 9 The staining results are summarized in Table 6.

[0287] Table 6 Summary of staining results of two staining methods

[0288]

[0289] Depend on Fig. 9 It can be seen that the diagnosis of ascites samples from the same source, combined with the clinical diagnosis of smear morphology and ascites cell wax block immunohistochemistry results, is consistent with: transformational carcinoma, female reproductive system (ovarian) source. This case shows that the signal intensity of liquid-based preparation ICC staining is higher than that of paraffin section IHC staining, especially in some nuclear staining projects. This is due to the fact that liquid-based preparation cell samples are fresher and have not undergone tedious steps such as paraffin embedding, so the antigens are better preserved.

[0290] Experimental Example 11

[0291] In this experimental example, the commercially available H brand cell washing solution (Medical Device Registration Certificate Number: Guoxiebei 20140214) and the cell washing solution in Example 1 were used to treat the concentrated bloody pleural effusion samples, and then the samples were stored in H brand cell preservation solution (Medical Device Registration Certificate Number: Guoxiebei 20140367) and cell preservation solution (Article No. TC1001), respectively. After liquid-based slide preparation, Papanicolaou staining was performed to compare the treatment effects. The specific steps are as follows:

[0292] The specific operating steps for treating samples using the commercially available H brand cell washing solution are described in the instructions, while the specific operating steps for treating samples using the cell washing solution of Example 1 are described in Experimental Example 2, and the subsequent staining is also described in Experimental Example 2.

[0293] For specific test results, see Fig.10 , Fig.11 As shown in the figure, the comparison of purulent pleural effusion samples after cell washing solution treatment is as follows Fig.14 The staining results are summarized in Table 7.

[0294] Table 7 Summary of staining results

[0295]

[0296] Experimental Example 12

[0297] In this experimental example, the commercially available H brand cell washing solution (Medical Device Registration Certificate Number: Guoxiebei 20140214) and the cell washing solution in Example 1 were used to treat the concentrated bloody pleural effusion samples, and then the samples were stored in H brand cell preservation solution (Medical Device Registration Certificate Number: Guoxiebei 20140367) and cell preservation solution (Article No. TC1001), respectively. After liquid-based slide preparation, immune cell staining was performed to compare the treatment effects. The specific steps are as follows:

[0298] The specific operating steps for treating samples using the commercially available H brand cell washing solution are described in the instructions, while the specific operating steps for treating samples using the cell washing solution of Example 1 are described in Experimental Example 2, and the subsequent staining is described in Experimental Example 7.

[0299] For detailed staining results, see Fig.12 , Fig.13 As shown in the figure, the comparison of purulent pleural effusion samples after cell washing solution treatment is as follows Fig.14 The staining results are summarized in Table 8.

[0300] Table 8 Summary of staining results

[0301]

[0302] After the bloody pleural effusion samples were treated with the commercially available H brand cell washing solution, the TTF-1 staining intensity and positive rate were significantly lower than those treated with the cell washing solution provided by the present invention, and the nuclear staining project was most obvious. Combined with the literature, it is considered that the commercially available H brand cell washing solution may inhibit the immunoreactivity of the antigen after pre-treatment of the cells.

[0303] The above description is only an illustrative embodiment of the present invention and is not intended to limit the scope of the present invention. Any equivalent changes and modifications made by a person of ordinary skill in the art without departing from the concept and principle of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A cell washing solution, characterized in that: The cell washing solution consists of component A and component B: the component A is an ethanol solution with a volume fraction of 40-50%; the component B consists of the following ingredients: 10 vol% formalin, 0.8-0.9 wt% ion maintainer, 0.095-0.190 wt% buffer, and the remainder water; the pH of the component B is 7.20-7.40; when used, the components A and B are mixed in a volume ratio of 1:

1.

2. The cell cleaning solution according to claim 1, characterized in that: The buffer includes one or two of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate; the ion maintaining agent includes one, two, or three of sodium chloride, potassium chloride, and ammonium chloride.

3. A reagent combination for treating serous cavity liquid-based cells, characterized in that: The reagent combination includes the cell washing solution and cell preservation solution according to claim 1 or 2; the cell preservation solution is composed of the following components: 18-22 vol% ethanol, 4-8 vol% ethylene glycol, 6-12 vol% isopropanol, 1-2 mM / L chelating agent, 1.0-1.2 wt% osmotic pressure maintaining agent, 0.05 vol% surfactant, and the solvent is a buffer solution; the pH of the cell preservation solution is 6.80-7.

20.

4. The reagent combination for treating serous cavity liquid-based cells according to claim 3, characterized in that: The buffer is one of PBS buffer, Tris-HCl buffer, PIPES buffer, acetic acid buffer, sodium acetate buffer, phosphate buffer, citric acid buffer, imidazole buffer and triethanolamine hydrochloride buffer.

5. The reagent combination for treating serous cavity liquid-based cells according to claim 4, characterized in that: The chelating agent is selected from one or two of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, sodium citrate, heparin, hepatokinase, streptokinase, and sodium citrate.

6. The reagent combination for treating serous cavity liquid-based cells according to claim 4, characterized in that: The osmotic pressure maintaining agent is selected from one or two of sodium chloride, potassium chloride and ammonium chloride; the surfactant is selected from one or two of Tween-20, Triton X-100 and ethylphenyl polyethylene glycol.

7. A serous cavity liquid-based cell staining kit, characterized in that: The serous cavity liquid-based cell staining kit comprises the serous cavity liquid-based cell treatment reagent combination according to any one of claims 3 to 6.

8. The serous cavity liquid-based cell staining kit according to claim 7, characterized in that: The serous cavity liquid-based cell staining kit comprises a peroxidase blocking agent, a primary antibody post-reagent, a polymer, a DAB substrate, a DAB buffer, and hematoxylin.

9. A method for staining serous cavity liquid-based cells, characterized in that: The following steps are involved: (1) centrifuging the serous cavity effusion sample to be sliced, adding the cell washing solution in the serous cavity fluid-based cell treatment reagent combination according to any one of claims 3 to 6 to the precipitate, shaking and mixing, centrifuging, and retaining the precipitate in the cell preservation solution in the serous cavity fluid-based cell treatment reagent combination according to any one of claims 3 to 6; (2) The sample stored in the cell preservation solution in step (1) is subjected to liquid-based slide preparation and subsequent staining.

10. The method for staining serous cavity liquid-based cells according to claim 9, characterized in that: The staining includes Papanicolaou staining and / or immunocytochemical staining.

Images