A rapid immunohistochemical staining method for ascites cytology

Abstract

The present application relates to the technical field of rapid immunohistochemistry, and particularly relates to a rapid immunohistochemical staining method for pleural effusion cytology. The method comprises the following steps: S1, taking the pleural effusion for processing to obtain cell sediment, drying the water of the cell group, and embedding; S2, placing the cell group on a freezing mold, wrapping with OCT embedding agent, and placing in a quick freezer to quickly freeze into a block, and then slicing; S3, taking several slices from the prepared cell group freezing block of S2, and using flowing water and PBS to clean the OCT embedding agent on the slices, and then placing the slices in a preheated citric acid buffer for medium-low fire repair; S4, after the slice repair is completed, using an OptiView DAB IHC Detection Kit to perform manual immunohistochemical staining on the slice. The rapid immunohistochemical staining technology for pleural effusion cytology established by the present application can obtain consistent diagnostic results with conventional paraffin methods within 2 hours, and provides reliable support for intraoperative rapid cytological pathological diagnosis.

Description

Technical Field

[0001] This invention relates to the field of rapid immunohistochemistry technology, specifically to a rapid immunohistochemical staining method for pleural and peritoneal fluid cytology. Background Technology

[0002] Pleural and peritoneal effusions are common pathological specimens, with complex and diverse causes such as infection, inflammation, and malignant tumors. Determining whether the effusion is neoplastic and identifying the primary tumor site is a key objective of pathological diagnosis, directly impacting clinical treatment strategies. In the development of cytological diagnosis of pleural and peritoneal effusions, early liquid-based cytology served as a fundamental tool, providing diagnostic evidence through cell morphology assessment. However, cell morphology alone is often insufficient to accurately determine the origin of the tumor.

[0003] Therefore, the combined application of liquid-based smears and immunohistochemical detection was introduced, improving diagnostic accuracy. However, this technique has limitations. Due to the different cell distribution in each smear, after HE staining to locate the tumor, the original HE staining needs to be removed before immunohistochemical staining, which is not only time-consuming but also prone to antigen loss. With technological advancements, pathology departments now widely use cell paraffin block preparation technology to enrich exfoliated cells from pleural and peritoneal fluid into cell paraffin blocks, enabling continuous sectioning and simultaneous detection of multiple biomarkers, thus improving diagnostic accuracy.

[0004] However, this method requires a 3-5 day preparation and diagnostic cycle, increasing the patient's time cost. Furthermore, clinicians sometimes need to submit cell samples intraoperatively to assist in surgical planning; traditional methods often cannot provide reliable identification of tumor cell type and origin, potentially failing to meet the need for rapid intraoperative diagnosis. Therefore, developing a staining technique that combines diagnostic accuracy and timeliness has significant clinical value.

[0005] In recent years, intraoperative rapid immunohistochemistry has shown significant advantages in histopathological diagnosis, but its application in cytology still lacks systematic research. This study aims to establish a frozen rapid immunohistochemistry technique system for fresh pleural and peritoneal effusion cytological specimens, and evaluate its feasibility as a rapid auxiliary diagnostic method by comparing it with the results of conventional paraffin-embedded multi-marker detection. Summary of the Invention

[0006] To address the shortcomings of existing rapid immunohistochemical techniques for cytology, this invention aims to provide a rapid, accurate, and reliable method for intraoperative rapid immunohistochemical staining of pleural and peritoneal effusion cytological specimens. Its core lies in unique improvements to the four steps of cytological immunohistochemistry: fixation, aggregation, antigen retrieval, and staining. Cell preservation solution is used to fix cells, maintaining their physiological state as they were in vitro. A combination of plasma and plasma thrombin is used to aggregate cell clusters. Microwave sectioning in citrate buffer at medium-low heat ensures sufficient exposure of the antigens to be detected in the sections. Finally, the OptiView DAB IHC Detection Kit is used under optimized conditions to achieve immunohistochemical staining of cellular antigens in pleural and peritoneal effusion cytological specimens.

[0007] To achieve the above objectives, the present invention provides a rapid immunohistochemical staining method for pleural and peritoneal fluid cytology, comprising the following steps: S1. Collect pleural and peritoneal fluid and process it to obtain cell sediment. Add a mixture of plasma and plasma thrombin to the cell sediment and stir until the cell sediment clumps together. Absorb the water from the cell clumps and wait for embedding. S2. Place the cell clusters treated in S1 on a freezing mold, wrap them with OCT embedding medium, and quickly freeze them into blocks in a freezer for slicing. S3. Take the frozen cell mass prepared in S2, cut several sections thinly, and fix them in ethanol. During this fixation period, preheat the citrate buffer. After fixation and preheating are completed, wash off the OCT embedding agent on the sections with running water and PBS one after the other. Then place the sections in the preheated citrate buffer for medium-low heat repair. S4. After the slides are repaired, the slides are manually stained with immunohistochemistry using the OptiView DAB IHC Detection Kit.

[0008] Preferably, S1 is as follows: S1.1. Take pleural and peritoneal fluid and centrifuge it in a centrifuge tube to enrich cells. Discard the supernatant and add cell preservation solution. Shake well to fix the cell sediment. Centrifuge again to discard the supernatant. Add physiological saline and stir to wash away the residual cell preservation solution. Shake well and transfer the liquid to an Eppendorf tube. Centrifuge to remove the supernatant. S1.2 Add the mixture of plasma and plasma thrombin to the cell sediment, stir to coagulate into clumps, wrap the coagulated cell clumps with paper towels to absorb the moisture, and then freeze-embed them.

[0009] Preferably, in S1.2, the volume ratio of plasma to plasma thrombin is 1:1.

[0010] Preferably, in S2, the temperature of the quick-freezing apparatus is -50℃.

[0011] Preferably, in S2, the combined mass of plasma and thrombin is in a 1:1 ratio to the mass of the cell clump precipitate.

[0012] Preferably, in S3, the prepared cell cluster frozen block is continuously sliced ​​into several thin slices with a thickness of 3.5 μm.

[0013] Preferably, in S4, the specific steps for manual immunohistochemical staining of the slides using the OptiView DAB IHC Detection Kit are as follows: S4.1. Stain the sections with OptiView Peroxidase Inhibitor reagent for 2-5 minutes, then wash several times with PBS; S4.2 Then, select the primary antibody to be used to stain the slides for 10 minutes, and then wash them several times with PBS; S4.3. Stain the PBS-washed sections with OptiView HQ Universal Linker reagent for 5 min, and then wash with PBS several more times. S4.4. Stain the sections treated in S4.3 with OptiView HRP Multimer reagent for 5 min, and wash several times with PBS; S4.5. Mix OptiView H2O2 and OptiView DAB reagent, and stain the sections treated in S4.4 for 1 min, then wash with water; S4.6. Stain the slices treated in S4.5 with hematoxylin and then rinse with water for a few seconds. S4.7 After staining, the sections are dehydrated and mounted according to standard procedures.

[0014] Preferably, in S4.5, the volume ratio of OptiView H2O2 to OptiView DAB reagent is 1:1.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) Cell preservation solution was used to fix the pleural and peritoneal fluid cell specimens, so that the cells could maintain their physiological state as far as possible after being removed from the body. At the same time, the cell preservation solution also lysed the red blood cells in the specimens, eliminating the interference of red blood cells on subsequent immunohistochemical staining.

[0016] (2) A combination of plasma and plasma thrombin was used to aggregate cell sediment. While aggregating cells, no excessive foreign antigens were added to interfere. Furthermore, because the cells were aggregated, continuous frozen sections could be better prepared, allowing for localization and comparison of different antibodies on multiple sections.

[0017] (3) Before performing rapid immunohistochemical staining, the frozen sections of cell clusters were repaired in a low-heat microwave oven with citrate buffer. This is a step that is lacking in the existing technology. Compared with rapid immunohistochemical staining that lacks this step, the background is cleaner and the immunohistochemical staining is more accurate.

[0018] (4) Through extensive experiments, the staining conditions for manual rapid cytological immunohistochemical staining using the OptiView DAB IHC Detection Kit were determined. While maintaining staining quality, the staining time was shortened, significantly reducing time costs for patients and clinicians (from 3-5 days to 1-2 hours). Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0020] In the attached diagram: Figure 1 Rapid immunohistochemical staining results of cells in the antigen-retrieval group and the non-antigen-retrieval group of pleural effusion specimens of lung adenocarcinoma (high magnification): 1a. Ber-EP4 (trypsin retrieval), 1b. MOC-31 (trypsin retrieval), 1c. TTF-1 (thermal retrieval), 1A. Ber-EP4 (non-retrieval), 1B. MOC-31 (non-retrieval), 1C. TTF-1 (non-retrieval), 1d. NapsinA (thermal retrieval), 1e. CK5 / 6 (thermal retrieval), 1f. D2-40 (thermal retrieval), 1D. NapsinA (non-retrieval), 1E. CK5 / 6 (non-retrieval), 1F. D2-40 (non-retrieval).

[0021] Figure 2 Rapid immunohistochemical staining results of cells in the antigen-repaired and non-antigen-repaired groups of serous adenocarcinoma ascites specimens (high magnification): 2a. Ber-EP4 (trypsin-repaired), 2b. MOC-31 (trypsin-repaired), 2c. PAX-8 (thermal-repaired), 2A. Ber-EP4 (non-repaired), 2B. MOC-31 (non-repaired), 2C. PAX-8 (non-repaired), 2d. P53 (thermal-repaired), 2e. CK-7 (thermal-repaired), 2f. CR (thermal-repaired), 2D. P53 (non-repaired), 2E. CK-7 (non-repaired), 2F. CR (non-repaired).

[0022] Figure 3Results of rapid immunohistochemistry and paraffin immunohistochemistry staining of pleural effusion specimens from lung adenocarcinoma (high magnification), as well as HE staining and liquid-based HE staining results using two methods (high magnification): 3a. Rapid immunohistochemistry HE, 3b. Paraffin immunohistochemistry HE, 3c. Liquid-based HE, 3d. Ber-EP4 (cell membrane / cytoplasm positive), 3e. MOC-31 (cell membrane / cytoplasm positive), 3f. TTF-1 (nuclear positive), 3D. Ber-EP4 (cell membrane / cytoplasm positive), 3E. MOC-31 (cell membrane / cytoplasm positive), 3F. TTF-1 (nuclear positive), 3g. Napsin A (cytoplasm positive), 3h. CK5 / 6 (cytoplasm positive), 3i. D2-40 (cell membrane positive), 3G. Napsin A (cytoplasm positive), 3H. CK5 / 6 (cytoplasm positive), 3I. D2-40 (cell membrane positive).

[0023] Figure 4 Results of rapid immunohistochemistry and paraffin immunohistochemistry staining of cytological specimens from serous adenocarcinoma ascites (high magnification), as well as HE staining and liquid-based HE staining results using both methods (high magnification): 4a. Rapid immunohistochemistry HE, 4b. Paraffin immunohistochemistry HE, 4c. Liquid-based HE, 4d. Ber-EP4 (cell membrane / plasma positive), 4e. MOC-31 (cell membrane / plasma positive), 4f. PAX-8 (nuclear positive), 4g. P53 (nuclear positive), 4D. Ber-EP 4 (Positive for cell membrane / cytoplasm), 4E.MOC-31 (Positive for cell membrane / cytoplasm), 4F.PAX-8 (Positive for cell nucleus), 4G.P53 (Positive for cell nucleus), 4h.CK7 (Positive for cell cytoplasm), 4i.CR (Positive for cell nucleus / cytoplasm), 4j.CK5 / 6 (Positive for cell cytoplasm), 4k.WT-1 (Positive for cell nucleus), 4H.CK7 (Positive for cell cytoplasm), 4I.CR (Positive for cell nucleus / cytoplasm), 4J.CK5 / 6 (Positive for cell cytoplasm), 4K.WT-1 (Positive for cell nucleus). Detailed Implementation

[0024] The following is in conjunction with the appendix Figures 1-4 The technical solutions of the present invention will be further described in detail below with specific embodiments. The examples given are only for explaining the present invention, but the present invention is not limited to the following technical solutions. In the following embodiments, the experimental materials used, unless otherwise specified, can be obtained from commercial channels. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Five pleural effusion and five ascites specimens of tumor-related pleural effusions were collected from the Department of Pathology, Peking University Shenzhen Hospital, between April and August 2025. Simultaneously, pleural and ascites specimens of confirmed adenocarcinoma and reactive mesothelial hyperplasia were collected as positive and negative controls, respectively.

[0026] Instruments and reagents (1) Instruments: Leica CM1950 cryostat, LBP-2601 liquid-based cytometer, Benchmark Ultra immunohistochemistry instrument, Anbiping JW-1024 low-speed centrifuge, Galanz P70F23P-G5 microwave oven, (2) Reagents: Citrate buffer (pH 6.0) was purchased from Beijing Zhongshan Jinqiao Company; primary antibody Ber-EP4, MOC-31, WT-1, TTF-1, D2-40, Napsin A, CK5 / 6, CDX2, and P53 were purchased from Fuzhou Maixin Company; P40 was purchased from Shenzhen Dameng Company; Pax-8, phosphate buffer, and cell preservation solution were purchased from Guangzhou Anbiping Company; OptiView DAB IHC Detection Kit and Protease 3 protease for cleaning sections were purchased from Shanghai Roche Company.

[0027] Classification: Each specimen was divided into three parts: one part was used for liquid-based HE staining, one part was made into a cell block for paraffin immunohistochemical staining, and one part was made into a cell frozen block for rapid cytological immunohistochemical staining. The results of paraffin immunohistochemical diagnosis were used as the "gold standard" to compare the consistency between the results of rapid cytological immunohistochemistry and paraffin immunohistochemistry. For pleural effusion samples, Ber-EP4, MOC-31, NapsinA, TTF-1, CK5 / 6, and WT-1 were selected for immunohistochemistry, and Ber-EP4, MOC-31, Pax-8, P53, CK-7, and CR were selected for immunohistochemistry.

[0028] Example 1: S1. Take 50ml of pleural and peritoneal fluid specimens and centrifuge at 2108r / min for 5min in a 50ml centrifuge tube to enrich cells. Discard the supernatant and add 10ml of cell preservation solution. Shake well to fix the cell sediment. Centrifuge again at 2108r / min for 5min and discard the supernatant. Add 1ml of physiological saline and stir to wash away the residual cell preservation solution. After shaking well, transfer the liquid to a 1.5ml Eppendorf tube and centrifuge at 800r / min for 30s. Discard the supernatant. Based on the principle that plasma thrombin can precipitate fibrinogen in plasma, add a 1:1 volume ratio of plasma and plasma thrombin mixture to the cell sediment and stir to form a clump. Wrap the clumps of cells with paper towels to absorb the moisture and then freeze-embed them.

[0029] S2. Place the processed cell clusters on a freezing mold, wrap them with OCT embedding agent, and quickly freeze them into blocks in a -50℃ freezer for later sectioning.

[0030] S3. Take the prepared frozen cell clusters and cut several thin sections (3.5 μm) continuously. Fix the sections in 95% ethanol. During fixation, preheat the sections in a microwave oven for 10 minutes using a water bath. After fixation and preheating, wash off the OCT embedding agent from the sections with running water and PBS, respectively. Then, place the sections in the preheated citrate buffer and microwave on low for 10 minutes for retrieval (for some antigens, such as Ber-EP4 and MOC-31 antigens, use trypsin digestion for 10 minutes for retrieval instead of heat retrieval; refer to the corresponding antibody reagent instructions for specific antigen retrieval options). After retrieval, place the heat-retrieved sections and the container in cold water for cooling, then wash the sections with PBS. For enzyme-retrieved sections, wash the sections directly with PBS.

[0031] S4. After section repair, perform manual immunohistochemical staining on the sections using the OptiView DAB IHC Detection Kit. The specific steps for manual immunohistochemical staining of the sections using the OptiView DAB IHC Detection Kit are as follows: S4.1. Stain the sections with OptiView Peroxidase Inhibitor reagent for 2-5 minutes, then wash several times with PBS; S4.2 Then, select the primary antibody to be used to stain the slides for 10 minutes, and then wash them several times with PBS; S4.3. Stain the PBS-washed sections with OptiView HQ Universal Linker reagent for 5 min, and then wash with PBS several more times. S4.4. Stain the sections treated in S4.3 with OptiView HRP Multimer reagent for 5 min, and wash several times with PBS; S4.5. Mix OptiView H2O2 and OptiView DAB reagent in a 1:1 ratio and stain the sections for 1 min, then wash with water; S4.6. Stain the slices treated in S4.5 with hematoxylin for 20-40 seconds, then rinse with water for a few seconds. S4.7 After staining, the sections are dehydrated and mounted according to standard procedures.

[0032] Comparative example: Cell paraffin immunohistochemistry: S1. Take 50ml of pleural and peritoneal fluid samples and centrifuge them in a 50ml centrifuge tube at 2108r / min for 5min to enrich cells. Discard the supernatant and add 10ml of cell preservation solution. Shake the cell sediment to fix it fully. Centrifuge again at 2108r / min for 5min and discard the supernatant. Add a 1:1 volume ratio of plasma and plasma thrombin mixture to the cell sediment and stir to form a clump. Wrap the clump of cells in a paper towel to absorb the moisture.

[0033] S2. The cell clusters were fixed using routine methods (soaked in 4% formalin for 4 hours), dehydrated in 85% ethanol for 1 hour, dehydrated in 95% ethanol for 2 hours, dehydrated in anhydrous ethanol for 2 hours, soaked in xylene for 2 hours, soaked in paraffin for 2 hours, and embedded in paraffin. S3. Take the prepared cell cluster wax block and cut several thin slices at 3.5μm. Bake in an 80℃ oven for 30 minutes. Then use a Benchmark Ultra immunohistochemistry instrument to detect the slices. Perform standard dehydration and mounting on the prepared slices.

[0034] result: 1. The effect of antigen retrieval on staining results Whether antigen retrieval is performed and the method of retrieval significantly affect the staining results. Slides without antigen retrieval show deeper background staining and some non-specific staining (e.g., Figure 1 1A-1F and Figure 2 (2A-2F), while slides that underwent antigen retrieval had a cleaner background and more specific staining (e.g., 2A-2F). Figure 1 1a-1f and Figure 2 2a-2f), especially heat repair, has the best effect (e.g. Figure 1 1c-1f and Figure 2 (2c-2f), but Ber-EP4 and MOC-31 target EpCAM, which is a fragile antigen. Heat retrieval can destroy the antigen, leading to false negatives. Therefore, both paraffin immunohistochemistry and frozen rapid immunohistochemistry for these antigens use trypsin digestion for retrieval. Trypsin digestion, compared to heat retrieval, results in slight background staining (e.g., ...). Figure 1 1a-1b and Figure 2 (2a-2b), so antigen retrieval is a crucial step in rapid cellular immunohistochemistry.

[0035] 2. Comparison of results between rapid immunohistochemical staining of cells and paraffin immunohistochemical staining of cells Table 1 shows the detection results of 5 pleural effusion specimens. Rapid cytological immunohistochemistry and paraffin immunohistochemistry showed that all detected markers exhibited completely consistent staining patterns: Ber-EP4, MOC-31, TTF-1, and Napsin A were all positive for tumor cells, while CK5 / 6 and D2-40 were both negative for tumor cells (with mesothelial cells as a positive control). The staining localization and staining intensity scores of the two methods were 100% consistent (Kappa=1, P＜0.01 ).

[0036] Table 1. Results of rapid immunohistochemical and paraffin immunohistochemical staining of pleural effusion specimens from five patients.

[0037] Taking a typical pleural effusion specimen of lung adenocarcinoma as an example, HE staining of three preparation methods—crystal block, paraffin block, and liquid-based cytology—all showed similar morphological characteristics. Figure 3 (3a-3c): Nested atypical tumor cells are visible against a background of mesothelial and inflammatory cells. The cell nucleus-cytoplasm ratio is increased, the nucleoli are prominent, and mitotic figures are visible. The morphology is consistent with adenocarcinoma.

[0038] Rapid immunohistochemical staining results showed that tumor cells were positive for Ber-EP4, MOC-31, TTF-1, and Napsin A, while mesothelial cells were negative. Figure 3 In the middle 3d-3g), mesothelial cells were positive for CK5 / 6 and D2-40, while tumor cells were negative. Figure 3 3h-3i). With cell paraffin immunohistochemistry ( Figure 3 The results of 3D-3I staining were consistent and all antibodies were accurately localized, supporting lung-derived adenocarcinoma.

[0039] Table 2 shows the detection results of 5 ascites specimens. Rapid cytological immunohistochemistry and paraffin immunohistochemistry showed that all detected markers exhibited completely consistent staining patterns: Ber-EP4, MOC-31, PAX-8, P53, CK7, and WT-1 were all positive for tumor cells, while CR and CK5 / 6 were both negative for tumor cells (with mesothelial cells as a positive control). The staining localization and staining intensity scores of the two methods were 100% consistent (Kappa=1, P＜0.01 ).

[0040] Table 2. Results of rapid immunohistochemical and paraffin immunohistochemical staining of ascites specimens from five patients.

[0041] Taking a typical serous adenocarcinoma ascites specimen as an example, HE staining of three preparation methods—cell frozen block, cell paraffin block, and liquid-based cytology—all showed similar morphological characteristics. Figure 44a-4c): Atypical cells are visible in nests or clusters, with moderate to abundant, eosinophilic cytoplasm, increased nuclear-cytoplasmic ratio, large, deeply stained nuclei with irregular karyotype, prominent nucleoli, and visible mitotic figures. The diagnosis is suspected tumor cells, requiring differentiation from mesothelioma, adenocarcinoma, and mesothelial cell hyperplasia. Rapid immunohistochemical results show that tumor cells are positive for Ber-EP4, MOC-31, PAX-8, P53, CK-7, and WT-1, while mesothelial cells are negative. Figure 4 (4d-4h and 4k), mesothelial cells were positive for CR and CK5 / 6, while tumor cells were negative. Figure 4 4i-4j). With cell paraffin immunohistochemistry ( Figure 4 The results of 4D-4K staining were consistent, and the antibody localization was accurate, suggesting serous adenocarcinoma.

[0042] The rapid immunohistochemistry and paraffin immunohistochemistry of the above 5 pleural effusion specimens and 5 ascites specimens showed completely consistent staining localization and intensity in all detected markers (Kappa=1, P＜0.01 This indicates that rapid cytological immunohistochemistry can be used as a rapid auxiliary testing method for cell paraffin immunohistochemistry during the examination of pleural and peritoneal fluid specimens.

[0043] 3. Comparison of the timeliness characteristics of the two methods Table 3. Timeliness comparison data of rapid immunohistochemistry and paraffin immunohistochemistry for pleural effusion specimens, ascites specimens, and all specimens.

[0044] Table 3 shows the detection times for two immunohistochemical methods in pleural effusion, ascites, and total pleural and peritoneal effusion specimens. The average detection time for the rapid cytological immunohistochemistry method was 1.36 ± 0.07 hours (CV = 3.3%), which was significantly shorter than that for the conventional paraffin immunohistochemistry method (95.6 ± 3.2 hours (CV = 3.3%). P＜0.001 The rapid immunohistochemistry method for cell biology shortens the traditional 3-5 day slide preparation cycle to within 2 hours, demonstrating the advantages of this technology.

[0045] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A rapid immunohistochemical staining method for pleural and peritoneal fluid cytology, characterized in that, Includes the following steps: S1. Collect pleural and peritoneal fluid and process it to obtain cell sediment. Add a mixture of plasma and plasma thrombin to the cell sediment and stir until the cell sediment clumps together. Absorb the water from the cell clumps and wait for embedding. S2. Place the cell clusters treated in S1 on a freezing mold, wrap them with OCT embedding medium, and quickly freeze them into blocks in a freezer for slicing. S3. Take the frozen cell mass prepared in S2, cut several sections thinly, and fix them in ethanol. During this fixation period, preheat the citrate buffer. After fixation and preheating are completed, wash off the OCT embedding agent on the sections with running water and PBS one after the other. Then place the sections in the preheated citrate buffer for medium-low heat repair. S4. After the slides are repaired, the slides are manually stained with immunohistochemistry using the OptiView DAB IHC Detection Kit.

2. The rapid immunohistochemical staining method for pleural and peritoneal fluid cytology according to claim 1, characterized in that, S1 specifically refers to: S1.

1. Take pleural and peritoneal fluid and centrifuge it in a centrifuge tube to enrich cells. Discard the supernatant and add cell preservation solution. Shake well to fix the cell sediment. Centrifuge again to discard the supernatant. Add physiological saline and stir to wash away the residual cell preservation solution. Shake well and transfer the liquid to an Eppendorf tube. Centrifuge to remove the supernatant. S1.2 Add the mixture of plasma and plasma thrombin to the cell sediment, stir to coagulate into clumps, wrap the coagulated cell clumps with paper towels to absorb the moisture, and then freeze-embed them.

3. The rapid immunohistochemical staining method for pleural and peritoneal fluid cytology according to claim 2, characterized in that, In S1.2, the volume ratio of plasma to plasma thrombin is 1:

1.

4. The rapid immunohistochemical staining method for pleural and peritoneal fluid cytology according to claim 3, characterized in that, In S2, the temperature of the quick-freezing apparatus is -50℃.

5. The rapid immunohistochemical staining method for pleural and peritoneal fluid cytology according to claim 4, characterized in that, In S2, the combined mass of plasma and thrombin is 1:1 with the mass of the cell clump precipitate.

6. The rapid immunohistochemical staining method for pleural and peritoneal fluid cytology according to claim 4, characterized in that, In S3, several slices were made from the prepared frozen cell mass at a thickness of 3.5 μm.

7. The rapid immunohistochemical staining method for pleural and peritoneal fluid cytology according to claim 5, characterized in that, In S4, the specific steps for manual immunohistochemical staining of the slides using the OptiView DAB IHC Detection Kit are as follows: S4.

1. Stain the sections with OptiView Peroxidase Inhibitor reagent for 2-5 minutes, then wash several times with PBS; S4.2 Then, select the primary antibody to be used to stain the slides for 10 minutes, and then wash them several times with PBS; S4.

3. Stain the PBS-washed sections with OptiView HQ Universal Linker reagent for 5 min, and then wash with PBS several more times. S4.

4. Stain the sections treated in S4.3 with OptiView HRP Multimer reagent for 5 min, and wash several times with PBS; S4.

5. Mix OptiView H2O2 and OptiView DAB reagent, and stain the sections treated in S4.4 for 1 min, then wash with water; S4.

6. Stain the slices treated in S4.5 with hematoxylin and then rinse with water for a few seconds. S4.7 After staining, the sections are dehydrated and mounted according to standard procedures.

8. The rapid immunohistochemical staining method for pleural and peritoneal fluid cytology according to claim 7, characterized in that, In S4.5, the volume ratio of OptiView H2O2 to OptiView DAB reagent is 1:1.

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