Sarcoma evaluation sections, methods of making and use thereof

Abstract

The application discloses a kind of for carcinosarcoma evaluation section and its preparation method and application, belong to pathological technical field.The preparation method includes the following steps: reticular fiber staining: silver staining method is carried out to the reticular fiber in section tissue and is dyed;Immunohistochemical staining: AE1 / AE3 protein in section tissue is marked and dyed by immunohistochemical staining method.The section obtained by the preparation method can present two kinds of staining on a tissue section, improve the expression effect of cancer, sarcoma, carcinosarcoma, reduce the identification difficulty, and be beneficial to tumor accurate typing.

Description

Technical Field

[0001] This invention relates to the field of pathology technology, and in particular to a slide for evaluating carcinosarcoma, its preparation method, and its application. Background Technology

[0002] Carcinosarcoma is a mixed malignant tumor composed of different types of abnormal cells, including cancer cells originating from epithelial tissue and sarcomatous cells originating from mesenchymal tissue. This tumor contains both malignant epithelial and malignant mesenchymal components, hence the name carcinosarcoma. Carcinosarcoma can occur in any part of the body, such as bone, muscle, nerve tissue, and blood vessels, and its occurrence may be related to various factors such as genetics and environment. Due to its dual characteristics of carcinoma and sarcoma, carcinosarcoma is highly malignant, difficult to treat, and generally has a poor prognosis.

[0003] Currently, pathologists typically observe the morphology, structure, arrangement, and mitotic figures of tumor cells under a microscope, combining this with techniques such as immunohistochemical staining, special staining, and gene testing to diagnose carcinosarcoma and differentiate the type, nature, and composition of the tumor. However, due to individual differences, tumor diversity, and inconsistencies in differentiation levels, pathologists often find it difficult to differentiate tumors based solely on morphology when making diagnostic classifications under a microscope, and there is also a degree of subjectivity between different pathologists. Commonly used auxiliary methods suffer from high technical costs and the difficulty of identifying and interpreting single staining patterns.

[0004] Therefore, accurately assessing the proportion of carcinoma and sarcoma in tumor tissue and more accurately determining tumor type is of great guiding significance for clinical decision-making, prognostic assessment and disease monitoring. Summary of the Invention

[0005] In response to the lack of a method for accurately assessing carcinosarcoma by staining tissue sections, this invention provides a method for preparing carcinosarcoma assessment sections. By presenting two staining methods on a single tissue section, the expression of carcinoma, sarcoma, and carcinosarcoma is improved, the difficulty of identification is reduced, and the process is beneficial for accurate tumor subtyping.

[0006] This invention discloses a method for preparing slides for carcinosarcoma evaluation, comprising the following steps:

[0007] Reticular fiber staining: The reticular fibers in the tissue section were stained using the silver staining method;

[0008] Immunohistochemical staining: Immunohistochemical staining is used to label and stain AE1 / AE3 proteins in tissue sections.

[0009] In the routine evaluation of carcinosarcoma, multiple immunohistochemical and special staining procedures are usually added for diagnostic differentiation. However, this requires repeated comparison of multiple slides to obtain an accurate conclusion, and it is particularly inefficient for paraffin blocks containing few lesion components. The inventors of this invention proposed to clearly display both components on the same slide, which would facilitate interpretation by the diagnostic physician and save on paraffin blocks.

[0010] Based on this, the inventors comprehensively considered the need to select a marker protein with broad coverage that can reflect different malignant epithelial components as a marker for identifying malignant epithelial components. This marker protein can accurately identify malignant mesenchymal components through reticular fiber staining. Furthermore, the inventors fully considered that two different staining methods should not only not interfere with each other in the same tissue section, but also have a clear contrast to facilitate interpretation. After comparing and screening various staining methods, the inventors finally chose silver staining for reticular fiber staining, combined with immunohistochemical staining of AE1 / AE3 proteins. This resulted in the ability to present two staining effects on the same tissue section, with the advantages of broad coverage for different cancer types and simple interpretation.

[0011] In some of these protocols, reticular fiber staining is performed first, followed by immunohistochemical staining.

[0012] In some of these schemes, the method for staining the reticular fibers is as follows: the tissue section is stained with silver ammonia staining solution.

[0013] In some of these protocols, the immunohistochemical staining procedure is as follows: take a tissue section, add AE1 / AE3 antibody, incubate with primary antibody, then add secondary antibody that binds to the above AE1 / AE3 antibody, incubate with secondary antibody, add a chromogenic agent targeting the above secondary antibody, and develop color.

[0014] In some of these schemes, the reticular fiber staining and immunohistochemical staining steps are followed by a hematoxylin staining step.

[0015] In some of these schemes, the procedure for hematoxylin staining is as follows: hematoxylin staining solution is added to the tissue sections for counterstaining.

[0016] In some of the above-mentioned schemes, in the dyeing step of the mesh fiber, the temperature of the silver ammonia dyeing solution is 20-40℃, preferably 37℃.

[0017] In some of the above-mentioned schemes, in the dyeing step of the mesh fiber, the time for the silver ammonia dye solution to drip is 5-20 min, preferably 5-15 min, and more preferably 15 min.

[0018] In some of the above-mentioned schemes, the chromogenic agent in the immunohistochemical staining step is a RED chromogenic agent.

[0019] In some of the above schemes, the operation method of staining the reticular fibers is as follows: take the dewaxed tissue sections, and oxidize them in sequence with potassium permanganate solution, bleach them with oxalic acid solution, mordant them with ammonium ferric sulfate solution, drop them with silver ammonia stain, reduce them with formaldehyde, adjust the color with gold chloride solution, and remove excess silver ions with sodium thiosulfate solution.

[0020] In some of the above methods, the oxidation treatment time of potassium permanganate solution is 3±1 min; the bleaching treatment time of oxalic acid solution is 1-2 min; the mordant treatment time of ammonium ferric sulfate solution is 3±1 min; the formaldehyde reduction treatment time is 30±10 s; the color-tuning treatment time of gold chloride solution is 1±0.5 min; and the treatment time of sodium thiosulfate solution is 2±1 min.

[0021] In some of the above-mentioned schemes, the immunohistochemical staining procedure is as follows: take a tissue section, add AE1 / AE3 antibody, incubate with primary antibody at 20-30℃ for 20±5 min, rinse with washing solution, add secondary antibody bound to the above AE1 / AE3 antibody, incubate with secondary antibody at 20-30℃ for 15±5 min, rinse with deionized water, add chromogenic agent against the above secondary antibody, develop color for 5±2 min, and rinse with deionized water.

[0022] In some of the above-mentioned schemes, a repair and blocking step is included before immunohistochemical staining. The repair and blocking operation method is as follows: the tissue sections are repaired and blocked sequentially; preferably, the antigen repair solution is used to repair at 100±20℃ for 15±5 min, and then treated with a peroxidase blocking agent for 15±5 min.

[0023] In some of the above-mentioned schemes, the procedure for hematoxylin staining is as follows: hematoxylin staining solution is added to the tissue section for 1 ± 0.5 min, followed by counterstaining.

[0024] On the other hand, the present invention also discloses tissue staining sections prepared by the above-described method for preparing sections for carcinosarcoma evaluation.

[0025] In some of these protocols, the stained tissue sections are used in the evaluation of carcinosarcomas for non-diagnostic treatment.

[0026] In some of these schemes, the carcinosarcoma assessment includes specific subtyping assessments of carcinoma, sarcoma, and carcinosarcoma, as well as assessments of the proportions of each component within the carcinosarcoma.

[0027] On the other hand, the present invention also discloses a kit for the evaluation of carcinosarcoma, comprising: a reticular fiber staining reagent and an immunohistochemical staining reagent, wherein,

[0028] The dyeing reagent for the reticulated fibers includes: silver ammonia dye solution;

[0029] The immunohistochemical staining reagent includes: AE1 / AE3 antibody, secondary antibody against AE1 / AE3 antibody, and chromogenic agent; the chromogenic agent is either RED chromogenic agent or DAB chromogenic agent.

[0030] In some of these solutions, the reticular fiber dyeing reagent further includes: potassium permanganate solution, oxalic acid solution, ferric ammonium sulfate solution, formaldehyde solution, gold chloride solution, and sodium thiosulfate solution.

[0031] In some of these schemes, the secondary antibody is an enzyme-labeled goat anti-mouse / rabbit IgG polymer.

[0032] In some of these schemes, the colorimetric agent is a RED colorimetric agent.

[0033] definition:

[0034] The "RED" mentioned above refers to BOND Polymer Refine Red Detection, a chromogenic agent widely used in immunohistochemical staining. It reacts with alkaline phosphatase (AP) to produce a red precipitate.

[0035] The aforementioned "DAB" refers to BOND Polymer Refine Detection, specifically 3,3'-diaminobenzidine, which is a chromogenic substrate for horseradish peroxidase. Its color development principle involves the loss of electrons under the action of hydrogen peroxide, resulting in color changes and accumulation, forming a brown insoluble product.

[0036] The term "cancer" as used above refers to malignant tumors originating from epithelial tissue.

[0037] The term "sarcoma" refers to a malignant tumor originating from mesenchymal tissue.

[0038] The term "carcinosarcoma" refers to a tumor that contains both carcinoma and sarcoma components.

[0039] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0040] The reagents and raw materials used in this invention are all commercially available.

[0041] The positive and progressive effects of this invention are as follows:

[0042] This invention discloses a method for preparing tissue sections for carcinosarcoma assessment. This method presents two staining methods on a single tissue section, improving the expression of carcinoma, sarcoma, and carcinosarcoma, reducing the difficulty of identification, facilitating accurate tumor subtyping, and minimizing results variability caused by differences in diagnostic personnel. Furthermore, it reduces tissue loss by avoiding multiple section preparations. Attached Figure Description

[0043] Figure 1 This is a typical photograph of AE1 / AE3 staining in Example 1.

[0044] Figure 2 This is a typical photograph of CK5 / 6 staining in Example 1.

[0045] Figure 3 This is a typical photograph of CK19 staining in Example 1.

[0046] Figure 4 This is a typical photograph of the dyeing of the reticular fibers in Example 1.

[0047] Figure 5 This is a typical photograph of VIM staining in Example 1.

[0048] Figure 6 This is a typical photograph of AE1 / AE3 staining and RED color development in Example 1.

[0049] Figure 7 These are typical photographs showing the color development of each group of mesh fibers in Example 1.

[0050] Figure 8 Typical photographs of the first group selected from the staining sequence in Example 1.

[0051] Figure 9 These are typical photographs of the second group selected from the staining sequence in Example 1. Detailed Implementation

[0052] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0053] AE1 / AE3 antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Product code: ZM-0069 ZM-0069UM.

[0054] CK5 / 6 Antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Generic Name: Cytokeratin 5 / 6 Antibody Reagent (Immunohistochemistry); Product Code: ZM-0313 ZM-0313UM

[0055] CK19 protein antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Product code: ZM-0074 ZA-0074UM

[0056] RED colorimetric reagent: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Product code: DS9390.

[0057] DAB colorimetric reagent: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Product code: DS9800.

[0058] Example 1

[0059] This embodiment develops a slice sample for carcinosarcoma assessment and examines and optimizes its preparation method.

[0060] I. Design Concept

[0061] For the interpretation of malignant epithelial components, immunohistochemical staining is considered, such as labeling components like AE1 / AE3, CK5 / 6, and CK19 proteins. For the interpretation of malignant mesenchymal components, reticular fiber staining or immunohistochemical VIM staining are considered.

[0062] II. Selection of Immunohistochemical Antibodies for Interpreting Malignant Epithelial Components

[0063] 1. Method

[0064] Ten cancer samples of different types (including squamous cell carcinoma, tubular adenocarcinoma, papillary thyroid carcinoma, urothelial carcinoma, and breast cancer samples from different locations) were selected from the historical samples of our unit. The cancer components were labeled with AE1 / AE3, CK5 / 6, and CK19 antibodies respectively according to the following method, and their expression effects were observed in order to screen for antibodies that have advantages in labeling unknown cancer components.

[0065] 1) Dewaxing: Dewax the tissue sections for 20 minutes.

[0066] 2) Repair and blockade: After repairing with conventional antigen repair solution at 100℃ for 15 min, rinse with cleaning solution, then treat with peroxidase blocker for 15 min, and then rinse with cleaning solution.

[0067] 3) Immunohistochemical staining: According to the product instructions, add 100u LAE1 / AE3 antibody, CK5 / 6 antibody or CK19 antibody respectively, incubate with primary antibody at room temperature (around 25℃) for 20min, rinse with washing buffer, incubate with secondary antibody at room temperature (around 25℃) for 15min, rinse with deionized water, add DAB chromogenic reagent for 5min, and rinse with deionized water.

[0068] 4) Hematoxylin staining: Add hematoxylin staining solution for counterstaining for 1 min, then rinse with deionized water.

[0069] 5) Dehydration and sealing: Following the conventional dehydration method, the tablets are treated sequentially with two tanks of 95% ethanol for 30 seconds each, two tanks of anhydrous ethanol for 30 seconds each, and three tanks of TO for 15 seconds each. Finally, the tablets are sealed with resin.

[0070] 2. Results

[0071] Typical slide images of different antibody labels, such as Figure 1-3 As shown, Figure 1 These are typical images of AE1 / AE3 staining, where A is a positive image of squamous cell carcinoma and B is a positive image of adenocarcinoma. Figure 2 These are typical images of CK5 / 6 staining. Image A shows a positive result for squamous cell carcinoma, while image B shows a negative result for adenocarcinoma, suggesting that CK5 / 6 staining cannot label adenocarcinoma. Figure 3 These are typical images of CK19 staining. Image A shows a positive result for adenocarcinoma, while image B shows a negative result for keratinizing squamous cell carcinoma, indicating that CK19 staining cannot label keratinizing squamous cell carcinoma. The expression results for each sample are shown in the table below.

[0072] Table 1. Interpretation of different antibody expression results

[0073]

[0074] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0075] The 10 samples from different cancer types mentioned above represent common clinical cancers. The results showed that AE1 / AE3 expression was 100%, CK5 / 6 expression was 40%, and CK19 expression was 60%. AE1 / AE3 can cover multiple keratin subtypes and can be used for epithelial tumor components from various sources. Considering the low differentiation degree and diverse possible origins of carcinosarcoma, AE1 / AE3 is a better choice for identifying malignant epithelial components due to its broad coverage and ability to effectively express them even when the cancer type is unknown.

[0076] III. Selection of Methods for Interpreting Interfoliar Components

[0077] Ten tissue samples of different types (including benign and malignant tissue samples from different sources) from our unit's historical samples were selected. The malignant mesenchymal components were labeled using reticular fiber staining and immunohistochemical VIM staining, respectively, and their expression effects were observed.

[0078] 1. Method

[0079] 1) Reticular fiber staining: Follow the instructions for the reticular fiber staining kit (Zhuhai Beso Biotechnology Co., Ltd., model B4BA4165) as follows:

[0080] Dewaxing: Place the tissue sections in three xylene baths for 10 min each, then place them in anhydrous ethanol for 2 min, anhydrous ethanol for 2 min, 95% ethanol for 2 min, and 80% ethanol for 2 min in sequence, and finally rinse with running water for 3-5 min.

[0081] Oxidation: The tissue sections were oxidized with potassium permanganate solution for 3 min, followed by rinsing with running water for 1 min.

[0082] Bleaching: Bleach the tissue sections with oxalic acid solution for 1-2 minutes, then rinse with running water for 1 minute.

[0083] Mordant: The tissue sections were mordanted with ferric ammonium sulfate solution for 3 min, followed by rinsing with deionized water for 10 s.

[0084] Silver ammonia staining: Stain tissue sections with silver ammonia staining solution (Ag(NH3)2OH) for 15 min (in a 37° oven), then rinse with deionized water for 10 s.

[0085] Reduction: Reduce the tissue sections with formaldehyde solution for 30 seconds, then rinse with running water for 1 minute.

[0086] Color adjustment: The tissue sections were colored with gold chloride solution for 1 min, followed by rinsing with running water for 1 min.

[0087] Silver removal: The tissue sections were treated with sodium thiosulfate solution for 2 minutes, followed by rinsing with running water for 1 minute to remove excess silver ions.

[0088] Microscopic quality control.

[0089] 2) Immunohistochemical VIM staining:

[0090] 1) Dewaxing: Dewax the tissue sections for 20 minutes.

[0091] 2) Repair and blockade: Repair at 100℃ for 15 minutes, rinse with cleaning solution, then treat with peroxidase blocker for 15 minutes, and then rinse with cleaning solution.

[0092] 3) Immunohistochemical staining: According to the instructions, add 100uL of VIM antibody, incubate with primary antibody at room temperature (around 25℃) for 20min, rinse with washing buffer, incubate with secondary antibody at room temperature (around 25℃) for 15min, rinse with deionized water, add DAB chromogenic reagent for 5min, and rinse with deionized water.

[0093] 4) Hematoxylin staining: Add hematoxylin staining solution for counterstaining for 1 min, then rinse with deionized water.

[0094] 5) Dehydration and sealing: Following the conventional dehydration method, the tablets are treated sequentially with two tanks of 95% ethanol for 30 seconds each, two tanks of anhydrous ethanol for 30 seconds each, and three tanks of TO for 15 seconds each. Finally, the tablets are sealed with resin.

[0095] 2. Results

[0096] Reticular fibers staining reveals a grayish-black texture, clearly showing the reticular fiber structure of normal tissue, and is characteristically distributed around cancer nests and sarcoma single cells in malignant tumors. It can effectively distinguish the distribution and growth patterns of carcinomas and sarcomas; a typical section is shown below. Figure 4 As shown, A is a section of non-malignant liver tissue, and B is a section of malignant uterine tumor.

[0097] VIM appears brownish-red and is expressed on mesenchymal components, but its expression is poor in bipolar or poorly differentiated tumors. It is also frequently expressed in endometrial glands, and carcinosarcomas in this location are relatively common, making differentiation difficult. A typical section looks like... Figure 5 As shown, section A is a malignant uterine tumor with weak positive expression, while section B is a non-malignant endometrial tumor, showing positive expression in both glands and stroma.

[0098] In summary, reticular fiber staining can reveal both epithelial-derived cancer nests and mesenchymal-derived sarcomas. While immunohistochemical VIM is typically expressed in mesenchymal-derived tumors, it has a significant disadvantage in distinguishing between these two components in carcinosarcomas. Therefore, reticular fiber staining was chosen as the method for interpreting malignant mesenchymal components in the assessment criteria for the proportion of carcinosarcoma components.

[0099] IV. Selection of Immunohistochemical Chromogenic Agents for Interpreting Malignant Epithelial Components

[0100] 1. Method

[0101] Ten cancer samples of different types were selected, and the expression effects were observed by using AE1 / AE3 as labeled antibodies and RED and DAB chromogenic agents respectively, following the above method.

[0102] 2. Results

[0103] Microscopic observation shows that the RED colorimetric photographs are as follows: Figure 6 As shown, A is a typical photograph of antibody expression in the cytoplasm / membrane, and B is a typical photograph of antibody expression in the cell nucleus. The results show that with RED chromogenic reagent, the cancerous components exhibit a bright red effect, clearly showing the distribution and content of the antigen; with DAB chromogenic reagent, the results are as follows: Figure 1 The cancerous components exhibit a brownish-brown color, which also clearly shows the distribution and content of the antigens.

[0104] However, considering that the reticular fibers appear grayish-black, in order to better distinguish the colors, the color difference values ​​of the above 10 cases were compared, and the results are shown in the table below.

[0105] Table 2. Statistical Table of Color Differences Between Color Development with Different Color Developers and Color Development with Reticulated Fibers

[0106]

[0107] The results show that the color difference between RED and mesh fiber is 48–65; the two colors are contrasting and have a sharp contrast. The color difference between DAB and mesh fiber is 8–15; the two colors are similar and have no obvious contrast.

[0108] Accordingly, RED colorimetry was selected as the colorimetric method for interpreting malignant epithelial components in this invention.

[0109] V. Optimization of dyeing conditions for reticular fibers

[0110] 1. Method

[0111] Ten different tissue samples were selected from the third item above, "Selection of methods for interpreting interleaf components," and reticular fiber staining was performed according to the method in the third item above. The staining quality was then observed under a microscope.

[0112] The samples were divided into 4 groups. Group 1 was stained with silver ammonia solution at 37℃ for 15 min, Group 2 was stained with silver ammonia solution at 37℃ for 5 min, Group 3 was stained with silver ammonia solution at room temperature (about 25℃) for 15 min, and Group 4 was stained with silver ammonia solution at room temperature (about 25℃) for 5 min.

[0113] 2. Results

[0114] Typical staining results for each group are as follows Figure 7 As shown, A represents a non-malignant liver tumor section from Group 1, B represents a malignant lung tumor section from Group 2, C represents a malignant bladder tumor section from Group 3, and D represents a benign ovarian tumor section from Group 4. The sections were graded according to the following method:

[0115] Scoring criteria: Scoring is based on the following basic standard table for the quality of reticular fiber stained sections. A score of ≥95 is considered excellent.

[0116] Table 3. Scoring criteria for the quality of reticular fiber stained sections

[0117]

[0118] Grading criteria: Total score ≥ 95 points is excellent; 85-94 points is good; 75-84 points is acceptable; 61-74 points is basically acceptable; ≤ 60 points (including sections with incorrect staining and positioning) is unacceptable and requires re-preparation. If it seriously affects the diagnosis, it will be directly rated as unacceptable.

[0119] Superior Grade Rate = (Number of Superior Grade Slices / Total Number of Slices) × 100%

[0120] Volatility coefficient = Standard deviation / Mean

[0121] Table 4. Slice Quality Scoring Table for Different Groups

[0122]

[0123] The results show that, under the conditions of 37℃ and 15 min, the silver ammonia staining solution in Group 1 produced the best quality of stained slides, the best overall quality, the smallest fluctuation coefficient, and the most stable experimental results.

[0124] VI. Staining order screening

[0125] 1. Method

[0126] Ten historical carcinosarcoma samples from our unit were selected and divided into two groups. Group 1 was stained with reticular fibers first and then with immunohistochemical staining, while Group 2 was stained with immunohistochemical staining first and then with reticular fibers. The samples were stained using the two methods mentioned above, while keeping other conditions constant, and the expression effect was observed.

[0127] The procedure for staining reticular fibers is the same as in item 3 above, and the procedure for immunohistochemical staining is the same as in item 2 above.

[0128] 2. Results

[0129] Typical slices from Group 1 and Group 2 are shown below. Figure 8-9 As shown. Figure 8 Images A and B are slides of samples C02 and C06 from group 1, respectively. The results show that under the microscope, the epithelial components appear as a bright red color with accurate localization, while the reticular fibers are grayish-black with clear texture, surrounding the cancer nests and individual sarcoma cells. The contrast between the two images is significant, clearly distinguishing the distribution of epithelial and mesenchymal tumor cells. Figure 9 Images A and B are photographs of sections from samples C02 and C06 in Group 1, respectively. The results show that under the microscope, the epithelial components appear bright red and are relatively accurately located, though some are disordered. The reticular fibers are grayish-black with slightly indistinct texture, surrounding the epithelial nests and individual sarcoma cells. The distinction between the two is not obvious; in some cases, the distribution of epithelial and mesenchymal tumor cells is clearly distinguishable, while in others it is difficult to identify.

[0130] Based on the following interpretation criteria, the judgment results of 10 samples were statistically analyzed, and the results are shown in the table below.

[0131] It can be accurately interpreted (marked with "√"): Reticular fiber staining and immunohistochemical staining are accurately located and do not interfere with each other, with obvious contrast, and can be clearly and effectively interpreted.

[0132] Slightly uncertain (marked as "○"): Reticular fiber staining and immunohistochemical staining are accurately located and do not interfere with each other. The contrast is relatively obvious and the results can be roughly interpreted.

[0133] Inaccurate interpretation (marked as "×"): The localization of reticular fiber staining and immunohistochemical staining is confused, and the color interference between the two is not obvious, making it impossible to clearly and effectively interpret the results.

[0134] Table 5. Interpretation of expression results in different groups

[0135]

[0136] The results showed that in Experiment 1, 90% of the samples could be accurately interpreted, 10% were slightly uncertain, and 0% could not be accurately interpreted. In Experiment 2, 50% of the samples could be accurately interpreted, 20% were slightly uncertain, and 30% could not be accurately interpreted. The better slide preparation results were achieved by performing reticular fiber staining first and then immunohistochemical staining in Experiment 1.

[0137] In summary, the key conditions for preparing the optimal slide samples for carcinosarcoma assessment in this embodiment are: labeling with AE1 / AE3 antibodies, RED staining to identify malignant epithelial components, and reticular fiber staining to identify malignant mesenchymal components. The silver ammonia staining solution is applied at a temperature of 37°C for 15 minutes. The optimal slide preparation results are achieved by first performing reticular fiber staining and then superimposing immunohistochemical staining.

[0138] Example 2

[0139] This embodiment validates the methodology of the preparation method of the above-mentioned slides for carcinosarcoma evaluation.

[0140] I. Material Selection

[0141] After the diagnosing physician searches the system for historical samples and verifies the applicability of the selected specimens, the archivist locates the corresponding tissue block and hands it over to the technician for preparation of the experiment.

[0142] The applicable criteria were as follows: the control group consisted of well-differentiated samples clearly diagnosed as carcinoma or sarcoma. The experimental group consisted of poorly differentiated malignant tumors that were difficult to distinguish under a microscope using HE staining and required further identification as carcinoma, sarcoma, or carcinosarcoma.

[0143] II. Experimental Methods

[0144] 1. Slicing and Baking

[0145] The tissue paraffin block specimens were routinely sectioned with a section thickness of 2 μm. The number of sections for each paraffin block was prepared according to the needs of reticular fiber staining and immunohistochemical staining. The sections were then baked in a 65℃ oven for 30 min.

[0146] 2. Dyeing of reticular fibers

[0147] After dewaxing and hydration of the sections, reticular fiber staining was performed according to the reticular fiber staining method in Example 1.

[0148] 3. Immunohistochemical staining

[0149] Immunohistochemistry was performed using a LEICA BOND-MAX fully automated immunohistochemical staining machine. The staining steps were as follows: AE1 / AE3 antibodies were added, and the primary antibody was incubated at room temperature (around 25°C) for 20 minutes. The mixture was then rinsed with washing buffer, and the secondary antibody was incubated at room temperature (around 25°C) for 15 minutes. The mixture was then rinsed with deionized water, and DAB chromogenic reagent was added for 5 minutes. The mixture was then rinsed with deionized water. (Except for the use of automated staining reagent, all other conditions were the same as those for AE1 / AE3 staining in Example 1.)

[0150] 4. Hematoxylin staining

[0151] Add hematoxylin staining solution for 1 minute for counterstaining, then rinse with deionized water.

[0152] 5. Dehydration and sealing: Following the conventional dehydration method, the tablets are treated sequentially with two tanks of 95% ethanol for 30 seconds each, two tanks of anhydrous ethanol for 30 seconds each, and three tanks of TO for 15 seconds each. Finally, the tablets are sealed with resin.

[0153] 6. Verification

[0154] One hundred samples of poorly differentiated malignant tumors from our unit's historical samples were selected and stained using the above method. The expression effect was observed. The basic information of the samples is shown in the table below.

[0155] Table 6. Basic Information of the Sample

[0156]

[0157] Cancer samples on the same slide showed reticular fibers as: reticular fiber carcinoma perinesia+, AE1 / AE3 carcinoma+;

[0158] The sarcoma sample on the same slide showed the following reticular fibers: reticular fibrosarcoma single cell periphery +, AE1 / AE3 sarcoma cells -;

[0159] Carcinosarcoma samples on the same slide showed the following reticular fibers: reticular fibers around cancer nests +, sarcoma single cells around cells +, AE1 / AE3 cancer nests +, sarcoma cells -.

[0160] The above samples were definitively diagnosed as carcinoma, sarcoma, or carcinosarcoma. Furthermore, 12 cases of carcinosarcoma were identified, and the proportion of each component was accurately assessed in all cases.

[0161] The pathologists assessed the differences in the proportion of samples diagnosed as carcinosarcoma in the above sample and compared the slide samples obtained by the method of this invention with those obtained by conventional methods.

[0162] The conventional method is as follows: under conventional HE staining, doctors make a preliminary assessment based on morphology and experience, or by combining multiple immunohistochemical tests with HE staining to assess the proportion of carcinoma and sarcoma components.

[0163] The method for assessing the proportion of carcinoma and sarcoma components using the method of this invention is as follows: The slide is prepared using the method of this invention, and the physician directly observes the reticular fiber staining and AE1 / AE3 color distribution on this single slide, combined with HE staining of the cut margin, to assess the proportion of carcinoma and sarcoma components. The results are shown in Tables 7-8 below.

[0164] Table 7. Assessment of the proportion of carcinoma and sarcoma components in slides obtained by conventional methods by 5 diagnostic physicians

[0165] (Percentage of cancerous components, %)

[0166]

[0167] Table 8. Evaluation of the proportion of carcinoma and sarcoma components in the slides obtained by the method of this embodiment by 5 diagnostic physicians.

[0168] (Percentage of cancerous components, %)

[0169]

[0170] The results showed that, using conventional methods, the difference between the proportion of carcinoma and sarcoma components in carcinosarcoma was 5-15% when pathologists assessed the samples. However, using the method described in this embodiment, the assessment difference was ≤5%.

[0171] The above results indicate that using a technique of first staining reticular fibers and then superimposing immunohistochemical AE1 / AE3 staining on the same slide to improve the expression of carcinosarcoma can help in the precise diagnosis and treatment of the disease and reduce the difference in results caused by differences in diagnostic personnel.

Claims

1. A method for preparing slides for evaluating carcinosarcoma, characterized in that, Includes the following steps: First, perform reticular fiber staining: use silver ammonia staining solution to stain the tissue sections. The temperature of the silver ammonia staining solution is 37℃ and the time is 15min. Immunohistochemical staining is then performed: Immunohistochemical staining is used to label and stain the AE1 / AE3 proteins in the tissue section. Specifically, AE1 / AE3 antibodies are added and incubated with the primary antibody. Then, secondary antibodies that bind to the above AE1 / AE3 antibodies are added and incubated with the secondary antibody. Finally, a chromogenic agent targeting the above secondary antibody is added for color development. The chromogenic agent for the secondary antibody is a RED chromogenic agent.

2. The method for preparing slides for carcinosarcoma evaluation as described in claim 1, characterized in that, The reticular fiber staining and immunohistochemical staining steps are followed by a hematoxylin staining step, wherein the hematoxylin staining operation method is as follows: hematoxylin staining solution is added to the tissue section for counterstaining.

3. The method for preparing slides for carcinosarcoma evaluation as described in claim 1, characterized in that, The procedure for staining the reticular fibers is as follows: take the dewaxed tissue sections, and successively oxidize with potassium permanganate solution, bleach with oxalic acid solution, mordant with ammonium ferric sulfate solution, stain with silver ammonia solution, reduce with formaldehyde, adjust the color with gold chloride solution, and remove excess silver ions with sodium thiosulfate solution.

4. The method for preparing slides for carcinosarcoma evaluation as described in claim 3, characterized in that, The oxidation treatment time with potassium permanganate solution is 3±1 min; the bleaching treatment time with oxalic acid solution is 1-2 min; the mordant treatment time with ammonium ferric sulfate solution is 3±1 min; and the formaldehyde reduction treatment time is 30±10 s. The treatment time for gold chloride solution was 1 ± 0.5 min; the treatment time for sodium thiosulfate solution was 2 ± 1 min.

5. The method for preparing slides for carcinosarcoma evaluation as described in claim 1, characterized in that, One or more of the following conditions must be met: (1) The immunohistochemical staining procedure is as follows: take tissue sections, add AE1 / AE3 antibody, incubate with primary antibody at 20-30℃ for 20±5 min, rinse with washing solution, add secondary antibody that binds to the above AE1 / AE3 antibody, incubate with secondary antibody at 20-30℃ for 15±5 min, rinse with deionized water, add chromogenic agent against the above secondary antibody, develop color for 5±2 min, and rinse with deionized water; (2) Before performing immunohistochemical staining, a repair and blocking step is also included. The operation method of the repair and blocking is: repairing and blocking the tissue sections in sequence; (3) The reticular fiber staining and immunohistochemical staining steps are followed by a hematoxylin staining step. The hematoxylin staining operation method is as follows: add hematoxylin staining solution to the tissue section for 1 ± 0.5 min and perform counterstaining.

6. The method for preparing slides for carcinosarcoma evaluation as described in claim 1, characterized in that, The antigen was repaired at 100±20℃ for 15±5 min with antigen retrieval solution, and then treated with peroxidase inhibitor for 15±5 min.

7. Tissue stained sections prepared by the method for preparing carcinosarcoma evaluation sections according to any one of claims 1-6.

8. The application of the tissue staining sections of claim 7 in the evaluation of carcinosarcoma for non-diagnostic treatment, wherein the carcinosarcoma evaluation includes the evaluation of specific subtypes of carcinoma, sarcoma and carcinosarcoma and the evaluation of the proportion of each component in carcinosarcoma.

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