Slice for evaluating cancer sarcoma as well as preparation method and application of slice

Abstract

The invention discloses a section for evaluating cancerosarcoma as well as a preparation method and application thereof, and belongs to the technical field of pathology. The preparation method comprises the following steps: reticular fiber dyeing: dyeing reticular fibers in section tissues by a silver staining method; and performing immunohistochemical staining: performing labeling staining on AE1 / AE3 protein in the section tissue by a non-histochemical staining method. According to the section obtained by the preparation method, two kinds of dyeing can be presented on one tissue section, the cancer, sarcoma and cancer and sarcoma expression effects are improved, the identification difficulty is reduced, and precise tumor typing is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of pathology, and in particular to a slice for evaluating carcinosarcoma, a preparation method thereof and an application thereof. Background Art

[0002] Carcinosarcoma is a mixed malignant tumor composed of different types of abnormal cells, including cancer cells derived from epithelial tissue and sarcoma cells derived from mesenchymal tissue. This tumor contains both malignant epithelial and mesenchymal components, so it is called carcinosarcoma. Carcinosarcoma can appear in any part of the body, such as bones, muscles, nerve tissue, blood vessels, etc., and its occurrence may be related to various factors such as genetics and environment. Because carcinosarcoma has the dual characteristics of cancer and sarcoma, it is highly malignant, difficult to treat, and the prognosis is usually poor.

[0003] At present, pathologists usually observe the morphology, structure, arrangement and nuclear division of tumor cells under a microscope, and combine immunohistochemical markers, special staining, genetic testing and other technical means to diagnose carcinosarcoma and identify the type, nature and proportion of tumor components. However, due to individual differences, tumor diversity and inconsistency in differentiation, it is difficult for pathologists to identify the tumor by morphology alone when performing diagnostic typing under the microscope, and there is also a certain degree of subjectivity between different doctors. Commonly used auxiliary means have problems such as high technical costs, single staining pattern identification and high difficulty in interpretation.

[0004] Therefore, how to accurately assess the respective proportions of cancer and sarcoma in tumor tissue and more accurately determine the tumor type has important guiding significance in clinical decision-making, prognosis assessment and disease monitoring. Summary of the invention

[0005] In view of the lack of a section staining method that can accurately evaluate carcinosarcoma, the present invention provides a method for preparing sections for carcinosarcoma evaluation, which improves the expression of cancer, sarcoma, and carcinosarcoma by presenting two stainings on a tissue section, reduces the difficulty of identification, and is conducive to accurate tumor classification.

[0006] The present invention discloses a method for preparing a slice for evaluating carcinosarcoma, comprising the following steps:

[0007] Reticular fiber staining: Silver staining is used to stain the reticular fibers in the sliced ​​tissue;

[0008] Immunohistochemical staining: Immunohistochemical staining was used to mark and stain the AE1 / AE3 proteins in the tissue sections.

[0009] In the routine evaluation of carcinosarcoma, multiple immunohistochemical and certain special staining are usually performed for diagnosis and identification, but it is necessary to repeatedly compare multiple sections to obtain an accurate conclusion, and it is extremely unfriendly to tissue wax blocks with fewer lesion components. The inventors consider clearly displaying the two components on the same section, which is convenient for diagnostic doctors to interpret on the one hand, and can also save tissue wax blocks on the other hand.

[0010] On this basis, the inventors comprehensively considered the need to select a marker protein that can reflect a wide coverage of different malignant epithelial components as a marker for the interpretation of malignant epithelial components, and a reticular fiber staining that can accurately interpret the malignant mesenchymal components. They also fully considered that the two different staining methods should not only not interfere with each other in the same tissue section, but also have to have a clear contrast to facilitate interpretation. After comparing and screening a variety of staining methods, they finally chose to use silver staining for reticular fiber staining, and combined it with immunohistochemical staining of AE1 / AE3 proteins, thus obtaining the ability to present two staining effects on the same tissue section, with the advantages of wide coverage of 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 the schemes, the operation method of reticular fiber staining is: drip staining the sliced ​​tissue with silver ammonia stain solution.

[0013] In some of the schemes, the immunohistochemical staining operation method is: take a tissue section, add AE1 / AE3 antibody, incubate with primary antibody, then add secondary antibody combined with the above AE1 / AE3 antibody, incubate with secondary antibody, add a color developer for the above secondary antibody, and develop color.

[0014] In some of the protocols, the reticular fiber staining and immunohistochemical staining steps further include a hematoxylin staining step.

[0015] In some of the schemes, the operation method of hematoxylin staining is: adding hematoxylin staining solution to the tissue section for counterstaining.

[0016] In some of the above schemes, in the mesh fiber dyeing step, the temperature of the silver ammonia dye dripping is 20-40°C, preferably 37°C.

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

[0018] In some of the above schemes, in the immunohistochemical staining step, the color developer is a RED color developer.

[0019] In some of the above schemes, the operation method of reticular fiber dyeing is: take the dewaxed section tissue, oxidize it with potassium permanganate solution, bleach it with oxalic acid solution, mordant it with ammonium ferric sulfate solution, drip dye it with silver ammonia dye, reduce it with formaldehyde, toner it with gold chloride solution and remove excess silver ions with sodium thiosulfate solution.

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

[0021] In some of the above schemes, the operation method of the immunohistochemical staining is: take a tissue section, add AE1 / AE3 antibody, incubate with the primary antibody at 20-30°C for 20±5min, rinse with cleaning solution, then add the secondary antibody combined with the above AE1 / AE3 antibody, incubate with the secondary antibody at 20-30°C for 15±5min, rinse with deionized water, add a color developer for the above secondary antibody, color for 5±2min, and rinse with deionized water.

[0022] In some of the above schemes, before immunohistochemical staining, a repair and blocking step is also included, and the operation method of the repair and blocking is: repairing and blocking the tissue sections in sequence; preferably, repairing with antigen repair solution at 100±20℃ for 15±5min, and then treating with peroxidase blocker for 15±5min.

[0023] In some of the above schemes, the operation method of hematoxylin staining is: dripping hematoxylin staining solution on the tissue section for 1±0.5min, and then re-staining.

[0024] On the other hand, the present invention also discloses a tissue staining section prepared by the above-mentioned method for preparing a section for evaluating carcinosarcoma.

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

[0026] In some of these protocols, the carcinosarcoma assessment includes assessment of specific types of carcinoma, sarcoma and carcinosarcoma and assessment of the proportion of each component in carcinosarcoma.

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

[0028] The reticular fiber staining reagent comprises: silver ammonia stain solution;

[0029] The immunohistochemical staining reagent comprises: AE1 / AE3 antibody, secondary antibody against AE1 / AE3 antibody, and color developer; the color developer is RED color developer or DAB color developer.

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

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

[0032] In some embodiments, the chromogenic agent is a RED chromogenic agent.

[0033] definition:

[0034] The “RED” mentioned above refers to BOND Polymer Refine Red Detection, which is a chromogen widely used in immunohistochemical staining. It can react with alkaline phosphatase (AP) to produce a red precipitate.

[0035] The above-mentioned "DAB" refers to BOND Polymer Refine Detection, namely 3,3'-diaminobenzidine, which is a chromogenic substrate of horseradish peroxidase. Its color development principle is that it loses electrons under the action of hydrogen peroxide and shows color change and accumulation, forming a brown insoluble product.

[0036] The above-mentioned "cancer" refers to a malignant tumor originating from epithelial tissue.

[0037] The above-mentioned "sarcoma" refers to a malignant tumor derived from mesenchymal tissue.

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

[0039] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0040] The reagents and raw materials used in the present invention are commercially available.

[0041] The positive and progressive effects of the present invention are:

[0042] The method for preparing a carcinosarcoma evaluation slice of the present invention presents two stainings on a tissue slice, improves the expression effect of cancer, sarcoma, and carcinosarcoma, reduces the difficulty of identification, is conducive to accurate tumor typing, and reduces the result difference caused by differences in diagnostic personnel. In addition, since multiple slice preparations are avoided, tissue loss is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0048] Figure 6 This is a typical photo of AE1 / AE3 RED staining in Example 1.

[0049] Figure 7 These are typical photos of the color development of each group of reticular fibers in Example 1.

[0050] Figure 8 This is a typical photo of the first group of staining sequence screening in Example 1.

[0051] Fig. 9 This is a typical photo of the second group of staining sequence screening in Example 1. DETAILED DESCRIPTION

[0052] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

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

[0054] CK5 / 6 antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Common name: Cytokeratin 5 / 6 antibody reagent (immunohistochemistry) Product number: ZM-0313 ZM-0313UM

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

[0056] RED color developer: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.; Product number: DS9390.

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

[0058] Example 1

[0059] In this example, a slice sample for carcinosarcoma assessment was developed, and its preparation method was investigated and optimized.

[0060] 1. Design Concept

[0061] For the interpretation of malignant epithelial components, immunohistochemical staining can be considered, such as labeling AE1 / AE3 protein, CK5 / 6 protein, and CK19 protein. For the interpretation of malignant mesenchymal components, reticular fiber staining or immunohistochemical VIM can be considered in special staining.

[0062] 2. Selection of immunohistochemical antibodies for interpretation of malignant epithelial components

[0063] 1. Methods

[0064] Ten different types of cancer samples (including squamous cell carcinoma, tubular adenocarcinoma, papillary thyroid carcinoma, urothelial cell carcinoma, and breast cancer samples from different sites) 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 to screen antibodies that have advantages in labeling unknown cancer components.

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

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

[0067] 3) Immunohistochemical staining: According to the product instructions, add 100uL of AE1 / AE3 antibody, CK5 / 6 antibody or CK19 antibody respectively, incubate with primary antibody at room temperature (about 25°C) for 20 minutes, rinse with cleaning solution, incubate with secondary antibody at room temperature (about 25°C) for 15 minutes, rinse with deionized water, add DAB color developer for color development for 5 minutes, and rinse with deionized water.

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

[0069] 5) Dehydration and sealing: According to the conventional dehydration method, use two cylinders of 95% ethanol for 30 seconds each, two cylinders of anhydrous ethanol for 30 seconds each, and three cylinders of TO for 15 seconds each, and finally seal the slides with gum.

[0070] 2. Results

[0071] Typical slides labeled with different antibodies Figure 1-3 As shown, Figure 1 The following are typical photos of AE1 / AE3 staining, where A is a photo of squamous cell carcinoma with positive expression, and B is a photo of adenocarcinoma with positive expression. Figure 2 These are typical photos of CK5 / 6 staining, where A is a photo of a squamous cell carcinoma sample with positive expression, but B is a photo of a sample with negative expression of adenocarcinoma, indicating that CK5 / 6 staining cannot mark adenocarcinoma. Figure 3 The following are typical photos of CK19 staining, where A is a positive expression photo of adenocarcinoma, but B is a negative expression sample photo of keratinizing squamous cell carcinoma, indicating that CK19 staining cannot mark keratinizing squamous cell carcinoma. The expression results of each sample are shown in the following table.

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

[0073]

[0074] Note: “+” indicates positive expression, and “-” indicates negative expression.

[0075] The above 10 samples of different cancer types are common clinical cancer types. The results show that AE1 / AE3 expression is 100%, CK5 / 6 expression is 40%, and CK19 expression is 60%. Among them, AE1 / AE3 can cover a variety of keratin subtypes and can be used for epithelial tumor components from a variety of sources. Considering that carcinosarcoma has a low degree of differentiation and a variety of possible sources of components, AE1 / AE3 is better for interpreting malignant epithelial components. It has the characteristics of wide coverage and can be effectively expressed when the cancer type is unknown.

[0076] 3. Selection of methods for interpreting interleaf components

[0077] Ten different types of tissue samples (including benign and malignant tissue samples from different sources) were selected from the historical samples of our unit. Reticular fiber staining and immunohistochemical VIM staining were used to mark the malignant mesenchymal components according to the following methods, and their expression effects were observed.

[0078] 1. Methods

[0079] 1) Reticular fiber staining was performed according to the instructions of the Reticular Fiber Staining Kit (Zhuhai Beso Biotechnology Co., Ltd., model B4BA4165), as follows:

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

[0081] Oxidation: The tissue sections were oxidized with potassium permanganate solution for 3 min and then rinsed with running water for 1 min.

[0082] Bleaching: Bleaching tissue sections with oxalic acid solution for 1-2 minutes, followed by rinsing in running water for 1 minute.

[0083] Mordanting: The tissue sections were mordanted with ammonium ferric sulfate solution for 3 min and then rinsed with deionized water for 10 s.

[0084] Silver ammonia staining: Tissue sections were stained with silver ammonia solution (Ag(NH 3 ) 2 OH) for 15 min (in a 37° oven), followed by rinsing with deionized water for 10 s.

[0085] Reduction: The tissue sections were reduced with formaldehyde solution for 30 seconds and then rinsed with running water for 1 minute.

[0086] Color correction: The tissue sections were colored with gold chloride solution for 1 min, and then rinsed with running water for 1 min.

[0087] Silver removal: Treat tissue sections with sodium thiosulfate solution for 2 minutes, then rinse with running water for 1 minute to remove excess silver ions.

[0088] Quality control under the microscope.

[0089] 2) Immunohistochemical VIM staining:

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

[0091] 2) Repair and blocking: After 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 VIM antibody, incubate with primary antibody at room temperature (about 25℃) for 20min, rinse with cleaning solution, incubate with secondary antibody at room temperature (about 25℃) for 15min, rinse with deionized water, add DAB color developer for 5min, and rinse with deionized water.

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

[0094] 5) Dehydration and sealing: According to the conventional dehydration method, use two cylinders of 95% ethanol for 30 seconds each, two cylinders of anhydrous ethanol for 30 seconds each, and three cylinders of TO for 15 seconds each, and finally seal the slides with gum.

[0095] 2. Results

[0096] Reticular fiber staining shows gray-black texture, clearly showing the reticular fiber structure of normal tissue, and characteristically distributed around cancer nests and around single cells of sarcoma in malignant tumors. It can effectively distinguish the distribution and growth pattern of cancer and sarcoma. Its typical sections are as follows Figure 4 As shown, A is a non-malignant tumor section of liver tissue, and B is a malignant tumor section of uterus.

[0097] VIM appears brown and is expressed in the mesenchymal components, but the expression is poor in bi-differentiated and poorly differentiated tumors. It is often expressed in the endometrial glands, and carcinosarcoma in this area is also relatively common, which makes it difficult to distinguish and identify. Its typical sections are as follows Figure 5 As shown, A is a section of uterine malignant tumor, showing weak positive expression, and B is a section of endometrial non-malignant tumor, showing positive expression in both glands and stroma.

[0098] In summary, reticular fiber staining can show both epithelial cancer nests and mesenchymal sarcomas. Immunohistochemical VIM is usually expressed in mesenchymal origin, but it has obvious disadvantages in distinguishing the two components in carcinosarcoma. Reticular fiber staining is selected as the method for interpreting malignant mesenchymal components in the evaluation criteria for the proportion of carcinosarcoma components.

[0099] IV. Selection of immunohistochemical colorants for interpretation of malignant epithelial components

[0100] 1. Methods

[0101] Ten cancer samples of different types were selected and developed using RED and DAB colorants, respectively, using AE1 / AE3 as labeled antibodies according to the above method to observe their expression effects.

[0102] 2. Results

[0103] Observed under the microscope, the RED color developer color photos are as follows Figure 6 As shown, A is a typical photo of cytoplasm / membrane expression of antibodies, and B is a typical photo of nucleus expression of antibodies. The results show that when the RED color developer is used, the cancer component presents a bright red effect, which can clearly show the distribution and content of the antigen; when the DAB color developer is used, the cancer component presents a bright red effect, which can clearly show the distribution and content of the antigen. Figure 1 , the cancer components appear brown in color, and the distribution and content of antigens can also be clearly shown.

[0104] However, considering that the reticular fibers appear gray-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 following table.

[0105] Table 2. Statistical table of color difference between different color developers and reticular fiber color development

[0106]

[0107] The above results show that the color difference between RED and reticular fiber is 48-65; the colors of the two are contrasting colors, and the contrast is sharp. The color difference between DAB and reticular fiber is 8-15; the colors of the two are similar to each other, and the contrast is not obvious.

[0108] Based on this, RED color development was selected as the color development method for interpreting malignant epithelial components of the present invention.

[0109] 5. Optimization of reticular fiber dyeing conditions

[0110] 1. Methods

[0111] Select 10 different tissue samples under the third item "Selection of methods for interpreting interleaf components" above, stain them according to the reticular fiber staining conditions according to the method in the third item above, and observe the staining quality under a microscope.

[0112] The samples were divided into 4 groups. The first group was stained with silver ammonia dye at 37°C for 15 minutes, the second group was stained with silver ammonia dye at 37°C for 5 minutes, the third group was stained with silver ammonia dye at room temperature (about 25°C) for 15 minutes, and the fourth group was stained with silver ammonia dye at room temperature (about 25°C) for 5 minutes.

[0113] 2. Results

[0114] Typical staining results of each group are shown in Figure 7 As shown, A is a non-malignant tumor section of liver tissue in Group 1, B is a malignant tumor section of lung tissue in Group 2, C is a malignant tumor section of bladder tissue in Group 3, and D is a benign ovarian tumor section in Group 4. The evaluation is performed according to the following method:

[0115] Scoring criteria: Scoring was performed according to the following basic quality standard table for reticular fiber staining sections, with scores ≥95 being excellent.

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

[0117]

[0118] Grading standard: total score ≥ 95 points, excellent grade; 85-94 points, good grade; 75-84 points, qualified; 61-74 points, basically qualified; ≤ 60 points (including sections with incorrect staining positioning) is unqualified and needs to be re-prepared. If it seriously affects the diagnosis, it will be directly rated as unqualified.

[0119] Excellent grade rate = (number of excellent slices) / total number of slices) × 100%

[0120] Coefficient of variation = standard deviation / mean

[0121] Table 4. Slice quality score table for different groups

[0122]

[0123] The above results show that in Experiment 1, the silver ammonia stain was dripped for 15 minutes at 37°C, and the staining preparation rate was the best, the quality was the best, the fluctuation coefficient was the smallest, and the experimental effect was the most stable.

[0124] 6. Staining sequence screening

[0125] 1. Methods

[0126] Ten historical samples of carcinosarcoma in our unit were selected and divided into two groups. The first group was stained with reticular fiber first and then immunohistochemical staining, and the second group was stained with immunohistochemical staining first and then reticular fiber staining. The samples were stained with the above two schemes respectively, while other conditions remained unchanged, and the expression effects were observed.

[0127] Among them, the reticular fiber staining operation method is the same as the third item above, and the immunohistochemical staining operation method is the same as the second item above.

[0128] 2. Results

[0129] The typical sections of Group 1 and Group 2 are shown in Figure 2. Figure 8-9 shown. Figure 8 A and B are the slice photos of the first group C02 and C06 samples, respectively. The results show that under the microscope, the epithelial components in the samples are bright red and accurately positioned, and the reticular fibers are gray-black with clear textures, surrounding the cancer nests and single sarcoma cells. The contrast between the two is obvious, and the distribution of epithelial and mesenchymal tumor cells can be clearly distinguished. Fig. 9 A and B are the slice photos of the first group C02 and C06 samples respectively. The results show that in the samples under the microscope, the epithelial components are bright red, with relatively accurate positioning, and some are chaotic. The reticular fibers are gray-black with slightly unclear textures, surrounding the epithelial nests and single sarcoma cells. The two are not obvious, and the distribution of epithelial and mesenchymal tumor cells can be clearly distinguished in some parts, but difficult to identify in others.

[0130] According to the following judgment criteria, the judgment results of 10 samples were statistically analyzed and the results are shown in the following table.

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

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

[0133] Unable to accurately interpret (marked as "×"): The positioning of reticular fiber staining and immunohistochemical staining is confused, and the color interference and contrast between the two are not obvious, so the results cannot be clearly and effectively interpreted.

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

[0135]

[0136] The results showed that in Experimental Group 1, 90% of the samples were accurately interpreted, 10% were slightly uncertain, and 0% were not accurately interpreted. In Experimental Group 2, 50% of the samples were accurately interpreted, 20% were slightly uncertain, and 30% were not accurately interpreted. In Experimental Group 1, the reticular fiber staining was performed first, and then the immunohistochemical staining was superimposed, which resulted in a better slide preparation effect.

[0137] In summary, the key conditions for the optimal preparation of slice samples for carcinosarcoma evaluation determined in this embodiment are: labeling with AE1 / AE3 antibodies, RED coloring, to achieve the interpretation of malignant epithelial components, and reticular fiber staining to achieve the interpretation of malignant mesenchymal components, wherein the temperature of silver ammonia staining is 37°C, the time is 15 minutes, and the order of reticular fiber staining first and then superimposing immunohistochemical staining is selected for preparation, which has the best preparation effect.

[0138] Example 2

[0139] This example methodologically validates the above-mentioned method for preparing slices for carcinosarcoma assessment.

[0140] 1. Material selection

[0141] The diagnostic physician searches the system for historical samples and verifies whether the selected specimens are suitable. The archivist then finds the corresponding tissue wax block and hands it over to the technician to prepare for the experiment.

[0142] Applicable criteria are: the control group selects samples that are well differentiated and clearly diagnosed as cancer or sarcoma. The experimental group selects samples of poorly differentiated malignant tumors that are difficult to identify morphologically under HE staining and require relevant means to further identify whether they are cancer, sarcoma, or carcinosarcoma.

[0143] 2. Experimental Methods

[0144] 1. Slice and bake

[0145] The tissue wax block specimens were routinely sectioned with a thickness of 2 μm. A corresponding number of sections were prepared for each wax block according to the needs of reticular fiber staining and immunohistochemical staining, and the sections were baked in a 65°C oven for 30 min.

[0146] 2. Reticular fiber dyeing

[0147] After the sections were dewaxed and hydrated, 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 automatic immunohistochemical staining machine, and the staining steps were as follows: adding AE1 / AE3 antibodies, incubating with the primary antibody at room temperature (about 25°C) for 20 min, rinsing with a cleaning solution, incubating with the secondary antibody at room temperature (about 25°C) for 15 min, rinsing with deionized water, adding DAB color developer for color development for 5 min, and rinsing with deionized water (except for staining with an automated stain, all other conditions were the same as those for immunohistochemical AE1 / AE3 staining in Example 1).

[0150] 4. Hematoxylin staining

[0151] Add hematoxylin solution for counterstaining for 1 min and rinse with deionized water.

[0152] 5. Dehydration and sealing: According to the conventional dehydration method, use two cylinders of 95% ethanol for 30 seconds each, two cylinders of anhydrous ethanol for 30 seconds each, and three cylinders of TO for 15 seconds each, and finally seal the slides with gum.

[0153] 6. Verification

[0154] 100 samples of poorly differentiated malignant tumors from the historical samples of our unit were selected and stained using the above method to observe the expression effect. The basic information of the samples is shown in the following table.

[0155] Table 6. Basic information of samples

[0156]

[0157] The reticular fibers of the cancer samples on the same slide were shown as: reticular fiber cancer peri-nest+, AE1 / AE3 cancer+;

[0158] The reticular fibers of the sarcoma samples on the same slide were shown as: reticulofibrosarcoma single cell Zhou+, AE1 / AE3 sarcoma cells-;

[0159] The reticular fibers in the carcinosarcoma sample on the same slide were shown as follows: reticular fiber carcinoma nests + around, sarcoma single cells + around, AE1 / AE3 carcinoma nests +, sarcoma cells -.

[0160] The above samples can be clearly diagnosed as cancer, sarcoma or carcinosarcoma. In addition, 12 cases of carcinosarcoma were screened out, and the proportion of each component can be accurately evaluated.

[0161] The samples diagnosed as carcinosarcoma in the above samples were counted, and the slice samples obtained by the method of the present invention and the conventional method were compared, and the pathologists evaluated the difference in proportion.

[0162] Among them, the conventional method is: under the conventional HE staining microscope, the doctor uses morphology combined with experience to make a preliminary assessment, or adds multiple immunohistochemical tests combined with HE for reference to evaluate the proportion of cancer and sarcoma components.

[0163] The method for evaluating the proportion of the present invention is as follows: the doctor directly observes the reticular fiber staining and AE1 / AE3 color distribution on this slice and evaluates the proportion of cancer and sarcoma components in combination with the HE staining margin. The results are shown in Tables 7-8 below.

[0164] Table 7. Evaluation of the proportion of cancer and sarcoma components in sections obtained by conventional methods by 5 diagnostic physicians

[0165] (Percentage of cancer components, %)

[0166]

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

[0168] (Percentage of cancer components, %)

[0169]

[0170] The results showed that, using the conventional method to obtain the slice samples, the difference in the proportion of cancer and sarcoma in the evaluation of the pathologists in the carcinosarcoma was 5-15%. After using the method of this embodiment, the evaluation difference presented was ≤5%.

[0171] The above results indicate that on the same section, the staining technique of first performing reticular fiber staining and then superimposing immunohistochemical AE1 / AE3 can improve the expression of carcinosarcoma, assist in the accurate diagnosis and treatment of the disease, and reduce the difference in results caused by differences in diagnostic personnel.

Claims

1. A method for preparing a slice for evaluating carcinosarcoma, characterized in that: The following steps are involved: Reticular fiber staining: Silver staining is used to stain the reticular fibers in the sliced ​​tissue; Immunohistochemical staining: Immunohistochemical staining was used to mark and stain the AE1 / AE3 proteins in the tissue sections.

2. The method for preparing a carcinosarcoma evaluation slice according to claim 1, characterized in that: Meet one or more of the following conditions: (1) Perform reticular fiber staining first, then immunohistochemical staining; (2) The operation method of the reticular fiber staining is: dripping the sliced ​​tissue with silver ammonia staining solution; (3) The immunohistochemical staining method is as follows: take a tissue section, drop AE1 / AE3 antibody, incubate with primary antibody, then drop secondary antibody that binds to the above AE1 / AE3 antibody, incubate with secondary antibody, add a color developer for the above secondary antibody, and develop color; (4) The reticular fiber staining and immunohistochemical staining steps further include a hematoxylin staining step; preferably, the hematoxylin staining operation method is: dripping hematoxylin staining solution on the tissue section for counterstaining.

3. The method for preparing a carcinosarcoma evaluation slice according to claim 2, characterized in that: Meet one or more of the following conditions: (1) In the step of dyeing the reticular fibers, the temperature of the silver ammonia dye solution is 20-40°C, preferably 37°C; (2) In the reticular fiber dyeing step, the silver ammonia dyeing liquid is dripped for 5-20 minutes, preferably 5-15 minutes, and more preferably 15 minutes; (3) In the immunohistochemical staining step, the color developer is RED color developer.

4. The method for preparing a slice for evaluating carcinosarcoma according to claim 2, characterized in that: The operation method of the reticular fiber dyeing is as follows: taking the dewaxed slice tissue, sequentially oxidizing with potassium permanganate solution, bleaching with oxalic acid solution, mordanting with ammonium ferric sulfate solution, dripping with silver ammonia dye, reducing with formaldehyde, toning with gold chloride solution and removing excess silver ions with sodium thiosulfate solution; Preferably, 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 mordanting treatment time of ammonium ferric sulfate solution is 3±1 min; and the formaldehyde reduction treatment time is 30±10 s; The coloring treatment time of gold chloride solution is 1±0.5min; the treatment time of sodium thiosulfate solution is 2±1min.

5. The method for preparing a slice for evaluating carcinosarcoma according to claim 2, characterized in that: Meet one or more of the following conditions: (1) The immunohistochemical staining method is as follows: take a tissue section, add AE1 / AE3 antibody, incubate with primary antibody at 20-30°C for 20±5min, rinse with cleaning solution, then add secondary antibody combined with the above AE1 / AE3 antibody, incubate with secondary antibody at 20-30°C for 15±5min, rinse with deionized water, add color developer for the above secondary antibody, color develop for 5±2min, and rinse with deionized water; (2) Before immunohistochemical staining, a repair and blocking step is also included, wherein the repair and blocking operation method is: repairing and blocking the tissue section in sequence; preferably, repairing with antigen repair solution at 100±20°C for 15±5 minutes, and then treating with peroxidase blocker for 15±5 minutes; (3) The operation method of the hematoxylin staining is: add hematoxylin staining solution to the tissue section for 1±0.5 min, and then perform re-staining.

6. A tissue staining section prepared by the method for preparing a section for evaluating carcinosarcoma according to any one of claims 1 to 5.

7. Use of the tissue stained section according to claim 6 in the evaluation of carcinosarcoma for non-diagnostic treatment.

8. The use according to claim 7, characterized in that The carcinosarcoma assessment includes the assessment of the specific classification of cancer, sarcoma and carcinosarcoma and the assessment of the proportion of each component in carcinosarcoma.

9. A kit for evaluating carcinosarcoma, characterized in that: include: Reticular fiber staining reagents and immunohistochemical staining reagents, wherein: The reticular fiber staining reagent comprises: silver ammonia stain solution; The immunohistochemical staining reagent comprises: AE1 / AE3 antibody, secondary antibody against AE1 / AE3 antibody, and color developer; the color developer is RED color developer or DAB color developer.

10. The kit for evaluating carcinosarcoma according to claim 9, characterized in that Meet one or more of the following conditions: (1) The reticular fiber dyeing reagent also includes: potassium permanganate solution, oxalic acid solution, ammonium ferric sulfate solution, formaldehyde solution, gold chloride solution and sodium thiosulfate solution; (2) The secondary antibody is an enzyme-labeled goat anti-mouse / rabbit IgG polymer; (3) The color developer is a RED color developer.

Images