Staining method for co-staining of three fibrous tissues

Abstract

The present invention discloses a staining method for co-staining of three fibrous tissues, which includes the steps of successively using Victoria blue staining and Gordon-Sweets silver ammonia solution staining on the processed tissue sections; the staining method of the present invention combines Victoria blue staining and silver ammonia solution staining, and can clearly present the staining of three fibrous tissues on the same section, clearly show the relationship between the cell nucleus structure and elastic fibers, significantly improve the diagnostic accuracy of the degree of tumor invasion, reduce the demand for tissue samples, and the staining method of the present invention has the advantages of clear color contrast, rich levels, accurate positioning, simple operation, etc., and has important significance for clinical pathological diagnosis and is worthy of popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of pathological diagnosis, and particularly to a staining method for co-staining three types of fibrous tissues. Background Art

[0002] Pathological diagnosis is to fix, dehydrate, impregnate with wax various specimens (including solid tissues, pleural effusion, ascites, blood, sputum, etc.) cut during surgery or obtained through autopsy, then embed them into wax blocks, and then section the wax blocks, perform HE staining, and then observe the tissue structure and cell lesions under a microscope. When necessary, immunohistochemistry, special staining, and gene detection are also required to diagnose diseases. Pathological diagnosis is often regarded as the "gold standard" for tumor diagnosis, which shows the importance of pathological work.

[0003] Connective tissue is widely distributed in various organs and tissues of the human body, playing functions such as support, connection, nutrition, defense, protection, and wound repair. Connective tissue can be further divided into elastic fibers, reticular fibers, and collagen fibers. Among them, elastic fibers are distributed in tissues such as the lungs, blood vessels, and skin, are elastic, and are not easily soluble. In the diagnosis of lung cancer, elastic fiber staining is an important indicator for observing the degree of tumor invasion. Elastic fibers are an important component of the pulmonary pleura. By combining Victoria blue staining with immunohistochemistry, it can be seen whether tumor cells break through the pleural layer, so as to make a correct judgment on whether the tumor cells have metastasized. Elastic fiber staining is mainly carried out with Victoria blue, resorcinol basic fuchsin, or weigert iron hematoxylin. However, since elastic fiber staining cannot show cell structure, it is necessary to repeatedly compare the tissue positions of different sections under the microscope to make a correct diagnosis of whether tumor cells invade the pleura. Reticular fibers are a type of fiber in reticular connective tissue, have no elasticity, can resist the corrosion of strong acids and alkalis, and are mainly distributed in hematopoietic organs and lymphoreticular organs. The morphology, distribution, quantity, breakage, or disintegration of reticular fibers are of great significance for pathological diagnosis, and are commonly used to distinguish carcinoma from sarcoma and to identify whether in situ carcinoma has early infiltration, etc. Currently, it is mainly stained by silver impregnation method. Collagen fibers are the most widely distributed and have the largest content, have great toughness, and are soluble in weak acids or boiling water, and are most abundant in the skin, sclera, and tendons. Collagen fiber staining is mainly used to distinguish from muscle fibers. In clinical pathological work, it is often necessary to observe these three types of fibers to determine the degree of tissue lesions and the repair situation. The classic staining is to use hematoxylin-eosin staining method (abbreviated as HE staining), but the three types of fibers are not easily distinguishable on HE sections, and special staining is required for identification. Currently, one or two fiber color development methods are commonly used in clinics, and the method for co-staining three types of fibrous tissues is relatively rare. Therefore, it is necessary to develop a method for co-staining three types of fibers. Summary of the Invention

[0004] In view of the problems in the background content, the present invention proposes a staining method for co-staining three types of fibrous tissues, which combines Victoria blue staining and silver ammonia solution staining, and can clearly present the staining of the three types of fibrous tissues on the same section, and can clearly show the relationship between the cell nucleus structure and elastic fibers.

[0005] For this reason, the present invention adopts the following technical solutions:

[0006] The present invention proposes a staining method for co-staining three types of fibrous tissues, including the steps of successively using Victoria blue staining and Gordon-Sweets silver ammonia solution staining on the processed tissue sections.

[0007] Furthermore, the above staining method specifically includes the following steps:

[0008] S1: Dewax, oxidize, and decolorize the tissue sections;

[0009] S2: After washing the tissue sections with alcohol, place them in a Victoria blue staining cylinder, and place the staining cylinder in a 37°C water bath for 10 - 60 min;

[0010] S3: Then differentiate the sections with alcohol and wash with water;

[0011] S4: Then treat with 4% ammonium ferric sulfate solution and wash with water;

[0012] S5: Drop Gordon-Sweets silver ammonia solution and let it act for 1 - 2 min, then wash with water;

[0013] S6: Drop 4% neutral formaldehyde solution to reduce for 1 - 3 min and wash with water;

[0014] S7: Dry by baking and mount the slides.

[0015] Furthermore, the tissue sections include, but are not limited to, sections from lung tissue, liver tissue, lymph nodes, and skin tissue.

[0016] Furthermore, the oxidation treatment method is to react with 0.5% potassium permanganate solution by mass concentration for 8 - 10 min, and then wash it clean with water.

[0017] Furthermore, the decolorization method is to add 2% oxalic acid until the tissue sections become colorless, and then wash it clean with water.

[0018] Furthermore, the alcohol concentration in steps S2 and S3 is 70%, and the treatment time is 1 - 10 s.

[0019] Furthermore, the above staining method more specifically includes the following steps:

[0020] S1: Dewax the tissue section, spin-dry the water, stain it with 0.5% potassium permanganate for 8 - 10 minutes of oxidation, and wash it with distilled water;

[0021] S2: Drop 2% oxalic acid for decolorization for 1 min, and wash it with distilled water;

[0022] S3: Rinse the section in 70% alcohol for a moment, then place the section in a Victoria blue staining cylinder, and place the staining cylinder in a 37°C water bath for 30 min for staining;

[0023] S4: Take out the section, wash it slightly with water, observe the staining situation, then place the section in 70% alcohol for differentiation for 1 - 10 seconds, and wash it with distilled water;

[0024] S5: Drop 4% ammonium ferric sulfate solution for 5 min, discard the liquid, and wash it with distilled water;

[0025] S6: Drop Gordon-Sweets silver ammonia solution and let it act for 1 min, discard the liquid, and wash it with distilled water;

[0026] S7: Drop 4% neutral formaldehyde for reduction for 1 - 2 min, and wash it with distilled water;

[0027] S8: Dry it by baking and mount the section.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The present invention combines Victoria blue staining and silver ammonia solution staining, and can stain elastic fibers, reticular fibers, and collagen fibers, clearly presenting them on the same section. The staining effect is remarkable, the staining target is accurately positioned, the staining contrast is clear, and the background is clean. In addition, it can clearly show the relationship between the cell nucleus structure and elastic fibers, which is beneficial for pathologists to judge whether tumor cells break through the elastic fiber layer or the reticular fiber layer on a single section, and the information provided is significantly better than traditional single staining. The staining method of the present invention solves the technical problem that the three types of fibers are not easily distinguishable on HE sections, and for puncture specimens with a small amount of specimens, in the case where special staining is often less done or not done, the staining method of co-coloring of the three types of fiber tissues can significantly reduce the demand for tissue samples. The staining method of the present invention has important significance for clinical pathological diagnosis work and is worthy of popularization and application in clinical pathological work. Description of the Drawings

[0030] Figure 1 It is a Victoria blue + silver ammonia solution staining diagram (100X) of the liver tissue in Example 2 of the present invention;

[0031] Figure 2 It is a Victoria blue staining diagram (100X) of the liver tissue in Comparative Example 1 of the present invention;

[0032] Figure 3 It is the ammoniacal silver solution staining diagram (100X) of the liver tissue in Comparative Example 3 of the present invention;

[0033] Figure 4 It is the Victoria blue + ammoniacal silver solution staining diagram (100X) of the lung tissue in Example 3 of the present invention;

[0034] Figure 5 It is the Victoria blue staining diagram (100X) of the lung tissue in Comparative Example 2 of the present invention. Detailed implementation manners

[0035] To make the purpose and technical solutions of the detailed implementation manners of the present invention clearer, the technical solutions of the detailed implementation manners of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention and the implementation examples of the detailed implementation manners. Obviously, the described detailed implementation manners are part of the detailed implementation manners of the present invention, rather than all of the detailed implementation manners.

[0036] The pathological specimens were from the Department of Pathology, the First Affiliated Hospital of Guangzhou Medical University, and were collected from January to December 2021. The pathological specimens include but are not limited to liver tissue specimens, lung tissue specimens, lymph node specimens, and skin tissue specimens.

[0037] Example 1 Preparation of main reagent solutions

[0038] (1) Victoria blue solution: Dissolve 2 g of Victoria blue, 0.5 g of dextrin, and 4 g of resorcinol in 200 mL of distilled water, dissolve and boil for 3 - 5 min, then add 25 mL of 30% ferric chloride and continue boiling for 3 min. After cooling, filter, bake the filter paper and sediment in an oven at 60°C until dry, add 400 mL of 70% alcohol to dissolve the sediment (it can be placed in a water bath at 80°C to promote dissolution). After the solution cools, add 4 mL of glacial acetic acid and 6 g of phenol, and it can be used after maturing at room temperature for one week.

[0039] (2) Gordon-Sweets ammoniacal silver solution: Dropwise add ammonium hydroxide solution into 2 mL of 10% silver nitrate aqueous solution while shaking. First, a precipitate appears. Continue to dropwise add ammonium hydroxide until the precipitate just completely dissolves. Dropwise add 2 mL of 3% sodium hydroxide, and a precipitate forms again. Continue to dropwise add ammonium hydroxide until the precipitate just dissolves again. Finally, add distilled water to 40 mL, store it in a brown sand bottle, place it in the refrigerator for storage, and take it out to return to room temperature before use.

[0040] Example 2 Staining of liver tissue specimens

[0041] 1. Dewax the liver tissue section A to water, shake off the water, dropwise stain with 0.5% potassium permanganate for 8 minutes, and wash slightly with distilled water;

[0042] 2. Add 2% oxalic acid solution to decolorize for 1 - 2 min until the tissue section becomes colorless, and wash it with distilled water;

[0043] 3. Place the section in 70% alcohol for a moment, then put the section into the Victoria blue staining cylinder, and place the staining cylinder in a 37°C water bath for staining for 30 min;

[0044] 4. Take out the section, wash it slightly with water, observe the staining condition, then place the section in 70% alcohol for differentiation for 5 seconds, and wash it with distilled water;

[0045] 5. Add 4% ammonium ferric sulfate solution and let it act for 5 min, discard the liquid, and wash it with distilled water;

[0046] 6. Add Gordon - Sweets silver ammonia solution and let it act for 1 min, discard the liquid, and wash it with distilled water;

[0047] 7. Reduce with 4% neutral formaldehyde for 1 - 2 min, and wash it with distilled water;

[0048] 8. Dry by baking and mount the section.

[0049] Example 3 Staining of Lung Tissue Specimen

[0050] 1. Dewax the lung tissue section A, shake off the moisture, add 0.5% potassium permanganate solution and oxidize for 8 minutes, and wash it slightly with distilled water;

[0051] 2. Add 2% oxalic acid solution to decolorize for 1 min until the tissue section becomes colorless, and wash it with distilled water;

[0052] 3. Place the section in 70% alcohol for a moment, then put the section into the Victoria blue staining cylinder, and place the staining cylinder in a 37°C water bath for staining for 30 min;

[0053] 4. Take out the section, wash it slightly with water, observe the staining condition, then place the section in 70% alcohol for differentiation for 5 seconds, and wash it with distilled water;

[0054] 5. Add 4% ammonium ferric sulfate solution and let it act for 5 min, discard the liquid, and wash it with distilled water;

[0055] 6. Add Gordon - Sweets silver ammonia solution and let it act for 1 min, discard the liquid, and wash it with distilled water;

[0056] 7. Reduce with 4% neutral formaldehyde for 1 - 2 min, and wash it with distilled water;

[0057] 8. Dry by baking and mount the section.

[0058] Comparative Example 1

[0059] 1. Dewax the liver tissue section B, shake off the moisture, add 0.5% potassium permanganate solution and oxidize for 10 minutes, and wash it with distilled water;

[0060] Decolorize with 2.2% oxalic acid for 1 min and wash with distilled water until clean;

[0061] 5. Rinse the section in 70% alcohol for a moment, then place the section in a Victoria blue staining jar and place the staining jar in a 37°C water bath for 30 min;

[0062] 4. Take out the section, wash it slightly with water, observe the staining condition, then differentiate the section in 70% alcohol for a few seconds and wash with distilled water until clean;

[0063] 5. Drain the water, drip eosin stain solution for counterstaining for 5 min, differentiate with 95% alcohol, and wash with distilled water until clean;

[0064] 6. Dry by baking and mount the slide.

[0065] Comparative Example 2

[0066] 1. Dewax the lung tissue section B, drain the water, drip 0.5% potassium permanganate for oxidation for 8 minutes, and wash slightly with distilled water;

[0067] 2. Decolorize with 2.2% oxalic acid for 1 min and wash with distilled water until clean;

[0068] 3. Rinse the section in 70% alcohol for a moment, then place the section in a Victoria blue staining jar and place the staining jar in a 37°C water bath for 30 min;

[0069] 4. Take out the section, wash it slightly with water, observe the staining condition, then differentiate the section in 70% alcohol for a few seconds and wash with distilled water until clean;

[0070] 5. Drain the water, drip eosin stain solution for counterstaining for 5 min, differentiate with 95% alcohol, and wash with distilled water until clean;

[0071] 6. Dry by baking and mount the slide.

[0072] Comparative Example 3

[0073] 1. Dewax the liver tissue section C, drain the water, drip 0.5% potassium permanganate for oxidation for 10 minutes, and wash slightly with distilled water;

[0074] 2. Dropwise add 2% oxalic acid for decolorization for 1 min and wash with distilled water until clean;

[0075] 3. Dropwise add 4% ammonium ferric sulfate solution for 5 min, discard the liquid, and wash with distilled water until clean;

[0076] 4. Dropwise add Gordon-Sweets silver ammonia solution and let it act for 1 min, discard the liquid, and wash with distilled water until clean;

[0077] 5. Dropwise add 4% neutral formaldehyde for reduction for 1 - 2 min and wash with distilled water until clean;

[0078] 6. Dry and mount the slides.

[0079] Comparative Example 4

[0080] 1. Dewax the lung tissue section C, spin-dry the water, stain with 0.5% potassium permanganate for 10 minutes for oxidation, and wash with distilled water;

[0081] 2. Add 2% oxalic acid dropwise for decolorization for 1 min, and wash with distilled water;

[0082] 3. Add 4% ammonium ferric sulfate solution dropwise for 5 min, discard the liquid, and wash with distilled water;

[0083] 4. Add Gordon-Sweets silver ammonia solution dropwise and let it act for 1 min, discard the liquid, and wash with distilled water;

[0084] 5. Add 4% neutral formaldehyde dropwise for reduction for 1 - 2 min, and wash with distilled water;

[0085] 6. Dry and mount the slides.

[0086] Evaluation of Staining Effect in Test Example 1

[0087] (1) Staining effect diagram

[0088] Take liver tissue specimens with the same wax block number, cut three consecutive sections, each 3 μm thick, and label them as section A, section B, and section C respectively. Stain the three liver tissue sections with the Victoria blue-silver ammonia solution staining method of the present invention, the Victoria blue single staining method, and the silver ammonia solution single staining method respectively. The specific staining processes correspond to Example 2, Comparative Example 1, and Comparative Example 3. The results of the three liver tissue sections after staining are as Figure 1 、 Figure 2 and Figure 3 shown.

[0089] Figure 1 is the staining diagram of liver tissue section A. It can be seen from Figure 1 that the section can show three kinds of fibrous tissues, among which elastic fibers are blue, reticular fibers are black, collagen fibers are yellowish-green, and cell nuclei are grayish-black to brownish-black.

[0090] Figure 2 is the staining diagram of liver tissue section B. It can be seen from Figure 2 that the section can only show that elastic fibers are purple-blue and other tissue structures are pink.

[0091] Figure 3 is the staining diagram of liver tissue section C. It can be seen from Figure 3 that the section shows that reticular fibers are black, collagen fibers are yellow, and cell nuclei are grayish-black.

[0092] Compare Figure 1 、Figure 2 and Figure 3 For the color rendering effect of the stained fibrous tissue, it can be clearly seen that the staining method of the present invention can stain elastic fibers, reticular fibers and collagen fibers simultaneously on the section and render the structure of the cell nucleus.

[0093] (2) Evaluation of staining effect

[0094] Evaluate the staining effect of the above-mentioned sections. After the sections were stained, two senior pathologists observed each section under the microscope using the single-blind method and scored them respectively from four aspects: accurate positioning, clear staining, clean background, and distinct contrast.

[0095] The statistical results were analyzed using SPSS 17.0 for the measurement data in this article. The statistical results are shown in Table 2.

[0096] 1. Scoring criteria

[0097] Table 1 Histological scoring criteria

[0098]

[0099] 2. Scoring results

[0100] Combined with the scoring criteria in Table 1, the staining, background, contrast and positioning of 50 lung tissue specimens were scored and statistically analyzed using the staining method of the present invention, Victoria blue single staining method, and silver ammonia solution single staining method respectively. The statistical results are shown in Table 2. It can be seen that there is no statistically significant difference in the scoring results (P>0.05). This shows that the Victoria blue-silver ammonia solution staining of the present invention has the same staining effect as the separate Victoria blue staining and silver ammonia solution staining.

[0101] Table 2 Scoring results

[0102]

[0103]

[0104] (3) Influence of staining on the relationship between tumor cells and elastic fibers

[0105] The sections of the same part of the lung tissue were stained using the staining method of the present invention and the Victoria blue single staining method respectively. The specific staining process is shown in Example 3 and Comparative Example 2. The staining results are as Figure 4 and Figure 5 shown.

[0106] Figure 4This is the effect picture after staining the lung tissue section A with the Victoria blue - silver ammonia solution staining method of the present invention. It can be seen from the figure that elastic fibers are blue, reticular fibers are black, collagen fibers are yellow - green, and cell nuclei are gray - black to brown - black. The relationship between the cell nucleus and elastic fibers can be well presented on a single tissue section, so as to determine whether tumor cells have broken through the elastic fiber layer or the reticular fiber layer.

[0107] Figure 5 This is the effect picture after staining the lung tissue section B with the Victoria blue staining method. It can be known from the figure that elastic fibers are purple - blue, cells, matrix and other fibrous tissues are pink and not easy to distinguish. After staining with Victoria blue, only elastic fibers can be shown, and the structure of tumor cell nuclei cannot be shown. Therefore, doctors need to repeatedly compare and diagnose under the microscope in combination with immunohistochemical sections.

[0108] (4) Influence of staining on the diagnostic rate of lung cancer invading the visceral pleura

[0109] Table 3 Comparison of different staining methods for improving the diagnostic rate of lung cancer invading the visceral pleura

[0110]

[0111] Without comparing the HE staining and immunohistochemical sections, two physicians independently interpreted the slides stained with Victoria blue single staining and Victoria blue + silver ammonia solution respectively to observe whether tumor cells invaded the pleura, and recorded the results as Result 1 and Result 2. The results showed that the accuracy rates of the two doctors' interpretations of tumor cells invading the visceral pleura in the Victoria blue - silver ammonia solution staining were both 100%, which was significantly higher than that of Victoria blue single staining, and the accuracy rates were 80% and 87% respectively. The difference was statistically significant (P < 0.05), indicating that the Victoria blue - silver ammonia solution staining could better meet the diagnostic needs of clinical pathologists, improve the diagnostic accuracy rate and reduce the missed diagnosis rate.

[0112] Therefore, the Victoria blue - silver ammonia solution staining method for co - staining of three fibrous tissues of the present invention can not only clearly present reticular fibers, collagen fibers and elastic fibers on a single slide, but also show the relationship between cell structure and fibrous tissues, more easily determine the relationship of whether tumor cells have broken through the elastic fiber layer, improve the diagnostic accuracy rate, and has important significance for clinical pathological diagnosis work and is worthy of popularization and application in clinical pathological work.

[0113] In the description of this specification, the specific features, structures, materials, or characteristics disclosed may be combined in any suitable manner in any one or more embodiments or examples. Although embodiments of the present invention have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents, and all belong to the scope of protection of the present invention.

Claims

1. A staining method for the co - staining of three fibrous tissues, characterized in that, It includes the steps of successively using Victoria blue staining and Gordon-Sweets silver ammonia solution staining on the processed tissue sections, staining three types of fibers, namely elastic fibers, reticular fibers, and collagen fibers, clearly presenting them on the same section, and showing the relationship between the cell nucleus structure and elastic fibers. Among them, elastic fibers are blue, reticular fibers are black, collagen fibers are yellowish-green, and cell nuclei are grayish-black to brownish-black. Specifically, it includes the following steps: S1: Dewax, oxidize, and decolorize the tissue sections; S2: After washing the tissue sections with 70% alcohol for 1 - 10 s, place them in a Victoria blue staining cylinder, and place the staining cylinder in a 37°C water bath for 10 - 60 min; S3: Then differentiate the sections with 70% alcohol for 1 - 10 s and wash with water; S4: Then treat with 4% ammonium ferric sulfate solution and wash with water; S5: Dropwise add Gordon-Sweets silver ammonia solution and let it act for 1 - 2 min, then wash with water; S6: Dropwise add 4% neutral formaldehyde solution to reduce for 1 - 3 min and wash with water; S7: Dry by baking and mount the slides.

2. The staining method for co-staining of three kinds of fibrous tissues according to claim 1, characterized in that, The tissue sections include sections of lung tissue, liver tissue, lymph nodes, and skin tissue.

3. The staining method for co-coloring three kinds of fibrous tissues according to claim 1, characterized in that, The method of oxidation is to react with 0.5% potassium permanganate solution by mass concentration for 8 - 10 min, and then wash it clean with water.

4. The staining method for co - staining of three fiber tissues according to claim 1, characterized in that, The method of decolorization is to add 2% oxalic acid until the tissue sections become colorless, and then wash it clean with water.

5. The staining method for co - staining of three fibrous tissues according to any one of claims 1 - 4, characterized in that, More specifically, it includes the following steps: S1: Dewax the tissue sections, drain the water, dropwise stain with 0.5% potassium permanganate for oxidation for 8 - 10 minutes, and wash with distilled water; S2: Dropwise add 2% oxalic acid for decolorization for 1 - 3 min until the tissue sections become colorless, and wash with distilled water; S3: Rinse the sections in 70% alcohol for a moment, then place the sections in a Victoria blue staining cylinder, and place the staining cylinder in a 37°C water bath for staining for 30 min; S4: Take out the sections, slightly wash with water, observe the staining situation, then place the sections in 70% alcohol for differentiation for 1 - 10 s, and wash with distilled water; S5: Dropwise add 4% ammonium ferric sulfate solution for 5 min, shake off the liquid, and wash with distilled water; S6: Dropwise add Gordon-Sweets silver ammonia solution and let it act for 1 min, shake off the liquid, and wash with distilled water; S7: Dropwise add 4% neutral formaldehyde to reduce for 1 - 2 min and wash with distilled water; S8: Dry by baking and mount the slides.

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