Tumor tissue immunohistochemical detection quality control reagent and application thereof

Abstract

The invention discloses a tumor tissue immunohistochemical detection quality control reagent and application thereof. The tumor tissue immunohistochemical detection quality control reagent comprises a positive quality control reagent, a negative quality control reagent and a color card, the positive quality control reagent comprises an anti-tumor tissue protein antibody, a buffer solution, a protein protective agent, a preservative, a pigment and purified water; the negative quality control reagent comprises non-immune immune globulin, a buffer solution, a protein protective agent, a preservative, a pigment and purified water; the color card is a standard example after immunohistochemical staining on tumor tissue by using a positive quality control reagent and a negative quality control reagent. The tumor tissue immunohistochemical detection quality control reagent can ensure the accuracy of immunohistochemical detection results.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor pathology detection, and relates to a tumor tissue immunohistochemical detection quality control reagent and application thereof. Background Art

[0002] The accuracy of immunohistochemistry (Immunohistochemistry, IHC) detection reagents for tumor tissues, especially companion diagnostic (CDx) reagents used to guide drug therapy, is crucial to the treatment of patients. Setting up appropriate quality control monitoring and detection processes in tumor tissue immunohistochemistry detection can ensure the reliability of the detection and avoid the adverse effects caused by erroneous results.

[0003] Clinically, immunohistochemical detection of tumor tissues often uses negative and positive quality control slices (usually tissue or cell slices) as quality control of the detection process during the detection process to avoid detection errors caused by improper operation during the detection process; at the same time, antibody diluent is used as a negative quality control reagent for detection of the test tissue to monitor whether there is non-specific staining of the sample during the detection process. Although the settings of these quality control slices or quality control reagents provide prompts for some errors in immunohistochemical detection, they cannot cover all possible problems. For example: protein loss caused by pretreatment or improper storage of samples; protein loss, non-specific staining, uneven staining and weakened staining signals caused by improper operation during the staining process. Therefore, the present invention provides a quality control reagent that can monitor the sample preparation, storage and detection of immunohistochemical staining of tumor tissues, provide comprehensive quality control information, and ensure the accuracy of the test results. Summary of the invention

[0004] In view of the deficiencies in the prior art and actual needs, the present invention provides a tumor tissue immunohistochemical detection quality control reagent and its application. The present invention arranges positive quality control reagents and negative quality control reagents for the entire process of sample preparation, preservation and detection, and monitors the entire detection process to ensure the accuracy of immunohistochemical detection.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a quality control reagent for immunohistochemical detection of tumor tissue, which comprises a positive quality control reagent, a negative quality control reagent and a color card; the positive quality control reagent comprises an anti-tumor tissue protein antibody, a buffer, a protein protectant, a preservative, a pigment and purified water; the negative quality control reagent comprises non-immune immunoglobulin, a buffer, a protein protectant, a preservative, a pigment and purified water; the color card is a standard example after immunohistochemical staining on tumor tissue using a positive quality control reagent and a negative quality control reagent.

[0007] The tumor tissue immunohistochemical detection quality control reagent of the present invention can ensure the accuracy of the immunohistochemical detection results. The positive quality control reagent can confirm whether there are abnormalities or false negative results in sample preparation, storage and immunohistochemical staining; the negative quality control reagent is used to exclude whether there are false positive results in samples and immunohistochemical staining; the color card can evaluate the staining deviation of the staining process.

[0008] Preferably, the tumor tissue protein antibodies include but are not limited to: anti-cytoskeleton protein antibodies, anti-cell surface glycoprotein antibodies, anti-cell cycle protein antibodies, anti-cell proliferation-related protein antibodies, anti-gene mutation protein antibodies, anti-tumor suppressor protein antibodies, anti-metastasis-related protein antibodies or anti-neural protein antibodies, any one or a combination of at least two of them.

[0009] Preferably, in breast cancer, the tumor tissue protein antibody includes but is not limited to any one or a combination of at least two of anti-VEGF, Ki-67, p53, ER, PR, HER2, EGFR, CK, MUC1, TRPS1 or SOX10 antibodies.

[0010] Preferably, in colorectal cancer, the tumor tissue protein antibody includes but is not limited to any one or a combination of at least two of anti-Ki-67, p53, CK20, CDX2, β-catenin, MLH1, MSH2, MSH6, PMS2 or MUC1 antibodies.

[0011] Preferably, in melanoma tumors, the tumor tissue protein antibodies include but are not limited to any one or a combination of at least two of the anti-VEGF, Ki-67, p53, S100, Tyrosinase or HMB-45 proteins.

[0012] Preferably, the final concentration of the tumor tissue protein antibody is 0.1-10 μg / mL (eg, 0.5 μg / mL, 1 μg / mL and 5 μg / mL).

[0013] Preferably, the non-immune immunoglobulin includes but is not limited to any one of mouse anti-human IgG protein, rabbit anti-human IgG protein or goat anti-human IgG protein, or a combination of at least two thereof.

[0014] Preferably, the final concentration of the non-immune immunoglobulin is 0.1-10 μg / mL (eg, 0.5 μg / mL, 1 μg / mL, and 5 μg / mL).

[0015] Preferably, the buffer comprises: any one of Tris-HCL buffer, TBS buffer, phosphate buffer, HEPES buffer, MES buffer, ACES buffer or citric acid buffer, or a combination of at least two thereof.

[0016] Preferably, the protein protective agent comprises: any one or a combination of at least two of bovine serum albumin, sucrose, gelatin, amino acids, Tween, casein, mannitol, sorbitol, inositol, thiol or polyethylene glycol.

[0017] Preferably, the protein protective agent is a combination of bovine serum albumin, sucrose, Triton X-100 and Tween-20.

[0018] Preferably, the mass percentage of the protein protectant in the positive quality control reagent and the negative quality control reagent is independently 0.1%-2% (eg, 0.1%, 0.2%, 0.4%, 0.8%, 1%, 1.5%, 2%).

[0019] Preferably, the preservative comprises any one of sodium azide, ProClin 300 or ProClin 950, or a combination of at least two thereof.

[0020] Preferably, the mass percentage of sodium azide in the positive quality control reagent and the negative quality control reagent is independently 0-0.1% (eg, 0.01%, 0.02%, 0.04%, 0.08%, 0.1%).

[0021] Preferably, the mass percentage of ProClin300 in the positive quality control reagent and the negative quality control reagent is independently 0-0.1% (eg, 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%).

[0022] Preferably, the mass percentage of ProClin 950 in the positive quality control reagent and the negative quality control reagent is independently 0-0.3% (eg, 0.01%, 0.02%, 0.04%, 0.08%, 0.1%, 0.2%, 0.3%).

[0023] Preferably, the pigment includes any one of tender leaf green, lemon yellow, brilliant blue or sunset yellow, or a combination of at least two thereof.

[0024] Preferably, the final concentration of the pigment is 0.01-0.03 mg / mL (eg, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL).

[0025] In a second aspect, the present invention provides a tumor tissue immunohistochemical detection kit, wherein the tumor tissue immunohistochemical detection kit contains the tumor tissue immunohistochemical detection quality control reagent described in the first aspect.

[0026] In a third aspect, the present invention provides the use of the tumor tissue immunohistochemistry detection quality control reagent described in the first aspect in antigen expression level detection and / or tissue damage detection.

[0027] In a fourth aspect, the present invention provides a method for immunohistochemical detection of tumor tissue, the method comprising: fixing, serially dehydrating, transparentizing, embedding, making continuous sections, staining, sealing and pathological interpretation of the sample to be tested, selecting at least 3 continuous sections for pathological interpretation, 2 of which are stained with the tumor tissue immunohistochemical detection quality control reagent described in the first aspect for the sample to be tested, and the remaining sections are stained with the antibody to be tested.

[0028] Preferably, the criteria for pathological interpretation are:

[0029] (1) First, the staining results of the positive quality control reagent and the negative quality control reagent are interpreted. If the result is that the positive quality control reagent shows positive, the negative quality control reagent shows negative, the color card comparison result shows that the staining characteristics are consistent with the color card, the quality control result shows that the section quality is reliable, and the staining process meets the requirements, then it is judged that the test is accurate and the specific test result can be guaranteed; if the result is any of the following, it is judged that the test result cannot guarantee accuracy and the test fails:

[0030] (a) The positive quality control reagent shows positive, the negative quality control reagent shows negative, the color card comparison result shows that the staining characteristics are inconsistent with the color card, and the quality control result shows that the section quality is abnormal or the staining process is abnormal;

[0031] (b) The positive quality control reagent shows positive, the negative quality control reagent shows positive, the color card comparison result shows that it does not meet the staining requirements, and the quality control result shows that the section quality is abnormal or the staining process is abnormal;

[0032] (c) The positive quality control reagent shows negative, the negative quality control reagent shows negative, the color card comparison result shows that it does not meet the staining requirements, and the quality control result shows that the section quality is abnormal or the staining process is abnormal;

[0033] (d) The positive quality control reagent shows negative, the negative quality control reagent shows positive, the color card comparison result does not meet the staining requirements, and the quality control result shows abnormal section quality or abnormal staining process;

[0034] (2) The interpretation standard of the staining results of the test antibody is: when the quality control results show that the test is accurate and the specific test results can be guaranteed, if a specific staining signal is present in the tissue cells, the test sample is judged to be positive; if there is no specific staining and background coloring in the tissue cells, the test sample is judged to be negative.

[0035] As a preferred technical solution, the present invention provides a method for immunohistochemical detection of tumor tissue, the method comprising the following steps:

[0036] (1) Sample preparation: The in vitro samples to be tested are fixed with neutral formalin for 24-48 hours. The fixed tissue samples are immersed in 50%, 70%, 80%, 90%, and 100% ethanol in turn. The immersion time for each alcohol concentration is 1-2 hours. The dehydrated tissue samples are immersed in xylene for 1-2 hours for transparency. The transparent tissue samples are immersed in molten paraffin and treated with an equal amount of paraffin and xylene mixed solution and pure paraffin for 3-4 times in turn. The total time is 3-4 hours. The wax-immersed samples are placed in an embedding box and then poured with molten paraffin to completely cover them. After the paraffin cools and solidifies, the samples will be fixed in the paraffin block. The paraffin-embedded tissue samples will be cut into 4 consecutive slices with a thickness of 4μm on a microtome, adhered to a slide that prevents shedding, and baked at 65℃ for 60-80min. The baked slices are stored at -20℃±5℃;

[0037] (2) HE staining: One of the consecutive sections was stained with HE, dried at 65-68°C for 30-40 min, soaked in xylene twice, each time for 15-20 min, soaked in 100% ethanol for 5-8 min, soaked in 100% ethanol for 5-8 min, soaked in 95-98% ethanol for 5-8 min, soaked in 75-78% ethanol for 5-8 min, washed with distilled water 2-3 times, each time for 2-3 min; stained with hematoxylin solution for 2-7 min, washed with running water for 2-4 min. times, differentiate with 1-2% hydrochloric acid ethanol for 2-3s, rinse with running water 2-3 times, 2-3s each time, soak in water for 2-5min to turn blue, soak in 80-85% ethanol for 15-20s, soak in eosin for 15-20s; soak in 95-98% ethanol for 15-20s, soak in 100% ethanol for 1-2min, soak in 100% ethanol for 1-2min; soak in xylene twice, 2-5min each time; use neutral gum to seal the slide, the seal must be flat, without bubbles or overflowing glue;

[0038] (3) Immunohistochemical staining: The remaining three sections in the serial sections were stained in parallel with positive quality control reagent, negative quality control reagent and the antibody to be tested;

[0039] (4) Staining interpretation:

[0040] First, the staining results of the positive quality control reagent and the negative quality control reagent are interpreted. If the result is that the positive quality control reagent shows positive, the negative quality control reagent shows negative, the color card comparison result shows that the staining characteristics are consistent with the color card, the quality control result shows that the section quality is reliable, and the staining process meets the requirements, then it is judged that the test is accurate and the specific test result can be guaranteed; if the result is any of the following, it is judged that the test result cannot guarantee accuracy and the test fails:

[0041] (a) The positive quality control reagent shows positive, the negative quality control reagent shows negative, the color card comparison result shows that the staining characteristics are inconsistent with the color card, and the quality control result shows that the section quality is abnormal or the staining process is abnormal;

[0042] (b) The positive quality control reagent shows positive, the negative quality control reagent shows positive, the color card comparison result shows that it does not meet the staining requirements, and the quality control result shows that the section quality is abnormal or the staining process is abnormal;

[0043] (c) The positive quality control reagent shows negative, the negative quality control reagent shows negative, the color card comparison result shows that it does not meet the staining requirements, and the quality control result shows that the section quality is abnormal or the staining process is abnormal;

[0044] (d) The positive quality control reagent shows negative, the negative quality control reagent shows positive, the color card comparison result does not meet the staining requirements, and the quality control result shows abnormal section quality or abnormal staining process;

[0045] When the quality control result is that the test is accurate and the specific test result can be guaranteed, and a specific staining signal is present in the tissue cells, the sample to be tested is judged to be positive;

[0046] When the quality control result is that the test is accurate and the specific test result can be guaranteed, and there is no specific staining and background coloring in the tissue cells, the sample to be tested is judged to be negative.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The tumor tissue immunohistochemical detection quality control reagent of the present invention uses a positive quality control reagent and a negative quality control reagent to perform quality control on the detection of the sample to be detected to ensure the accuracy of the immunohistochemical detection result. The positive quality control reagent can confirm whether the sample preparation, storage and immunohistochemical staining have abnormalities or false negative results; the negative quality control reagent is used to exclude whether the sample and immunohistochemical staining have false positive results; the color card can evaluate the staining deviation of the staining process. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a staining diagram of the positive quality control reagent in colorectal cancer tissue in Example 1;

[0050] Figure 2 This is a staining diagram of the negative quality control reagent in colorectal cancer tissue in Example 1;

[0051] Figure 3 This is the staining image of MMR (MLH1, PMS2, MSH2 and MSH6) in colorectal cancer tissue in Example 1. DETAILED DESCRIPTION

[0052] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0053] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0054] Example 1

[0055] This embodiment provides a quality control reagent for colorectal cancer tissue immunohistochemistry detection, including a positive quality control reagent, a negative quality control reagent and a color card, as follows:

[0056] Positive quality control reagents include mouse anti-human CK20 antibody with a final concentration of 0.3 μg / mL, mouse anti-human Ki-67 antibody with a final concentration of 0.3 μg / mL, mouse anti-human MLH1 antibody with a final concentration of 0.3 μg / mL, 0.137 M NaCl, 2.7 mM KCl, 1.8 mM sodium dihydrogen phosphate, 0.01 M sodium dihydrogen phosphate, 1% bovine serum albumin, 0.25% sucrose, 0.05% Triton X-100, 0.05% Tween-20, 0.05% sodium azide, 0.01 mg / mL young leaf green and process water;

[0057] Negative quality control reagents include mouse anti-human IgG antibody with a final concentration of 0.9 μg / mL, 0.137 M NaCl, 2.7 mM KCl, 1.8 mM sodium dihydrogen phosphate, 0.01 M sodium dihydrogen phosphate, 1% bovine serum albumin, 0.25% sucrose, 0.05% Triton X-100, 0.05% Tween-200, 0.05% sodium azide, 0.01 mg / mL brilliant blue and process water;

[0058] The color chart is a standard example of immunohistochemical staining of rectal cancer tissue using positive and negative control reagents.

[0059] Example 2

[0060] This embodiment provides a parathyroid carcinoma tissue immunohistochemical detection quality control reagent, including a positive quality control reagent, a negative quality control reagent and a color card, as follows:

[0061] Positive quality control reagents include mouse anti-human GATA3 antibody with a final concentration of 0.6 μg / mL, mouse anti-human PR antibody with a final concentration of 0.3 μg / mL, 10 mM Tris, 40 mM Tris-HCL, 1% bovine serum albumin, 0.05% ProClin300, 0.02 mg / mL tartrazine, and process water;

[0062] Negative quality control reagents include mouse anti-human IgG antibody with a final concentration of 0.9 μg / mL, 10 mM Tris, 40 mM Tris-HCL, 1% bovine serum albumin, 0.05% ProClin300, 0.02 mg / mL brilliant blue and process water.

[0063] The color chart is a standard example of immunohistochemical staining of parathyroid carcinoma tissue using positive and negative control reagents.

[0064] Example 3

[0065] This embodiment provides a quality control reagent for immunohistochemistry detection of melanoma tumor tissue, including a positive quality control reagent, a negative quality control reagent and a color card, as follows:

[0066] Positive quality control reagents include rabbit anti-human S100 antibody with a final concentration of 0.3μg / mL, mouse anti-human Tyrosinase antibody with a final concentration of 0.3μg / mL, mouse anti-human HMB-45 antibody with a final concentration of 0.3μg / mL, 0.137MNaCl, 2.7mM KCl, 1.8mM sodium dihydrogen phosphate, 0.01M sodium dihydrogen phosphate, 0.1% ProClin 950, 0.03mg / mL sunset yellow and process water;

[0067] Negative quality control reagents include rabbit anti-human IgG antibody with a final concentration of 0.3 μg / mL, mouse anti-human IgG antibody with a final concentration of 0.6 μg / mL, 0.137 M NaCl, 2.7 mM KCl, 1.8 mM sodium dihydrogen phosphate, 0.01 M sodium dihydrogen phosphate, 0.1% ProClin 950, 0.03 mg / mL brilliant blue and process water.

[0068] The color chart is a standard example of immunohistochemical staining of melanoma tumor tissue using positive and negative quality control reagents.

[0069] Example 4

[0070] This embodiment provides quality control reagents for immunohistochemistry detection of breast cancer tumor tissue, including positive quality control reagents, negative quality control reagents and color cards, as follows:

[0071] Positive quality control reagents include rabbit anti-human VEGF antibody, 0.2μg / mL rabbit anti-human Ki-67 antibody, 0.1μg / mL mouse anti-human p53 antibody, 0.2μg / mL rabbit anti-human ER antibody, 0.1μg / mL mouse anti-human PR antibody, 0.1μg / mL rabbit anti-human HER2 antibody, 0.2μg / mL rabbit anti-human EGFR antibody, 0.1μg / mL mouse anti-human CK5 / 6 antibody, 0.2μg / mL rabbit anti-human MUC1 antibody, 0.2μg / mL rabbit anti-human TRPS1 antibody, 0.2μg / mL rabbit anti-human SOX10 antibody, 0.137M NaCl, 2.7mM KCl, 1.8mM sodium dihydrogen phosphate, 0.01M sodium dihydrogen phosphate, 1% gelatin, 0.05% sodium azide, 0.01mg / mL sunset yellow and process water;

[0072] Negative quality control reagents include rabbit anti-human IgG antibody with a final concentration of 1.5 μg / mL, mouse anti-human IgG antibody with a final concentration of 0.3 μg / mL, 0.137M NaCl, 2.7mM KCl, 1.8mM sodium dihydrogen phosphate, 0.01M sodium dihydrogen phosphate, 1% gelatin, 0.05% sodium azide, 0.01mg / mL brilliant blue and process water.

[0073] The color chart is a standard example of immunohistochemical staining of breast cancer tumor tissue using positive and negative control reagents.

[0074] Test Example 1

[0075] The tumor tissue immunohistochemical detection quality control reagent of Example 4 was used to perform PD-L1 immunohistochemical detection of breast cancer tumor tissue.

[0076] 1. Sample Preparation

[0077] (1) The breast cancer specimens removed by surgery were cut into pieces of 10 mm*5 mm*3 mm in size and fixed in 20 volumes of 10% neutral formalin for 48 h within 30 min of ex vivo treatment.

[0078] (2) The fixed tissue samples were immersed in 50%, 70%, 80%, 90%, and 100% ethanol in turn to remove moisture from the samples. The immersion time for each alcohol concentration was 1 h.

[0079] (3) Soak the dehydrated tissue samples in xylene for 2 h to remove the alcohol and make the tissue transparent.

[0080] (4) The transparent tissue sample was immersed in molten paraffin and treated with an equal amount of a mixture of paraffin and xylene and pure paraffin in turn. The pure paraffin treatment was repeated three times to ensure that the paraffin fully penetrated into the tissue, for a total of 3 hours.

[0081] (5) Place the wax-soaked sample in an embedding box and then pour in molten paraffin to completely cover it. Once the paraffin cools and solidifies, the sample will be fixed in the paraffin block.

[0082] (6) The paraffin-embedded tissue sample was cut into four consecutive sections with a thickness of 4 μm using a microtome and adhered to a non-shedding slide.

[0083] (7) Bake the slices at 65°C for 60 min.

[0084] 2. HE staining

[0085] One of the consecutive sections was stained with HE.

[0086] (1) Dewaxing and hydration: bake the slides at 65°C for 30 min, soak in xylene twice for 15 min each time, soak in 100% ethanol for 5 min, soak in 100% ethanol for 5 min, soak in 95% ethanol for 5 min, soak in 75% ethanol for 5 min, and wash with distilled water three times for 3 min each time.

[0087] (2) Staining: Stain with hematoxylin solution for 7 min, wash the hematoxylin with running water, rinse 4 times, differentiate with 1% hydrochloric acid ethanol for 3 s, rinse with running water 3 times, 3 s each time, soak in water for 5 min to turn blue, soak in 80% ethanol for 20 s, and soak in eosin for 20 s.

[0088] (3) Dehydration: soak in 95% ethanol for 20 seconds, soak in 100% ethanol for 2 minutes, and soak in 100% ethanol for 2 minutes.

[0089] (4) Soak in xylene twice, 5 min each time.

[0090] (5) Sealing: Use neutral gum to seal the slide. The seal must be flat without bubbles or glue overflow.

[0091] 3. Immunohistochemical Staining

[0092] The remaining three sections in the serial sections were stained in parallel with positive quality control reagent, negative quality control reagent and PD-L1 antibody.

[0093] (1) Reagent preparation

[0094] Cleaning Solution EZ Prep Concentrate (10×): Use distilled or deionized water (reagent grade water) to dilute Cleaning Solution EZ Prep Concentrate (10×) at a ratio of 1:10 to prepare enough Cleaning Solution EZ Prep Concentrate (1×) for use in the cleaning step.

[0095] Reaction Buffer Concentrate (10×): Use distilled or deionized water (reagent grade water) to dilute the Reaction Buffer Concentrate (10×) at a ratio of 1:10 to prepare enough Reaction Buffer Concentrate (1×) for the cleaning step.

[0096] (2) Set up the staining program

[0097] Set the program on the Ventana fully automated immunohistochemistry staining system BenchMarkULTRA, check "Antibody Titration" and set the primary antibody incubation time to 20 minutes. The staining program settings are shown in Table 1 below.

[0098] Table 1

[0099]

[0100]

[0101] (3) Sample preparation before loading

[0102] Place the sample slices in a 65°C oven for 60 minutes, take them out and cool them to room temperature, soak them in 10% whole milk for 10 minutes, and rinse them with tap water twice. Select the above staining program, print the labels, and prepare for staining on the machine.

[0103] (4) On-machine testing

[0104] Label the three sections and load them onto the instrument. Run the instrument. When the instrument reaches the primary antibody incubation step, manually add 150 μL of reagent, one using the positive quality control reagent, one using the negative quality control reagent, and one using the PD-L1 antibody reagent.

[0105] (5) Sealing

[0106] After the staining process is completed, the sections are removed and the oil film is washed off; after dehydration, transparency, and sealing, they are stored at room temperature away from light.

[0107] 4. Staining Interpretation

[0108] (1) HE staining

[0109] The sections should be complete, without bubbles, breaks, warping or falling off, with natural color and uniform staining, clear tissue structure, no obvious background impurities, and the tissue should contain no less than 100 living tumor cells.

[0110] (2) Positive quality control reagent staining

[0111] In tissues stained with positive quality control reagents, tumor cells and / or immune cells should show yellow, brown cell membrane and / or cytoplasm and / or nucleus staining signals, proving that no protein is lost during the preparation, preservation and staining of tissue sections, and that the staining process is not affected by manipulation or results in false negatives.

[0112] (3) Negative quality control reagent staining

[0113] The tissue cells stained with negative control reagents had no specific staining and background coloring, proving that the tissue staining was not interfered by nonspecific staining and there was no false positive caused by manipulation during the staining process.

[0114] (4) Color card comparison

[0115] Compare the stained sections of (2) and (3) with the color chart. The stained cell types and locations should be consistent with the color chart.

[0116] (5) PD-L1 antibody reagent staining

[0117] After the staining of steps (1) to (4) is completely consistent, the result of PD-L1 antibody reagent staining is the accurate PD-L1 expression of the tissue.

[0118] Results: The tumor tissue immunohistochemical detection quality control reagents of Examples 1-4 of the present invention can ensure the accuracy of the immunohistochemical detection results. The positive quality control reagents can confirm whether there are abnormalities or false negative results in sample preparation, storage and immunohistochemical staining; the negative quality control reagents are used to exclude whether there are false positive results in samples and immunohistochemical staining; the color card can evaluate the staining deviation of the staining process.

[0119] The detection results of the positive quality control reagent in colorectal cancer tissue in Example 1 are as follows: Figure 1 As shown, the detection results of the negative quality control reagent in colorectal cancer tissue in Example 1 are as follows Figure 2 The detection results of MMR (MLH1, PMS2, MSH2 and MSH6) in colorectal cancer tissue in Example 1 are shown as follows: Figure 3 As shown, the detection result of this sample is MSI-H.

[0120] In summary, the tumor tissue immunohistochemical detection quality control reagent of the present invention can ensure the accuracy of the immunohistochemical detection results. The positive quality control reagent can confirm whether there are abnormalities or false negative results in sample preparation, storage and immunohistochemical staining; the negative quality control reagent is used to exclude whether there are false positive results in samples and immunohistochemical staining; the color card can evaluate the staining deviation of the staining process.

[0121] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A quality control reagent for immunohistochemical detection of tumor tissue, characterized in that: The tumor tissue immunohistochemistry detection quality control reagents include positive quality control reagents, negative quality control reagents and color cards; The positive quality control reagent includes anti-tumor tissue protein antibody, buffer, protein protectant, preservative, pigment and purified water; The negative quality control reagent includes non-immune immunoglobulin, buffer, protein protective agent, preservative, pigment and purified water; The color card is a standard example of immunohistochemical staining on tumor tissue using positive quality control reagents and negative quality control reagents.

2. The tumor tissue immunohistochemistry detection quality control reagent according to claim 1, characterized in that: The anti-tumor tissue protein antibodies include but are not limited to: any one or a combination of at least two of anti-cytoskeleton protein antibodies, anti-cell surface glycoprotein antibodies, anti-cell cycle protein antibodies, anti-cell proliferation-related protein antibodies, anti-gene mutation protein antibodies, anti-tumor suppressor protein antibodies, anti-metastasis-related protein antibodies or anti-neural protein antibodies; Preferably, in breast cancer, the tumor tissue protein antibody includes but is not limited to any one or a combination of at least two of anti-VEGF, Ki-67, p53, ER, PR, HER2, EGFR, CK, MUC1, TRPS1 or SOX10 antibodies; Preferably, in colorectal cancer, the tumor tissue protein antibody includes but is not limited to any one or a combination of at least two of anti-Ki-67, p53, CK20, CDX2, β-catenin, MLH1, MSH2, MSH6, PMS2 or MUC1 antibodies; Preferably, in melanoma tumors, the tumor tissue protein antibodies include but are not limited to any one or a combination of at least two of anti-VEGF, Ki-67, p53, S100, Tyrosinase or HMB-45 proteins; Preferably, the final concentration of the tumor tissue protein antibody is 0.1-10 μg / mL; Preferably, the non-immune immunoglobulin includes but is not limited to any one or a combination of at least two of mouse anti-human IgG protein, rabbit anti-human IgG protein or goat anti-human IgG protein; Preferably, the final concentration of the non-immune immunoglobulin is 0.1-10 μg / mL.

3. The tumor tissue immunohistochemistry detection quality control reagent according to claim 1 or 2, characterized in that: The buffer comprises any one of Tris-HCL buffer, TBS buffer, phosphate buffer, HEPES buffer, MES buffer, ACES buffer or citric acid buffer, or a combination of at least two thereof.

4. The tumor tissue immunohistochemistry detection quality control reagent according to any one of claims 1 to 3, characterized in that: The protein protective agent comprises: any one or a combination of at least two of bovine serum albumin, sucrose, gelatin, amino acids, Tween, casein, mannitol, sorbitol, inositol, thiol or polyethylene glycol; Preferably, the protein protective agent comprises a combination of bovine serum albumin, sucrose, TritonX-100 and Tween-20; Preferably, the mass percentage of the protein protective agent in the positive quality control reagent and the negative quality control reagent is independently 0.1%-2%.

5. The tumor tissue immunohistochemistry detection quality control reagent according to any one of claims 1 to 4, characterized in that: The preservative includes: any one of sodium azide, ProClin 300 or ProClin 950, or a combination of at least two thereof; Preferably, the mass percentage of sodium azide in the positive quality control reagent and the negative quality control reagent is independently 0-0.1%; Preferably, the mass percentage of ProClin300 in the positive quality control reagent and the negative quality control reagent is independently 0-0.1%; Preferably, the mass percentage of ProClin 950 in the positive quality control reagent and the negative quality control reagent is independently 0-0.3%.

6. The tumor tissue immunohistochemistry detection quality control reagent according to any one of claims 1 to 5, characterized in that: The pigment includes any one of tender leaf green, lemon yellow, brilliant blue or sunset yellow, or a combination of at least two thereof; Preferably, the final concentration of the pigment is 0.01-0.03 mg / mL.

7. A tumor tissue immunohistochemical detection kit, characterized in that: The tumor tissue immunohistochemistry detection kit contains the tumor tissue immunohistochemistry detection quality control reagent according to any one of claims 1 to 6.

8. Use of the tumor tissue immunohistochemistry detection quality control reagent according to any one of claims 1 to 6 in antigen expression level detection and / or tissue damage detection.

9. A method for immunohistochemical detection of tumor tissue, characterized in that: The method comprises: fixing, serially dehydrating, transparentizing, embedding, preparing continuous sections, staining, sealing and pathological interpretation of the sample to be tested, selecting at least 3 continuous sections for pathological interpretation, wherein 2 of the sections are stained with the tumor tissue immunohistochemistry detection quality control reagent described in any one of claims 1 to 6 for the sample to be tested, and the remaining sections are stained with the antibody to be tested.

10. The tumor tissue immunohistochemical detection method according to claim 9, characterized in that: The criteria for pathological interpretation are: (1) First, the staining results of the positive quality control reagent and the negative quality control reagent are interpreted. If the result is that the positive quality control reagent shows positive, the negative quality control reagent shows negative, the color card comparison result shows that the staining characteristics are consistent with the color card, the quality control result shows that the section quality is reliable, and the staining process meets the requirements, then it is judged that the test is accurate and the specific test result can be guaranteed; if the result is any of the following, it is judged that the test result cannot guarantee accuracy and the test fails: (a) The positive quality control reagent shows positive, the negative quality control reagent shows negative, the color card comparison result shows that the staining characteristics are inconsistent with the color card, and the quality control result shows that the section quality is abnormal or the staining process is abnormal; (b) The positive quality control reagent shows positive, the negative quality control reagent shows positive, the color card comparison result shows that it does not meet the staining requirements, and the quality control result shows that the section quality is abnormal or the staining process is abnormal; (c) The positive quality control reagent shows negative, the negative quality control reagent shows negative, the color card comparison result shows that it does not meet the staining requirements, and the quality control result shows that the section quality is abnormal or the staining process is abnormal; (d) The positive quality control reagent shows negative, the negative quality control reagent shows positive, the color card comparison result does not meet the staining requirements, and the quality control result shows abnormal section quality or abnormal staining process; (2) The interpretation standard of the staining results of the test antibody is: when the quality control result is accurate and the specificity test result can be guaranteed, if a specific staining signal is present in the tissue cells, the test sample is judged to be positive; If there is no specific staining and background coloring in tissue cells, the sample to be tested is judged to be negative.

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