Preparation method of PD-L1 immunohistochemical detection quality control product and product

Abstract

The invention discloses a preparation method of a PD-L1 immunohistochemical detection quality control product and a product, and relates to the technical field of biology. The quality control product comprises cell sections of human breast cancer cells, human prostate cancer cells and human breast duct cancer cells. The preparation method comprises the step of soaking the cell sections with a 3-7% BSA solution for 20-40 min. The pretreatment step of soaking the cell slices with bovine serum albumin is added, so that the dyeing effect is enhanced, non-specific dyeing is reduced, and the storage stability is improved. The PD-L1 quality control sheet provided by the invention contains cells expressing PD-L1 protein in different degrees, the cells are high in density and good in form, and the cells can be used as sensitivity control of PD-L1 protein expression.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and product of a PD-L1 immunohistochemistry detection quality control product. Background Art

[0002] PD-L1 is a negative immune regulator. PD-L1 is the ligand of PD-1. The combination of PD-1 and PD-L1 induces tumor cell immune escape by inhibiting T cell activation and proliferation. PD-L1 is highly expressed on the surface of many tumor cells and can also induce immune cells (especially helper T lymphocytes) to secrete immunosuppressive factors, further inhibiting the killing effect of anti-tumor immunity.

[0003] Immunohistochemistry (IHC) uses the basic principle of immunology, that is, the specific binding between antibodies and antigens for pathological detection. Immunohistochemistry is widely used in tumor diagnosis and classification due to its high accuracy and strong specificity. Immunohistochemistry is mainly divided into immunoenzyme histochemistry, immunofluorescence histochemistry, affinity immunohistochemistry and other immunohistochemistry. Immunoenzyme histochemistry is a type of detection technology that directly uses tissue sections or cells as antigens and uses enzyme-linked immunosorbent assay to detect the presence or location of antigens or antibodies. Its characteristics are accurate signal positioning, long-term preservation of specimens and simple methods. The principle of immunofluorescence histochemistry is to label the inhibitory antibody with fluorescein, and then use this fluorescent antibody to detect the corresponding antigen in the tissue or cell. Affinity immunohistochemistry uses the strong affinity between biotin and substances such as avidin or avidin.

[0004] Chinese patent CN115508168A discloses a method for preparing an immunohistochemical quality control product and its application, comprising the following steps: S1: culturing tumor cell lines separately; S2: preparing cell blocks; S3: preparing corresponding color block; S4: fixing and dehydrating the cell blocks and color block, and then waxing and embedding the cell blocks and color block together according to a color block corresponding to each tumor cell block, so as to obtain a cell wax block quality control product. In this invention, tumor cells with stable sources are used as culture objects to make cell wax block quality control products with stable quality and long-term use, which can achieve the purpose of histological control: after verification, the staining results meet the actual expectations, and can help readers confirm the authenticity of the immunohistochemical results (true and false negative, positive) in real time. The cell wax block of the invention also includes a color block, which can more clearly define the cell morphological positioning characteristics (nucleus, plasma, membrane), which is convenient for readers to distinguish characteristic identification or morphology. However, the stability of the quality control product prepared by it has not been improved.

[0005] Currently, there are few finished products for PD-L1 immunohistochemistry kits. When developing the kit, it is necessary to establish a relatively stable quality control product. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation method and product of a PD-L1 immunohistochemistry detection quality control product.

[0007] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: On the one hand, the present invention provides a method for preparing a PD-L1 immunohistochemistry quality control product, wherein the quality control product includes cell sections of human breast cancer cells, human prostate cancer cells, and human ductal mammary cancer cells, and the preparation method includes: soaking the cell sections in a 3-7% BSA solution for 20-40 minutes.

[0008] Furthermore, the preparation method includes: soaking the cell slices in 5% BSA solution for 30 minutes.

[0009] Specifically, the cell sections of human breast cancer cells, human prostate cancer cells and human ductal mammary cancer cells include human breast cancer cells, human prostate cancer cells and human ductal mammary cancer cells.

[0010] Specifically, the intensities of immunohistochemical staining of cell sections of human breast cancer cells, human prostate cancer cells, and human ductal breast cancer cells are 3+, 2+, and 0, respectively.

[0011] Specifically, the immunohistochemical staining ratios of the cell sections of the human breast cancer cells, human prostate cancer cells, and human ductal mammary cancer cells are TC>10%, 1%≤TC<10%, and TC<1%, respectively.

[0012] Specifically, the raw materials for preparing the cell slices include agarose, tissue fixative and BSA.

[0013] Furthermore, the agarose is low melting point agarose; the concentration of BSA is 3-7%.

[0014] Furthermore, the method for preparing the PD-L1 immunohistochemistry quality control product comprises the following steps: S1: Tumor cell lines were cultured separately; S2: prepare low melting point agarose solution with color marker; S3: prepare corresponding cell blocks; S4: Fixing, dehydrating, wax-immersing and embedding the cell block to obtain a cell wax block; S5: Slice the cell wax block and soak it in 3-7% BSA solution for 20-40 minutes to obtain the cell wax block quality control product.

[0015] Furthermore, step S1 specifically comprises culturing tumor cell lines with PD-L1 of 0, 2+, and 3+ respectively; centrifuging the cultured cells to remove the supernatant to obtain cell pellets respectively, and resuspending the cell pellets respectively to obtain corresponding cell suspensions.

[0016] Furthermore, the tumor cell lines with PD-L1 of 0, 2+, and 3+ were human ductal breast cancer cell lines, human prostate cancer cell lines, and human breast cancer cell lines, respectively.

[0017] Furthermore, the number of cells in the cultured cell mass of tumor cells should be (6-8)×10 7 The cultured cells need to be digested with enzymes, fixed with a 10% formalin solution, and then centrifuged.

[0018] Furthermore, the amount of agarose added in step S2 is 1.5%-2%.

[0019] Furthermore, step S2 specifically includes selecting a corresponding number of color markers of different colors according to the number of tumor cell types in S1, adding a certain proportion of low melting point agarose to the color markers of different colors, mixing them evenly and setting them aside.

[0020] Furthermore, step S3 specifically includes removing the supernatant after centrifugation of the cells and then wrapping them with low melting point agarose of a corresponding color marker to obtain corresponding cell blocks, and trimming the cell blocks into a certain size.

[0021] Furthermore, step S4 specifically involves fixing, dehydrating, wax-immersing and embedding the cell block in step S3 to obtain a cell wax block quality control product.

[0022] Furthermore, the fixation includes the following steps: placing the cell block in a 10wt% neutral formalin solution for fixation for 7-9h, and then placing it in a beaker and soaking it in running water for 2h.

[0023] Furthermore, the dehydration includes the following steps: placing the fixed cell block in 60wt%, 75wt%, 85wt%, and 95wt% ethanol in sequence for preliminary dehydration, with the dehydration time for each concentration being 1-2h; then placing it in anhydrous ethanol for dehydration twice, each time for 1-2h; then placing it in xylene for dehydration twice, each time for 1-2h; then placing it in 60°C paraffin for dehydration twice, the first time for 1-2h; the second time for 2-3h.

[0024] Furthermore, the corresponding cell blocks are respectively embedded into cell wax blocks, and a tissue chip preparation instrument is used to take points to make quality control chip wax blocks.

[0025] Furthermore, the slicing condition in step S5 is 3-5 μm.

[0026] Furthermore, in step S5, the cell wax block is sliced ​​and then immersed in a 5% BSA solution for 30 minutes.

[0027] Furthermore, the preparation method of the PD-L1 immunohistochemistry quality control product may also include other cell wax block sectioning steps, including but not limited to: sectioning, baking, dewaxing and hydration, staining, dehydration, and sealing.

[0028] Specifically, the staining step is hematoxylin and eosin staining.

[0029] Specifically, the baking conditions are 65° C., 30 min.

[0030] Specifically, the dewaxing and hydrating steps are: xylene twice, 15 min each time; 100% ethanol twice, 5 min; 95% ethanol, 5 min; 75% ethanol, 5 min; and distilled water washing 2-3 times, 3 min each time.

[0031] Specifically, the staining steps are: hematoxylin for 7 minutes; washing with flowing distilled water for 3-4 times; differentiation with 1% hydrochloric acid ethanol for 2-3 seconds; washing with flowing distilled water for 3-4 times; soaking in water for 5 minutes; 80% ethanol for 20 seconds; soaking in eosin for 15-20 seconds.

[0032] Specifically, the dehydration steps are: 95% ethanol, 20 s; 100% ethanol twice, 2 min; xylene twice, 5 min each time.

[0033] Specifically, the sealing film is a neutral gum sealing film, which is dried at room temperature.

[0034] In another aspect, the present invention provides a PD-L1 immunohistochemistry quality control product prepared by the above-mentioned preparation method.

[0035] In yet another aspect, the present invention provides use of the above-mentioned PD-L1 immunohistochemistry quality control product in the preparation of a PD-L1 immunohistochemistry kit.

[0036] In yet another aspect, the present invention provides a PD-L1 immunohistochemistry kit, wherein the PD-L1 immunohistochemistry kit comprises the above-mentioned PD-L1 immunohistochemistry quality control product.

[0037] Specifically, the PD-L1 immunohistochemistry kit also includes other reagents for immunohistochemistry detection, including one or more of xylene, ethanol, antigen repair solution, hydrogen peroxide, immunochromogenic reagent, DAB chromogenic reagent, hematoxylin, and neutral gum.

[0038] The beneficial effects of the present invention are: 1. The present invention adds a pre-treatment step of soaking the cell sections in bovine serum albumin, which enhances the staining effect, reduces non-specific staining and improves storage stability.

[0039] 2. The present invention provides HCC1954, PC-3, and T47D quality control products. These cells are easy to obtain and can be used multiple times to evaluate the effectiveness of PD-L1 antibodies and diluents.

[0040] 3. The PD-L1 quality control sheet provided by the present invention contains cells expressing PD-L1 protein to varying degrees, with high cell density and good morphology, and can be used as a sensitivity control for PD-L1 protein expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 These are the H&E staining results of each sample in Example 1.

[0042] Figure 2 The staining results of immunohistochemical detection of HCC1954, PC-3, and T47D sample sections that passed the H&E staining in Example 1, where 1 is PC-3, 2 is HCC1954, and 3 is T47D.

[0043] Figure 3 Results of immunohistochemistry testing of quality control products using a Leica stainer.

[0044] Figure 4 The test results were obtained by storing the cell slices at 30±5℃ on day 0 and one month.

[0045] Figure 5 The results are obtained when cell sections were stored at 30±5℃ for 2 months.

[0046] Figure 6 The results are obtained when cell slices were stored at 30±5℃ for 3 months.

[0047] Figure 7 The results are shown for cell sections stored at 30±5℃ for 4 months.

[0048] Figure 8 The results are shown for cell sections stored at 30±5℃ for 5 months.

[0049] Fig. 9 The results are shown for cell sections stored at 30±5℃ for 6 months.

[0050] Fig.10 The test results are for cell sections stored at 30±5℃ for more than 6 months.

[0051] Fig.11 The cell sections were stored at 37°C for 0 days and 3 days.

[0052] Fig.12 The cell sections were stored at 37°C for 7 days for detection results.

[0053] Fig.13 The cell sections were stored at 37°C for 14 days for testing results.

[0054] Fig.14 The results are shown for cell slices immersed in 5% BSA for 30 min and those for slices not immersed in 5% BSA for 30 min.

[0055] Fig.15 The results of human breast ductal carcinoma cell sections immersed in 3% BSA for 20-40 minutes.

[0056] Fig.16 These are the test results of human breast ductal carcinoma cell sections immersed in 5% BSA for 20-40 minutes.

[0057] Fig.17 These are the test results of human breast ductal carcinoma cell sections immersed in 7% BSA for 20-40 minutes.

[0058] Fig.18 The results are shown for human prostate cancer cell sections immersed in 3% BSA for 20-40 minutes.

[0059] Fig.19 The results are shown for human prostate cancer cell sections immersed in 5% BSA for 20-40 minutes.

[0060] Fig. 20 The results are shown for human prostate cancer cell sections immersed in 7% BSA for 20-40 minutes.

[0061] Fig.21 These are the test results of human breast cancer cell sections immersed in 3% BSA for 20-40 minutes.

[0062] Fig. 22 These are the test results of human breast cancer cell sections immersed in 5% BSA for 20-40 minutes.

[0063] Fig.23 These are the test results of human breast cancer cell sections immersed in 7% BSA for 20-40 minutes.

[0064] Fig.24 These are the test results of cell sections immersed in 5% BSA solution and 10% BSA solution.

[0065] Fig.25 These are the test results of cell sections immersed in 5% BSA solution and 5% skimmed milk powder solution. DETAILED DESCRIPTION

[0066] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0067] Example 1 Selection and staining of quality control products The specific information of the sample used in this example is as follows: Table 1

[0068] The dyeing requirements are as follows: Table 2

[0069] Preparation of PD-L1 immunohistochemistry quality control: S1: Cultivate tumor cell lines with PD-L1 of 0, 2+, and 3+, respectively, where the tumor cell lines with 0, 2+, and 3+ are human ductal breast cancer cells, human prostate cancer cells, and human breast cancer cells, respectively; digest the cultured cells with 2 ml of trypsin cell digestion solution for 1-2 minutes, and fix them with a formalin solution with a mass concentration of 10%; centrifuge to remove the supernatant, obtain cell pellets, and resuspend the cell pellets with 75% ethanol solution to obtain corresponding cell suspensions; S2: According to the number of tumor cell types in S1, select the corresponding color markers of different colors, that is, select 50 μL of red, yellow, and blue color markers (corresponding to human breast cancer cells, human prostate cancer cells, and human ductal mammary cancer cells, respectively), add 2 mL of 1.5% low-melting point agarose to the color markers of different colors, mix well, and set aside; S3: After the cells are centrifuged and the supernatant is removed, they are wrapped with low melting point agarose of the corresponding color standard agent to obtain corresponding cell blocks, and the cell blocks are trimmed into cell blocks with a thickness of 0.5 cm and a diameter of 0.8 cm; S4: Fix, dehydrate, wax-impregnate and embed the cell block in step S3 to obtain a cell wax block quality control product.

[0070] The fixation and dehydration process of the cell block is as follows: the cell block is fixed in a 10wt% neutral formalin solution for 7-9h, then placed in a beaker and soaked in running water for 2h, then placed in 60wt%, 75wt%, 85wt%, and 95wt% ethanol for preliminary dehydration, and the dehydration time for each concentration is 1h; then placed in anhydrous ethanol for dehydration twice, each time for 1h; then placed in xylene for dehydration twice, each time for 1h; then placed in 60℃ paraffin for dehydration twice, the first time for 1h; the second time for 2h; finally, the corresponding cell blocks are embedded into cell wax blocks, and the tissue chip preparation instrument is used to take points to make quality control chip wax blocks.

[0071] S5: The collected cell samples were sliced ​​at 3-5 µm. After baking, the slices were soaked in 5% BSA solution for 30 min, then dewaxed and hydrated, stained with hematoxylin and eosin (H&E), dehydrated, sealed, observed under a microscope, and cell morphology was evaluated.

[0072] The specific steps are as follows: Baking: 65℃, 30 min; Dewaxing and hydration: xylene twice, 15 min each time → 100% ethanol twice, 5 min → 95% ethanol, 5 min → 75% ethanol, 5 min → distilled water wash 2-3 times, 3 min each time; Staining: Hematoxylin for 7 min → Wash with flowing distilled water for 3-4 times → Differentiation with 1% hydrochloric acid ethanol for 2-3 s → Wash with flowing distilled water for 3-4 times → Soak in water for 5 min → 80% ethanol for 20 s → Soak in eosin for 15-20 s; Dehydration: 95% ethanol, 20 s→100% ethanol twice, 2 min→xylene twice, 5 min each time; Sealing: Seal with neutral gum and dry at room temperature; Observation: Observe the cell staining depth, transmittance, cell integrity, etc. under a microscope to determine whether the staining film meets the requirements.

[0073] The HE staining results of each cell are shown in Figure 1 .

[0074] The results of HE staining of cell chip are shown in Figure 2 .

[0075] HE staining results showed that all cell sections could be successfully stained and the cells in the sections were intact and undamaged.

[0076] Example 2 Evaluation of the fully automated immunohistochemistry platform using quality control products The Leica BOND series fully automatic stainer commonly used in hospitals was selected for immunohistochemistry testing of quality control products. The antibody selected was PD-L1 (E1L3N) rabbit monoclonal antibody.

[0077] The staining procedure of the Leica stainer BOND-Ш is as follows: Table 3

[0078] The staining results are shown in Figure 3 .

[0079] The results showed that the staining intensity of the positive control was 3+ for HCC1954 and 2+ for PC-3. There was no clear staining on the negative control.

[0080] Example 3 Storage stability of quality control products The long-term storage stability test was performed on the quality control sample slices.

[0081] Quality control samples with different PD-L1 expression ratios and different staining intensities were selected, sliced ​​serially, and stored at 37°C. Immunohistochemical staining was performed on a Leica stainer BOND-Ш using a PD-L1 detection kit (immunohistochemistry method). The BOND-Ш staining procedure was based on Example 2. After staining, the slices were removed, dehydrated, transparentized, and sealed, and the results were observed under a microscope.

[0082] Results Figure 4-Figure 10 The quality control sample prepared in Example 3 was stored in an oven at 30±5°C for 0 days, 1 month, 2 months, 3 months, 4 months, 5 months, and 6 months. The staining results did not decrease, and after 6 months, the staining results were still in line with expectations. Therefore, it was determined that the sample could be stored at 37°C for up to 14 days, supporting a shelf life of 2 years at 2-8°C.

[0083] Results Figure 11-13 The quality control sample prepared in Example 3 was stored at 37°C for 0 days, 3 days, 7 days, and 14 days. The staining did not decrease after 14 days, and there was no significant difference in the staining results. Therefore, it was determined that the sample could be stored at 37°C for up to 14 days, supporting a shelf life of 2 years when stored at 2-8°C.

[0084] Results Fig.14 The staining results of the control sample of Example 3 were compared between the sections soaked in 5% BSA solution for 30 minutes and the sections without soaking (the rest of the steps were the same). The staining results showed that the sections soaked in 5% BSA solution for 30 minutes had a stronger staining effect than the sections without soaking. This indicates that soaking the sample in 5% BSA solution for 30 minutes can enhance the staining effect, reduce nonspecific staining and improve storage stability.

[0085] Example 4 BSA blocking buffer immersion experiment of quality control products Quality control samples with different PD-L1 expression ratios and different staining intensities were selected, and 9 consecutive slices were sliced. Nine experimental conditions were set, including immersion in 3% BSA blocking buffer for 20 min, 30 min, and 40 min; immersion in 5% BSA blocking buffer for 20 min, 30 min, and 40 min; and immersion in 7% BSA blocking buffer for 20 min, 30 min, and 40 min. The 9 quality control chips were baked and immersed in the 9 experimental conditions for immunohistochemical staining. The staining results showed that the quality control chips had better staining effect when immersed in 3-7% BSA blocking buffer for 20-40 min. The results are shown in Figure 2. Figure 15-Figure 23 .

[0086] The present invention creatively uses different tumor cells as raw materials for quality control products, which can be prepared efficiently and quickly, and uses 3-7% BSA to soak the slices, ensuring the long-term stable production of quality control products while reducing the staining background, overcoming the limitation of using human tissue samples as a source. In addition, the preparation of cell slices containing different proportions and different staining intensities can ensure the quality control of antibody staining and provide guidance for accurate interpretation, that is, to provide comprehensive and accurate quality control and interpretation guidance for antibody staining, effectively exclude false negatives and false positives, which is of great clinical significance; or used for effective PD-L1 immunohistochemistry detection process charges; or as a reference for PD-L1 immunohistochemistry inter-laboratory quality assessment.

[0087] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the concentration of BSA is different. The concentration of BSA in Example 1 is 5%, and the concentration of BSA in Comparative Example 1 is 10%. The rest are the same. From the staining results, the staining effect of 5% BSA concentration is better than that of 10% BSA concentration. The staining results are shown in Fig.24 .

[0088] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the solution for soaking the cell slices is different. The solution for soaking the cell slices in Example 1 is 5% BSA buffer solution, and the solution for soaking the cell slices in Comparative Example 1 is 5% skimmed milk powder. The rest is the same. From the staining results, the staining effect of 5% BSA concentration is better than that of 5% skimmed milk powder. The staining results are shown in FIG. Fig.25 .

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a PD-L1 immunohistochemistry quality control product, characterized in that: The quality control products include cell slices of human breast cancer cells, human prostate cancer cells, and human ductal carcinoma cells. The preparation method includes: soaking the cell slices in a 3-7% BSA solution for 20-40 minutes.

2. The preparation method according to claim 1, characterized in that: The preparation method comprises: soaking the cell slices in a 5% BSA solution for 30 minutes.

3. The preparation method according to claim 1, characterized in that: The intensities of immunohistochemical staining of the cell sections of the human breast cancer cells, human prostate cancer cells, and human ductal mammary cancer cells were 3+, 2+, and 0, respectively.

4. The preparation method according to claim 1, characterized in that: The immunohistochemical staining ratios of the cell sections of the human breast cancer cells, human prostate cancer cells, and human ductal mammary cancer cells are TC>10%, 1%≤TC<10%, and TC<1%, respectively.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1: Tumor cell lines were cultured separately; S2: prepare agarose solution with color marker; S3: prepare corresponding cell blocks; S4: Fixing, dehydrating, wax-immersing and embedding the cell block to obtain a cell wax block; S5: Slice the cell wax block and soak it in 3-7% BSA solution for 20-40 minutes to obtain the cell wax block quality control product.

6. The preparation method according to claim 5, characterized in that: The amount of agarose added in step S2 is 1.5%-2%.

7. The preparation method according to claim 5, characterized in that: The fixation described in step S4 includes the following steps: placing the cell block in a neutral formalin solution for fixation for 7-9 hours.

8. The preparation method according to claim 5, characterized in that: The dehydration described in step S4 includes the following steps: placing the fixed cell block in 60wt%, 75wt%, 85wt%, and 95wt% ethanol in sequence for preliminary dehydration, and the dehydration time for each concentration is 1-2h; then placing it in anhydrous ethanol for dehydration twice, each time for 1-2h; placing it in xylene for dehydration twice, each time for 1-2h; placing it in 60°C paraffin for dehydration twice, the first time for 1-2h, and the second time for 2-3h.

9. The preparation method according to claim 5, characterized in that: The preparation method of the PD-L1 immunohistochemistry quality control product also includes other steps, including: slicing, baking, dewaxing and hydrating, staining, dehydrating, and sealing.

10. The PD-L1 immunohistochemistry quality control product prepared by the preparation method according to any one of claims 1 to 9.

11. Use of the PD-L1 immunohistochemistry quality control product according to claim 10 in the preparation of a PD-L1 immunohistochemistry kit.

12. A PD-L1 immunohistochemistry kit, characterized in that: The PD-L1 immunohistochemistry kit comprises the PD-L1 immunohistochemistry quality control product according to claim 10.

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