PD-L1 immunohistochemical quality control product and preparation method thereof

Abstract

The invention discloses a PD-L1 immunohistochemical quality control product and a preparation method thereof, and relates to the technical field of biology. The PD-L1 immunohistochemical quality control product comprises cell slices prepared from a plurality of different types of tumor cells, the cell section comprises a positive quality control product and a negative quality control product which are marked by different colors. In the invention, different tumor cells are creatively adopted as raw materials of the quality control product, the quality control product can be efficiently and quickly prepared, and the limitation of the source of a human tissue sample is overcome; a BSA sealing buffer solution is used for soaking the section, so that long-term stable production of a quality control product can be realized, the dyeing background is reduced, and the dyeing effect is enhanced; besides, by preparing the cell slices with various proportions and different staining intensities, the quality control on the staining of the antibody can be ensured, and guidance can also be provided for accurate interpretation, namely comprehensive and accurate quality control and interpretation guidance are provided for the staining of the antibody, false negative and false positive are effectively eliminated, and the method has great clinical significance.

Description

Technical Field

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

[0002] PD-L1 is a protein expressed on the surface of many tumor cells. It binds to the PD-1 receptor on the surface of T cells, exerts a negative regulatory effect, inhibits the activation, differentiation and proliferation of T cells, and allows tumor cells to escape T cell killing and obtain immune escape. It is a mechanism for tumors to escape immune surveillance. By detecting the expression level of PD-L1 in tumor tissues, it can help evaluate the patient's potential response to immune checkpoint inhibitors (such as anti-PD-1 or anti-PD-L1 antibodies). Patients with high PD-L1 expression are considered potential candidates for immunotherapy.

[0003] Immunohistochemistry (IHC) is an effective method for evaluating the expression status of PD-L1 in tumor tissues. It is widely used in a variety of malignant tumors including non-small cell lung cancer. Immunohistochemistry uses specific antibodies against PD-L1 to perform qualitative and quantitative analysis of PD-L1 expression in tumor tissues. The technical core of the IHC detection method is to visualize the location, intensity and proportion of positive cells of PD-L1 protein through staining technology, providing potential evaluation basis for tumor immunotherapy in clinic.

[0004] At present, the IHC detection method requires the provision of stable quality control products for reference and comparison during the detection process; however, the quality of PD-L1 immunohistochemistry kits used as quality control products on the market varies greatly, and some quality control products have low quality control accuracy, and some quality control products can only be used for qualitative reference; at the same time, most quality control samples are mainly derived from tumor tissues of cancer patients, and the sample source is limited.

[0005] Therefore, how to solve the above technical problems is a technical problem currently faced by general technicians in this field.

[0006] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0007] In view of the above technical problems, an embodiment of the present invention provides a PD-L1 immunohistochemistry quality control product and a preparation method thereof to solve the problems raised in the above background technology.

[0008] A PD-L1 immunohistochemistry quality control product includes cell sections prepared from multiple different types of tumor cells; the cell sections include positive quality control products and negative quality control products marked with different colors.

[0009] Preferably, the positive quality control includes cell sections with immunohistochemical staining intensity of 3+ and 2+ respectively; the negative quality control includes cell sections with immunohistochemical staining intensity of 0.

[0010] Preferably, the tumor cells in the positive quality control product are selected from human breast cancer cells HCC1954 and human prostate cancer cells PC-3; the tumor cells in the negative quality control product are selected from human ductal breast cancer cells T47D; The immunohistochemical staining intensity of the cell sections prepared from human breast cancer cells HCC1954 was 3+; the immunohistochemical staining intensity of the cell sections prepared from human prostate cancer cells PC-3 was 2+; and the immunohistochemical staining intensity of the cell sections prepared from human ductal breast cancer cells T47D was 0.

[0011] Specifically, the immunohistochemical staining ratios of cell section samples of human breast cancer cells HCC1954, human prostate cancer cells PC-3, and human ductal breast cancer cells T47D were TC>10%, 1%≤TC<10%, and TC<1%, respectively.

[0012] Preferably, the cell slices are immersed in a blocking buffer; the blocking buffer is one or more of PBS buffer solution, purified water, and bovine serum albumin (BSA); further, the concentration of the BSA solution is preferably 5%; and the immersion time is preferably 30 minutes.

[0013] Preferably, the blocking buffer is a 3-7% BSA solution; the cell slices are immersed in the 3-7% BSA solution for 20-40 minutes.

[0014] A method for preparing the PD-L1 immunohistochemistry quality control product as described above comprises the following steps: S61: Cultivate different types of tumor cell lines for preparing positive quality control products and negative quality control products; S62: Prepare agarose solution with different color markers; S63: After the cultured tumor cells are centrifuged and the supernatant is removed, the cell pellets are respectively encapsulated with agarose solutions containing different color markers to obtain corresponding cell blocks; S64: fixing, dehydrating, wax-immersing and embedding the cell block to obtain a cell wax block; S65: Slice the cell wax block and soak it in a 3-7% BSA solution for 20-40 minutes to obtain a PD-L1 immunohistochemistry quality control product; further, the concentration of the BSA solution is preferably 5%; and the soaking time is preferably 30 minutes.

[0015] Specifically, the preparation method of PD-L1 immunohistochemistry quality control product also includes: slicing, baking, dewaxing and hydrating, staining, dehydrating, and sealing; the slicing condition thickness is preferably 3-5 µm; and the staining step is performed using hematoxylin and eosin staining.

[0016] Preferably, the tumor cells in the positive quality control product are selected from human breast cancer cells HCC1954 and human prostate cancer cells PC-3; the tumor cells in the negative quality control product are selected from human ductal breast cancer cells T47D; The immunohistochemical staining intensity of the cell sections prepared from human breast cancer cells HCC1954 was 3+; the immunohistochemical staining intensity of the cell sections prepared from human prostate cancer cells PC-3 was 2+; and the immunohistochemical staining intensity of the cell sections prepared from human ductal breast cancer cells T47D was 0.

[0017] Preferably, the method for fixing and dehydrating the cell blocks in step S64 includes: after the cells are enzymatically digested, they are fixed with a formalin solution with a mass concentration of 10% for 7-9 hours; then they are placed in a beaker and soaked in running water for 2 hours, and then placed in 60wt%, 75wt%, 85wt%, and 95wt% ethanol in sequence for preliminary dehydration, and the dehydration time for each concentration is 1-2 hours; then they are placed in anhydrous ethanol for dehydration twice, each time for 1-2 hours; they are placed in xylene for dehydration twice, each time for 1-2 hours; they are placed in 60°C paraffin for dehydration twice, the first time for 1-2 hours, and the second time for 2-3 hours.

[0018] A PD-L1 immunohistochemistry kit, comprising the PD-L1 immunohistochemistry quality control product described above; or comprising the PD-L1 immunohistochemistry quality control product prepared by the preparation method of the PD-L1 immunohistochemistry quality control product described above.

[0019] Preferably, the PD-L1 immunohistochemistry kit also includes other reagents for IHC detection; other reagents for IHC detection include: one or more of xylene, ethanol, antigen repair solution, hydrogen peroxide, immunochromogenic reagent, DAB chromogenic reagent, hematoxylin, and neutral gum.

[0020] The PD-L1 immunohistochemistry quality control product and preparation method thereof provided in the embodiment of the present invention have the following beneficial effects: 1. In the present invention, different tumor cells are creatively used as raw materials for quality control products, and quality control products can be prepared efficiently and quickly, overcoming the limitation of using human tissue samples as the source; 2. Soaking the sections in BSA blocking buffer can achieve long-term stable production of quality control products, reduce staining background and enhance staining effect; 3. In addition, by preparing cell sections containing various proportions and different staining intensities, the quality control of antibody staining can be guaranteed, and guidance can also be provided for accurate interpretation, that is, comprehensive and accurate quality control and interpretation guidance can be provided for antibody staining, effectively eliminating false negatives and false positives, which is of great clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the H&E staining results of each sample in Example 1.

[0022] Figure 2 The staining results of IHC 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.

[0023] Figure 3 Results of IHC testing of quality control products for the Leica stainer.

[0024] Figure 4 The test results were obtained by storing the cell slices at 30±5℃ on day 0 and one month.

[0025] Figure 5 The results are obtained when cell sections were stored at 30±5℃ for 2 months.

[0026] Figure 6 The results are obtained when cell slices were stored at 30±5℃ for 3 months.

[0027] Figure 7 The results are shown for cell sections stored at 30±5℃ for 4 months.

[0028] Figure 8 The results are shown for cell sections stored at 30±5℃ for 5 months.

[0029] Fig. 9 The results are shown for cell sections stored at 30±5℃ for 6 months.

[0030] Fig.10 The test results are for cell sections stored at 30±5℃ for more than 6 months.

[0031] Fig.11 The cell sections were stored at 37°C for 0 days and 3 days.

[0032] Fig.12 The cell sections were stored at 37°C for 7 days for detection results.

[0033] Fig.13 The cell sections were stored at 37°C for 14 days for testing results.

[0034] Fig.14The 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. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0036] In view of the above technical problems, an embodiment of the present invention provides a PD-L1 immunohistochemistry quality control product and a preparation method thereof to solve the problems raised in the above background technology.

[0037] Example 1: Cultivation and acquisition of different tumor cell samples Tumor cell lines with PD-L1 immunohistochemical staining intensities of 0, 2+, and 3+ were cultured, and the tumor cell lines with 0, 2+, and 3+ were human ductal breast cancer cells, human prostate cancer cells, and human breast cancer cells, respectively; it should be noted here that previous studies have confirmed that the immunohistochemical staining intensities of human ductal breast cancer cells, human prostate cancer cells, and human breast cancer cells are 0, 2+, and 3+, respectively; The cultured tumor cells were digested and fixed with a formalin solution having a mass concentration of 10%; the supernatant was then removed by centrifugation to obtain a cell precipitate, and the cell precipitate was resuspended with a 75% ethanol solution to obtain a corresponding cell suspension; According to the number of different tumor cell types, select the corresponding color markers of different colors (such as red, yellow, and blue color markers corresponding to human breast cancer cells, human prostate cancer cells, and human ductal mammary cancer cells, respectively), 50 μL each, add 2 mL of 1.5% agarose to the color markers of different colors, mix well, and set aside; The cell pellet was wrapped with agarose of the corresponding color marker to obtain the corresponding cell block, and the obtained cell block was trimmed into a cell block with a thickness of 0.5 cm and a diameter of 0.8 cm; The prepared cell block is fixed, dehydrated, wax-impregnated and embedded to obtain a cell wax block quality control product.

[0038] 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.

[0039] Example 2: Selection and staining of quality control products The specific information of the samples used in this embodiment is shown in Table 1: Table 1

[0040] Staining requirements are shown in Table 2: Table 2

[0041] The collected cell samples were sliced ​​at a thickness of 3-5 µm. After baking, the slices were dewaxed and hydrated, stained with hematoxylin and eosin (H&E), dehydrated, sealed, observed under a microscope, and cell morphology was evaluated. 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.

[0042] The HE staining results of each cell are shown in Figure 1 .

[0043] The results of HE staining of cell chip are shown in Figure 2 .

[0044] HE staining results showed that all cell sections could be successfully stained and the cells in the sections were intact and undamaged.

[0045] Example 3: 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.

[0046] The staining procedure of Leica stainer BOND-Ш is shown in Table 3: Table 3

[0047] The staining results are shown in Figure 3 .

[0048] Figure 3 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.

[0049] Example 4: Storage stability of quality control products The long-term storage stability test was performed on the quality control sample slices.

[0050] Quality control samples with different PD-L1 expression ratios and different staining intensities were selected, sliced ​​serially, and stored at 37°C. IHC staining was performed on a Leica stainer BOND-Ш using a PD-L1 detection kit (immunohistochemistry). 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.

[0051] 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.

[0052] 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.

[0053] Results Fig.14The quality control sample of Example 3 was soaked in 5% BSA solution for 30 minutes and the staining results of the sections without soaking were compared. 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. Therefore, it was determined that soaking the sample in 5% BSA solution for 30 minutes can enhance the staining effect, reduce nonspecific staining and improve storage stability.

[0054] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A PD-L1 immunohistochemistry quality control product, characterized in that: Includes cell sections prepared from multiple different types of tumor cells; the cell sections include positive quality control products and negative quality control products marked with different colors.

2. The PD-L1 immunohistochemistry quality control product according to claim 1, characterized in that: Positive quality control products include cell sections with immunohistochemical staining intensity of 3+ and 2+ respectively; negative quality control products include cell sections with immunohistochemical staining intensity of 0.

3. The PD-L1 immunohistochemistry quality control product according to claim 2, characterized in that: The tumor cells in the positive quality control product are selected from human breast cancer cells and human prostate cancer cells; the tumor cells in the negative quality control product are selected from human ductal mammary cancer cells; The immunohistochemical staining intensity of cell sections prepared from human breast cancer cells was 3+; The immunohistochemical staining intensity of cell sections prepared from human prostate cancer cells was 2+; The immunohistochemical staining intensity of the cell sections prepared from human ductal breast carcinoma cells was 0.

4. The PD-L1 immunohistochemistry quality control product according to claim 1, characterized in that: Cell sections were immersed in blocking buffer.

5. The PD-L1 immunohistochemistry quality control product according to claim 4, characterized in that: The blocking buffer is 3-7% BSA solution; the cell sections are immersed in the 3-7% BSA solution for 20-40 minutes.

6. A method for preparing a PD-L1 immunohistochemistry quality control product according to claim 1, characterized in that: The following steps are involved: S61: Cultivate different types of tumor cell lines for preparing positive quality control products and negative quality control products; S62: Prepare agarose solution with different color markers; S63: After the cultured tumor cells are centrifuged and the supernatant is removed, the cell pellets are respectively encapsulated with agarose solutions containing different color markers to obtain corresponding cell blocks; S64: fixing, dehydrating, wax-immersing and embedding the cell block to obtain a cell wax block; S65: Slice the cell wax block and soak it in 3-7% BSA solution for 20-40 minutes to obtain PD-L1 immunohistochemistry quality control product.

7. The method for preparing the PD-L1 immunohistochemistry quality control product according to claim 6, characterized in that: The tumor cells in the positive quality control product are selected from human breast cancer cells and human prostate cancer cells; the tumor cells in the negative quality control product are selected from human ductal mammary cancer cells; The immunohistochemical staining intensity of cell sections prepared from human breast cancer cells was 3+; The immunohistochemical staining intensity of cell sections prepared from human prostate cancer cells was 2+; The immunohistochemical staining intensity of the cell sections prepared from human ductal breast carcinoma cells was 0.

8. The method for preparing the PD-L1 immunohistochemistry quality control product according to claim 6, characterized in that: The method for fixing and dehydrating the cell block in step S64 includes: after the cells are digested by enzymes, they are fixed with a formalin solution with a mass concentration of 10% for 7-9 hours; then they are placed in a beaker and soaked in running water for 2 hours, and then they are placed in 60wt%, 75wt%, 85wt%, and 95wt% ethanol in sequence for preliminary dehydration, and the dehydration time for each concentration is 1-2 hours; then they are placed in anhydrous ethanol for dehydration twice, each time for 1-2 hours; they are placed in xylene for dehydration twice, each time for 1-2 hours; they are placed in 60°C paraffin for dehydration twice, the first time for 1-2 hours, and the second time for 2-3 hours.

9. A PD-L1 immunohistochemistry kit, characterized in that: Including the PD-L1 immunohistochemistry quality control product according to any one of claims 1 to 5; or including the PD-L1 immunohistochemistry quality control product prepared by the preparation method of the PD-L1 immunohistochemistry quality control product according to any one of claims 6 to 8.

10. The PD-L1 immunohistochemistry kit according to claim 9, characterized in that: Other reagents for IHC detection are also included.

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