Preparation method of PD-L1 immunohistochemical liquid cytology quality control product and special glass slide
By preparing PD-L1 immunohistochemical liquid cytology quality control materials and slides, the problems of cumbersome operation and high cost of existing quality control materials are solved, and the detection efficiency and accuracy are improved, making them suitable for PD-L1 immunohistochemical detection.
Patent Information
- Application Number
- CN202511235930.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-01
- Publication Date
- 2025-12-05
AI Technical Summary
The availability of PD-L1 quality control materials is limited, the operation is cumbersome and expensive, resulting in inconsistent test results and affecting testing efficiency and accuracy.
To prepare PD-L1 immunohistochemical liquid cytology quality control, a stable cell suspension quality control was obtained through steps such as culturing tumor cells, hypoxia treatment, trypsin digestion, fixation, and dehydration. Control circles were set on a glass slide to mark positive and negative areas to ensure the reliability of the detection.
We provide simple and easy-to-use quality control products to improve detection efficiency and accuracy, ensure the reliability and consistency of PD-L1 immunohistochemical detection, and are suitable for mainstream IHC platforms.
Smart Images
Figure CN121068290A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of in vitro diagnostic reagent quality control technology, and specifically relates to a method for preparing PD-L1 immunohistochemical liquid cytology quality control material and a glass slide. Background Technology
[0002] PD-L1 is a membrane protein expressed on the surface of various tumor cells. It exerts a negative regulatory effect by binding to the PD-1 receptor on the surface of T cells, inhibiting T cell activation, differentiation, and proliferation, thus enabling tumor cells to escape immune surveillance. This is a crucial mechanism for tumors to evade immune surveillance. Detecting the expression level of PD-L1 in tumor tissue can assess a patient's potential response to immune checkpoint inhibitors (such as anti-PD-1 or anti-PD-L1 antibodies), and patients with high expression levels are considered potential candidates for immunotherapy.
[0003] Immunohistochemistry (IHC) is a commonly used clinical method for evaluating PD-L1 expression status. However, the quality of IHC testing is easily affected by factors such as antibodies, substrates, detection systems, and specimen preparation conditions. Therefore, providing stable quality control materials for reference and comparison during the testing process is crucial. Given the direct impact of PD-L1 expression on clinical treatment decisions, multiple expert consensuses recommend that PD-L1 testing require strict internal quality control materials.
[0004] However, in current clinical applications, most PD-L1 quality control samples are derived from tumor tissue or tonsils of cancer patients, limiting the availability of samples. Due to the lack of standardized quality control materials, comparisons and mutual recognition of results are impossible between different hospitals and different antibody models. Furthermore, commercially available cell line quality control materials are mostly paraffin-embedded tissues, requiring pre-sectioning, which is cumbersome and expensive. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to provide a method for preparing PD-L1 immunohistochemical liquid cytology quality control material and a glass slide. Compared with traditional cell block quality control materials, the cell suspension quality control material prepared by the present invention has significant advantages such as simple operation, on-demand use, and convenient use. It can effectively improve detection efficiency and accuracy, and provide a more reliable quality control method for PD-L1 immunohistochemical detection.
[0006] This invention provides a method for preparing PD-L1 immunohistochemical liquid cytology quality control material, comprising the following steps:
[0007] (1) Culture tumor cells and transfer positive cell lines to a hypoxia incubator; discard the normal culture medium, wash the cells with PBS, and then add sugar-free and serum-free culture medium for hypoxia treatment.
[0008] (2) Cells that have undergone hypoxia were treated by trypsin digestion and the supernatant was removed by centrifugation to obtain cell pellet.
[0009] (3) Wash the cell pellet with PBS, then fix with 10% neutral formalin at room temperature for 1 hour;
[0010] (4) Pre-cooled cells at -20℃ with 75% ethanol for dehydration and preservation;
[0011] (5) After centrifugation to remove the supernatant, cell pellet was obtained; then, a working solution was prepared using a 40% ethanol-PBS mixture, and the cell concentration was adjusted to 10. 4 PD-L1 immunohistochemical liquid cytology quality control was obtained by measuring 1 unit / ul.
[0012] Preferably, the tumor cells in step (1) are selected from breast cancer cells.
[0013] Preferably, the hypoxia treatment in step (1) is performed in a hypoxia device pre-introduced with a mixture of bodily fluids. Positive cell lines are induced to undergo hypoxia treatment, resulting in an immunohistochemical staining intensity of 3. + The staining intensity of negative strains is 0 to ensure clear differences in characteristics.
[0014] Preferably, the gas mixture is composed of 95% N2 and 5% CO2.
[0015] There are two methods for cell fixation in step (2):
[0016] Option 1: Fix 75% ethanol pre-cooled at -20℃ for 24 hours.
[0017] Option 2: Fix with 10% neutral formalin at room temperature for 1 hour.
[0018] Results Comparison: Samples prepared with two different fixatives were used in stability experiments, tested weekly for four consecutive weeks. Samples fixed with 75% ethanol showed lighter staining and, after approximately three weeks, showed indistinct cell membrane localization, transitioning to the cell membrane itself, while the cytoplasm remained positive. The recommended fixative protocol was 10% neutral formalin, fixed at room temperature for 1 hour.
[0019] Selection of dehydration solution:
[0020] Option 1: Normal gradient dehydration
[0021] Centrifuge the fixed cell suspension at 2000 rpm for 3 min with 70% ethanol, discard the supernatant, add 70% ethanol, gently resuspend the cells, and incubate at 4°C for 1 hour.
[0022] 80% ethanol: Centrifuge, discard the supernatant, add 80% ethanol, suspend the cells, and incubate at 4°C for 1 hour.
[0023] 90% ethanol: Centrifuge, discard the supernatant, add 80% ethanol, suspend the cells, and incubate at 4°C for 1 hour.
[0024] 95% ethanol: Centrifuge, discard the supernatant, add 80% ethanol, suspend the cells, and incubate at 4°C for 1 hour.
[0025] 100% ethanol: Centrifuge, suspend in 100% ethanol, and let stand at 4°C for 1 hour.
[0026] Option 2: Simplified dehydration
[0027] Centrifuge the fixed cell suspension at 2000 rpm for 3 min with 75% ethanol, discard the supernatant, add pre-cooled 75% ethanol at -20℃, gently resuspend the cells, and incubate at 4℃ for 1 hour.
[0028] Preferably, the dehydration method in step (4) is simplified dehydration with 75% ethanol. Compared to cells dehydrated using a standard gradient method, these cells shrink, become smaller, and the structures between the cell membrane, nucleus, and cytoplasm are less clear. Simplified dehydration does not affect the subsequent PD-L1 staining effect, but the overall cell structure is better, which is more conducive to PD-L1 staining. Cells suspended in pre-cooled ethanol can be stored at 4°C for about 3 months, and can be stored at -20°C for 1 year.
[0029] Preferably, the gradient alcohol-PBS mixture in step (5) is a 40% ethanol-PBS mixture. At this concentration, adding 1 μL of cell sample results in a full, round shape with little or no permeation. Above this ethanol concentration, cell permeation occurs; lower ethanol concentrations are unsuitable for preserving cell morphology and antigenicity.
[0030] Preferably, for all centrifugation steps, the selected centrifugal force is 200×g, approximately 1000rpm, and the centrifugation time is 3–5 minutes.
[0031] The present invention also provides a glass slide based on PD-L1 immunohistochemistry liquid cytology quality control. The prepared PD-L1 immunohistochemistry liquid cytology quality control is dropped into the quality control ring on the glass slide and allowed to air dry naturally. A white cell membrane is visible to the naked eye. The glass slide is then dried and used directly for immunohistochemical detection.
[0032] Preferably, the back of the glass slide is provided with control rings for identifying positive control samples and negative control samples, respectively.
[0033] Preferably, the area of the quality control ring is designed to match the penetration range of the quality control product, ensuring that the quality control product is uniformly distributed within the quality control ring.
[0034] Preferably, the quality control ring is made by screen printing a layer of black PTFE hydrophobic ring to mark the quality control area and prevent the quality control sample from leaking into the test area and causing cross-contamination. The slide size is fully compatible with mainstream IHC platforms such as Ventana Benchmark ULTRA and DakoAutostainer Link 48, and can be directly used on the instrument.
[0035] Beneficial effects
[0036] Compared to traditional cell block quality control products, the cell suspension quality control products prepared in this invention have significant advantages such as simple operation, on-demand application, and ease of use. They can effectively improve detection efficiency and accuracy, providing a more reliable quality control method for PD-L1 immunohistochemical detection. This invention prepares a homogeneous, stable, and standardized cell suspension from engineered cell lines, possessing clearly defined PD-L1 protein expression characteristics. It is highly similar to the target biomarker in the tissue sample being tested, with stable and controllable expression abundance. Furthermore, its simple operation and on-demand application make it convenient to use, effectively meeting the quality control requirements for PD-L1 immunohistochemical detection and improving detection reliability. Attached Figure Description
[0037] Figure 1 Image of HE-stained cell blocks from positive cell lines.
[0038] Figure 2 Image of PD-L1 staining on cell blocks from positive cell lines.
[0039] Figure 3 Image of HE staining of a positive cell line suspension.
[0040] Figure 4 Image of PD-L1 staining in cell suspension of positive cell line.
[0041] Figure 5 Image of HE staining of a negative cell line suspension.
[0042] Figure 6 Image of PD-L1 staining in a suspension of negative cell line.
[0043] Figure 7 Images of PD-L1 staining used to test the stability of positive cell lines one month after their inception.
[0044] Figure 8 Images of PD-L1 staining used to test the stability of positive cell lines 3 months after initiation.
[0045] Figure 9 Images of PD-L1 staining used to test the stability of positive cell lines after 6 months.
[0046] Figure 10This is a schematic diagram of the carrier chip of the present invention; wherein, 1 is the glass head, 2 is the quality control area, 3 is the area to be inspected, and 4 is the quality control ring. Detailed Implementation
[0047] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
[0048] Example 1
[0049] This embodiment provides a PD-L1 immunohistochemical liquid cytology quality control, which uses breast cancer cell lines as the basic framework. The preparation process of this quality control is as follows:
[0050] 1. Cell Culture and Hypoxia Induction: When tumor cells reach 80% cell density, the positive cell lines are transferred to a hypoxia incubator. The normal culture medium is discarded first, the cells are washed 2-3 times with PBS, then sugar-free and serum-free culture medium is added, and the cells are placed in a hypoxia device pre-purified with a mixture of 95% N2 and 5% CO2 gas for hypoxia treatment.
[0051] 2. Cell digestion and collection: Cultured tumor cells were treated with trypsin digestion, and the cell pellet was obtained after centrifugation to remove the supernatant.
[0052] 3. Cell fixation: After washing the cell pellet once with PBS, fix it with 10% neutral formalin at room temperature for 1 hour.
[0053] 4. Cell dehydration and preservation: Centrifuge the fixed cell suspension at 2000 rpm for 3 min, discard the supernatant, add 75% ethanol pre-cooled at -20℃, gently suspend the cells, and incubate at 4℃ for 1 hour.
[0054] 5. Suspension Preparation and Adjustment: The tumor cells were centrifuged to remove the supernatant, resuspended in 1 ml of 75% ethanol, and cell counts were performed. Subsequently, a working solution was prepared using a 40% ethanol-PBS mixture, and the cell concentration was adjusted to 10-1. 4 Cells per 1 μL are used to ensure cell viability and stability, resulting in PD-L1 immunohistochemical liquid cytology quality control, i.e., cell suspension.
[0055] Depend on Figure 1 , 2 and Figure 3 , 4 The comparison shows that, compared with the positive cell line cell suspension prepared by the existing positive cell line FFPE-embedded cell block preparation method, there is no significant difference in staining intensity and membrane localization of PD-L1 expression.
[0056] Depend on Figure 3-4 , Figure 5-6 It can be seen that the PD-L1 staining of the cell suspension of the positive cell line is clearly positive, while the PD-L1 staining of the cell suspension of the negative cell line is clearly negative.
[0057] Depend on Figure 7-9 It can be seen that the positive cell line suspension of the present invention has good stability.
[0058] like Figure 10 As shown, this embodiment also provides a slide for PD-L1 immunohistochemistry liquid cytology quality control, including a glass head, a quality control area, and a test area. The back of the slide has quality control rings for identifying positive and negative quality controls, located within the quality control area. The area of the quality control rings is designed to match the permeability range of the quality control samples (1μl-2μl), ensuring a uniform distribution of the quality control samples within the rings. A black PTFE hydrophobic ring is screen-printed onto the quality control rings to identify the quality control area and prevent leakage of the quality control samples into the test area, thus preventing cross-contamination. The slide size is fully compatible with mainstream IHC platforms such as Ventana Benchmark ULTRA and Dako Autostainer Link 48, allowing for direct instrumentation. The prepared PD-L1 immunohistochemistry liquid cytology quality control is dropped into the quality control rings on the slide and allowed to air dry naturally until a white cell membrane is visible to the naked eye. The slide is then dried and used directly for immunohistochemical detection.
[0059] By innovatively setting quality control zones on the slide, researchers can clearly identify the areas of quality control samples, enabling them to quickly and accurately locate the samples under a microscope. This significantly improves detection efficiency, ensures a smooth quality control process, and enhances the practicality and reliability of liquid cell quality controls in immunohistochemical detection.
Claims
1. A preparation method of PD-L1 immunohistochemical liquid cytology quality control product and a special slide, comprising the following steps: (1) culturing tumor cells, moving the positive cell strain to a low-oxygen incubator; first discarding the normal culture solution, washing the cells with PBS, then adding a sugar-free and serum-free culture medium to perform hypoxic treatment; (2) treating the cells subjected to hypoxic treatment by using a trypsin digestion method, obtaining cell precipitates after centrifugation to remove supernatants; (3) washing the cell precipitates with PBS, and then fixing them with 10% neutral formalin at room temperature for 1 hour; (4) dehydrating and storing the cells by using 75% ethanol pre-cooled at -20°C; (5) After removing the supernatant by centrifugation, the cell precipitate was obtained; then a 40% ethanol-PBS mixture was used to prepare a working solution, and the cell concentration was adjusted to 10 4 cells / ul to obtain the PD-L1 immunohistochemical liquid cytology quality control product.
2. The method of claim 1, wherein: the tumor cells in the step (1) are selected from breast cancer cells.
3. The method of claim 1, wherein: the hypoxic treatment in the step (1) is performed by placing the tumor cells in a hypoxic device pre-filled with a gas mixture.
4. The method of claim 3, wherein: the gas mixture is mixed by 95% N2 and 5% CO2.
5. A slide based PD-L1 immunohistochemistry liquid cytology quality control product, characterized in that: the PD-L1 immunohistochemical liquid cytology quality control product prepared according to claim 1 is added dropwise into a quality control circle on the slide, naturally air-dried, and white cell membranes are formed under naked eyes; then the slide is directly used for immunohistochemical detection after being baked and dried.
6. The slide of claim 5, wherein: the back of the slide is provided with a quality control circle for identifying positive quality control products and a quality control circle for identifying negative quality control products.
7. The slide of claim 5, wherein: the area of the quality control circle is designed to match the penetration range of the quality control product, so as to ensure that the quality control product is uniformly distributed in the quality control circle.
8. The slide of claim 5, wherein: the quality control circle is provided with a layer of black PTFE hydrophobic ring by silk screen printing, which is used for identifying the quality control area and preventing the quality control sample from leaking into the detection area to cause cross contamination.