Four-in-one reference material for her2 immunohistochemistry detection and application thereof

Abstract

The application discloses a four-in-one reference material for HER2 immunohistochemical detection and application thereof. The reference material comprises four screened and verified cell lines, which stably present 0+, 1+, 2+ and 3+ immunohistochemical staining characteristics of HER2 protein expression respectively, and exhibit good staining stability and reproducibility. The above cell lines have been preserved in the China General Microbiological Culture Collection Center (CGMCC). The four-in-one reference material can realize synchronous acquisition of full-spectrum HER2 expression control in a single staining, and significantly improve the standardization, precision and comparability of HER2 detection in a gastric cancer background, and is suitable for precise diagnosis of HER2 related diseases such as gastric cancer.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay, specifically relating to a four-in-one reference material for HER2 immunohistochemical detection and its application. Background Technology

[0002] In the clinical diagnosis and treatment of malignant tumors such as gastric cancer, the expression status of human epidermal growth factor receptor 2 (HER2) is a key biomarker guiding targeted therapy (such as trastuzumab). Immunohistochemistry (IHC) is used as a primary screening method for HER2 detection, and the interpretation of results relies on a semi-quantitative scoring system of 0+, 1+, 2+, and 3+. However, IHC testing is easily affected by factors such as antibody batch, staining platform, operator, and laboratory conditions, resulting in poor repeatability and consistency of results, especially with significant subjective differences in the interpretation of weak positives (1+, 2+).

[0003] Currently, most commercially available or literature-reported HER2 immunohistochemical quality control materials are based on breast cancer cell lines, whose tissue origin, membrane structure characteristics, and antigen epitope exposure patterns differ significantly from those of gastric cancer cells. Furthermore, according to current clinical guidelines (such as the Chinese consensus on HER2 testing in gastric cancer), the interpretation criteria for HER2 in gastric cancer are not entirely the same as those for breast cancer. Therefore, using quality control materials derived from breast cancer often results in staining pattern mismatches or interpretation biases in gastric cancer sample testing, failing to accurately reflect the HER2 status of gastric cancer samples and severely impacting testing accuracy and treatment decisions.

[0004] Furthermore, existing quality control materials are mostly cell lines or tissue samples with single expression levels, making it difficult to simultaneously provide a complete 0+ to 3+ control gradient on the same staining plate. Moreover, existing reference materials have limited cross-validation capabilities across different detection platforms, failing to meet the urgent need for standardized reference materials. Therefore, there is an urgent need for a novel reference material based on gastric cancer cell background, integrating four HER2 expression levels, exhibiting stable staining performance, and applicable to multiple IHC platforms, to achieve standardization, precision, and comparability in gastric cancer HER2 detection. Summary of the Invention

[0005] In view of this, the present invention provides a four-in-one reference material for HER2 immunohistochemical detection and its application.

[0006] The present invention achieves the above objectives using the following technical solution: In this invention, the inventors used various basic gastric cancer cell lines (such as SNU5, AGS, etc.) with negative or weakly positive levels of endogenous HER2 antigen expression as starting materials. They constructed lentiviral expression vectors carrying the full-length coding sequence of human HER2 through genetic engineering. Combined with promoter regulation strategies of different strengths, they finely regulated the expression level of exogenous HER2 protein. For each parental cell line, this invention independently screened and obtained a series of cell lines with different levels of exogenous HER2 expression and morphological characteristics of monoclonal cell lines that were highly consistent with the corresponding parental cells.

[0007] In this invention, the HER2 expression levels (0, 1+, 2+, 3+) of each monoclonal strain were determined using a dual confirmation system: primarily immunohistochemical (IHC) visual grading, supplemented by qPCR transcriptional level verification. The core basis for this system is as follows: referring to the clinical interpretation standards for HER2 in gastric cancer, multiple pathologists performed double-blind microscopic scoring of the cell membrane integrity and staining intensity after IHC staining to ensure that the staining gradient at the pathological level matches the actual clinical testing scenario; simultaneously, qPCR was used to determine the relative transcription fold of HER2 mRNA in each strain (2... -ΔΔCT The IHC grading and qPCR CT values ​​were used as objective quantitative standards for screening the "best combination". A series of cell lines that maintained stable IHC grading and qPCR CT values ​​during continuous passage were selected to form multi-level quality control products. This process aims to construct a multi-level quality control system covering the complete staining gradient, particularly addressing the current practical need for stable and reproducible weak positive controls (1+ and 2+) in immunohistochemical assays.

[0008] The inventors further deposited the selected staining results, which met the criteria of 0+, 1+, 2+, and 3+ and exhibited stability and reproducibility, HER2-G0, HER2-G1, HER2-G2, and HER2-G3, into the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date was April 3, 2026, and the accession numbers were CGMCC No. 46797, CGMCC No. 46798, CGMCC No. 46799, and CGMCC No. 46800, respectively.

[0009] A first aspect of the present invention provides a multi-stage control cell composition for immunohistochemical detection of human epidermal growth factor receptor 2, said composition comprising at least two of the following four cell types: The first cell showed a HER2 protein expression level of 0+ as verified by immunohistochemistry, with no membrane staining. The second cell showed that the HER2 protein expression level was 1+ as verified by immunohistochemistry, and it was weakly or faintly visible as membrane staining. The third cell, whose HER2 protein expression level was verified to be 2+ by immunohistochemistry, showed weak to moderate basal side membrane, side membrane or complete membrane staining. The fourth cell showed a HER2 protein expression level of 3+ as verified by immunohistochemistry, exhibiting strong staining of the basal side membrane, side membrane, or complete membrane.

[0010] The immunohistochemical verification results and membrane staining grading standards for 0+, 1+, 2+, and 3+ were determined with reference to the interpretation criteria in the "Guidelines for HER2 Detection in Gastric Cancer" or the ASCO / CAP Guidelines for HER2 Detection in Gastroesophageal Adenocarcinoma.

[0011] The first to fourth cells were named HER2-G0, HER2-G1, HER2-G2, and HER2-G3, respectively, and have been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 46797, CGMCC No. 46798, CGMCC No. 46799, and CGMCC No. 46800, respectively.

[0012] In some embodiments, the composition comprises a combination of first to fourth cells.

[0013] A second aspect of the present invention provides a HER2 immunohistochemical multi-stage quality control paraffin block, the paraffin block being prepared from the multi-stage control cell composition described in the first aspect.

[0014] In some embodiments, the wax block includes cellular regions expressing at least two of the first to fourth cells in the first aspect.

[0015] In some embodiments, the wax block includes cellular regions expressing a combination of the first to fourth cells in the first aspect.

[0016] The third aspect of the present invention provides a method for preparing the wax block described in the second aspect, the method comprising the steps of: collecting the first to fourth cells in the first aspect and fixing them, and preparing the fixed cells into a wax block.

[0017] In some embodiments, those skilled in the art may determine the number of cells collected in this invention according to the specific purpose of use. Depending on the purpose of the experiment, 1 10×10 7 (1×10 7 2×10 7 3×10 7 4×10 7 5×10 7 6×10 7 7×107 8×10 7 9×10 7 10×10 7 The number of cells is appropriate.

[0018] In some embodiments, the preparation method includes the following steps: (1) Take samples and fix them. Culture the first to fourth cells described in the first aspect, collect the cell pellets, and fix them with fixative for 24 to 48 h.

[0019] In some embodiments, the fixative is 10% neutral formalin.

[0020] In this invention, the main function of the fixative is to fix the cell structure and maintain the integrity of the cell DNA and proteins. The volume of the fixative is at least 5, 10, 20 times or more of the cell volume.

[0021] A suitable fixative does not impair antigenicity and can preserve the structure of tissues and cells while allowing macromolecules to enter cells and bind to antigens. The main fixative groups, classified according to their mechanism of action, include aldehydes, alcohols, oxidants, mercuric chlorides, and picrates, wherein the aldehydes include C1... C6 alkyl chain or C1 C8 alkylene dialdehyde, wherein the alcohol includes ethylene glycol, propylene glycol, glycerol, sorbitol and mannitol.

[0022] In some embodiments, the stationary liquid is paraformaldehyde, which is a polymeric form of formaldehyde that depolymerizes upon heating to provide formalin.

[0023] In a specific embodiment of the present invention, the fixative is formaldehyde, preferably 10% neutral buffered formalin (NBF).

[0024] The fixative solution described in this invention can be prepared using the following buffer solutions: phosphate buffer, bicarbonate buffer, malate buffer, carboxylate buffer, and florfenicol buffer.

[0025] In some embodiments, the fixing step is performed at a varying temperature or a constant temperature.

[0026] The temperature change refers to the alternating hot and cold operation process of the reagent. A typical approach is to first place it in a low-temperature environment of <15°C to facilitate the penetration of the fixative into the cells, and then transfer it to a high-temperature environment of >20°C to enhance the cross-linking reaction kinetics.

[0027] In some embodiments, the temperature range of the constant temperature is 20℃ 37℃.

[0028] In some embodiments, when fixing is performed at room temperature, the duration of the fixing process can be 12 hours. 48 hours, preferably about 24 hours.

[0029] (2) Centrifuge to remove fixative, add cell gel to the first to fourth cells after fixation, and prepare cell column.

[0030] In some embodiments, the cell gel may be selected from any one or more combinations of xanthan gum, sodium alginate, pectin, propylene glycol alginate, carboxymethyl cellulose, gum arabic, guar gum, polysaccharides, or conventional gels. In a specific embodiment of the present invention, the cell gel is a polysaccharide, and the polysaccharide is agarose.

[0031] Furthermore, the agarose concentration is 1%. 3%.

[0032] Furthermore, the volume of the agarose is 1 / 5 to 1 / 2 of the volume of the cell precipitate.

[0033] (3) The prepared cell columns were subjected to the following treatments in sequence: 70% ethanol for 2 h, 85% ethanol for 2 h, 95% ethanol for 1 h, 95% ethanol for 1 h, anhydrous ethanol for 1 h, anhydrous ethanol for 1 h, anhydrous ethanol for 30 min, anhydrous ethanol: xylene in a ratio of 1:1, xylene for 30 min, xylene for 30 min, paraffin high temperature treatment for 1 h, paraffin high temperature treatment for 1 h, paraffin high temperature treatment for 1 h.

[0034] In some embodiments, xylene in step (3) may be replaced by any combination of one or more of the following reagents: ethanol, n-butanol, methanol, isopropanol, acetone, benzoic acid. 2 Ethylhexyl ester, palmitic acid 2 Ethylhexyl ester, coconut oil acid 2 Ethylhexyl ester, stearic acid 2 Ethylhexyl ester, acetic acid 2 Ethylhexyl ester, benzyl benzoate, polyethylene glycol methyl ether methacrylate, tetrahydroxypropyl ethylenediamine solution, limonene.

[0035] (4) Spatial positioning assembly of at least two of the cell columns of the first to fourth cells to form a composite cell column.

[0036] In some embodiments, the combination of cell columns of the first to fourth cells is spatially positioned and assembled to form a four-in-one composite cell column.

[0037] In some embodiments, the arrangement of the spatial positioning assembly can be linear, rectangular / square, concentric circle, marked orientation arrangement, or other asymmetrical but identifiable arrangements (such as L-shaped, T-shaped, etc.) as required by those skilled in the art.

[0038] (5) The embedding machine embeds the wax blocks into multi-stage quality control blocks.

[0039] In some embodiments, the embedding cassette in the preparation of the wax block can be of any shape. Commonly used embedding cassettes in the art include cubes and cuboids.

[0040] The fourth aspect of the present invention provides a multi-stage control slide for HER2 immunohistochemical detection, wherein the slide is obtained by sequentially slicing the paraffin block described in the second aspect.

[0041] In some embodiments, the thickness of the slice is 3-5 μm.

[0042] In some embodiments, the single slice contains at least two of the cellular regions of the first to fourth cells.

[0043] In some embodiments, the single slice contains a cellular region comprising a combination of the first to fourth cells.

[0044] A fifth aspect of the present invention provides a HER2 immunohistochemical standard reference material, wherein the standard reference material is selected from any of the following forms: 1) The multi-stage control cell composition described in the first aspect; 2) The multi-stage quality control wax block described in the second aspect; 3) The multi-level control sections mentioned in the fourth aspect.

[0045] In this invention, the standard reference material, also known as the quality control material or quality control substance, refers to a substance used to monitor the quality of the testing process. Its concentration or characteristics are similar to those of clinical samples, and it is used to evaluate the stability, accuracy, and precision of the testing system.

[0046] In some embodiments, when the standard reference material is in the form of a slice, it is fixed on a glass slide.

[0047] In some embodiments, the slices are sealed.

[0048] In this invention, the glass slide can be selected from conventional glass slides, coated glass slides, plastic glass slides, charged glass slides, grid glass slides, and fluorescently marked glass slides as needed.

[0049] The sixth aspect of the present invention provides a HER2 immunohistochemical detection system, the detection system comprising a quality control module and a detection module; the quality control module comprises the standard reference material described in the fifth aspect; the detection module comprises a monoclonal antibody that specifically recognizes the HER2 antigen.

[0050] In some embodiments, the monoclonal antibody, under a standard immunohistochemical procedure, can generate staining signals corresponding to the expression levels of 0+, 1+, 2+, and 3+ cell regions in the quality control module.

[0051] In some embodiments, the clone number of the monoclonal antibody is 4B5.

[0052] In a specific embodiment of the present invention, the HER2 monoclonal antibody with clone number 4B5 was produced by Roche Diagnostics.

[0053] The seventh aspect of the present invention provides a kit for HER2 immunohistochemical detection, comprising the standard reference material described in the fifth aspect or the detection system described in the sixth aspect.

[0054] In some embodiments, the kit further includes one or more of the following reagents: blocking solution, antigen retrieval solution, chromogenic substrate, washing buffer, and counterstaining solution.

[0055] The chromogenic substrates include DAB staining solution, AEC staining solution, 4-chloro-1-naphthol chromogenic solution, etc. The blocking solutions include protein blocking solutions, such as bovine serum albumin (BSA) or normal goat serum (NGS); hydrogen peroxide, levamisole, avidin / biotin blockers, or other commercial blocking solutions. The washing buffers include phosphate-buffered saline (PBS), Tris buffer (including Tris-HCl buffer, TBS), Karasson-Schwlt's phosphate buffer, dimethylarsinate buffer, etc. The antigen retrieval solutions include sodium citrate buffer, EDTA-Tris buffer, etc. The counterstaining solutions include hematoxylin, methyl green, etc.

[0056] In some embodiments, the kit further includes one or more of the following: primary antibody, secondary antibody, chromogenic agent, washing solution, diluent, antigen retrieval solution, stop solution, and lining agent.

[0057] In some embodiments, the kit further includes instructions for use, which describe the use of the standard reference material for calibrating, validating, or quality-controlling HER2 immunohistochemical detection results.

[0058] The eighth aspect of the present invention provides for any of the following applications of the multi-stage control cell composition described in the first aspect, the multi-stage quality control paraffin block described in the second aspect, and / or the multi-stage control slide described in the fourth aspect: 1) As an internal quality control material for HER2 immunohistochemical detection; 2) Used for the development, validation, or standardization of HER2 immunohistochemical detection platforms, antibodies, or procedures; 3) Prepare pathological specimens, digital pathological image databases, or automated interpretation training samples for teaching purposes.

[0059] The ninth aspect of this invention provides for any of the following applications of the standard reference material described in the fifth aspect, the detection system described in the sixth aspect, and / or the reagent kit described in the seventh aspect: 1) Internal quality control or external proficiency testing within the laboratory; 2) Quality control products for preclinical research and companion diagnostic reagent development; 3) Standardized input data sources in medical education, pathologist training, or artificial intelligence algorithm training.

[0060] In some embodiments, the pathological specimens used for teaching include physical slide specimens or digital slide files generated by whole-slide scanning.

[0061] Internal quality control, also known as internal quality control, refers to the internal quality control process required for each immunohistochemical test in a clinical laboratory to ensure the accuracy and consistency of the test results. In this invention, the multi-level control cell composition, multi-level quality control paraffin blocks, or multi-level control slides described in aspect eight, or the standard reference materials, detection system, and reagent kits described in aspect nine, can be used as internal quality control samples.

[0062] Specifically, the 0+, 1+, 2+, and 3+ four-in-one quality control slides provided by this invention can be processed in parallel with the clinical samples to be tested in the same staining process. By evaluating indicators such as whether the staining intensity of the positive control cells (1+, 2+, and 3+) meets the expected gradient and whether the negative control cells (0+) have no non-specific staining, it can be determined whether the detection system is under control.

[0063] External proficiency testing, also known as interlaboratory quality assessment, refers to a quality assessment process used to confirm the consistency and comparability of HER2 immunohistochemical detection results between different laboratories. In this invention, the aforementioned standard reference materials (such as multi-level control slides) can be distributed to multiple participating laboratories, and each laboratory performs staining and interpretation according to its standard operating procedures. Through centralized analysis of the returned results, the differences between laboratories in antibody performance, platform stability, and interpretation standards can be objectively evaluated, thereby promoting cross-institutional standardization of testing quality.

[0064] In some embodiments, the pathological specimens used for teaching include physical slide specimens or digital slide files generated by whole-slide scanning.

[0065] In some embodiments, the device refers to an intelligent detection device prepared by immobilizing antigen information from multi-level control slides onto a specific carrier. This device can perform quantitative or qualitative analysis of antigens in a sample using optical, electrochemical, or other detection methods. The device can be a portable detection device.

[0066] In some embodiments, the cell model refers to a functional in vitro model constructed based on the positive control cells (e.g., 1+, 2+, 3+) and / or negative control cells (0+) described in the first aspect. This model can be used to construct a high-throughput drug screening platform. Through automated sample loading and imaging systems, the effects of a large number of candidate compounds on HER2 expression levels, localization, or signaling pathways can be tested, thereby rapidly identifying therapeutic candidate molecules with the potential to regulate HER2, serving targeted drug development and mechanism research.

[0067] Advantages and beneficial effects of this invention: This invention provides for the first time a four-in-one reference material, which contains four screened and validated cell lines that stably exhibit 0+, 1+, 2+, and 3+ immunohistochemical staining characteristics of HER2 protein expression, demonstrating good staining stability and reproducibility. It enables simultaneous acquisition of full-lineage HER2 expression controls in a single staining, significantly improving the standardization, accuracy, and comparability of HER2 detection in gastric cancer, and is suitable for the accurate diagnosis of HER2-related diseases such as gastric cancer. Attached Figure Description

[0068] Figure 1 The images show representative staining results of cell lines with low HER2 expression, where A is SNU5, B is KATO III, C is HER2-G1, D is SNU16, E is HUTU 80, F is MKN45, G is SNU1, H is AGS, and I is HER2(G)-0.

[0069] Figure 2 The image shows representative staining results of cell lines with high HER2 expression, where A represents HER2-G2, B represents NUGC4, and C represents HER2-G3.

[0070] Figure 3 The chart shows the statistical results of CT values ​​for cloned cell lines, where HER2-G0 is A, HER2-G1 is B, HER2-G2 is C, and HER2-G3 is D.

[0071] Figure 4 The images show representative staining results of HER2-G0, HER2-G1, HER2-G2, and HER2-G3 on the Zhongshan platform (Roche antibody clone number 4B5), corresponding to A, B, C, and D respectively.

[0072] Figure 5The images show representative staining results of HER2-G0, HER2-G1, HER2-G2, and HER2-G3 on the Roche platform (Roche antibody clone number 4B5), corresponding to A, B, C, and D respectively.

[0073] Figure 6 The images show representative staining results of HER2-G0, HER2-G1, HER2-G2, and HER2-G3 on the Zhongshan platform (Zhongshan antibody clone number UMAB36), corresponding to A, B, C, and D respectively. Detailed Implementation

[0074] The reagents, raw materials, and experimental consumables used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are typically performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following examples are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0075] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0076] Example I. Construction of cell lines 1. Source of cell line screening This invention uses basic gastric cancer cell lines (such as SNU5 and AGS) with negative or weakly positive levels of endogenous HER2 antigen expression as starting materials. Through genetic engineering, a lentiviral expression vector carrying the full-length coding sequence of human HER2 is constructed. Combined with promoter regulation strategies of different strengths, the expression level of exogenous HER2 protein is finely regulated. For each parental cell line, this invention independently screens and obtains a series of cell lines with different levels of exogenous HER2 expression and morphological characteristics of monoclonal cell lines that are highly consistent with the corresponding parental cells.

[0077] In this invention, the HER2 expression levels (0, 1+, 2+, 3+) of each monoclonal strain were determined using a dual confirmation system: primarily immunohistochemical (IHC) visual grading, supplemented by qPCR transcriptional level verification. The core basis for this system is as follows: referring to the clinical interpretation standards for HER2 in gastric cancer, multiple pathologists performed double-blind microscopic scoring of the cell membrane integrity and staining intensity after IHC staining to ensure that the staining gradient at the pathological level matches the actual clinical testing scenario; simultaneously, qPCR was used to determine the relative transcription fold of HER2 mRNA in each strain (2... -ΔΔCT The IHC grading and qPCR CT values ​​were used as objective quantitative standards for screening the "best combination". A series of cell lines that maintained stable IHC grading and qPCR CT values ​​during continuous passage were selected to form multi-level quality control products. This process aims to construct a multi-level quality control system covering the complete staining gradient, particularly addressing the current practical need for stable and reproducible weak positive controls (1+ and 2+) in immunohistochemical assays.

[0078] During the screening process, the growth characteristics of each clone were first standardized and evaluated, including cell morphology, adhesion ability, proliferation rate, and culture stability. Simultaneously, cell density was accurately counted using a cell counter or hemocytometer to ensure consistent cell numbers for each experiment and that all candidate lines were in good and consistent physiological condition. Subsequently, multiple rounds of immunohistochemical staining were conducted for initial screening, grading cells according to the interpretation criteria of 0+ (negative), 1+ (weakly positive), 2+ (moderately positive), and 3+ (strongly positive), with particular focus on screening for the 1+ and 2+ phenotypes. Weakly positive and moderately positive cells often exhibit weak signals, large batch-to-batch fluctuations, or blurred staining boundaries in conventional quality control materials. Therefore, this invention focuses on retaining clones with clear staining characteristics, well-defined membrane localization, clean backgrounds, and no significant drift in expression levels after continuous passage.

[0079] The immunohistochemical verification results and membrane staining grading standards for 0+, 1+, 2+, and 3+ were determined with reference to the interpretation criteria in the "Guidelines for HER2 Detection in Gastric Cancer" or the ASCO / CAP Guidelines for HER2 Detection in Gastroesophageal Adenocarcinoma.

[0080] Table 1. Interpretation and scoring criteria for HER2 immunohistochemical detection results in gastric cancer specimens

[0081] After further screening and verification, two cell lines were finally obtained that stably exhibited typical 1+ (named HER2-G1) and 2+ (named HER2-G2) staining patterns, respectively. They showed excellent reproducibility and antigen accessibility under different staining platforms, making them very suitable as standardized controls for weakly positive levels.

[0082] Simultaneously, through parallel screening, a negative control cell (0+, named HER2-G0) with virtually no HER2 expression and no nonspecific staining was obtained, along with a positive control cell (3+, named HER2-G3) with high HER2 expression and strong, uniform staining. These four cell lines (0+, 1+, 2+, and 3+) together constitute a complete four-in-one reference material combination.

[0083] 2. Cell line expression level detection Primers were designed to perform qPCR experiments on the screened cell lines to detect HER2 molecule expression in different cell lines. Cell lines with different HER2 molecule expression levels were selected as control groups. Primer sequence information is shown in Table 2, and reaction preparation system is shown in Table 3.

[0084] Table 2 Primer sequence information

[0085] Table 3 qPCR reaction system

[0086] The selected cell lines were subjected to qPCR experiments, and their CT values ​​are shown in Table 4.

[0087] Table 4 CT value results of cell lines

[0088] As shown in Table 4 and Figure 1 As shown, using known low-expression cells in the prior art as a comparative example, it can be seen that in cell lines with low HER2 molecule expression, except for HER2-G0, sporadic cell staining is observed in all others, with some exhibiting non-specific cytoplasmic and nuclear staining. For example, KATO III cells, whose molecule expression level is higher than SNU16 and MKN45, show weaker HER2 protein expression than other cell lines, with only sporadic cells showing weak staining. HER2-G0 cells, on the other hand, show negative staining with no specific background staining, revealing an unexpected result.

[0089] As shown in Table 4 and Figure 2 As shown, cells with known high expression levels in existing technologies were used as control examples. It is evident that NUGC4 expression was low, but immunohistochemistry showed 2+ staining and significant heterogeneity, with some cells exhibiting inaccurate localization after whole-cell staining. HER2-G2 expression was moderate, showing moderately positive membrane staining, resulting in better results.

[0090] II. Immunostaining Detection 1. The four selected cell lines were prepared into paraffin blocks. The process for preparing the cell paraffin blocks is as follows: 1) Cell collection and fixation: Collect cells and fix them in 10% formalin for 24-48 h; 2) Centrifuge to remove the fixative, add cell gel to the fixed cells, and prepare a cell column; 3) The composite cell column was subjected to the following treatments in sequence: 70% ethanol for 2 h; 85% ethanol for 2 h; 95% ethanol for 1 h; 95% ethanol for 1 h; anhydrous ethanol for 1 h; anhydrous ethanol for 1 h; anhydrous ethanol:xylene (1:1) for 30 min; xylene for 30 min; xylene for 30 min; paraffin for 1 h; paraffin for 1 h; paraffin for 1 h; 4) The cell columns of the four types of cells are spatially positioned and assembled to form a four-in-one composite cell column, which is then embedded into a cell wax block using an embedding machine.

[0091] 2. After the cell paraffin blocks are prepared, immunohistochemical staining is performed. The staining process uses Roche antibodies and the Zhongshan platform for fully automated staining. The specific staining process is as follows: 1) Sectioning: Use a microtome to section the paraffin block, completely cut out the required parts, adjust the section thickness to 3-5 μm, start slicing, place it in a tissue spreader to spread the slide, then take it out with a glass slide, and label it with the name and date; 2) Drying the film: Place the film in an oven (60℃) and dry for 1~2 hours; 3) Immunohistochemistry procedure: Select the antibody to be stained and the corresponding staining program name: HER2: Roche antibody, clone number 4B5, selection program 20, 30.

[0092] 4) Unloading: After staining, click the button to unload the slide holder, remove the slides, dehydrate them with gradient alcohol, and then clear and mount them.

[0093] 3. Further, to verify the universality of HER2-G0, HER2-G1, HER2-G2, and HER2-G3 as reference materials in HER2 antigen immunohistochemical staining, the following was performed: 1) Staining was performed on the Roche platform benchmark ultra: Roche antibody, clone number 4B5 ( Figure 5 ); 2) Staining on the Zhongshan platform: Zhongshan antibody, clone number UMAB36 ( Figure 6 ).

[0094] According to the staining results, when using the HER2 antibody with clone number UMAB36, the staining of 2+ cells was close to that of 3+ cells, resulting in poor staining effect. However, when using the HER2 antibody with clone number 4B5, good staining stability and reproducibility were achieved on different staining platforms, and the staining results for different cell regions fully met the judgment criteria in Table 1, making it a suitable reference material for HER2 immunohistochemical detection.

[0095] To ensure the reproducibility and industrial applicability of this invention, HER2-G0, HER2-G1, HER2-G2, and HER2-G3 have been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on April 3, 2026, with accession numbers CGMCC No. 46797, CGMCC No. 46798, CGMCC No. 46799, and CGMCC No. 46800, respectively. This complies with the relevant provisions of the Budapest Treaty on the International Recognition of the Preservation of Microorganisms for Patent Proceedings.

[0096] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A multi-stage control cell composition for immunohistochemical detection of human epidermal growth factor receptor 2, characterized in that, The composition comprises at least two of the following four cell types: The first cell showed a HER2 protein expression level of 0+ as verified by immunohistochemistry, with no membrane staining. The second cell showed that the HER2 protein expression level was 1+ as verified by immunohistochemistry, and it was weakly or faintly visible as membrane staining. The third cell, whose HER2 protein expression level was verified to be 2+ by immunohistochemistry, showed weak to moderate basal side membrane, side membrane or complete membrane staining. The fourth cell showed that its HER2 protein expression level was 3+ as verified by immunohistochemistry, and it exhibited strong staining of the basal side membrane, side membrane, or complete membrane. The first to fourth cells were named HER2-G0, HER2-G1, HER2-G2, and HER2-G3, respectively, and have been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 46797, CGMCC No. 46798, CGMCC No. 46799, and CGMCC No. 46800, respectively. Preferably, the composition comprises a combination of the first to fourth cells.

2. A HER2 immunohistochemical multi-level quality control paraffin block, characterized in that, The wax block was prepared from the multi-stage control cell composition described in claim 1; Preferably, the wax block includes cellular regions expressing at least two of the first to fourth cells of claim 1; Preferably, the wax block includes cellular regions expressing a combination of the first to fourth cells of claim 1.

3. The method for preparing the wax block according to claim 2, characterized in that, The preparation method includes the following steps: collecting the first to fourth cells of claim 1 and fixing them, and preparing the fixed cells into a wax block; Preferably, the preparation method includes the following steps: (1) Take samples and fix them. Culture the first to fourth cells as described in claim 1 respectively, collect the cell pellets, and fix them with fixative for 24 to 48 h. Preferably, the fixative is 10% neutral formalin; (2) Centrifuge to remove fixative, add cell gel to the first to fourth cells after fixation, and prepare cell columns; Preferably, the cell gel is a polysaccharide; (3) The cell column was subjected to the following treatments in sequence: 70% ethanol for 2 h, 85% ethanol for 2 h, 95% ethanol for 1 h, 95% ethanol for 1 h, anhydrous ethanol for 1 h, anhydrous ethanol for 1 h, anhydrous ethanol for 30 min, anhydrous ethanol: xylene in a ratio of 1:1, xylene for 30 min, xylene for 30 min, paraffin high temperature treatment for 1 h, paraffin high temperature treatment for 1 h, paraffin high temperature treatment for 1 h; (4) Spatial positioning assembly of at least two of the cell columns of the first to fourth cells to form a composite cell column; Preferably, the cell columns of the first to fourth cells are spatially assembled to form a four-in-one composite cell column; (5) The embedding machine embeds the wax blocks into multi-stage quality control blocks.

4. A multi-stage control slide for HER2 immunohistochemical detection, characterized in that, The slices are obtained by continuously slicing the wax block as described in claim 2. Preferably, the thickness of the slice is 3-5 μm; Preferably, the single slice contains at least two types of cellular regions from the first to the fourth cells; Preferably, the single slice contains a cellular region comprising a combination of the first to fourth cells.

5. A HER2 immunohistochemical standard reference material, characterized in that, The standard reference material is selected from any of the following forms: 1) The multi-stage control cell composition according to claim 1; 2) The multi-stage quality control wax block as described in claim 2; 3) The multi-level control slices as described in claim 4; Preferably, when the standard reference material is in the form of a slice, it is fixed on a glass slide; Preferably, the slices are sealed.

6. A HER2 immunohistochemical detection system, characterized in that, The detection system includes a quality control module and a detection module; The quality control module includes the standard reference material as described in claim 5; The detection module contains a monoclonal antibody that specifically recognizes the HER2 antigen; Preferably, the monoclonal antibody, under standard immunohistochemical procedures, can generate staining signals corresponding to the expression levels of 0+, 1+, 2+, and 3+ cell regions in the quality control module; Preferably, the monoclonal antibody has a clone number of 4B5.

7. A kit for HER2 immunohistochemical detection, characterized in that, It includes the standard reference material as described in claim 5 or the detection system as described in claim 6; Preferably, the kit further includes one or more of the following reagents: blocking solution, antigen retrieval solution, chromogenic substrate, washing buffer, and counterstaining solution; Preferably, the kit further includes an instruction manual, which describes the use of the standard reference material for calibrating, verifying, or quality-controlling HER2 immunohistochemical detection results.

8. Any of the following applications of the multi-stage control cell composition of claim 1, the multi-stage quality control paraffin block of claim 2, and / or the multi-stage control slide of claim 4: 1) As an internal quality control material for HER2 immunohistochemical detection; 2) Used for the development, validation, or standardization of HER2 immunohistochemical detection platforms, antibodies, or procedures; 3) Prepare pathological specimens, digital pathological image databases, or automated interpretation training samples for teaching purposes.

9. Any of the following applications of the standard reference material of claim 5, the detection system of claim 6, and / or the kit of claim 7: 1) Internal quality control or external proficiency testing within the laboratory; 2) Quality control products for preclinical research and companion diagnostic reagent development; 3) Standardized input data sources in medical education, pathologist training, or artificial intelligence algorithm training.

10. The application according to any one of claims 8-9, characterized in that, The pathological specimens used for teaching include physical slide specimens or digital slide files generated by scanning a whole slide.

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