Dyeing method of immunohistochemistry multi-staining combined reagent and application thereof

Abstract

The invention relates to the technical field of biology, and particularly discloses a staining method and application of an immunohistochemistry multi-staining combined reagent, the combined reagent comprises a first antibody combination and a second antibody combination, the first antibody combination comprises a myoepithelium specific marker Calponin and p16 with myoepithelium expression characteristics and a cell cycle regulation function; and the second antibody combination comprises an epithelial marker E-cadherin. According to the invention, three markers of Calponin, p16 and E-cadherin are subjected to immunohistochemical multi-staining combination, so that whether muscle epithelial cells exist or not and the attributes of gland epithelial cells can be synchronously displayed on the same tissue slice at the same site; mutual-verification and complementary morphological and molecular basis is provided for indirectly prompting whether the basilar membrane is damaged or not through the integrity of the muscular epithelial layer, and the detection rate of the micro-invasive cancer is increased. Meanwhile, spatial distribution information of the three types of key markers can be obtained through one dyeing process, tissue samples are saved, and the method is suitable for accurate diagnosis of biopsy specimens.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to staining methods and applications of immunohistochemical multiple staining combination reagents. Background Technology

[0002] Immunohistochemistry (IHC) is an indispensable tool in pathological diagnosis and research. It locates and visualizes specific proteins in tissue sections through antigen-antibody specific binding reactions, thereby providing key evidence for cell typing, tumor origin determination, and biological behavior assessment.

[0003] In the pathological diagnosis of breast diseases, accurately distinguishing between ductal carcinoma in situ (DCIS) and minimally invasive carcinoma (MIC) is crucial, as it directly affects the choice of clinical treatment strategies and patient prognosis assessment. However, lesions of minimally invasive carcinoma are usually extremely small and often lack typical morphological features in routine hematoxylin-eosin (HE) stained sections. Especially in the presence of fibrosis, tissue compression, or inflammatory background, relying solely on HE staining for interpretation can easily lead to missed diagnoses or diagnostic uncertainty (reported as "suspicious invasion"), posing a significant challenge to the consistency and accuracy of diagnosis.

[0004] For example, the invention disclosed in application publication number CN113466015A, concerning a staining reagent for distinguishing normal cells from cancer cells, its preparation method, and its application, uses an orange-red G solution as a carbon source and prepares an orange-red G carbon dot solution via electrolysis. This orange-red G carbon dot solution serves as the staining reagent for distinguishing normal cells from cancer cells. By placing the staining reagent-treated cell fluid and normal cell fluid under a fluorescence differential interference microscope and comparing the cell morphology, it is possible to determine whether the tested cells have undergone cancerous transformation. However, this patent provides a preliminary detection of whether a single cell is cancerous and is applicable to a wide range of tissue cells, making its application in identifying breast disease cells relatively limited.

[0005] To overcome the limitations of HE staining, immunohistochemical staining techniques using single biomarkers are widely used in auxiliary diagnosis. For example, myoepithelial markers such as Calponin are used to show the integrity of the basement membrane: if the myoepithelial layer is continuous, it tends to indicate carcinoma in situ; if there are gaps or interruptions, it suggests invasion.

[0006] However, single antibody staining has obvious limitations: First, it provides limited information dimensions and cannot simultaneously reflect the properties of epithelial cells and the state of myoepithelial cells. Diagnosis requires pathologists to compare and comprehensively analyze different sections, which is a cumbersome process and prone to misjudgment due to differences in tissue sections. Second, some markers (such as p16) are enhanced in high-grade lesions, but when used alone, they may lead to false negative or false positive results in the interpretation of myoepithelial cells due to problems such as staining background, cytoplasmic staining, or technical artifacts.

[0007] In recent years, immunohistochemical double staining techniques have been developed, which can simultaneously display two target proteins on the same tissue section, improving the efficiency of information integration. However, existing double staining protocols are mostly limited to combinations of two antibodies, and their diagnostic sensitivity and specificity in complex pathological scenarios remain insufficient.

[0008] When differentiating high-grade ductal carcinoma in situ with microinvasiveness, combining only myoepithelial markers and one epithelial marker may still not be enough to fully reveal the complete picture of the lesion, and it may still be difficult to give a clear conclusion for some "suspicious" or "borderline" lesions.

[0009] Therefore, there is an urgent need in this field to develop a more efficient and precise immunohistochemical multiple staining technique. Summary of the Invention

[0010] The purpose of this invention is to provide a staining method and its application using immunohistochemical multiple staining reagents, in order to solve the problem mentioned in the background art that the current methods for distinguishing between ductal carcinoma in situ and microinvasive carcinoma of the breast in complex pathological scenarios are insufficient.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] The combined reagents include a first antibody combination and a second antibody combination. The first antibody combination includes the myoepithelial marker Calponin and p16, which has both myoepithelial expression characteristics and cell cycle regulation functions. The second antibody combination includes the epithelial marker E-cadherin.

[0013] Preferably, the first antibody combination is developed using a DAB colorimetric system, and the second antibody combination is developed using an AP-Red colorimetric system.

[0014] Preferably, the Calponin monoclonal antibody, p16 monoclonal antibody, and E-cadherin monoclonal antibody are all ready-to-use monoclonal antibodies, and the mixing ratio of the Calponin monoclonal antibody to the p16 monoclonal antibody is 1:1.

[0015] The application of the combination reagent in the preparation of a detection kit for distinguishing cell types in breast tissue samples, the kit being suitable for a fully automated immunohistochemical staining instrument with a pre-set staining program corresponding to the combination reagent.

[0016] The staining method using immunohistochemical multiplex staining reagents includes the following steps:

[0017] 1) Sample preparation: Select diseased tissue, fix the diseased tissue with 4% neutral formaldehyde solution, and then embed it in paraffin to prepare tissue sections with a thickness of 4μm. Bake the tissue sections at 65℃ for 1 hour.

[0018] 2) Antigen retrieval: Place the baked tissue sections obtained in step 1) in an environment of 100℃ for antigen retrieval for 64 minutes.

[0019] 3) Multi-antibody stepwise staining: Staining was performed using an antibody combination consisting of Calponin monoclonal antibody, p16 monoclonal antibody, and E-cadherin monoclonal antibody.

[0020] The specific operations include:

[0021] ① First antibody incubation and color development: Calponin monoclonal antibody and p16 monoclonal antibody were mixed and incubated on the antigen-repaired tissue sections at a constant temperature of 37°C for 32 minutes; after incubation, the first color development reaction was performed using the DAB color development system.

[0022] ② Second antibody incubation and color development: Add E-cadherin monoclonal antibody to the tissue sections after the first color development and incubate at 37°C for 24 minutes; after incubation, perform the second color development reaction using the AP-Red color development system;

[0023] 4) Counterstaining and mounting: After the tissue sections in step 3) have undergone two staining processes, the cell nuclei are counterstained with hematoxylin solution. After counterstaining, the sections are rinsed and then mounted to obtain stained sections of the lesion tissue. The presence of breast microinvasive carcinoma in the lesion tissue cells is determined by observing the stained sections.

[0024] Immunohistochemical multiple staining reagents are used to observe the presence of missing myoepithelial cells and the distribution of glandular epithelial cells in breast lesions.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] This invention combines immunohistochemical multiplexing of three biomarkers—Calponin, p16, and E-cadherin—to simultaneously and at the same site on a single tissue section, revealing the presence or absence of myoepithelial cells and the properties of glandular epithelial cells. This provides mutually corroborating and complementary morphological and molecular evidence for indirectly indicating basement membrane damage through the integrity of the myoepithelial layer, significantly improving the detection rate of microinvasive carcinoma. Furthermore, this invention obtains the spatial distribution information of these three key biomarkers in a single staining process, saving tissue samples and making it suitable for the precise diagnosis of biopsy specimens.

[0027] The method of this invention is compatible with fully automated immunohistochemical staining instruments. Through preset programmed operation, it reduces the variation of manual operation steps, making it easy to promote and apply on a large scale in clinical pathology departments and ensuring the stability of diagnostic quality. Attached Figure Description

[0028] Figure 1 This is a diagram illustrating the pathological analysis of microinvasive cancer cells in an embodiment of the present invention.

[0029] Figure 2 This is a diagram illustrating in situ cancer cells analyzed in a pathological embodiment of the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1:

[0032] Ninety-eight surgical specimens of breast lesions received by the Department of Pathology of our hospital from January to June 2025 were selected. All samples met the following criteria: ① The patients were female, aged 31-80 years (median age 53 years); ② Exclusion criteria included history of breast malignant tumor surgery, tumor resection biopsy, and neoadjuvant chemotherapy; ③ Preliminary diagnosis by HE staining included 47 cases of ductal carcinoma in situ, 30 cases with suspected invasive lesions, and 21 cases of microinvasive carcinoma; ④ All samples were fixed with 4% neutral formaldehyde solution, with no tissue necrosis, autolysis, or other conditions affecting the staining effect.

[0033] Reagents and Consumables

[0034] Antibodies: Calponin monoclonal antibody, p16 monoclonal antibody, and E-cadherin monoclonal antibody (all ready-to-use, manufactured by Maixin Biotechnology Co., Ltd., Fuzhou, China, catalog numbers MAB-0516, MAB-0111, and MAB-0529, respectively).

[0035] Colorimetric systems: DAB colorimetric kit and AP-Red colorimetric kit (both from Roche, catalog numbers 760964 and 760974, respectively);

[0036] Auxiliary reagents: 4% neutral formaldehyde solution (Sinopharm Chemical Reagent Co., Ltd.), hematoxylin stain (Beijing Solarbio Technology Co., Ltd.), graded ethanol (75%, 85%, 95%, anhydrous ethanol, Sinopharm Group), xylene (Sinopharm Group), paraffin (Shanghai Hualing Rehabilitation Equipment Co., Ltd.);

[0037] Consumables: Pathology slide scalpel (Leica, Germany), anti-detachment slides (Jiangsu Shitai Experimental Equipment Co., Ltd.), coverslips (Jiangsu Shitai).

[0038] Sample preparation

[0039] Fixation: The surgically removed breast lesion tissue was cut into 1cm×1cm×0.3cm tissue blocks and fixed in 4% neutral formaldehyde solution at room temperature for 24 hours, changing the fixative once during the period to ensure adequate fixation;

[0040] Dehydration and embedding: The fixed tissue blocks were sequentially immersed in 75% ethanol (2 hours), 85% ethanol (2 hours), 95% ethanol (2 hours, twice), and anhydrous ethanol (1 hour, twice) for gradient dehydration. Then, they were cleared with xylene (1 hour, twice) and finally immersed in molten paraffin (60°C, 3 hours). The embedding was completed using a paraffin embedding machine, and the tissue blocks were prepared after cooling.

[0041] Sectioning and baking: The tissue paraffin block was cut into continuous sections with a thickness of 4μm using a microtome. The sections were then laid flat on a glass slide to prevent detachment and baked in a constant temperature oven at 65℃ for 1 hour to remove the paraffin from the sections and enhance the adhesion between the sections and the glass slide.

[0042] Antigen repair

[0043] Place the baked slides into an antigen retrieval instrument, add citrate buffer (pH 6.0) as the retrieval solution, set the retrieval conditions to 100℃ for 64 minutes, allow them to cool naturally to room temperature after retrieval, and rinse three times with PBS buffer (pH 7.4) for 3 minutes each time.

[0044] Multi-antibody stepwise staining (performed using the BenchMark Ultra fully automated immunohistochemical staining system)

[0045] Blocking: Add 3% hydrogen peroxide solution to the surface of the slide and incubate at room temperature for 10 minutes to block endogenous peroxidase activity. Rinse three times with PBS for three minutes each time.

[0046] First antibody incubation and color development: Mix Calponin monoclonal antibody and p16 monoclonal antibody at a volume ratio of 1:1, and evenly drop the mixed antibody solution (about 50 μL per slide) onto the slide. Set the staining instrument incubation parameters to 37°C and 32 minutes.

[0047] After incubation, rinse three times with PBS for three minutes each time, add the chromogenic solution from the DAB chromogenic kit, incubate at room temperature for five minutes, observe the chromogenic effect under a microscope (stop when brown appears), and rinse with distilled water to stop the chromogenic process.

[0048] Second antibody incubation and color development: Add E-cadherin monoclonal antibody solution (about 50 μL per slide) to the slide, and set the staining instrument incubation parameters to 37℃ for 24 minutes;

[0049] After incubation, rinse three times with PBS for three minutes each time, add the chromogenic solution from the AP-Red chromogenic kit, incubate at room temperature for eight minutes, observe under a microscope until red appears, and then rinse with distilled water to stop the chromogenic process.

[0050] Counterstaining: Place the sections that have undergone two staining processes into hematoxylin staining solution and incubate at room temperature for 3 minutes to counterstain the cell nuclei. Then rinse with distilled water, and then differentiate in 1% hydrochloric acid ethanol for 30 seconds. Finally, rinse with running water for 10 minutes to return to blue.

[0051] Dehydration and clearing: The sections were placed in 95% ethanol (2 minutes, 2 times) and anhydrous ethanol (2 minutes, 2 times) for dehydration, and then cleared with xylene (5 minutes, 2 times).

[0052] A drop of neutral resin is placed in the center of the slide, covered with a coverslip to avoid air bubbles, and placed in a fume hood to air dry naturally, resulting in a stained slide of breast lesion tissue.

[0053] According to the 2012 WHO classification of breast tumors, this study used a maximum invasive lesion diameter of no more than 1 mm as the diagnostic threshold for microinvasive carcinoma. All pathological sections were reviewed and diagnosed by two senior pathologists using a double-blind method. The following characteristic features were observed in the immunohistochemical multiplex staining assay using Calponin, p16 (dual antibody combination), and E-cadherin: ductal carcinoma in situ and normal ductal epithelial cells showed red cytoplasmic staining; myoepithelial cells surrounding non-invasive glandular tissue showed brown staining of both cytoplasm and nucleus. Diagnostic criteria: When continuous or discontinuous brown myoepithelial cells surround the red glandular epithelium, it is considered DCIS; if the myoepithelial cells surrounding the glandular epithelium lack staining and the maximum diameter of the tumor cell nest is ≤1 mm, it is diagnosed as microinvasive breast carcinoma. (See details for further information.) Figure 1 and Figure 2 As shown.

[0054] clinicopathological features

[0055] A total of 123 female patients were included, aged 29-58 years (median age 51 years), and all were pathologically diagnosed with breast proliferative lesions. Another 98 female patients, aged 31-80 years (median age 53 years), were preliminarily diagnosed with ductal carcinoma in situ of the breast by HE staining and immunohistochemical examination (some of whom had suspicious invasive or microinvasive features). Specific clinicopathological features are shown in Table 1.

[0056] Table 1. Clinicopathological characteristics of 98 patients with breast tumors (n, %)

[0057]

[0058] Optimal antibody combination and test results

[0059] Calponin / p16 combined with E-cadherin was selected as the optimal detection method. In 98 cases initially diagnosed as ductal carcinoma in situ (LCIS), with suspected invasive or microinvasive carcinoma, a double staining method using Calponin / p16 and E-cadherin was employed for analysis. Results showed that in the double staining group, 48 cases (48.98%) were LCIS, 11 cases (11.22%) were LCIS with suspected invasive involvement, and 39 cases (39.80%) were microinvasive carcinoma. In contrast, in the initial pathological diagnosis, 47 cases (47.96%) were LCIS, 30 cases (30.61%) had suspected invasive involvement, and 21 cases (21.43%) were microinvasive carcinoma. The double staining method significantly improved the detection rate of microinvasive carcinoma compared to the initial diagnosis (from 21.43% to 39.80%), while significantly reducing the reporting rate of suspected microinvasive carcinoma (from 30.61% to 11.22%). The triple antibody double staining technique (using a triple marker immunohistochemical multiple staining combination of Calponin, p16 (double antibody combination) and E-cadherin) showed a statistically significant difference compared with the single staining method (P < 0.05).

[0060] Table 2. Analysis of double staining detection and initial pathological diagnosis results of 98 breast tumor specimens (n, %)

[0061]

[0062] Traditional single-marker immunohistochemical staining has limitations in determining whether microinvasiveness is present in ductal carcinoma in situ (DCIS). Calponin, as a myoepithelial cell marker, is often used to identify the presence of an intact myoepithelial layer around the ducts, thereby determining the invasive state. p16 is often overexpressed in breast cancer, especially in high-grade DCIS, and has auxiliary diagnostic value. E-cadherin is retained in most ductal breast cancers, which helps to confirm the ductal origin of the tumor. Double staining of Calponin and p16 on the same tissue section can simultaneously show the absence of myoepithelial cells and the abnormal proliferation of tumor cells. Combined with co-expression analysis of E-cadherin, it can more comprehensively reflect the histological characteristics of the lesion.

[0063] This multi-target simultaneous detection not only saves tissue samples but also improves information integration efficiency, avoiding misdiagnosis caused by differences in sampling between different sections. The results showed a significant increase in the detection rate of microinvasive carcinoma, indicating that some lesions with microinvasive involvement were underestimated or missed during traditional initial diagnosis. This is related to the small size and atypical morphology of invasive lesions under conventional HE staining, especially in areas with fibrotic background or significant compression effect, which are difficult to identify under conventional microscopy. However, through double staining, the discontinuity of the myoepithelial layer can be visually presented through Calponin-negative areas. Combined with strong p16 positivity indicating high-grade intraepithelial lesions and E-cadherin positivity confirming ductal origin, these three factors work synergistically to provide pathologists with clearer diagnostic criteria, thus transforming previously "suspicious" cases into definitively diagnosed microinvasive carcinomas.

[0064] In conclusion, the Calponin / p16 combined with E-cadherin double staining technique significantly improves the detection rate of breast microinvasive carcinoma and reduces diagnostic uncertainty by integrating tissue structure and molecular phenotypic information, demonstrating good clinical applicability and promotional value.

[0065] It should be understood that numerous specific implementation decisions can be made during the development of any practical implementation, such as in any engineering or design project. Such development efforts may be complex and time-consuming, but for those skilled in the art who benefit from this disclosure, the development effort will be a routine work of design, manufacturing, and production without requiring much experimentation.

[0066] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An immunohistochemical multiplex staining reagent, characterized in that, The combined reagents include a first antibody combination and a second antibody combination. The first antibody combination includes the myoepithelial marker Calponin and p16, which has both myoepithelial expression characteristics and cell cycle regulation functions. The second antibody combination includes the epithelial marker E-cadherin.

2. The immunohistochemical multiplex staining reagent according to claim 1, characterized in that: The first antibody combination was developed using the DAB colorimetric system, and the second antibody combination was developed using the AP-Red colorimetric system.

3. The immunohistochemical multiplex staining reagent according to claim 1, characterized in that: The Calponin monoclonal antibody, p16 monoclonal antibody, and E-cadherin monoclonal antibody are all ready-to-use monoclonal antibodies, and the mixing ratio of the Calponin monoclonal antibody to the p16 monoclonal antibody is 1:

1.

4. The use of the combined reagent according to any one of claims 1 to 3 in the preparation of a detection kit for distinguishing cell types in breast tissue samples, wherein the kit is suitable for a fully automated immunohistochemical staining instrument, the instrument having a pre-set staining program corresponding to the combined reagent.

5. The staining method using the immunohistochemical multiplex staining reagent according to any one of claims 1 to 3, characterized in that: Includes the following steps: 1) Sample preparation: Select diseased tissue, fix the diseased tissue with 4% neutral formaldehyde solution, and then embed it in paraffin to prepare tissue sections with a thickness of 4μm. Bake the tissue sections at 65℃ for 1 hour. 2) Antigen retrieval: Place the baked tissue sections obtained in step 1) in an environment of 100℃ for antigen retrieval for 64 minutes. 3) Multi-antibody stepwise staining: Staining was performed using an antibody combination consisting of Calponin monoclonal antibody, p16 monoclonal antibody, and E-cadherin monoclonal antibody. The specific operations include: ① First antibody incubation and color development: Calponin monoclonal antibody and p16 monoclonal antibody were mixed and incubated on the antigen-repaired tissue sections at a constant temperature of 37°C for 32 minutes; after incubation, the first color development reaction was performed using the DAB color development system. ② Second antibody incubation and color development: Add E-cadherin monoclonal antibody to the tissue sections after the first color development and incubate at 37°C for 24 minutes; after incubation, perform the second color development reaction using the AP-Red color development system; 4) Counterstaining and mounting: After the tissue sections in step 3) have undergone two staining processes, the cell nuclei are counterstained with hematoxylin solution. After counterstaining, the sections are rinsed and then mounted to obtain stained sections of the lesion tissue. The presence of breast microinvasive carcinoma in the lesion tissue cells is determined by observing the stained sections.

6. The staining method of the immunohistochemical multiple staining reagent combination according to claim 5 is applied to observe the presence or absence of myoepithelial cells and the distribution status of glandular epithelial cells in breast lesion tissue.

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