Establishment and application of immunohistochemical positive controls: Based on tissue fragment suspensions and tissue microarrays

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Methods A variety of residual human tissue samples (including tonsil, appendix, stomach, liver, kidney, intestine, thyroid, placenta, breast, and tumor tissues with known positive target antigens) were collected from June 2023 to August 2023. After standardized processing, tissue paraffin blocks were prepared. Internal laboratory positive controls were constructed using tissue fragment suspension technology and tissue microarray technology, respectively. In subsequent IHC assays, test tissues and corresponding positive control tissues were mounted on the same glass slide for simultaneous staining. The two methods were evaluated in terms of preparation efficiency, operational convenience, staining consistency, antigen stability, and quality control reliability. Results A long-term usable internal quality control reference for the laboratory was successfully established. Tissue fragment suspensions saved time and effort, significantly improving work efficiency; tissue microarrays, when applied to IHC detection related to targeted therapy, remarkably enhanced the accuracy of test results. Conclusion The IHC positive control quality control system established based on tissue fragment suspensions and tissue microarrays is economical, convenient, efficient, and reliable. It not only effectively ensures the accuracy of IHC results but also realizes the standardization of internal quality control. Particularly suitable for promotion and application in daily diagnostic work, this system holds important clinical value and practical significance. Immunohistochemistry Positive control Tissue fragment suspension Tissue microarray Quality control Figures Figure 1 Figure 2 Introduction In the daily work of the pathology deparTMAent, immunohistochemical technology is an indispensable technical tool for pathological diagnosis. Especially in neoplastic lesions, IHC can be used to study specific clinical and biological characteristics, playing a crucial role in tumor classification and typing, diagnosis and differential diagnosis, prognosis judgment, and identification of immunotherapeutic targets[ 1 – 3 ]. However, the IHC technical process is complex, involving tissue fixation, embedding, sectioning, antigen retrieval, antibody incubation, and color development. Operations in each step can affect the final results, leading to the risk of misdiagnosis or missed diagnosis. The lack of standardization and reference control often results in result deviations[ 4 ]. Establishing a reliable positive control system is therefore essential to ensure the accuracy and reproducibility of IHC results. In the IHC quality control system, normal tissues or tissue samples with specific antigen expression are required as control tissues. The establishment of positive control tissues is the key to monitoring the standardization of the entire experimental process and ensuring the accuracy and reliability of experimental results. Positive control methods for IHC vary among laboratories, including internal tissue controls, amniotic membrane roll multi-tissue controls, and single-tissue controls. However, not all antigens have suitable internal controls in the test tissue, and both multi-tissue and single-tissue positive controls require large-scale sectioning, making them unsuitable for laboratories with heavy workloads. Based on the above practical needs, this study established two positive control methods: tissue fragment suspensions and tissue microarrays. Through systematic collection of various residual human tissue samples, a sustainable internal quality control system was successfully constructed. Positive controls using tissue suspensions are particularly suitable for daily high-volume, multi-antibody detection work; positive controls using tissue microarrays, which integrate tissue microarrays with different expression intensities into a single paraffin block, can provide a complete gradient reference in a single section experiment, making them especially suitable for semi-quantitative analysis of target-related markers. The selection of positive control tissues was based on recommendations from the Nordic Immunohistochemical Quality Control (NordiQC) and relevant antibody instructions, followed by preliminary experimental verification[ 5 – 6 ]. Since the establishment of the positive control system in 2022, our laboratory has widely applied it to daily internal IHC quality control. Practice has proven that the establishment of tissue fragment suspensions and tissue microarrays is economical, convenient, efficient, and accurate. This paper aims to systematically introduce the establishment methods of these two positive controls in detail and explore their application value in depth, providing a validated and feasible technical reference for the construction of quality control systems in IHC laboratories. Materials and methods Materials A variety of residual human tissue samples after pathological diagnosis were collected from June 2023 to August 2023, mainly including tonsil, appendix, stomach, liver, kidney, intestine, thyroid, placenta, breast, or tumor tissues with known positive target antigens. All tissues were fixed with 3.7% buffered neutral formalin, and paraffin blocks were prepared through standardized processes such as dehydration, transparency, wax infiltration, and embedding. This study was approved by the Ethics Committee of Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University). Main reagents and instruments Reagents: All primary antibodies for IHC involved in this study, IHC secondary antibody staining kits, xylene, absolute ethanol, 80% ethanol, and HE staining kits. Instruments: Leica RM2235 paraffin microtome, tissue microarray gun, flotation machine, baking machine, Roche automatic IHC stainer, optical microscope, low-speed centrifuge, oscillator, Sakura automatic coverslipper, etc. Methods Preparation of tissue fragment suspensions Sectioning: Paraffin blocks of positive control tissues were sectioned according to the corresponding IHC markers. Taking tonsil tissue as an example, 5 tonsil tissue paraffin blocks were selected for continuous sectioning on a Leica microtome at a thickness of 3–4 µm, totaling 300–400 sections. Crushing and dewaxing: The wax sections were collected in a clean 100 ml glass beaker and gently crushed with a glass rod. Approximately 2 ml of xylene was added halfway until a rice paste-like turbid liquid was formed. Subsequently, 100 ml of xylene was added, the beaker mouth was sealed, and the mixture was allowed to stand for 1 hour. The tissue suspension was transferred to a 50 ml centrifuge tube, balanced, and centrifuged at 3000 r/min for 4 minutes. At this point, tissue fragments were observed to precipitate at the bottom of the tube. The supernatant was carefully discarded, and this step was repeated twice until the supernatant was clear to ensure complete dissolution of paraffin. Replacement and dehydration: 50 ml of absolute ethanol was added to the precipitated tissue fragments, and the mixture was allowed to stand for 30 minutes. It was then centrifuged at 3000 r/min for 4 minutes, and the supernatant was discarded. This step was repeated twice to ensure the tissue fragments were free of xylene and moisture. Preservation: The final precipitated tissue fragments were mixed with 80% ethanol at a volume ratio of 1:50 to form a suspension, which was transferred to multiple sterile centrifuge tubes. The tissue type and preparation date were clearly marked, and the tubes were stored in a 4°C medical refrigerator. Concentration adjusTMAent and quality control: Before use, 1 µL of the tissue suspension was dropped onto a glass slide multiple times for HE staining to observe cell distribution. If the cells were too scattered, centrifugation was repeated to increase the concentration of the tissue suspension; if cell overlap was severe, centrifugation was repeated to dilute the concentration. AdjusTMAents were made until the cells were uniformly distributed with clear morphology. Application of tissue fragment suspensions Before use, the stored suspension was taken out of the 4°C medical refrigerator and thoroughly mixed on an oscillator for 30 seconds. A portion of the suspension was transferred to a new 6 ml primary antibody reagent bottle (or similar container) for recent use, and most of the remaining suspension was stored in centrifuge tubes to reduce storage risks. When fishing test tissue sections, according to the laboratory's positive control antibody classification table (Table 1 ), antibody markers requiring the same tissue suspension as a positive control on the same day were grouped together for centralized application to improve work efficiency. Using a pipette, approximately 1 µL of the tissue suspension was accurately dropped at the upper left corner and lower right corner of the test tissue, with a distance of about 0.5 cm from the tissue edge to prevent cross-contamination. The tissue suspension and test tissue were mounted on the same section and subjected to the IHC staining process simultaneously to implement quality control throughout the entire process. In the early stage of establishing the laboratory's positive control antibody classification table, the laboratory conducted multiple effective experimental verifications for all antibody markers corresponding to positive control suspensions. Before using each new batch of suspension, performance verification was also performed to ensure the positive control exhibited the expected positive effect before being put into the daily quality control system. Preparation of tissue microarrays For IHC related to targeted therapy, the laboratory established tissue microarray positive controls (Table 2 ). Taking HER-2 as an example, breast cancer tissue paraffin blocks with known target antigen expression results were selected: 1 case each of 0, 2+ (FISH-confirmed), and 3+[ 7 – 8 ]. Localization: Corresponding to HE sections and IHC sections, two senior pathologists marked representative areas under a microscope and identified the corresponding positions in the paraffin blocks[ 9 ]. According to the marked positions, tissues were accurately extracted using a tissue microarray gun (2 mm in diameter) and classified as 0, 2+, or 3+. Embedding: The 0, 2+, and 3 + tissues were embedded in the same embedding mold in sequence to prepare a HER-2 tissue microarray positive control paraffin block, which was stored routinely. Sectioning for standby use: The HER-2 tissue microarray positive control paraffin block was routinely sectioned at a thickness of 3–4 µm, and the sections were mounted on one end of an IHC anti-slip glass slide. To ensure uniform quality, the first, middle, and last sections were mounted on the same glass slide for HER-2 IHC staining. Under a microscope, the staining intensity of tissues at the same site on these three sections was evaluated for consistency and tissue integrity. Only microarray batches where the three sections showed highly consistent staining intensity and no obvious tissue loss were deemed qualified for subsequent experiments. Verified qualified microarray sections should be stored in a cool, dry, and light-protected slide box or refrigerated at 4°C. To avoid antigen attenuation due to long-term exposure, it is recommended that the number of sections prepared at one time meet the one-week usage requirement. Application of tissue microarrays For IHC detection of targeted therapy-related markers such as PD-L1 (SP263), ALK (D5F3), and HER-2, tissue microarray positive control slides were taken from standby sections. The test tissue was directly mounted on the other end of the slide with pre-prepared tissue microarray tissues, ensuring a distance of at least 0.5 cm between the test tissue and the microarray tissues. This "two-in-one" section was subjected to simultaneous IHC staining. Tissue microarray positive controls can serve as direct references for interpreting staining intensity, especially for indicators requiring semi-quantitative scoring such as HER-2 and PD-L1, greatly improving the accuracy and consistency of interpretation. Observation indicators On the same section, the positive control tissue was considered positive if it achieved the expected positive result. Statistical analysis Statistical analysis was performed using SPSS 25.0 software. The excellent and good rates of the two groups of positive controls were compared using the chi-square test (Fisher's exact test). A P value less than 0.05 was considered statistically significant. Results The total amount of tissue fragment suspension obtained through one standardized preparation could be stably used for more than 12 months under routine workload, providing reliable internal quality control guarantee for over 130,000 IHC sections. Compared with the traditional method of using a separate positive control section for each test section, this method saved more than 90% of control tissue consumption and corresponding sectioning time, with simple operation. Periodic sampling inspections (at 3, 6, 9, and 12 months) of suspensions stored at 4°C for up to 12 months showed no difference in IHC staining intensity compared with newly prepared suspensions, and no significant attenuation of antigenicity was observed (Fig. 1 ), demonstrating good long-term stability. Tissue microarrays targeting HER2 included samples exhibiting a gradient of expression levels, ranging from negative to weakly positive and strongly positive (Fig. 2 ), weakly positive to strongly positive, which not only verified the effectiveness of the experiment but also assisted technicians in accurately grasping the cell membrane/cytoplasmic characteristics of different staining intensities, ensuring the accurate application of scoring standards. Evaluation of more than 130,000 IHC sections using positive controls from September 2023 to September 2024 showed that 126,790 cases used tissue fragment suspensions with an excellent and good rate of 99.9% (including 105 false negatives, mainly due to the large distance between control tissue and test tissue), and 3,501 cases used tissue microarrays with an excellent and good rate of 99.9% (including 3 false negatives, mainly due to instrument failure). There was no statistical significance between the two groups of data (χ² = 0.057, P > 0.05), indicating that both methods could provide reliable guarantee for internal IHC quality control (Table 3 ). During daily internal quality control, staining problems could be detected in a timely manner, ensuring the accuracy and reliability of IHC experimental results. Discussion Immunohistochemistry is a technology that uses the principle of specific binding between antigens and antibodies to visualize labeled antibodies through chemical reactions, thereby enabling the localization, qualitative, and quantitative study of specific antigens in tissues or cells. IHC can label and detect cell lineage markers, therapeutic targets, and prognostic markers[ 10 ]. Although the practice of IHC has reshaped diagnostic pathology and promoted improvements in cancer treaTMAent, it still faces widespread challenges in standardization and reproducibility[ 11 – 14 ]. The experimental process is affected by various factors such as tissue fixation, experimental steps, human factors, and equipment factors. Without standardized quality control standards[ 15 ], it is difficult to obtain ideal test results[ 16 ]. In the IHC staining process, positive controls are the core tool for IHC standardization[ 17 ]. Through samples with known reactivity, positive controls ensure the reliability of IHC detection from antibody performance to operational procedures, helping to standardize staining, reduce experimental differences, and ensure the accuracy and reliability of experimental results through quality control throughout the IHC process[ 18 ]. This study detailed and evaluated the establishment methods and comprehensive application effects of IHC positive controls based on tissue fragment suspensions and tissue microarrays. Through systematic tissue collection and standardized processing, an internal quality control reference system for IHC laboratories with a wide coverage and complete targets was successfully constructed. Covering 15 different types of normal tissues and lesion tissues, it can provide reliable positive control support for more than 100 clinically commonly used IHC antibodies. Practice has proven that these two methods together form a scientific, economical, efficient, and reliable internal laboratory quality control solution, realizing "self-sufficiency" and "customization" of positive controls. Tissue fragment suspensions, characterized by convenience, efficiency, and economy, serve as quality control guarantees for daily high-volume, multi-index labeling. Our long-term stability data confirm that tissue fragment suspensions can achieve satisfactory IHC experimental results when stored under appropriate conditions (80% ethanol preservation solution, 4°C) for more than one year. Tissue microarrays, with the advantages of precision and intuition, focus on quality control guarantees for IHC markers related to targeted therapy, becoming increasingly prominent in the era of precision medicine. They not only verify the accuracy of experimental results but also serve as a reliable compass for result interpretation. The combination of the two ensures comprehensive internal quality control of the laboratory from routine diagnosis to cutting-edge detection. It is worth noting that performance verification emphasized in this study is the cornerstone of ensuring the effective operation of the entire system. Both the use of new batches of suspensions and the construction of new tissue microarrays must go through strict internal verification processes. In addition, combining these two internal quality control methods with participation in external quality control evaluations (such as PQCC, NordiQC, CAP, etc.) constitutes the best practice for quality management in IHC laboratories[19]. The establishment of positive controls using tissue fragment suspensions and tissue microarrays not only significantly improves the results of single IHC tests but also realizes the long-term stability and continuous improvement of laboratory quality control through standardized and reproducible operational procedures. By effectively monitoring the experimental process, it reduces instrument and human errors and improves operational standardization. Ultimately, it makes pathological diagnosis results more accurate and reliable. We strongly recommend the application of this comprehensive quality control system in daily internal quality control of pathology deparTMAents, especially in units with heavy daily diagnostic tasks. The combined use of tissue fragment suspensions and tissue microarrays, featuring convenience, efficiency, precision, and reliability, is of great significance for comprehensively improving the quality control of IHC, enhancing the overall quality of pathological diagnosis, and ensuring patient medical safety. Table 1 classification table of antibodies for tissue fragment suspension positive controls in IHC laboratory Control tissue Recommended antibodies Tonsil Proliferation and apoptosis-related: Ki67, Bcl-2, Bcl-6 T cell and related: CD2, CD3, CD4, CD5, CD7, CD8, TIA-1, CD45RO B cell and related: CD10, CD19, CD20, CD21, CD22, CD23, MUM1, CD79a Myeloid/histiocyte: CD15, CD68, CD163 Other leukocytes: CD38, CD43, CD45, CD57, CD123, CD138 Epithelial and mesenchymal markers: CK (HMW), CK (Pan), C5/6, Vimentin, P63 Mismatch repair proteins: MLH1, MSH2, MSH6, PMS2 Immunoglobulins: Kappa, Lambda, IgG Appendix Epithelial markers: CK8/18, CK20, CEA, Villin Mesenchymal/vascular markers: CD31, CD34, F8, FN, S-100, SMA, Desmin, Actin Gastrointestinal-specific marker: CDX-2 Lung cancer EGFR, SP-B, TTF-1, NapsinA, CK7, CK (LMW) Pheochromocytoma Neuroendocrine universal markers: CgA, Syn, NSE, CD56 Thyroid papillary carcinoma with BRAF V600E mutation BRAF V600E, Galectin-3, CK19, Tg, Ber-EP4 Liver cancer AFP, HepPar-1, Arginase-1, Glypican-3, GS Placenta CD163, GLUT-3, HPL, S-100P Breast cancer GATA3, TRPS1, E-cadherin, GCDFP-15 Cervix ER, PR Gastrointestinal stromal tumor CD117, DOG1 Clear cell renal cell carcinoma RCC, CAIX, TFE-3 Known positive urothelial carcinoma Uroplakin Ⅱ Kidney WT-1, PAX-8 Prostate cancer PSA, AR, P504S Table 2 Classification table of antibodies for tissue microarray positive controls in IHC laboratory Control tissue Recommended antibodies Breast cancer HER-2 0、2+、3+ HER-2 Appendix ALK(D5F3) Placenta PD-L1(SP263) Tissue with known positive target antigen CD30 Known positive pancreatic neuroendocrine tumor SSTR2 Gastric tissue Claudin18.2 Known Positive lung adenocarcinoma tissue c-MET Table 3 Expression analysis of two positive control methods Control method Positive(sections) False negative(sections) Total Excellent and good rate(%) χ² Value P value TFS 126790 105 126895 99.9 0.057 > 0.05 TMA 3501 3 3504 99.9 Total 130291 108 130399 99.9 Abbreviations IHC Immunohistochemistry PQCC National Pathology Quality Control Center NordiQC Nordic Immunohistochemical Quality Control CAP College of American Pathologists PD-L1 Programmed Death-Ligand 1 TFS Tissue fragment suspension TMA Tissue microarray Declarations Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate The study was approved by the Ethics Committee of the Hunan Provincial People's Hospital and The first-affiliated hospital of Hunan Normal University. The study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. As the study was a retrospective design and did not include any potentially identifiable patient data, informed consent was waived by the Ethics Committee of the Hunan Provincial People's Hospital and The first-affiliated hospital of Hunan Normal University. Consent for publication Not applicable. Conflict of interest The authors declare no competing interests. References Tsutsumi Y. Pitfalls and Caveats in Applying Chromogenic Immunostaining to Histopathological Diagnosis. Cells. 2021;10(6):1501. 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Reduced Number of Thymoma CTLA4-Positive Cells Is Associated With a Higher Probability of Developing Myasthenia Gravis. Neurol Neuroimmunol Neuroinflamm. 2023;10(2):e200085. Published 2023 Jan 25. doi:10.1212/NXI.0000000000200085 van Kempen S, Gerritsen WJG, Nguyen TQ, et al. Monitoring Immunohistochemical Staining Variations Using Artificial Intelligence on Standardized Controls. Lab Invest. 2025;105(5):104105. doi:10.1016/j.labinv.2025.104105 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 30 Jan, 2026 Editor assigned by journal 27 Jan, 2026 Editor invited by journal 06 Jan, 2026 Submission checks completed at journal 05 Jan, 2026 First submitted to journal 05 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8401699","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":583224861,"identity":"217570bc-bb09-4743-95e9-c5ff4815010b","order_by":0,"name":"Xiaoying Liu","email":"","orcid":"","institution":"Hunan Provincial People's Hospital and The first-affiliated hospital of Hunan normal university","correspondingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Liu","suffix":""},{"id":583224864,"identity":"78669b46-cfe4-4d7c-afdc-fe5b3eee37cc","order_by":1,"name":"Kun Wang","email":"","orcid":"","institution":"Hunan Provincial People's Hospital and The first-affiliated hospital of Hunan normal university","correspondingAuthor":false,"prefix":"","firstName":"Kun","middleName":"","lastName":"Wang","suffix":""},{"id":583224866,"identity":"1752fc66-8ca3-4e24-a23e-68f8a4b95818","order_by":2,"name":"Zhe Zhang","email":"","orcid":"","institution":"Hunan Provincial People's Hospital and The first-affiliated hospital of Hunan normal university","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Zhang","suffix":""},{"id":583224868,"identity":"f993935f-b115-434c-9850-4f5231f415d9","order_by":3,"name":"Penghui Dai","email":"","orcid":"","institution":"Hunan Provincial People's Hospital and The first-affiliated hospital of Hunan normal university","correspondingAuthor":false,"prefix":"","firstName":"Penghui","middleName":"","lastName":"Dai","suffix":""},{"id":583224869,"identity":"d36b2a58-6f50-492d-b8da-0a7a512edeab","order_by":4,"name":"Jie Zhao","email":"","orcid":"","institution":"Hunan Provincial People's Hospital and The first-affiliated hospital of Hunan normal university","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhao","suffix":""},{"id":583224871,"identity":"71017b7d-ba45-4b2b-b057-f34d0f75125f","order_by":5,"name":"Zhihong Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACCQY2ECnHL//4GEzMgBgtFsaSDWlpJGmpSNxwIMeMOC3ys5ufPfi4QwKo5cy3x7w77iQ2sDdvk2CouYNTC+OcY+aGM89IGM882LvdmPfMs8QGnmNlEgzHnuHUwiyRwybN2yYh23eYdxuQcTixQSLHTIKx4TBOLWwgLX/bgGqO8TyDaJF/g18LD0gLY5uE4oQzPGxQW3jwa5GQSDOT7G2TMJacwWYmObftsHEbT1qxRcIx3FrkZyQ/k/jZVifHL8H8TOJt22HZfvbDG298qMGtBYvvQEQCCRpGwSgYBaNgFGACAJb9UGu4WQnFAAAAAElFTkSuQmCC","orcid":"","institution":"Hunan Provincial People's Hospital and The first-affiliated hospital of Hunan normal university","correspondingAuthor":true,"prefix":"","firstName":"Zhihong","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-12-19 07:09:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8401699/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8401699/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101762929,"identity":"5895ca48-a655-4df1-822a-51a6bc896535","added_by":"auto","created_at":"2026-02-03 11:34:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":231628,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of immunohistochemical staining effects of CD117, CD20 and Ki-67 in tissue fragment suspension stored for 3, 6, 9 and 12 months.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8401699/v1/0658dc9a3c69657971ac4daa.png"},{"id":101762928,"identity":"b76c1d21-1d9e-460b-bf7c-207b87c0c7e1","added_by":"auto","created_at":"2026-02-03 11:34:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":326669,"visible":true,"origin":"","legend":"\u003cp\u003eIHC staining of HER-2 tissue microarrays. A: HER-2 negative; B: HER-2 1+; C: HER-2 2+; D: HER-2 3+\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8401699/v1/d213b772ddffc820cc05f82c.png"},{"id":104808097,"identity":"fcb7e229-c64c-4129-a4f6-75b4e99c7784","added_by":"auto","created_at":"2026-03-17 12:13:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1156416,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8401699/v1/7076c335-96a8-4a76-a787-dc2a736cff88.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment and application of immunohistochemical positive controls: Based on tissue fragment suspensions and tissue microarrays","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the daily work of the pathology deparTMAent, immunohistochemical technology is an indispensable technical tool for pathological diagnosis. Especially in neoplastic lesions, IHC can be used to study specific clinical and biological characteristics, playing a crucial role in tumor classification and typing, diagnosis and differential diagnosis, prognosis judgment, and identification of immunotherapeutic targets[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the IHC technical process is complex, involving tissue fixation, embedding, sectioning, antigen retrieval, antibody incubation, and color development. Operations in each step can affect the final results, leading to the risk of misdiagnosis or missed diagnosis. The lack of standardization and reference control often results in result deviations[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Establishing a reliable positive control system is therefore essential to ensure the accuracy and reproducibility of IHC results. In the IHC quality control system, normal tissues or tissue samples with specific antigen expression are required as control tissues. The establishment of positive control tissues is the key to monitoring the standardization of the entire experimental process and ensuring the accuracy and reliability of experimental results. Positive control methods for IHC vary among laboratories, including internal tissue controls, amniotic membrane roll multi-tissue controls, and single-tissue controls. However, not all antigens have suitable internal controls in the test tissue, and both multi-tissue and single-tissue positive controls require large-scale sectioning, making them unsuitable for laboratories with heavy workloads.\u003c/p\u003e \u003cp\u003eBased on the above practical needs, this study established two positive control methods: tissue fragment suspensions and tissue microarrays. Through systematic collection of various residual human tissue samples, a sustainable internal quality control system was successfully constructed. Positive controls using tissue suspensions are particularly suitable for daily high-volume, multi-antibody detection work; positive controls using tissue microarrays, which integrate tissue microarrays with different expression intensities into a single paraffin block, can provide a complete gradient reference in a single section experiment, making them especially suitable for semi-quantitative analysis of target-related markers. The selection of positive control tissues was based on recommendations from the Nordic Immunohistochemical Quality Control (NordiQC) and relevant antibody instructions, followed by preliminary experimental verification[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Since the establishment of the positive control system in 2022, our laboratory has widely applied it to daily internal IHC quality control. Practice has proven that the establishment of tissue fragment suspensions and tissue microarrays is economical, convenient, efficient, and accurate. This paper aims to systematically introduce the establishment methods of these two positive controls in detail and explore their application value in depth, providing a validated and feasible technical reference for the construction of quality control systems in IHC laboratories.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eA variety of residual human tissue samples after pathological diagnosis were collected from June 2023 to August 2023, mainly including tonsil, appendix, stomach, liver, kidney, intestine, thyroid, placenta, breast, or tumor tissues with known positive target antigens. All tissues were fixed with 3.7% buffered neutral formalin, and paraffin blocks were prepared through standardized processes such as dehydration, transparency, wax infiltration, and embedding. This study was approved by the Ethics Committee of Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMain reagents and instruments\u003c/h3\u003e\n\u003cp\u003eReagents: All primary antibodies for IHC involved in this study, IHC secondary antibody staining kits, xylene, absolute ethanol, 80% ethanol, and HE staining kits. Instruments: Leica RM2235 paraffin microtome, tissue microarray gun, flotation machine, baking machine, Roche automatic IHC stainer, optical microscope, low-speed centrifuge, oscillator, Sakura automatic coverslipper, etc.\u003c/p\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of tissue fragment suspensions\u003c/h2\u003e \u003cp\u003eSectioning: Paraffin blocks of positive control tissues were sectioned according to the corresponding IHC markers. Taking tonsil tissue as an example, 5 tonsil tissue paraffin blocks were selected for continuous sectioning on a Leica microtome at a thickness of 3\u0026ndash;4 \u0026micro;m, totaling 300\u0026ndash;400 sections. Crushing and dewaxing: The wax sections were collected in a clean 100 ml glass beaker and gently crushed with a glass rod. Approximately 2 ml of xylene was added halfway until a rice paste-like turbid liquid was formed. Subsequently, 100 ml of xylene was added, the beaker mouth was sealed, and the mixture was allowed to stand for 1 hour. The tissue suspension was transferred to a 50 ml centrifuge tube, balanced, and centrifuged at 3000 r/min for 4 minutes. At this point, tissue fragments were observed to precipitate at the bottom of the tube. The supernatant was carefully discarded, and this step was repeated twice until the supernatant was clear to ensure complete dissolution of paraffin. Replacement and dehydration: 50 ml of absolute ethanol was added to the precipitated tissue fragments, and the mixture was allowed to stand for 30 minutes. It was then centrifuged at 3000 r/min for 4 minutes, and the supernatant was discarded. This step was repeated twice to ensure the tissue fragments were free of xylene and moisture. Preservation: The final precipitated tissue fragments were mixed with 80% ethanol at a volume ratio of 1:50 to form a suspension, which was transferred to multiple sterile centrifuge tubes. The tissue type and preparation date were clearly marked, and the tubes were stored in a 4\u0026deg;C medical refrigerator. Concentration adjusTMAent and quality control: Before use, 1 \u0026micro;L of the tissue suspension was dropped onto a glass slide multiple times for HE staining to observe cell distribution. If the cells were too scattered, centrifugation was repeated to increase the concentration of the tissue suspension; if cell overlap was severe, centrifugation was repeated to dilute the concentration. AdjusTMAents were made until the cells were uniformly distributed with clear morphology.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eApplication of tissue fragment suspensions\u003c/h3\u003e\n\u003cp\u003eBefore use, the stored suspension was taken out of the 4\u0026deg;C medical refrigerator and thoroughly mixed on an oscillator for 30 seconds. A portion of the suspension was transferred to a new 6 ml primary antibody reagent bottle (or similar container) for recent use, and most of the remaining suspension was stored in centrifuge tubes to reduce storage risks. When fishing test tissue sections, according to the laboratory's positive control antibody classification table (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), antibody markers requiring the same tissue suspension as a positive control on the same day were grouped together for centralized application to improve work efficiency. Using a pipette, approximately 1 \u0026micro;L of the tissue suspension was accurately dropped at the upper left corner and lower right corner of the test tissue, with a distance of about 0.5 cm from the tissue edge to prevent cross-contamination. The tissue suspension and test tissue were mounted on the same section and subjected to the IHC staining process simultaneously to implement quality control throughout the entire process. In the early stage of establishing the laboratory's positive control antibody classification table, the laboratory conducted multiple effective experimental verifications for all antibody markers corresponding to positive control suspensions. Before using each new batch of suspension, performance verification was also performed to ensure the positive control exhibited the expected positive effect before being put into the daily quality control system.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of tissue microarrays\u003c/h2\u003e \u003cp\u003eFor IHC related to targeted therapy, the laboratory established tissue microarray positive controls (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Taking HER-2 as an example, breast cancer tissue paraffin blocks with known target antigen expression results were selected: 1 case each of 0, 2+ (FISH-confirmed), and 3+[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Localization: Corresponding to HE sections and IHC sections, two senior pathologists marked representative areas under a microscope and identified the corresponding positions in the paraffin blocks[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. According to the marked positions, tissues were accurately extracted using a tissue microarray gun (2 mm in diameter) and classified as 0, 2+, or 3+. Embedding: The 0, 2+, and 3\u0026thinsp;+\u0026thinsp;tissues were embedded in the same embedding mold in sequence to prepare a HER-2 tissue microarray positive control paraffin block, which was stored routinely. Sectioning for standby use: The HER-2 tissue microarray positive control paraffin block was routinely sectioned at a thickness of 3\u0026ndash;4 \u0026micro;m, and the sections were mounted on one end of an IHC anti-slip glass slide. To ensure uniform quality, the first, middle, and last sections were mounted on the same glass slide for HER-2 IHC staining. Under a microscope, the staining intensity of tissues at the same site on these three sections was evaluated for consistency and tissue integrity. Only microarray batches where the three sections showed highly consistent staining intensity and no obvious tissue loss were deemed qualified for subsequent experiments. Verified qualified microarray sections should be stored in a cool, dry, and light-protected slide box or refrigerated at 4\u0026deg;C. To avoid antigen attenuation due to long-term exposure, it is recommended that the number of sections prepared at one time meet the one-week usage requirement.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eApplication of tissue microarrays\u003c/h3\u003e\n\u003cp\u003eFor IHC detection of targeted therapy-related markers such as PD-L1 (SP263), ALK (D5F3), and HER-2, tissue microarray positive control slides were taken from standby sections. The test tissue was directly mounted on the other end of the slide with pre-prepared tissue microarray tissues, ensuring a distance of at least 0.5 cm between the test tissue and the microarray tissues. This \"two-in-one\" section was subjected to simultaneous IHC staining. Tissue microarray positive controls can serve as direct references for interpreting staining intensity, especially for indicators requiring semi-quantitative scoring such as HER-2 and PD-L1, greatly improving the accuracy and consistency of interpretation.\u003c/p\u003e\n\u003ch3\u003eObservation indicators\u003c/h3\u003e\n\u003cp\u003eOn the same section, the positive control tissue was considered positive if it achieved the expected positive result.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using SPSS 25.0 software. The excellent and good rates of the two groups of positive controls were compared using the chi-square test (Fisher's exact test). A P value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe total amount of tissue fragment suspension obtained through one standardized preparation could be stably used for more than 12 months under routine workload, providing reliable internal quality control guarantee for over 130,000 IHC sections. Compared with the traditional method of using a separate positive control section for each test section, this method saved more than 90% of control tissue consumption and corresponding sectioning time, with simple operation. Periodic sampling inspections (at 3, 6, 9, and 12 months) of suspensions stored at 4\u0026deg;C for up to 12 months showed no difference in IHC staining intensity compared with newly prepared suspensions, and no significant attenuation of antigenicity was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), demonstrating good long-term stability. Tissue microarrays targeting HER2 included samples exhibiting a gradient of expression levels, ranging from negative to weakly positive and strongly positive (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), weakly positive to strongly positive, which not only verified the effectiveness of the experiment but also assisted technicians in accurately grasping the cell membrane/cytoplasmic characteristics of different staining intensities, ensuring the accurate application of scoring standards. Evaluation of more than 130,000 IHC sections using positive controls from September 2023 to September 2024 showed that 126,790 cases used tissue fragment suspensions with an excellent and good rate of 99.9% (including 105 false negatives, mainly due to the large distance between control tissue and test tissue), and 3,501 cases used tissue microarrays with an excellent and good rate of 99.9% (including 3 false negatives, mainly due to instrument failure). There was no statistical significance between the two groups of data (χ\u0026sup2; = 0.057, \u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05), indicating that both methods could provide reliable guarantee for internal IHC quality control (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). During daily internal quality control, staining problems could be detected in a timely manner, ensuring the accuracy and reliability of IHC experimental results.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImmunohistochemistry is a technology that uses the principle of specific binding between antigens and antibodies to visualize labeled antibodies through chemical reactions, thereby enabling the localization, qualitative, and quantitative study of specific antigens in tissues or cells. IHC can label and detect cell lineage markers, therapeutic targets, and prognostic markers[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although the practice of IHC has reshaped diagnostic pathology and promoted improvements in cancer treaTMAent, it still faces widespread challenges in standardization and reproducibility[\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The experimental process is affected by various factors such as tissue fixation, experimental steps, human factors, and equipment factors. Without standardized quality control standards[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], it is difficult to obtain ideal test results[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the IHC staining process, positive controls are the core tool for IHC standardization[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Through samples with known reactivity, positive controls ensure the reliability of IHC detection from antibody performance to operational procedures, helping to standardize staining, reduce experimental differences, and ensure the accuracy and reliability of experimental results through quality control throughout the IHC process[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study detailed and evaluated the establishment methods and comprehensive application effects of IHC positive controls based on tissue fragment suspensions and tissue microarrays. Through systematic tissue collection and standardized processing, an internal quality control reference system for IHC laboratories with a wide coverage and complete targets was successfully constructed. Covering 15 different types of normal tissues and lesion tissues, it can provide reliable positive control support for more than 100 clinically commonly used IHC antibodies. Practice has proven that these two methods together form a scientific, economical, efficient, and reliable internal laboratory quality control solution, realizing \"self-sufficiency\" and \"customization\" of positive controls.\u003c/p\u003e \u003cp\u003eTissue fragment suspensions, characterized by convenience, efficiency, and economy, serve as quality control guarantees for daily high-volume, multi-index labeling. Our long-term stability data confirm that tissue fragment suspensions can achieve satisfactory IHC experimental results when stored under appropriate conditions (80% ethanol preservation solution, 4\u0026deg;C) for more than one year. Tissue microarrays, with the advantages of precision and intuition, focus on quality control guarantees for IHC markers related to targeted therapy, becoming increasingly prominent in the era of precision medicine. They not only verify the accuracy of experimental results but also serve as a reliable compass for result interpretation. The combination of the two ensures comprehensive internal quality control of the laboratory from routine diagnosis to cutting-edge detection. It is worth noting that performance verification emphasized in this study is the cornerstone of ensuring the effective operation of the entire system. Both the use of new batches of suspensions and the construction of new tissue microarrays must go through strict internal verification processes. In addition, combining these two internal quality control methods with participation in external quality control evaluations (such as PQCC, NordiQC, CAP, etc.) constitutes the best practice for quality management in IHC laboratories[19].\u003c/p\u003e \u003cp\u003eThe establishment of positive controls using tissue fragment suspensions and tissue microarrays not only significantly improves the results of single IHC tests but also realizes the long-term stability and continuous improvement of laboratory quality control through standardized and reproducible operational procedures. By effectively monitoring the experimental process, it reduces instrument and human errors and improves operational standardization. Ultimately, it makes pathological diagnosis results more accurate and reliable. We strongly recommend the application of this comprehensive quality control system in daily internal quality control of pathology deparTMAents, especially in units with heavy daily diagnostic tasks. The combined use of tissue fragment suspensions and tissue microarrays, featuring convenience, efficiency, precision, and reliability, is of great significance for comprehensively improving the quality control of IHC, enhancing the overall quality of pathological diagnosis, and ensuring patient medical safety.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eclassification table of antibodies for tissue fragment suspension positive controls in IHC laboratory\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl tissue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecommended antibodies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTonsil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProliferation and apoptosis-related: Ki67, Bcl-2, Bcl-6\u003c/p\u003e \u003cp\u003eT cell and related: CD2, CD3, CD4, CD5, CD7, CD8, TIA-1, CD45RO\u003c/p\u003e \u003cp\u003eB cell and related: CD10, CD19, CD20, CD21, CD22, CD23, MUM1, CD79a\u003c/p\u003e \u003cp\u003eMyeloid/histiocyte: CD15, CD68, CD163\u003c/p\u003e \u003cp\u003eOther leukocytes: CD38, CD43, CD45, CD57, CD123, CD138\u003c/p\u003e \u003cp\u003eEpithelial and mesenchymal markers: CK (HMW), CK (Pan), C5/6, Vimentin, P63\u003c/p\u003e \u003cp\u003eMismatch repair proteins: MLH1, MSH2, MSH6, PMS2\u003c/p\u003e \u003cp\u003eImmunoglobulins: Kappa, Lambda, IgG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAppendix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEpithelial markers: CK8/18, CK20, CEA, Villin\u003c/p\u003e \u003cp\u003eMesenchymal/vascular markers: CD31, CD34, F8, FN, S-100, SMA, Desmin, Actin\u003c/p\u003e \u003cp\u003eGastrointestinal-specific marker: CDX-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLung cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEGFR, SP-B, TTF-1, NapsinA, CK7, CK (LMW)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePheochromocytoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNeuroendocrine universal markers: CgA, Syn, NSE, CD56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThyroid papillary carcinoma with BRAF V600E mutation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBRAF V600E, Galectin-3, CK19, Tg, Ber-EP4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAFP, HepPar-1, Arginase-1, Glypican-3, GS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlacenta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD163, GLUT-3, HPL, S-100P\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBreast cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATA3, TRPS1, E-cadherin, GCDFP-15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCervix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eER, PR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGastrointestinal stromal tumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD117, DOG1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClear cell renal cell carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRCC, CAIX, TFE-3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKnown positive urothelial carcinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUroplakin Ⅱ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT-1, PAX-8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProstate cancer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePSA, AR, P504S\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClassification table of antibodies for tissue microarray positive controls in IHC laboratory\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl tissue\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRecommended antibodies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBreast cancer HER-2 0、2+、3+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHER-2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAppendix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eALK(D5F3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlacenta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePD-L1(SP263)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTissue with known positive target antigen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKnown positive pancreatic neuroendocrine tumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSSTR2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGastric tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClaudin18.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKnown Positive lung adenocarcinoma tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ec-MET\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExpression analysis of two positive control methods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive(sections)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFalse negative(sections)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExcellent and good rate(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eχ\u0026sup2; Value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTFS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e126790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e126895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003e\u0026gt;\u003c/em\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTMA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e130291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e130399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e99.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePQCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNational Pathology Quality Control Center\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNordiQC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNordic Immunohistochemical Quality Control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCollege of American Pathologists\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePD-L1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProgrammed Death-Ligand 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTissue fragment suspension\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTissue microarray\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of the Hunan Provincial People\u0026apos;s Hospital and The first-affiliated hospital of Hunan Normal University. The study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. As the study was a retrospective design and did not include any potentially identifiable patient data, informed consent was waived by the Ethics Committee of the Hunan Provincial People\u0026apos;s Hospital and The first-affiliated hospital of Hunan Normal University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTsutsumi Y. Pitfalls and Caveats in Applying Chromogenic Immunostaining to Histopathological Diagnosis. Cells. 2021;10(6):1501. Published 2021 Jun 15. doi:10.3390/cells10061501\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHavnar C, Hotzel K, Espiritu C, Lo A, Webster JD. Standardized Processing for Formalin-Fixed, Paraffin-Embedded Cell Pellet Immunohistochemistry Controls. J Vis Exp. 2022;(185):10.3791/64276. Published 2022 Jul 27. doi:10.3791/64276\u003c/li\u003e\n \u003cli\u003eKnudsen BS, Jadhav A, Perry LJ, et al. A Pipeline for Evaluation of Machine Learning/Artificial Intelligence Models to Quantify Programmed Death Ligand 1 Immunohistochemistry. Lab Invest. 2024;104(6):102070. doi:10.1016/j.labinv.2024.102070\u003c/li\u003e\n \u003cli\u003eWang J, Xia YC, Tian BX, et al. Novel quantitative immunohistochemistry method using histone H3, family 3B as the internal reference standard for measuring human epidermal growth factor receptor 2 expression in breast cancer. Cancer. 2024;130(S8):1424-1434. doi:10.1002/cncr.35176\u003c/li\u003e\n \u003cli\u003eVyberg M, Nielsen S. Proficiency testing in immunohistochemistry--experiences from Nordic Immunohistochemical Quality Control (NordiQC). Virchows Arch. 2016;468(1):19-29. doi:10.1007/s00428-015-1829-1\u003c/li\u003e\n \u003cli\u003eTorlakovic EE, Nielsen S, Francis G, et al. Standardization of positive controls in diagnostic immunohistochemistry: recommendations from the International Ad Hoc Expert Committee. Appl Immunohistochem Mol Morphol. 2015;23(1):1-18. doi:10.1097/PAI.0000000000000163\u003c/li\u003e\n \u003cli\u003eBaez-Navarro X, van Bockstal MR, Nawawi D, et al. Interobserver Variation in the Assessment of Immunohistochemistry Expression Levels in HER2-Negative Breast Cancer: Can We Improve the Identification of Low Levels of HER2 Expression by Adjusting the Criteria? An International Interobserver Study. Mod Pathol. 2023;36(1):100009. doi:10.1016/j.modpat.2022.100009\u003c/li\u003e\n \u003cli\u003eFurrer D, Sanschagrin F, Jacob S, Diorio C. Advantages and disadvantages of technologies for HER2 testing in breast cancer specimens. Am J Clin Pathol. 2015 Nov;144(5):686-703. doi: 10.1309/AJCPT41TCBUEVDQC. PMID: 26486732.\u003c/li\u003e\n \u003cli\u003eChen KJ, Jin RM, Shi CC, et al. The prognostic value of Niemann-Pick C1-like protein 1 and Niemann-Pick disease type C2 in hepatocellular carcinoma. J Cancer. 2018;9(3):556-563. Published 2018 Jan 1. doi:10.7150/jca.19996\u003c/li\u003e\n \u003cli\u003ePhipps WS, Kilgore MR, Kennedy JJ, Whiteaker JR, Hoofnagle AN, Paulovich AG. Clinical Proteomics for Solid Organ Tissues. Mol Cell Proteomics. 2023;22(11):100648. doi:10.1016/j.mcpro.2023.100648\u003c/li\u003e\n \u003cli\u003eHornick JL. Replacing molecular testing with next-generation immunohistochemistry: I can diagnose that soft tissue tumor with a single antibody!. Mod Pathol. Published online October 12, 2025. doi:10.1016/j.modpat.2025.100912\u003c/li\u003e\n \u003cli\u003eBogen SA, Dabbs DJ, Miller KD, et al. A Consortium for Analytic Standardization in Immunohistochemistry. Arch Pathol Lab Med. 2022;147(5):584-590. doi:10.5858/arpa.2022-0031-RA\u003c/li\u003e\n \u003cli\u003eTaylor CR. Quantitative in situ proteomics; a proposed pathway for quantification of immunohistochemistry at the light-microscopic level. Cell Tissue Res. 2015;360(1):109-120. doi:10.1007/s00441-014-2089-0\u003c/li\u003e\n \u003cli\u003eTaylor CR. An exaltation of experts: concerted efforts in the standardization of immunohistochemistry. Hum Pathol. 1994;25(1):2-11. doi:10.1016/0046-8177(94)90164-3\u003c/li\u003e\n \u003cli\u003eCanadian Association of Pathologists-Association canadienne des pathologistes National Standards Committee, Torlakovic EE, Riddell R, et al. Canadian Association of Pathologists-Association canadienne des pathologistes National Standards Committee/Immunohistochemistry: best practice recommendations for standardization of immunohistochemistry tests. Am J Clin Pathol. 2010;133(3):354-365. doi:10.1309/AJCPDYZ1XMF4HJWK\u003c/li\u003e\n \u003cli\u003eLiu, Xiaoying et al. \u0026ldquo;Application of the PDCA cycle in companion diagnostic PD-L1 SP263 immunohistochemical testing.\u0026rdquo; BMC cancer vol. 25,1 1167. 12 Jul. 2025, doi:10.1186/s12885-025-14572-4\u003c/li\u003e\n \u003cli\u003e\u0026Aacute;lvarez-Velasco R, Dols-Icardo O, El Bounasri S, et al. Reduced Number of Thymoma CTLA4-Positive Cells Is Associated With a Higher Probability of Developing Myasthenia Gravis. Neurol Neuroimmunol Neuroinflamm. 2023;10(2):e200085. Published 2023 Jan 25. doi:10.1212/NXI.0000000000200085\u003c/li\u003e\n \u003cli\u003evan Kempen S, Gerritsen WJG, Nguyen TQ, et al. Monitoring Immunohistochemical Staining Variations Using Artificial Intelligence on Standardized Controls. Lab Invest. 2025;105(5):104105. doi:10.1016/j.labinv.2025.104105\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Immunohistochemistry, Positive control, Tissue fragment suspension, Tissue microarray, Quality control","lastPublishedDoi":"10.21203/rs.3.rs-8401699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8401699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aims to explore the establishment of positive controls for immunohistochemistry (IHC), involving two distinct methods\u0026mdash;tissue fragment suspensions and tissue microarrays\u0026mdash;and evaluate their application value in improving the accuracy and reliability of pathological diagnosis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA variety of residual human tissue samples (including tonsil, appendix, stomach, liver, kidney, intestine, thyroid, placenta, breast, and tumor tissues with known positive target antigens) were collected from June 2023 to August 2023. After standardized processing, tissue paraffin blocks were prepared. Internal laboratory positive controls were constructed using tissue fragment suspension technology and tissue microarray technology, respectively. In subsequent IHC assays, test tissues and corresponding positive control tissues were mounted on the same glass slide for simultaneous staining. The two methods were evaluated in terms of preparation efficiency, operational convenience, staining consistency, antigen stability, and quality control reliability.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA long-term usable internal quality control reference for the laboratory was successfully established. Tissue fragment suspensions saved time and effort, significantly improving work efficiency; tissue microarrays, when applied to IHC detection related to targeted therapy, remarkably enhanced the accuracy of test results.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe IHC positive control quality control system established based on tissue fragment suspensions and tissue microarrays is economical, convenient, efficient, and reliable. It not only effectively ensures the accuracy of IHC results but also realizes the standardization of internal quality control. Particularly suitable for promotion and application in daily diagnostic work, this system holds important clinical value and practical significance.\u003c/p\u003e","manuscriptTitle":"Establishment and application of immunohistochemical positive controls: Based on tissue fragment suspensions and tissue microarrays","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 11:34:06","doi":"10.21203/rs.3.rs-8401699/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-01-30T17:47:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-27T07:45:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-07T04:47:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-06T00:21:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2026-01-06T00:16:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"999e3564-b5d6-4479-9478-48e6183e1549","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-03T11:34:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-03 11:34:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8401699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8401699","identity":"rs-8401699","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}