New Method for Cytological Evaluation Using Direct Nipple Discharge without Aspiration

New Method for Cytological Evaluation Using Direct Nipple Discharge without Aspiration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article New Method for Cytological Evaluation Using Direct Nipple Discharge without Aspiration Jiang Zhu, Han Cong, Xiaotong Zhang, Xiaoya Dong, Song Zhao, Chaolu Hu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5177988/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Conventional smear cytology (CSC) is a specific method used for breast tumor diagnosis in patients with nipple discharge. However, CSC tends to miss diagnose or even misdiagnose due to contaminating blood cells and other impurities. Thus, it is critical to develop more accurate and sensitive methods for clinical evaluation. Using nipple discharge collected directly from patients without aspiration, we performed liquid-based cytology (LBC) to analyze 111 collected samples for cytological evaluation for the first time. Following centrifugation to remove blood cells and protein contaminants, the sample of each nipple discharge was analyzed by light microscopy. In parallel, CSC was performed for comparison. Our results showed LBC has better diagnostic sensitivity than CSC (40.00% vs 22.22%, χ2 = 6.636, P = 0.01). The specificity was improved (LBC 100% vs CSC 95.2%) and area under the curve was also enhanced (AUC LBC =0.700 vs AUC csc =0.587). Moreover, LBC images have a cleaner background, clearer field of view, uniform cell arrangement, and improved colour contrast, with an overall image quality significantly better than CSC. Therefore, LBC provides a new and better diagnostic method for early detection of breast tumors with nipple discharge. Biological sciences/Cancer/Breast cancer Biological sciences/Biological techniques/Cytological techniques Biological sciences/Biological techniques/Cytological techniques/Tissue culture Liquid-based cytology diagnosis breast cancer nipple discharge Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Nipple discharge is a common complaint among patients presenting with breast problems. The causes of discharge are divided into functional and pathological. Functional discharge is usually a bilateral porous discharge, often caused by increased Prolactin associated with lactation, pituitary adenoma, or antipsychotics. Pathological discharge is spontaneous and intermittent, unrelated to pregnancy or lactation, and lasts from several months to several years. The most common cause of pathological discharge is intraductal papilloma, a typical precancerous lesion. The second is breast cancer, mostly intraductal carcinoma [ 1 ]. Sometimes, benign hyperplastic lesions can also lead to nipple discharge. At present, specific clinical examinations of nipple discharge include ductoscopy, galactography, analysis of tumor markers in nipple discharge, cytological examination, etc. The nature and composition of nipple discharge vary greatly from patient to patient. Abnormal cell detection using cytological examination is a qualitative diagnosis, showing the nature of the lesion, assisting surgeons to identify the need for surgery, and design a surgical plan if necessary. However, conventional smear cytology (CSC) poses several issues. First, the cell detection rate is low (30.8%-83%) [ 2 – 4 ] and, second, the image quality is poor because of overlapping cells and blood cell contamination [ 5 ], either of which can lead to false-positive or false-negative results. Liquid-based cytology (LBC) was invented by Hologic Gen-Probe, with Hutchinson first reporting its application to cervical cytology in 1991 [ 6 ]. LBC involves the use of automatic cell dyeing and high-adhesion slides to examine cells based on their physical properties. Some advantages of using this method include simple operation, clear background, contrastive color imaging, uniform distribution of cells, and feasibility for immunochemical staining and flow cytometry [ 7 ]. Besides automatic imaging, settling is a commonly used manual imaging method for LBC and involves the use of a special device that enriches the cells on the slide upon centrifugation, which improves the detection rate [ 8 ]. At present, LBC has been used in non-gynecological fields for the diagnosis of lung cancer, thyroid nodules, and other diseases [ 9 – 11 ], as well as for the evaluation of nipple aspirate fluid [ 12 ]. No direct application in nipple discharge has been reported. Here, we describe the first application of LBC for direct nipple discharge analysis and compare diagnostic efficacy and image quality between LBC and CSC, illustrating that LBC can be used as an effective non-invasive diagnostic method for analyzing disease-associated nipple discharge. Materials and methods Patient selection Inclusion criteria: Female patients with nipple discharge. Had not undergone any invasive examination (e.g., mammography, ductoscopy or surgery) before cytology examination. Informed consent was obtained. Exclusion criteria: Male patients. Patients were pregnant or lactating. Had taken oral antipsychotic medications over the past 6 months. Had undergone any invasive examination before cytology examination. 5) Rejected informed consent. A total of 101 patients with nipple discharge had received surgical treatment in the Department of Breast Surgery, Qilu Hospital of Shandong University. Among them, 10 had bilateral nipple discharge. A total of 111 nipple discharge samples were collected. Approval for this study was obtained from the ethics committee of Qilu Hospital of Shandong University. Sample collection At the time of sample collection, the affected nipple was scrubbed with 75% alcohol and then dried with a sterile cotton swab. Nipple discharge is gently squeezed from around the breast. Finally, a sterile Eppendorf duct is used to collect nipple discharge. Half of the nipple discharge was taken for CSC, the other half for LBC (Fig. 1). Liquid-based cytology examination Drain the discharge from the sterile EP tube into a centrifuge tube and add the cell preservation solution (Hanhe Medical Devices Co., LTD., Jinan, Shandong, China) to 1 ml. Add lymphocyte separation medium to 1:1 ratio. Put into a TD5Z-WS centrifuge (XIANGZHI Centrifuge Instrument Co., LTD., Changsha, Hunan, China), 2,000 r/min for 5 min. Aspirate the nucleated cell layer and the upper layer of the original fluid to another centrifuge tube. Put the centrifuge tube into TD4Z-WS auto-balancing centrifuge (XIANGZHI Centrifuge Instrument Co., LTD., Changsha, Hunan, China), 900 r/min for 4 min to obtain a glass slide. The glass slides were coded and placed on a staining rack. Add Wright-giemsa staining solution A + pitonate buffer (7:10) and stain for 25 min. Wash slides and dry. The slide was reviewed under a microscope. Conventional Smear cytologic examination A) Make smear manually with glass slides and place on the staining rack after coding. B) Add Wright-giemsa staining solution A + pitonate buffer (7:10) and stain for 25 min. C) Wash slides and dry. D) Review the slide under a microscope. Report Two professional cytopathologists evaluated slides, reported observed cell types (ductal epithelial cells, blood cells, etc.) and morphological characteristics, defined detected cancer cells or atypical hyperplasia cells as positive results, and if not, defined as negative results. Data Analysis 1) According to paraffin pathology, the lesions were divided into three groups: breast cancer, precancerous lesions (intraductal papilloma, atypical hyperplasia), and benign hyperplastic lesions (mammary fibroadenoma, sclerosing adenosis, mammary hyperplasia). Clinical data of all patients were collected to clarify the relationship between clinical features and lesion types. P values were calculated by paired χ2 test or Fisher exact test, and P < 0.05 was considered statistically significant. 2) All reports were reviewed and tabulated to present the types of cells in the reports. A Venn diagram was made to show the detection of ductal epithelial cells in all cases, breast cancer, and precancerous lesions (intraductal papilloma, atypical hyperplasia). 3) Cancer cells and atypical cells were defined as pathological cells. If pathological cells had been detected, the results were positive, otherwise, they were negative. In clinical treatment, both cancer and precancerous lesions had strong surgical indications. The two were combined and distinguished from benign hyperplastic lesions. The diagnostic validity of the two cytological examination methods was determined to include sensitivity, specificity, PPV, NPV and Kappa consistency test, drawn ROC curve, and performed ROC analysis. SPSS 25.0 software was used for data processing and statistical analysis. Results Clinical and pathological data of patients There were 111 nipple discharge samples from 101 patients included in this study (10 patients with bilateral nipple discharge). The mean age of the patients was 46.6 years (23-81 years, SD=10.6 y). There were 25 samples from premenopausal patients and 86 samples from postmenopausal patients; 46 palpable breast lumps and 65 unpalpable. Nipple discharge with lumps was an indication of malignancy (P=0.002). Of the 111 cases of discharge, there were 36 breast cancer, 54 precancerous lesions (51 intraductal papillomas, 3 atypical hyperplasia), and 21 benign proliferative lesions (7 fibroadenomas, 14 adenopathy and general hyperplasia). Among the 36 breast cancers, the most common pathological type was DCIS (86.1%, 31/36). There were 40 cases of bloody discharge, 51 cases of serous discharge, 17 cases of clear water discharge, and 3 cases of milk discharge. The characteristics of discharge were not correlated with specific diseases (P=0.019). There were no significant differences in age, number of ductal pores, menstrual status, family history, and breastfeeding history between breast cancer, precancerous lesions, and benign hyperplastic lesions (Table 1). Cytological assessment of nipple discharge 1. Fewer miscellaneous cells in LBC The cell types reported are shown in Figure 2. The areas marked in green blocks show cell detection in LBC, while orange blocks marked cell detection in CSC. The LBC method could detect fewer blood cells and squamous epithelial cells, in another word, fewer miscellaneous cells. Among 111 samples, ductal epithelial cells (including cancer cells, atypical cells, and normal ductal epithelial cells) were detected in 65 cases using LBC and in 34 cases using CSC. However, no ductal epithelial cells were detected by either method in 44 cases (Figure 3. (a, b)). For breast cancer samples (n=36), LBC identified ductal epithelial cells in 22 cases while CSC identified them in 14 cases (Figure 3. (c,d)). For intraductal papilloma samples (n=51), LBC detected ductal epithelial cells in 34 cases compared to CSC which found them in only 17 cases (Figure 3. (e, f)). As shown in the Venn diagram, there is concordance between these two methods. However, the ability of LBC to detect ductal epithelial cells was superior to that of CSC, particularly in breast cancer. 2. The diagnostic validity of LBC is better In all the samples (n=111), 36 cases of pathological cells were detected using LBC, and 21 cases were detected using CSC. Among samples from breast cancer (n=36), 19 cases of pathological cells were detected with LBC, while only 11 were detected using CSC. Among samples from intraductal papilloma (n=51), 17 cases of pathological cells were detected using LBC compared to nine using CSC. None of the atypical hyperplasia (n=3) samples showed any pathological cell detection by either method. Among samples from benign hyperplasia lesions (n=21), CSC detected pathological cells from one sample of bloody discharge, however, no case of pathological cells was detected using LBC. When diagnostic validity was evaluated, we found LBC had higher sensitivity than CSC for breast tumors (52.78% vs. 30.56% for breast cancer; and 31.48% vs. 16.67% for intraductal papilloma). Breast cancer, intraductal papilloma, and atypical hyperplasia were grouped and distinguished from benign hyperplastic lesions. Based on this distinction, the diagnostic validity of these two methods were calculated. For LBC: sensitivity = 40%, specificity = 100%, positive predictive value = 100%, negative predictive value = 28%, and Kappa value = 0.201. For CSC: sensitivity = 22.22%, specificity = 95.22%, positive predictive value = 95.20%, negative predictive value = 22%, and Kappa value = 0 .077 (Table 2). Chi-square test of the two groups of rate was performed: Sensitivity χ2=6.636, P=0.01. That is, the sensitivity, specificity, positive predictive value, negative predictive value, and Kappa value of the LBC test were higher than those of CSC, and the difference in sensitivity was statistically significant. In receiver operating characteristic (ROC) curves of two methods (Figure 4), AUC LBC = 0.700 (p = 0.004, 95% CI: 0.598~0.802). AUC CSC = 0.587 (p = 0.214, 95% CI: 0.463~0.711). The diagnostic validity of LBC is, therefore, better than CSC. 3. The image quality of LBC is better The accuracy of diagnosis is related to the image quality, which can be greatly reduced by blood cells and cell debris in the sample. A bloody discharge of a breast cancer patient showed cells with large and deeply stained nuclei (Figure 5. a-b). While the CSC image showed more blood cells in the background, damaged cell morphology, and many overlapped cells (a), the LBC image had a clean background, good morphological integrity, and no cell debris (b). In a bloody discharge of a patient with intraductal papilloma (Figure 5. c-d), big ductal epithelial cells with increased nucleocytoplasmic ratio could be identified by both LBC and CSC. In the background of the CSC image, a large quantity of blood cells were arranged in clusters and the image was crowded, which sometimes obscured cancer cells. Moreover, the distribution of cancer cells was not concentrated, and the color contrast was not strong (c). However, the background of LBC is clean, cell distribution is concentrated, and the color is contrastive (d). In a sero-like discharge of intraductal papilloma (Figure 5. e-f), the sample is rich in protein. In CSC, protein impurities adhere to the slide and appear purplish, which would shade cells (e). In LBC, the protein impurities are no longer attached to the slide after centrifugation (f). Overall, the cell preservation and image quality of LBC are superior to CSC. Discussion Breast cancer accounts for approximately 5–12% of pathologic nipple discharge in patients undergoing surgery [ 13 ], most of which are DCIS [ 1 ]. For patients with nipple discharge, cytolgical examination is presently the predominant method for clinical evaluation. Since the report of CSC in 1969 [ 14 ], it has been widely used for pathologic diagnosis. Although the sensitivity and specificity of cytology-based tests are comparable to that of imaging, cytology evaluation cannot replace imaging and is not recommended alone for clinical diagnosis [ 3 , 15 , 16 ]. LBC is an emerging and more sensitive cytological examination method than CSC, as reported here, with removal of impurities such as blood cells and mucus proteins during sample preparation to obtain a clearer image background. To date, LBC has been applied to nipple aspirate analysis using ductoscopy. The ductal epithelia could be damaged, resulting in ductal adhesion and disappearance of nipple discharge, making it difficult to position tumor locations. Moreover, ductoscopy often misses lesions in the terminal ductal lobular unit (TDLU). In this report, we successfully applied LBC for direct analysis of nipple discharge, and showed it represents a more sensitive diagnosis than CSC. Two advantages of LBC over CSC include: 1) The image quality of LBC is superior to that of CSC; and 2) the diagnostic validity of LBC is better than that of CSC. In LBC, centrifugation helps remove impurities such as blood cells and mucus. When nipple discharge is bloody, LBC produces a cleaner image than CSC. Moreover, during LBC examination, the cell preservation solution is added immediately after collecting nipple discharge that preserves better cell morphology, especially the intactness of the nucleus necessary for observing any atypic changes. After settling, the distribution of cells is more concentrated and uniform. For specimens with less cells, ductal epithelial cells can also be significantly enriched. In contrast, during CSC examination, nipple discharge is collected and placed directly onto the slide. When not treated in time, the specimen will dry out and lead to cell deformation and fragmentation. Cell overlapping and scattered distribution in CSC tend to fatigue the reader, resulting in bias. All the above factors lead to improved image quality of LBC. In our analysis, the positive results of LBC did not completely cover match the CSC results. Two samples were positive for CSC but negative for LBC. The reasons may be: 1) The number of cells shed by discharge was limited, and there was sampling error in the examination process; 2) some background cells and proteins eliminated after centrifugation may provide clues to lesions; and 3) as LBC was not completely automated, some operations may be affected by artificial errors. Some reasons for flash negative cytology may be: 1) The nipple discharge and breast lesion do not originate from the same mammary ductal system; 2) some of the lesion cells were not shed by discharge, which might be related to the differential expression of tumor adhesion factors; 3) Invasive ductal carcinoma is difficult to shed through the duct; and 4) ductal epithelial cells which entered nipple discharge might be out of shape, however the cognition of morphology features of pathological cells is still limited to tissue. Therefore, multiple samplings and multiple deliveries can be applied to reduce sampling errors. In addition, automatic staining and AI-aided automated cytology systems can also be used. AI can perform initial screening and then cytopathologists make the diagnosis of abnormal cells, with the potential to reduce artificial references and improve work efficiency. At the same time, cytopathologists need to receive special training to refresh their cognition and improve the sensitivity of diagnosis. The sensitivity, specificity, PPV, NPV and AUC value of LBC were higher than those of CSC, as shown by compared diagnostic consistency of the cytology examination and pathology. Although Kappa values of the LBC were higher than those of CSC, neither method have poor diagnostic consistency with paraffin pathology due to both results < 0.4. Therefore, it is necessary to combine other specific tests in nipple discharge, such as the Thomsen-Freidenreich antigen test [ 17 ] and detection of tumor markers (CEA, CA153[ 18 ], miRNA[ 19 ]., etc). In conclusion, LBC can be used as a new diagnostic method for cytological evaluation using direct nipple discharge without aspiration. However, due to sampling errors, LBC cannot completely replace CSC. If the sample volume was enough, patients should receive both examination methods to avoid misdiagnosis. If discharge volume was limited, LBC seems to be more effective to diagnose pathological nipple discharge. Moreover, LBC is also more conducive to the preservation of genetic material including protein, RNA and DNA [ 20 , 21 ], which may be used for subsequent molecular and immunocytochemical examination, while its application potential needs to be verified by follow-up experiments. Declarations Acknowledgments We would like to thank Prof. Cheng-Ming Chiang and Prof. Yunyun Gong for language editing and valuable comments. Funding statement This work was supported by grants from Shandong Provincial Natural Science Foundation, China (No. ZR2022MH248), the Postdoctoral Innovation Foundation of Shandong Province, China (No. 202103004). Author contributions statements Jiang Zhu, Han Cong and Xiaotong Zha ng (first author): Data Curation and Analysis, Methodology, Investigation and Writing-Original Draft. Rong Ma, Pengyu Li (Correspondence) and Jiang Zhu : Conceptualization, Methodology, Funding Acquisition, Supervision and Writing-Review＆Editing. Shuxin Yan and Jianli Wang : Cytopathological evaluation and diagnosis. JUDE RANCHU MAIMO : Language editing. Song Zhao, Chaolu Hu, Yawen Wang and Kai Zhang : Patients admission and management, Conceptualization, Resources and Validation. All authors reviewed the manuscript. Conflict of interest All authors have no conflicts of interest. Data availability statement The datasets generated during this study are available from the corresponding author on reasonable request. Ethics declaration This study was reviewed by the Ethics Committee of Qilu Hospital of Shandong University (No.2021-088). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study. References Morrogh, M., Park, A., Elkin, E. B. & King, T. A. Lessons learned from 416 cases of nipple discharge of the breast. Am. J. Surg. 200 (1), 73–80. 10.1016/j.amjsurg.2009.06.021 (2010). Epub 2010/01/19. Dolan, R. T., Butler, J. S., Kell, M. R., Gorey, T. F. & Stokes, M. A. Nipple discharge and the efficacy of duct cytology in evaluating breast cancer risk. surgeon: J. Royal Colleges Surg. Edinb. Irel. 8 (5), 252–258 (2010). PubMed PMID: 20709281. Jiwa, N. et al. Diagnostic Accuracy of Nipple Discharge Fluid Cytology: A Meta-Analysis and Systematic Review of the Literature. Annals of surgical oncology. ;29(3):1774-86. Epub 2021/11/29. doi: (2022). 10.1245/s10434-021-11070-2 . PubMed PMID: 34839426; PubMed Central PMCID: PMCPMC8627297 detection of breast cancer under the supervision of D. R. Leff and Z. Takats. The remaining authors have no conflicts of interest. Grunwald, S. et al. Mammary ductoscopy for the evaluation of nipple discharge and comparison with standard diagnostic techniques. J. Minim. Invasive. Gynecol. 13 (5), 418–423. 10.1016/j.jmig.2006.05.004 (2006). Epub 2006/09/12. Clark, S. E. et al. The investigation and management of unilateral nipple discharge. Ann. R. Coll. Surg. Engl. 102 (5), 369–374. 10.1308/rcsann.2020.0036 (2020). Epub 2020/04/03. Hutchinson, M. L., Cassin, C. M. & Ball, H. G. 3 The efficacy of an automated preparation device for cervical cytology. Am. J. Clin. Pathol. 96 (3), 300–305. 10.1093/ajcp/96.3.300 (1991). Epub 1991/09/01. Pathuthara, S. et al. Conventional versus Liquid-based Cytology: Man versus Machine. J. Cytol. 40 (4), 169–176. 10.4103/joc.joc_54_23 (2023). Epub 2023/12/07. Makde, M. M. & Sathawane, P. Liquid-based cytology: Technical aspects. CytoJournal . 19 , 41 (2022). Epub 2022/08/06. doi: 10.25259/cmas_03_16_2021. PubMed PMID: 35928530; PubMed Central PMCID: PMCPMC9345114. Kim, S. Y. et al. Combined use of conventional smear and liquid-based preparation versus conventional smear for thyroid fine-needle aspiration. Endocrine. ;53(1):157 – 65. Epub 2015/12/31. doi: (2016). 10.1007/s12020-015-0835-z . PubMed PMID: 26714459. Hashimoto, S. et al. Diagnostic efficacy of liquid-based cytology for solid pancreatic lesion samples obtained with endoscopic ultrasound-guided fine-needle aspiration: Propensity score-matched analysis. Dig. endoscopy: official J. Japan Gastroenterological Endoscopy Soc. 29 (5), 608–616. 10.1111/den.12827 (2017). Epub 2017/02/06. Cao, C. et al. Diagnostic role of liquid-based cytology of bronchial lavage fluid in addition to bronchial brushing specimens in lung cancer. Tumori . 107 (4), 325–328. 10.1177/0300891620960218 (2021). Epub 2020/09/26. Masood, S. & Khalbuss, W. E. Nipple fluid cytology. Clinics in laboratory medicine. ;25(4):787 – 94, vii-viii. Epub 2005/11/26. doi: (2005). 10.1016/j.cll.2005.08.010 . PubMed PMID: 16308092. Patel, B. K., Falcon, S. & Drukteinis, J. Management of nipple discharge and the associated imaging findings. Am. J. Med. 128 (4), 353–360. 10.1016/j.amjmed.2014.09 (2015). Epub 2014/12/03. Matsuda, M. & Koyama, H. [Cytologic diagnosis of the abnormal nipple discharge]. Gan no rinsho Japan J. cancer Clin. 15 (12), 1039–1046 (1969). Epub 1969/12/01. PubMed PMID: 5392544. Jiwa, N. et al. Nipple aspirate fluid and its use for the early detection of breast cancer. Annals of medicine and surgery 2022;77:103625. Epub 2022/06/01. doi: (2012). 10.1016/j.amsu.2022.103625 . PubMed PMID: 35638006; PubMed Central PMCID: PMCPMC9142541. Carvalho, M. J. et al. What is the diagnostic value of nipple discharge cytology and galactography in detecting duct pathology? Eur. J. Gynaecol. Oncol. 30 (5), 543–546 (2009). Epub 2009/11/11. PubMed PMID: 19899412. Deutscher, S. L. et al. Carbohydrate antigens in nipple aspirate fluid predict the presence of atypia and cancer in women requiring diagnostic breast biopsy. BMC cancer . 10 , 519. 10.1186/1471-2407-10-519 (2010). Epub 2010/10/06. Zhao, S. et al. Levels of CEA, CA153, CA199, CA724 and AFP in nipple discharge of breast cancer patients. Int. J. Clin. Exp. Med. 8 (11), 20837–20844 (2015). Epub 2016/02/18. PubMed PMID: 26885008; PubMed Central PMCID: PMCPMC4723853. Zhang, K. et al. Identification of microRNAs in Nipple Discharge as Potential Diagnostic Biomarkers for Breast Cancer. Annals of surgical oncology. ;22 Suppl 3:S536-44. Epub 2015/05/16. doi: (2015). 10.1245/s10434-015-4586-0 . PubMed PMID: 25976861. Konofaos, P. et al. The role of ThinPrep cytology in the investigation of ki-67 index, p53 and HER-2 detection in fine-needle aspirates of breast tumors. J. BUON: official J. Balkan Union Oncol. 18 (2), 352–358 (2013). Epub 2013/07/03. PubMed PMID: 23818345. Sauer, T., Ebeltoft, K., Pedersen, M. K. & Kåresen, R. Liquid based material from fine needle aspirates from breast carcinomas offers the possibility of long-time storage without significant loss of immunoreactivity of estrogen and progesterone receptors. CytoJournal . 7 , 24. 10.4103/1742-6413.75665 (2010). Epub 2011/02/08. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 29 Nov, 2024 Reviews received at journal 26 Nov, 2024 Reviews received at journal 16 Nov, 2024 Reviewers agreed at journal 04 Nov, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviewers invited by journal 21 Oct, 2024 Editor assigned by journal 21 Oct, 2024 Editor invited by journal 21 Oct, 2024 Submission checks completed at journal 16 Oct, 2024 First submitted to journal 30 Sep, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5177988","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":366817300,"identity":"4cab262a-ac3b-49e5-b1cf-9407ba28a09b","order_by":0,"name":"Jiang Zhu","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Zhu","suffix":""},{"id":366817301,"identity":"669a2511-72df-4fb9-89b3-e3d2ca7650f5","order_by":1,"name":"Han Cong","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Cong","suffix":""},{"id":366817302,"identity":"44c548d7-f979-416d-9dde-2d3b0b3f7e96","order_by":2,"name":"Xiaotong Zhang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaotong","middleName":"","lastName":"Zhang","suffix":""},{"id":366817303,"identity":"e6419cb3-41cf-40bf-85ee-996463e07df2","order_by":3,"name":"Xiaoya Dong","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoya","middleName":"","lastName":"Dong","suffix":""},{"id":366817304,"identity":"7784bacb-92e2-497c-a41e-acb345a428c9","order_by":4,"name":"Song Zhao","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Song","middleName":"","lastName":"Zhao","suffix":""},{"id":366817305,"identity":"a87b691c-389e-48eb-a9f9-94fbef1b9c23","order_by":5,"name":"Chaolu Hu","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Chaolu","middleName":"","lastName":"Hu","suffix":""},{"id":366817306,"identity":"da00dc9e-6a65-4609-be11-0835515f1332","order_by":6,"name":"JUDE RANCHU MAIMO","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"JUDE","middleName":"RANCHU","lastName":"MAIMO","suffix":""},{"id":366817307,"identity":"c62644f6-9603-4095-86a8-348d80421578","order_by":7,"name":"Yawen Wang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Wang","suffix":""},{"id":366817308,"identity":"be6d9a5e-6b68-4be8-aead-6393536f6bc2","order_by":8,"name":"Kai Zhang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zhang","suffix":""},{"id":366817309,"identity":"fa26af12-3b61-4120-9f5c-3411fc5d8703","order_by":9,"name":"Jianli Wang","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Jianli","middleName":"","lastName":"Wang","suffix":""},{"id":366817310,"identity":"c9a42b05-7fa1-43b0-9799-c952cbb1d7de","order_by":10,"name":"Pengyu Li","email":"","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":false,"prefix":"","firstName":"Pengyu","middleName":"","lastName":"Li","suffix":""},{"id":366817311,"identity":"5503e106-90fa-4998-ba53-133a635614b5","order_by":11,"name":"Rong Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYBADOTZmxsYHCRU1xGsx5mdvPmzw4Mwx4rUkzuw5lib5sIWZsFLd9rMPHxf8Osy44UaOWUViAxsDf3t3Al4tZmfSjY1n9h1mNgBquZG4Q4ZB4szZDfi1HEhjk+btuc0G0XKGjcFAIpeAlvPPwFp4QFoKEtuYidByA2gLz4/bEpJA7zMQqeUZszFvw38DUCBLJJw5xkPYL+fTGB/z/EmrbwNG5ccfFTVy/O29+LWAAWMbgs1DWDkY/CFS3SgYBaNgFIxMAAB+ckyi+ODMggAAAABJRU5ErkJggg==","orcid":"","institution":"Qilu Hospital of Shandong University","correspondingAuthor":true,"prefix":"","firstName":"Rong","middleName":"","lastName":"Ma","suffix":""}],"badges":[],"createdAt":"2024-09-30 05:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5177988/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5177988/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-88456-9","type":"published","date":"2025-02-04T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":67285929,"identity":"f15fcc39-a08c-4c26-89ca-b906e286856f","added_by":"auto","created_at":"2024-10-23 09:29:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":323002,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5177988/v1/2c1ba5ee69b8002e008d9d64.png"},{"id":67286785,"identity":"ea47f762-470b-4224-a0c1-bb975479340b","added_by":"auto","created_at":"2024-10-23 09:37:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1482346,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5177988/v1/bc7e10ce7464bb55bca44fe3.png"},{"id":67285930,"identity":"7d593f91-fe11-4c06-90db-78e952a80ec9","added_by":"auto","created_at":"2024-10-23 09:29:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":448257,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5177988/v1/be8670f7ee8027a0900f09b0.png"},{"id":67285932,"identity":"8cf19700-9287-4d7c-a131-73df069e0198","added_by":"auto","created_at":"2024-10-23 09:29:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":209911,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5177988/v1/70a7deac8a47997de1fddbe3.png"},{"id":67285933,"identity":"a2df1f75-4d23-4731-a85e-b0e52ed1aac3","added_by":"auto","created_at":"2024-10-23 09:29:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":601767,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5177988/v1/f5e22735b76ab922e47bc5a5.png"},{"id":75929948,"identity":"0473ed08-b079-46d1-8875-334e8e45adaf","added_by":"auto","created_at":"2025-02-10 16:07:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3649380,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5177988/v1/8384ff55-c14b-4866-b7a8-b774daf440c4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"New Method for Cytological Evaluation Using Direct Nipple Discharge without Aspiration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNipple discharge is a common complaint among patients presenting with breast problems. The causes of discharge are divided into functional and pathological. Functional discharge is usually a bilateral porous discharge, often caused by increased Prolactin associated with lactation, pituitary adenoma, or antipsychotics. Pathological discharge is spontaneous and intermittent, unrelated to pregnancy or lactation, and lasts from several months to several years. The most common cause of pathological discharge is intraductal papilloma, a typical precancerous lesion. The second is breast cancer, mostly intraductal carcinoma [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Sometimes, benign hyperplastic lesions can also lead to nipple discharge.\u003c/p\u003e \u003cp\u003eAt present, specific clinical examinations of nipple discharge include ductoscopy, galactography, analysis of tumor markers in nipple discharge, cytological examination, etc. The nature and composition of nipple discharge vary greatly from patient to patient. Abnormal cell detection using cytological examination is a qualitative diagnosis, showing the nature of the lesion, assisting surgeons to identify the need for surgery, and design a surgical plan if necessary. However, conventional smear cytology (CSC) poses several issues. First, the cell detection rate is low (30.8%-83%) [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and, second, the image quality is poor because of overlapping cells and blood cell contamination [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], either of which can lead to false-positive or false-negative results.\u003c/p\u003e \u003cp\u003eLiquid-based cytology (LBC) was invented by Hologic Gen-Probe, with Hutchinson first reporting its application to cervical cytology in 1991 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. LBC involves the use of automatic cell dyeing and high-adhesion slides to examine cells based on their physical properties. Some advantages of using this method include simple operation, clear background, contrastive color imaging, uniform distribution of cells, and feasibility for immunochemical staining and flow cytometry [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Besides automatic imaging, settling is a commonly used manual imaging method for LBC and involves the use of a special device that enriches the cells on the slide upon centrifugation, which improves the detection rate [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. At present, LBC has been used in non-gynecological fields for the diagnosis of lung cancer, thyroid nodules, and other diseases [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], as well as for the evaluation of nipple aspirate fluid [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. No direct application in nipple discharge has been reported. Here, we describe the first application of LBC for direct nipple discharge analysis and compare diagnostic efficacy and image quality between LBC and CSC, illustrating that LBC can be used as an effective non-invasive diagnostic method for analyzing disease-associated nipple discharge.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePatient selection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInclusion criteria:\u0026nbsp;\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eFemale patients with nipple discharge.\u003c/li\u003e\n \u003cli\u003eHad not undergone any invasive examination (e.g., mammography, ductoscopy or surgery) before cytology examination.\u003c/li\u003e\n \u003cli\u003eInformed consent was obtained.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eExclusion criteria:\u0026nbsp;\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eMale patients.\u003c/li\u003e\n \u003cli\u003ePatients\u0026nbsp;were pregnant or lactating.\u003c/li\u003e\n \u003cli\u003eHad taken oral antipsychotic medications over the past 6 months.\u003c/li\u003e\n \u003cli\u003eHad undergone any invasive examination before cytology examination.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e5)\u0026nbsp;Rejected informed consent.\u003c/p\u003e\n\u003cp\u003eA total of 101 patients with nipple discharge had received surgical treatment in the Department of Breast Surgery, Qilu Hospital of Shandong University. Among them, 10 had bilateral nipple discharge. A total of 111 nipple discharge samples were collected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eApproval for this study was obtained from the ethics committee of Qilu Hospital of Shandong University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the time of sample collection, the affected nipple was scrubbed with 75% alcohol and then dried with a sterile cotton swab. Nipple discharge is gently squeezed from around the breast. Finally, a sterile Eppendorf duct is used to collect nipple discharge. Half of the nipple discharge was taken for CSC, the other half for LBC (Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiquid-based cytology examination\u003c/strong\u003e\u003c/p\u003e\n\u003col style=\"list-style-type: upper-alpha;\"\u003e\n \u003cli\u003eDrain the discharge from the sterile EP tube into a centrifuge tube and add the cell preservation solution (Hanhe Medical Devices Co., LTD., Jinan, Shandong, China) to 1 ml.\u003c/li\u003e\n \u003cli\u003eAdd lymphocyte separation medium to 1:1 ratio.\u003c/li\u003e\n \u003cli\u003ePut into a TD5Z-WS centrifuge (XIANGZHI Centrifuge Instrument Co., LTD., Changsha, Hunan, China), 2,000 r/min for 5 min.\u003c/li\u003e\n \u003cli\u003eAspirate the nucleated cell layer and the upper layer of the original fluid to another centrifuge\u0026nbsp;tube.\u003c/li\u003e\n \u003cli\u003ePut the centrifuge tube into TD4Z-WS auto-balancing centrifuge (XIANGZHI Centrifuge Instrument Co., LTD., Changsha, Hunan, China), 900 r/min for 4 min to obtain a glass slide.\u003c/li\u003e\n \u003cli\u003eThe glass slides were coded and placed on a staining rack.\u003c/li\u003e\n \u003cli\u003eAdd Wright-giemsa staining solution A + pitonate buffer (7:10) and stain for 25 min.\u003c/li\u003e\n \u003cli\u003eWash slides and dry.\u003c/li\u003e\n \u003cli\u003eThe slide was reviewed under a microscope.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eConventional Smear cytologic examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) Make smear manually with glass slides and place on the staining rack after coding.\u003c/p\u003e\n\u003cp\u003eB) Add Wright-giemsa staining solution A + pitonate buffer (7:10) and stain for 25 min.\u003c/p\u003e\n\u003cp\u003eC) Wash slides and dry.\u003c/p\u003e\n\u003cp\u003eD) Review the slide under a microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReport\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo professional cytopathologists evaluated slides, reported observed cell types (ductal epithelial cells, blood cells, etc.) and morphological characteristics, defined detected cancer cells or atypical hyperplasia cells as positive results, and if not, defined as negative results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1) According to paraffin pathology, the lesions were divided into three groups: breast cancer, precancerous lesions (intraductal papilloma, atypical hyperplasia), and benign hyperplastic lesions (mammary fibroadenoma, sclerosing adenosis, mammary hyperplasia). Clinical data of all patients were collected to clarify the relationship between clinical features and lesion types. P values were calculated by paired \u0026chi;2 test or Fisher exact test, and P \u0026lt; 0.05 was considered statistically significant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2) All reports were reviewed and tabulated to present the types of cells in the reports. A Venn diagram was made to show the detection of ductal epithelial cells in all cases, breast cancer, and precancerous lesions (intraductal papilloma, atypical hyperplasia).\u003c/p\u003e\n\u003cp\u003e3) Cancer cells and atypical cells were defined as pathological cells. If pathological cells had been detected, the results were positive, otherwise, they were negative. In clinical treatment, both cancer and precancerous lesions had strong surgical indications. The two were combined and distinguished from benign hyperplastic lesions. The diagnostic validity of the two cytological examination methods was determined to include sensitivity, specificity, PPV, NPV and Kappa consistency test, drawn ROC curve, and performed ROC analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSPSS 25.0 software was used for data processing and statistical analysis.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eClinical\u0026nbsp;and pathological data\u0026nbsp;of patients\u003c/h2\u003e\n\u003cp\u003eThere were 111 nipple discharge samples from 101 patients included in this study (10 patients with bilateral nipple discharge). The mean age of the patients was 46.6 years (23-81 years, SD=10.6 y). There were 25 samples from premenopausal patients and 86 samples from postmenopausal patients; 46 palpable breast lumps and 65 unpalpable. Nipple discharge with lumps was an indication of malignancy (P=0.002). Of the 111 cases of discharge, there were 36 breast cancer, 54 precancerous lesions (51 intraductal papillomas, 3 atypical hyperplasia), and 21 benign proliferative lesions (7 fibroadenomas, 14 adenopathy and general hyperplasia). Among the 36 breast cancers, the most common pathological type was DCIS (86.1%, 31/36). There were 40 cases of bloody discharge, 51 cases of serous discharge, 17 cases of clear water discharge, and 3 cases of milk discharge. The characteristics of discharge were not correlated with specific diseases (P=0.019). There were no significant differences in age, number of ductal pores, menstrual status, family history, and breastfeeding history between breast cancer, precancerous lesions, and benign hyperplastic lesions (Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytological assessment of nipple discharge\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1. Fewer miscellaneous cells in LBC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cell types reported are shown in Figure 2. The areas marked in green blocks show cell detection in LBC, while orange blocks marked cell detection in CSC. The LBC method could detect fewer blood cells and squamous epithelial cells, in another word, fewer miscellaneous cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong 111 samples, ductal epithelial cells\u0026nbsp;(including cancer cells, atypical cells, and normal ductal epithelial cells)\u0026nbsp;were detected in 65 cases using LBC and in 34 cases using CSC. However, no ductal epithelial cells were detected by either method in 44 cases (Figure 3. (a, b)). For breast cancer samples (n=36), LBC identified ductal epithelial cells in 22 cases while CSC identified them in 14 cases (Figure 3. (c,d)). For intraductal papilloma samples (n=51), LBC detected ductal epithelial cells in 34 cases compared to CSC which found them in only 17 cases (Figure 3. (e, f)). As shown in the Venn diagram, there is concordance between these two methods. However, the ability of LBC to detect ductal epithelial cells was superior to that of CSC, particularly in breast cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. The diagnostic validity of LBC is better\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn all the samples (n=111), 36 cases of pathological cells were detected using LBC, and 21 cases were detected using CSC. Among\u0026nbsp;samples\u0026nbsp;from breast cancer (n=36), 19 cases of pathological cells were detected with LBC, while only 11 were detected using CSC. Among samples from intraductal papilloma (n=51), 17 cases of pathological cells were detected using LBC compared to nine using CSC. None of the atypical hyperplasia (n=3) samples showed any pathological cell detection by either method. Among samples from benign hyperplasia lesions (n=21), CSC detected pathological cells from one sample of bloody discharge, however, no case of pathological cells was detected using LBC.\u003c/p\u003e\n\u003cp\u003eWhen diagnostic validity was evaluated, we found LBC had higher sensitivity than CSC for breast tumors (52.78% vs. 30.56% for breast cancer; and 31.48% vs. 16.67% for intraductal papilloma). Breast cancer, intraductal papilloma, and atypical hyperplasia were grouped and distinguished from benign hyperplastic lesions. Based on this distinction, the diagnostic validity of these two methods were calculated. For LBC: sensitivity = 40%, specificity = 100%, positive predictive value = 100%, negative predictive value = 28%, and Kappa value = 0.201. For CSC: sensitivity = 22.22%, specificity = 95.22%, positive predictive value = 95.20%, negative predictive value = 22%, and Kappa value = 0 .077 (Table 2). Chi-square test of the two groups of rate was performed: Sensitivity \u0026chi;2=6.636, P=0.01. That is, the sensitivity, specificity, positive predictive value, negative predictive value, and Kappa value of the LBC test were higher than those of CSC, and the difference in sensitivity was statistically significant. In receiver operating characteristic (ROC) curves of two methods (Figure 4), AUC\u003csub\u003eLBC\u0026nbsp;\u003c/sub\u003e= 0.700 (p = 0.004, 95% CI: 0.598~0.802). AUC\u003csub\u003eCSC\u0026nbsp;\u003c/sub\u003e= 0.587 (p = 0.214, 95% CI: 0.463~0.711). The diagnostic validity of LBC is, therefore, better than CSC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. The image quality of LBC is better\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe accuracy of diagnosis is related to the image quality, which can be greatly reduced by blood cells and cell debris in the sample. A bloody discharge of a breast cancer patient showed cells with large and deeply stained nuclei (Figure 5. a-b). While the CSC image showed more blood cells in the background, damaged cell morphology, and many overlapped cells (a), the LBC image had a clean background, good morphological integrity, and no cell debris (b). In a bloody discharge of a patient with intraductal papilloma (Figure 5. c-d), big ductal epithelial cells with increased nucleocytoplasmic ratio could be identified by both LBC and CSC. In the background of the CSC image, a large quantity of blood cells were arranged in clusters and the image was crowded, which sometimes obscured cancer cells. Moreover, the distribution of cancer cells was not concentrated, and the color contrast was not strong (c). However, the background of LBC is clean, cell distribution is concentrated, and the color is contrastive (d). In a sero-like discharge of intraductal papilloma (Figure 5. e-f), the sample is rich in protein. In CSC, protein impurities adhere to the slide and appear purplish, which would shade cells (e). In LBC, the protein impurities are no longer attached to the slide after centrifugation (f). Overall, the cell preservation and image quality of LBC are superior to CSC.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBreast cancer accounts for approximately 5\u0026ndash;12% of pathologic nipple discharge in patients undergoing surgery [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], most of which are DCIS [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. For patients with nipple discharge, cytolgical examination is presently the predominant method for clinical evaluation. Since the report of CSC in 1969 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], it has been widely used for pathologic diagnosis. Although the sensitivity and specificity of cytology-based tests are comparable to that of imaging, cytology evaluation cannot replace imaging and is not recommended alone for clinical diagnosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. LBC is an emerging and more sensitive cytological examination method than CSC, as reported here, with removal of impurities such as blood cells and mucus proteins during sample preparation to obtain a clearer image background. To date, LBC has been applied to nipple aspirate analysis using ductoscopy. The ductal epithelia could be damaged, resulting in ductal adhesion and disappearance of nipple discharge, making it difficult to position tumor locations. Moreover, ductoscopy often misses lesions in the terminal ductal lobular unit (TDLU). In this report, we successfully applied LBC for direct analysis of nipple discharge, and showed it represents a more sensitive diagnosis than CSC.\u003c/p\u003e \u003cp\u003eTwo advantages of LBC over CSC include: 1) The image quality of LBC is superior to that of CSC; and 2) the diagnostic validity of LBC is better than that of CSC. In LBC, centrifugation helps remove impurities such as blood cells and mucus. When nipple discharge is bloody, LBC produces a cleaner image than CSC. Moreover, during LBC examination, the cell preservation solution is added immediately after collecting nipple discharge that preserves better cell morphology, especially the intactness of the nucleus necessary for observing any atypic changes. After settling, the distribution of cells is more concentrated and uniform. For specimens with less cells, ductal epithelial cells can also be significantly enriched. In contrast, during CSC examination, nipple discharge is collected and placed directly onto the slide. When not treated in time, the specimen will dry out and lead to cell deformation and fragmentation. Cell overlapping and scattered distribution in CSC tend to fatigue the reader, resulting in bias. All the above factors lead to improved image quality of LBC.\u003c/p\u003e \u003cp\u003eIn our analysis, the positive results of LBC did not completely cover match the CSC results. Two samples were positive for CSC but negative for LBC. The reasons may be: 1) The number of cells shed by discharge was limited, and there was sampling error in the examination process; 2) some background cells and proteins eliminated after centrifugation may provide clues to lesions; and 3) as LBC was not completely automated, some operations may be affected by artificial errors.\u003c/p\u003e \u003cp\u003eSome reasons for flash negative cytology may be: 1) The nipple discharge and breast lesion do not originate from the same mammary ductal system; 2) some of the lesion cells were not shed by discharge, which might be related to the differential expression of tumor adhesion factors; 3) Invasive ductal carcinoma is difficult to shed through the duct; and 4) ductal epithelial cells which entered nipple discharge might be out of shape, however the cognition of morphology features of pathological cells is still limited to tissue. Therefore, multiple samplings and multiple deliveries can be applied to reduce sampling errors. In addition, automatic staining and AI-aided automated cytology systems can also be used. AI can perform initial screening and then cytopathologists make the diagnosis of abnormal cells, with the potential to reduce artificial references and improve work efficiency. At the same time, cytopathologists need to receive special training to refresh their cognition and improve the sensitivity of diagnosis.\u003c/p\u003e \u003cp\u003eThe sensitivity, specificity, PPV, NPV and AUC value of LBC were higher than those of CSC, as shown by compared diagnostic consistency of the cytology examination and pathology. Although Kappa values of the LBC were higher than those of CSC, neither method have poor diagnostic consistency with paraffin pathology due to both results\u0026thinsp;\u0026lt;\u0026thinsp;0.4. Therefore, it is necessary to combine other specific tests in nipple discharge, such as the Thomsen-Freidenreich antigen test [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and detection of tumor markers (CEA, CA153[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], miRNA[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]., etc).\u003c/p\u003e \u003cp\u003eIn conclusion, LBC can be used as a new diagnostic method for cytological evaluation using direct nipple discharge without aspiration. However, due to sampling errors, LBC cannot completely replace CSC. If the sample volume was enough, patients should receive both examination methods to avoid misdiagnosis. If discharge volume was limited, LBC seems to be more effective to diagnose pathological nipple discharge. Moreover, LBC is also more conducive to the preservation of genetic material including protein, RNA and DNA [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], which may be used for subsequent molecular and immunocytochemical examination, while its application potential needs to be verified by follow-up experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Prof. Cheng-Ming Chiang and Prof. Yunyun Gong for language editing and valuable comments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from Shandong Provincial Natural Science Foundation, China (No. ZR2022MH248), the Postdoctoral Innovation Foundation of Shandong Province, China (No. 202103004).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJiang Zhu, Han Cong\u0026nbsp;\u003c/em\u003eand \u003cem\u003eXiaotong Zha\u003c/em\u003e\u003cem\u003eng\u0026nbsp;\u003c/em\u003e(first author): Data Curation and Analysis, Methodology, Investigation and Writing-Original Draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRong Ma,\u0026nbsp;\u003c/em\u003e\u003cem\u003ePengyu Li\u0026nbsp;\u003c/em\u003e(Correspondence) and\u0026nbsp;\u003cem\u003eJiang Zhu\u003c/em\u003e: Conceptualization, Methodology, Funding Acquisition, Supervision and Writing-Review＆Editing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eShuxin Yan and Jianli Wang\u003c/em\u003e: Cytopathological evaluation and diagnosis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eJUDE RANCHU MAIMO\u003c/em\u003e: Language editing.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSong Zhao, Chaolu Hu, Yawen Wang\u003c/em\u003e and \u003cem\u003eKai Zhang\u003c/em\u003e: Patients admission and management, Conceptualization, Resources and Validation.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during\u0026nbsp;this\u0026nbsp;study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was reviewed by the Ethics Committee of Qilu Hospital of Shandong University\u0026nbsp;(No.2021-088).\u003c/p\u003e\n\u003cp\u003eAll procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5). Informed consent was obtained from all patients for being included in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMorrogh, M., Park, A., Elkin, E. B. \u0026amp; King, T. A. Lessons learned from 416 cases of nipple discharge of the breast. \u003cem\u003eAm. J. Surg.\u003c/em\u003e \u003cb\u003e200\u003c/b\u003e (1), 73\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.amjsurg.2009.06.021\u003c/span\u003e\u003cspan address=\"10.1016/j.amjsurg.2009.06.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010). Epub 2010/01/19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDolan, R. T., Butler, J. S., Kell, M. R., Gorey, T. F. \u0026amp; Stokes, M. A. Nipple discharge and the efficacy of duct cytology in evaluating breast cancer risk. \u003cem\u003esurgeon: J. Royal Colleges Surg. Edinb. Irel.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (5), 252\u0026ndash;258 (2010). PubMed PMID: 20709281.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiwa, N. et al. Diagnostic Accuracy of Nipple Discharge Fluid Cytology: A Meta-Analysis and Systematic Review of the Literature. Annals of surgical oncology. ;29(3):1774-86. Epub 2021/11/29. doi: (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1245/s10434-021-11070-2\u003c/span\u003e\u003cspan address=\"10.1245/s10434-021-11070-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 34839426; PubMed Central PMCID: PMCPMC8627297 detection of breast cancer under the supervision of D. R. Leff and Z. Takats. The remaining authors have no conflicts of interest.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrunwald, S. et al. Mammary ductoscopy for the evaluation of nipple discharge and comparison with standard diagnostic techniques. \u003cem\u003eJ. Minim. Invasive. Gynecol.\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e (5), 418\u0026ndash;423. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jmig.2006.05.004\u003c/span\u003e\u003cspan address=\"10.1016/j.jmig.2006.05.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006). Epub 2006/09/12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eClark, S. E. et al. The investigation and management of unilateral nipple discharge. \u003cem\u003eAnn. R. Coll. Surg. Engl.\u003c/em\u003e \u003cb\u003e102\u003c/b\u003e (5), 369\u0026ndash;374. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1308/rcsann.2020.0036\u003c/span\u003e\u003cspan address=\"10.1308/rcsann.2020.0036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020). Epub 2020/04/03.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHutchinson, M. L., Cassin, C. M. \u0026amp; Ball, H. G. 3 The efficacy of an automated preparation device for cervical cytology. \u003cem\u003eAm. J. Clin. Pathol.\u003c/em\u003e \u003cb\u003e96\u003c/b\u003e (3), 300\u0026ndash;305. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/ajcp/96.3.300\u003c/span\u003e\u003cspan address=\"10.1093/ajcp/96.3.300\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1991). Epub 1991/09/01.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePathuthara, S. et al. Conventional versus Liquid-based Cytology: Man versus Machine. \u003cem\u003eJ. Cytol.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e (4), 169\u0026ndash;176. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/joc.joc_54_23\u003c/span\u003e\u003cspan address=\"10.4103/joc.joc_54_23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023). Epub 2023/12/07.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMakde, M. M. \u0026amp; Sathawane, P. Liquid-based cytology: Technical aspects. \u003cem\u003eCytoJournal\u003c/em\u003e. \u003cb\u003e19\u003c/b\u003e, 41 (2022). Epub 2022/08/06. doi: 10.25259/cmas_03_16_2021. PubMed PMID: 35928530; PubMed Central PMCID: PMCPMC9345114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, S. Y. et al. Combined use of conventional smear and liquid-based preparation versus conventional smear for thyroid fine-needle aspiration. Endocrine. ;53(1):157\u0026thinsp;\u0026ndash;\u0026thinsp;65. Epub 2015/12/31. doi: (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12020-015-0835-z\u003c/span\u003e\u003cspan address=\"10.1007/s12020-015-0835-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 26714459.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHashimoto, S. et al. Diagnostic efficacy of liquid-based cytology for solid pancreatic lesion samples obtained with endoscopic ultrasound-guided fine-needle aspiration: Propensity score-matched analysis. \u003cem\u003eDig. endoscopy: official J. Japan Gastroenterological Endoscopy Soc.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (5), 608\u0026ndash;616. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/den.12827\u003c/span\u003e\u003cspan address=\"10.1111/den.12827\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017). Epub 2017/02/06.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao, C. et al. Diagnostic role of liquid-based cytology of bronchial lavage fluid in addition to bronchial brushing specimens in lung cancer. \u003cem\u003eTumori\u003c/em\u003e. \u003cb\u003e107\u003c/b\u003e (4), 325\u0026ndash;328. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1177/0300891620960218\u003c/span\u003e\u003cspan address=\"10.1177/0300891620960218\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021). Epub 2020/09/26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasood, S. \u0026amp; Khalbuss, W. E. Nipple fluid cytology. Clinics in laboratory medicine. ;25(4):787\u0026thinsp;\u0026ndash;\u0026thinsp;94, vii-viii. Epub 2005/11/26. doi: (2005). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.cll.2005.08.010\u003c/span\u003e\u003cspan address=\"10.1016/j.cll.2005.08.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 16308092.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel, B. K., Falcon, S. \u0026amp; Drukteinis, J. Management of nipple discharge and the associated imaging findings. \u003cem\u003eAm. J. Med.\u003c/em\u003e \u003cb\u003e128\u003c/b\u003e (4), 353\u0026ndash;360. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.amjmed.2014.09\u003c/span\u003e\u003cspan address=\"10.1016/j.amjmed.2014.09\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015). Epub 2014/12/03.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsuda, M. \u0026amp; Koyama, H. [Cytologic diagnosis of the abnormal nipple discharge]. \u003cem\u003eGan no rinsho Japan J. cancer Clin.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e (12), 1039\u0026ndash;1046 (1969). Epub 1969/12/01. PubMed PMID: 5392544.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiwa, N. et al. Nipple aspirate fluid and its use for the early detection of breast cancer. Annals of medicine and surgery 2022;77:103625. Epub 2022/06/01. doi: (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.amsu.2022.103625\u003c/span\u003e\u003cspan address=\"10.1016/j.amsu.2022.103625\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 35638006; PubMed Central PMCID: PMCPMC9142541.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarvalho, M. J. et al. What is the diagnostic value of nipple discharge cytology and galactography in detecting duct pathology? \u003cem\u003eEur. J. Gynaecol. Oncol.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e (5), 543\u0026ndash;546 (2009). Epub 2009/11/11. PubMed PMID: 19899412.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeutscher, S. L. et al. Carbohydrate antigens in nipple aspirate fluid predict the presence of atypia and cancer in women requiring diagnostic breast biopsy. \u003cem\u003eBMC cancer\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e, 519. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1471-2407-10-519\u003c/span\u003e\u003cspan address=\"10.1186/1471-2407-10-519\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010). Epub 2010/10/06.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, S. et al. Levels of CEA, CA153, CA199, CA724 and AFP in nipple discharge of breast cancer patients. \u003cem\u003eInt. J. Clin. Exp. Med.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e (11), 20837\u0026ndash;20844 (2015). Epub 2016/02/18. PubMed PMID: 26885008; PubMed Central PMCID: PMCPMC4723853.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, K. et al. Identification of microRNAs in Nipple Discharge as Potential Diagnostic Biomarkers for Breast Cancer. Annals of surgical oncology. ;22 Suppl 3:S536-44. Epub 2015/05/16. doi: (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1245/s10434-015-4586-0\u003c/span\u003e\u003cspan address=\"10.1245/s10434-015-4586-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PubMed PMID: 25976861.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKonofaos, P. et al. The role of ThinPrep cytology in the investigation of ki-67 index, p53 and HER-2 detection in fine-needle aspirates of breast tumors. \u003cem\u003eJ. BUON: official J. Balkan Union Oncol.\u003c/em\u003e \u003cb\u003e18\u003c/b\u003e (2), 352\u0026ndash;358 (2013). Epub 2013/07/03. PubMed PMID: 23818345.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSauer, T., Ebeltoft, K., Pedersen, M. K. \u0026amp; K\u0026aring;resen, R. Liquid based material from fine needle aspirates from breast carcinomas offers the possibility of long-time storage without significant loss of immunoreactivity of estrogen and progesterone receptors. \u003cem\u003eCytoJournal\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e, 24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/1742-6413.75665\u003c/span\u003e\u003cspan address=\"10.4103/1742-6413.75665\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010). Epub 2011/02/08.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Liquid-based cytology, diagnosis, breast cancer, nipple discharge","lastPublishedDoi":"10.21203/rs.3.rs-5177988/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5177988/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eConventional smear cytology (CSC) is a specific method used for breast tumor diagnosis in patients with nipple discharge. However, CSC tends to miss diagnose or even misdiagnose due to contaminating blood cells and other impurities. Thus, it is critical to develop more accurate and sensitive methods for clinical evaluation. Using nipple discharge collected directly from patients without aspiration, we performed liquid-based cytology (LBC) to analyze 111 collected samples for cytological evaluation for the first time. Following centrifugation to remove blood cells and protein contaminants, the sample of each nipple discharge was analyzed by light microscopy. In parallel, CSC was performed for comparison. Our results showed LBC has better diagnostic sensitivity than CSC (40.00% vs 22.22%, χ2\u0026thinsp;=\u0026thinsp;6.636, P\u0026thinsp;=\u0026thinsp;0.01). The specificity was improved (LBC 100% vs CSC 95.2%) and area under the curve was also enhanced (AUC\u003csub\u003eLBC\u003c/sub\u003e=0.700 vs AUC\u003csub\u003ecsc\u003c/sub\u003e=0.587). Moreover, LBC images have a cleaner background, clearer field of view, uniform cell arrangement, and improved colour contrast, with an overall image quality significantly better than CSC. Therefore, LBC provides a new and better diagnostic method for early detection of breast tumors with nipple discharge.\u003c/p\u003e","manuscriptTitle":"New Method for Cytological Evaluation Using Direct Nipple Discharge without Aspiration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-23 09:29:41","doi":"10.21203/rs.3.rs-5177988/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-29T08:26:33+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-26T16:19:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-16T13:21:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108906973188375357426507777461049346330","date":"2024-11-04T12:15:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338206773409787423641487711249302030056","date":"2024-10-23T13:54:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-21T13:22:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-21T13:19:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-21T09:32:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-16T13:53:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-30T05:25:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d5255786-8df9-4bd8-8367-031b9e589e37","owner":[],"postedDate":"October 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39030332,"name":"Biological sciences/Cancer/Breast cancer"},{"id":39030333,"name":"Biological sciences/Biological techniques/Cytological techniques"},{"id":39030334,"name":"Biological sciences/Biological techniques/Cytological techniques/Tissue culture"}],"tags":[],"updatedAt":"2025-02-10T15:59:01+00:00","versionOfRecord":{"articleIdentity":"rs-5177988","link":"https://doi.org/10.1038/s41598-025-88456-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-02-04 15:56:58","publishedOnDateReadable":"February 4th, 2025"},"versionCreatedAt":"2024-10-23 09:29:41","video":"","vorDoi":"10.1038/s41598-025-88456-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-88456-9","workflowStages":[]},"version":"v1","identity":"rs-5177988","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5177988","identity":"rs-5177988","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}