Cost-Effective and Practical Manual Microdissection Method for Accurate High-Purity Tissue Sampling

Cost-Effective and Practical Manual Microdissection Method for Accurate High-Purity Tissue Sampling | 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 Cost-Effective and Practical Manual Microdissection Method for Accurate High-Purity Tissue Sampling Yasuko Fujita, Masataka Haneda, Waki Hosoda This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5654096/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Tissue-based molecular analyses have become a mainstay of biomedical research, including high-throughput DNA and RNA sequencing, and for appropriate results, microdissection is necessary to enrich the target cells. Many types of microdissection methods have been developed, among which laser capture microdissection (LCM) is the most precise. However, LCM is expensive and requires specialized equipment and membrane slides. Herein, we present a modified manual microdissection method that is simple, inexpensive, and does not require special equipment. On a wet condition after microtome sectioning of non-stained formalin-fixed paraffin-embedded tissue, dissection of targeted foci of as small as 0.02 square millimeters was possible using a needle under stereomicroscopic observation. Three representative cases with 16 foci were demonstrated, with successful detection of KRAS and/or GNAS mutations by direct sequencing and Cycleave polymerase chain reaction (PCR) assays. This modified manual microdissection method is simple and efficient, allowing us to obtain materials with a high tumor purity in any laboratory. Biological sciences/Cancer Health sciences/Medical research Health sciences/Oncology Microdissection Manual microdissection Formalin-fixed paraffin-embedded tissue Laser microdissection Tumor purity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Recent advances in molecular profiling have accelerated our understanding of many diseases. The improvements in high-throughput sequencing technologies for formalin-fixed paraffin-embedded (FFPE) tissues have enabled the analysis of clinical materials [1]. These advances have significantly affected many fields of biomedical research and clinical molecular diagnostics, including cancer. However, cancer tissues are composed of tumor and non-tumor cells, such as stromal, vascular, or inflammatory cells. The enrichment of the cancer cell ratio plays a vital role in molecular analysis [2]. Various kinds of dissection methods have been described, depending on the purpose of the investigation [3-5]. Manual microdissection is a simple and inexpensive dissection method for FFPE and frozen sections using a sharp instrument under a microscope [3]. The surrounding non-neoplastic tissue is scraped from the section before the target tissue is collected (Fig. 1a). Manual microdissection allows the investigators to obtain an almost pure target tissue at minimal cost, for performing many molecular analyses, and thus has become the most common method of tissue enrichment in research fields. However, macro-dissection [5] provides enrichment of tumor purity in clinical diagnostics. This method uses serial hematoxylin and eosin (H&E) sections as a guide instead of a microscope. This allows for an easy rough dissection. Laser capture microdissection (LCM) is a well-established cell separation technique that enables to obtain specific tissues with high purity [6, 7]. This method is most effective if the surrounding normal or stromal cells can affect the molecular analysis of the target tissue or if microscopic precursor or multifocal lesions need to be investigated separately. The samples obtained by LCM have been used in different types of research, including next-generation sequencing, expression arrays, and proteomic techniques [8]. In the field of clinical molecular diagnostics, the LCM method may be used for molecular tests on a special occasion [9]. There are several obstacles that hamper the routine application of LCM in clinical diagnostics, such as expensive equipment must be installed and the entire process is time-consuming and labor-intensive. We aimed to establish a low-cost method that could achieve precision close to that of LCM. Herein, we developed a modified manual microdissection technique for FFPE sections using equipment and instruments that most pathology laboratories have, with minimal additional costs. Results Protocol of modified manual microdissection The scheme and workflow are illustrated in Figs. 1b and 2, respectively. Step 1. Section the FFPE tissue into 5–15 μ m thick sections The FFPE tissues were sectioned using a microtome. Although the thickness of the sections depends on the required amount of nucleic acid, the sections that are too thin are easily broken to be picked up, and those that are too thick do not allow for the identification of target areas during microdissection. Approximately 10-μm thick sections are the best, although 5–15 μm thickness can also be used for successful target identification and microdissection. The representative section in Fig. 2 was 10-μm thick, whereas the dissected section in Fig. 3 was 15-μm thick. Step 2. Float the sections on warm water This step smoothens and stretches sections. When the section is not smooth, the dissection process in Step 5 can be difficult because of the shallow depth of focus under a stereomicroscope. Step 3. Place the tissue onto a non-coating slide Glass slides used in routine practice can be used in this method. The use of a new slide for each dissection can reduce the risk of contamination. Step 4. Wipe the underside of the slide without drying the section This is the most important step. Slides are typically air-dried using conventional manual microdissection. However, it is difficult to pick up the dissected section once the tissue firmly adheres to the glass slide. Therefore, in this method, dissection was performed without drying. The section was kept wet, creating a condition in which it slightly floated on a thin layer of water on a glass slide. The area of interest can be easily collected after sectioning. However, the underside of the slide must not be wet. The water between the slide and stage of the stereomicroscope interferes with the visualization of the FFPE section. Step 5. Dissect the section using a needle under a stereomicroscope and collect it into a tube or tube cap First, dissect the non-dried section on the slide with a sharp instrument, such as using a disposable hypodermic needle, and a serial hematoxylin and eosin (H&E) section as a guide. Then, section the area of interest under a stereomicroscope (SZ-61; Olympus, Tokyo, Japan) with lighting from the side. The illumination from the side creates a contrast between the tissue structures in an FFPE section without any staining. While dissecting, another needle or finger of the other hand with a clean globe should hold the edge of the section. After cutting the entire circumference, the fragment should be collected in a sample tube. When the dissected section is small, it is better placed inside the tube cap, where a small amount of extraction reagent is applied. This manipulation may require skill, and we prepared a movie of Step 5, shown in Supplementary Movie 1. Representative cases A representative case of duodenal neoplasm is shown in Fig 3. To obtain tumor-rich material, the area indicated by yellow arrowheads was dissected (Fig. 3a, b). The non-stained FFPE sections were observed under a stereomicroscope before (Fig. 3c) and after (Fig. 3d) dissection, and DNA was extracted from the sections. Direct sequencing detected the GNAS c.601C>T (p.R201C) mutation (Fig 3e). We demonstrated that multiple foci in a single section can be dissected separately using this method. The second case had multiple pancreatic intraepithelial neoplastic (PanIN) lesions (Fig. 4), and each PanIN lesion was separately dissected and analyzed by direct sequencing and/or cycleavePCR for KRAS mutations. Although each lesion was minute (0.0667–0.686 mm 2 in area) (Supplementary Table 2), we detected KRAS c.34G>C (p.G12R) in two areas and KRAS c.35G>A (p.G12D) in the remaining areas. The second lesion was collected from multiple serial sections (Supplementary Fig. 1). The amounts of extracted DNA are presented in Supplementary Table 2. The third case had intraductal papillary mucinous neoplasm (IPMN) (Fig. 5). A broader area could be dissected using this method (Fig. 5a–e) and was analyzed for KRAS and GNAS mutations. Both KRAS c.35G>A (p.G12D) (Supplementary Fig. 3f) and GNAS c.601C>T (p.R201C) (Fig. 5g) mutations were present in IPMN. Discussion Tumor purity is a crucial factor that leads to appropriate results in molecular analyses. Although several dissecting methods have been used in research and clinical fields [3-9], precise dissection methods such as LCM require expensive equipment, and inexpensive methods such as macro-dissection make it difficult to achieve fine dissection. This study developed a simple and inexpensive modified manual microdissection method that enabled precise dissection. Macrodissection is the easiest dissection method, wherein the FFPE section is dissected using instruments, such as a razor blade, without the aid of a microscope [5]. In this method, the target area is roughly dissected using serial HE sections as a guide. Unlike other microdissection methods, this method does not allow for precise dissection. In conventional manual microdissection [3], the tissue sections are attached to glass slides and non-tumor tissue is scraped away to obtain the targeted tumor tissue. In this method, deparaffinized and stained tissues are dissected under a microscope, allowing for fine separation. However, because the target tissue is collected after scraping off the surrounding tissue, it is possible that the tissue fragments may be mixed, which requires time and effort. In addition, it is difficult to confirm the sampled area retrospectively. Our modified manual microdissection method was an improvement over the conventional manual microdissection method. The key feature of our method was to cut the FFPE sections under wet conditions before deparaffinization. Before drying, the sections were peeled off the glass slides. Therefore, this method enabled the removal and collection of the target tissue in one piece without scraping the surrounding tissue (Fig. 1a and b). This may have reduced the risk of contamination. Using our method, the section can be cut into any shape or size by LCM with a sharp needle (Supplementary Movie 1). It was also possible to sample a small area or different areas separately (Fig. 4). For small sections, static electricity may have caused the sample to be lost when collected in tubes. This can be avoided by cutting off the tube cap and applying a small amount of the extract solution before dissection, as shown in step 5. This enabled the collection of samples under a stereomicroscope (Supplementary Fig. 1 and Supplementary Movie 1). By performing H&E staining on the sections remaining after sample collection (Fig. 3b), the collected area could also be retrospectively confirmed by comparison with a serial H&E section (Fig. 3a). One disadvantage of this method, which is performed before deparaffinization, is that staining to identify tissue structures is impossible. However, it is possible to recognize tissue morphology even in unstained sections, like the presented case (Fig. 3c, 4, and 5c), by making appropriate preparations and observing under a stereomicroscope. Side lighting and semi-wet conditions make this possible. Most equipment used in this method was inexpensive and readily available in ordinary laboratories. Although it was necessary to prepare a stereomicroscope, it was performed using instruments that are generally used in clinical practice, such as injection needles and glass slides, and did not require expensive equipment, such as an LCM [6]. If more amount of DNA is needed, a larger area (Fig. 5) or greater number of sections (Supplementary Fig. 1) can be dissected. This method can also be used for whole-exome sequencing, similar to LCM [10]. The DNA quality obtained using this method is also suitable for molecular analysis. As shown in the present case (Fig. 3e), even small samples were successfully analyzed using Sanger sequencing, which is not highly sensitive. This was probably because the samples were collected before deparaffinization. This may prevent the denaturation and degradation of nucleic acids before extraction. In addition, the electropherogram showed that we could collect the samples with high tumor purity. However, this method had some limitations. First, although this is an inexpensive and easy dissection method that can be performed in any laboratory, it requires precise handling and skill to precisely cut small areas of interest. Second, to collect samples without deparaffinization, deparaffinization must be performed at the time of extraction. However, if a dried specimen is deparaffinized on a slide, unnecessary areas can be removed using this method to increase tumor purity. In conclusion, we described a modified method for manual microdissection of FFPE sections. This method allows investigators to perform microdissection under a stereomicroscope to collect highly enriched target tissues in a manner similar to LCM. In general, clinical specimens have low tumor purity as they contain mixed various tissues; hence, their molecular analyses may yield inaccurate results. However, our method is versatile because it can collect tumor-enriched specimens not only by isolating and obtaining tumor tissue but also by removing non-tumor tissue and gathering the remaining tumor tissue. In addition, our method can separate tissues from different parts of the same tumor in a single section, which allows for examining molecular changes in tumor progression and genetic differences between histologically different parts of the tumor. Therefore, our method is useful in both clinical and research settings. Methods Case selection This study included three cases with duodenal or pancreatic neoplasia in which surgery was performed at Aichi Cancer Center between 2018 and 2021. The resected specimens were fixed in formalin and embedded in paraffin. A total of 16 lesions were microdissected by present method from FFPE sections and subjected to the molecular analyses described below. The uniqueness of this modified manual microdissection is summarized in two points: a sectioned tissue was floated on a layer of water on a glass slide, and the tissue was microdissected under wet conditions using a needle under microscopic observation. No special equipment was required, except for a stereomicroscope. The scheme and workflow are illustrated in Figs. 1b and 2, respectively. This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Aichi Cancer Center (Approval No. R011032). DNA extraction DNA was extracted from the collected sections using a deparaffinization solution (Qiagen, Venlo, Netherlands) and QIAamp DNA FFPE Tissue Kit (Qiagen, Venlo, Netherlands), according to the manufacturer’s protocol. The amount of extracted DNA was measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Massachusetts, U.S.). Sanger sequencing and Cycleave polymerase chain reaction (PCR) We tested whether the DNA extracted using this dissection protocol could be used for molecular analysis. We examined GNAS exon 8 and KRAS exon 2 using direct sequencing and/or cycleave-PCR. The methods for both the assays and primers have been described previously [11-13]. Declarations Acknowledgments We thank the technical staff members, Noriko Shibata and Motoko Nimura, of the Department of Pathology and Molecular Diagnostics, Aichi Cancer Center, for their technical assistance. This work was supported by JSPS KAKEN-HI (grant number 20K16179). We would like to thank Editage (www.editage.jp) for English language editing. Author contributions Yasuko Fujita designed the study, collected samples, performed molecular analysis, wrote the manuscript, and prepared the figures and movies. Masataka Haneda contributed to the improvement of the method. Waki Hosoda organized the study and revised the manuscript. All the authors have read and approved the final manuscript. Competing interests: The authors declare that there are no conflicts of interest. References Mathieson W, Thomas GA. Why formalin-fixed, paraffin-embedded biospecimens must be used in genomic medicine: an evidence-based review and conclusion. J Histochem Cytochem 68:543–552. https://doi.org/10.1369/0022155420945050 (2020) Kogo M, Fujimoto D, Hosoya K, Nagata K, Nakagawa A, Tachikawa R, Yamashita D, Kitamura Y, Imai Y, Tomii K. Tumour content ratio matters for detecting epidermal growth factor receptor mutation by cobas test in small biopsies; a retrospective study. BMC Cancer 20:104. https://doi.org/10.1186/s12885-020-6603-3 (2020) Hunt JL, Finkelstein SD. Microdissection techniques for molecular testing in surgical pathology. Arch Pathol Lab Med 128:1372–1378. https://doi.org/10.5858/2004-128-1372-MTFMTI (2014) Walsh EM, Halushka MK. A comparison of tissue dissection techniques for diagnostic, prognostic, and theragnostic analysis of human disease. Pathobiology 1–10. https://doi.org/10.1159/000525979 (2022) Wisner L, Larsen B, Maguire A. Enhancing tumor content through tumor microdissection. J Vis Exp. https://doi.org/10.3791/62961 (2022) Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA. Laser capture microdissection. Science 274:998–1001. https://doi.org/10.1126/science.274.5289.998 (1996) Schütze K, Lahr G. Identification of expressed genes by laser-mediated manipulation of single cells. Nat Biotechnol 16:737–742. https://doi.org/10.1038/nbt0898-737 (1998) Datta S, Malhotra L, Dickerson R, Chaffee S, Sen CK, Roy S. Laser capture microdissection: Big data from small samples. Histol Histopathol. 30:1255–1269. https://doi.org/10.14670/HH-11-622 (2015) Higuchi R, Goto T, Nakagomi T, Hirotsu Y, Oyama T, Amemiya K, Mochizuki H, Omata M. Discrimination between primary lung cancer and lung metastases by genomic profiling. JTO Clin Res Rep 2:100255. https://doi.org/10.1016/j.jtocrr.2021.100255 (2021) Noë M, Niknafs N, Fischer CG et al. Genomic characterization of malignant progression in neoplastic pancreatic cysts. Nat Commun 11:4085. https://doi.org/10.1038/s41467-020-17917-8 (2020) Yatabe Y, Hida T, Horio Y, Kosaka T, Takahashi T, Mitsudomi T. A rapid, sensitive assay to detect EGFR mutation in small biopsy specimens from lung cancer. J Mol Diagn 8:335–341. https://doi.org/10.2353/jmoldx.2006.050104 (2006) Hosoda W, Sasaki E, Murakami Y, Yamao K, Shimizu Y, Yatabe Y. GNAS mutation is a frequent event in pancreatic intraductal papillary mucinous neoplasms and associated adenocarcinomas. Virchows Arch 466:665–674. https://doi.org/10.1007/s00428-015-1751-6 (2015) Hosoda W, Takagi T, Mizuno N, Shimizu Y, Sano T, Yamao K, Yatabe Y. Diagnostic approach to pancreatic tumors with the specimens of endoscopic ultrasound-guided fine needle aspiration. 60:358–364. https://doi.org/10.1111/j.1440-1827.2010.02527.x (2010) Additional Declarations No competing interests reported. Supplementary Files Supplementarymovie1.mp4 SupplementaryTable1.docx SupplementaryTable2.docx Supplementaryfigure1.tif SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted 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-5654096","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":397063782,"identity":"1e035d19-d563-4f82-8c01-57c4ef22ff69","order_by":0,"name":"Yasuko Fujita","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYFACxgcMEgYMDPwMjA3MDAZgoQQCWpgNgFoMGCQbSNLCAFRqcADIJMpZBgeY2SQsCv7IGx8/3Pi5oIBBnr+B4dkDglqADjPcdiaxWXqGAYPhjAMM6Qb4tfAfA2lh3HYgsY2Zx+B/AlB5mgQxtthv7n8I0sJAvJbEDRKJRGqRPMzMbCFhYJw848bDZmkekF8OE/AL3/FmxtsSf+Rs+/vTH37m+QMMsfaetAf4tCgcBkYHqjuYedLw6WCQbwAmmQ+oYuzH8GoZBaNgFIyCEQcAh+c9u8YKmLEAAAAASUVORK5CYII=","orcid":"","institution":"Aichi Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Yasuko","middleName":"","lastName":"Fujita","suffix":""},{"id":397063783,"identity":"bea62538-a3db-4cf9-a585-c330cae45915","order_by":1,"name":"Masataka Haneda","email":"","orcid":"","institution":"Aichi Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Masataka","middleName":"","lastName":"Haneda","suffix":""},{"id":397063784,"identity":"1a90fa76-e3f5-4167-abf0-2450e40b8f98","order_by":2,"name":"Waki Hosoda","email":"","orcid":"","institution":"Aichi Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Waki","middleName":"","lastName":"Hosoda","suffix":""}],"badges":[],"createdAt":"2024-12-16 13:08:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5654096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5654096/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81963518,"identity":"15662ca9-8480-4349-8252-0cc787f65049","added_by":"auto","created_at":"2025-05-05 11:19:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":956634,"visible":true,"origin":"","legend":"\u003cp\u003eSchemes of manual microdissection. (a) Conventional manual microdissection, (b) present method of modified manual microdissection.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/65fccf6c8f932b13cb2fd08c.png"},{"id":81963519,"identity":"7a3c3e51-ea79-4421-bd8c-52bf525208f7","added_by":"auto","created_at":"2025-05-05 11:19:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":882483,"visible":true,"origin":"","legend":"\u003cp\u003eA workflow of the present method of manual microdissection.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/cb40cfa077cb2aa7bc981a20.png"},{"id":81962427,"identity":"35c55d5f-afe0-4427-becc-a6583cce9585","added_by":"auto","created_at":"2025-05-05 11:11:52","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3165487,"visible":true,"origin":"","legend":"\u003cp\u003eHistologic and stereomicroscopic images and \u003cem\u003eGNAS\u003c/em\u003e molecular analysis of the representative duodenal neoplasm. (a) Histology of the serial section, (b) remaining section after microdissection, (c) stereomicroscopic images before and (d) after dissection. The yellow arrow heads indicate the dissected area of the section in (a)–(d). (e) The electropherogram of the \u003cem\u003eGNAS\u003c/em\u003e gene using forward primer. A red allow shows the altered nucleotide, c.601C\u0026gt;T (p.R201C).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/ba08e12c57ad3392eb0ead20.jpg"},{"id":81963521,"identity":"74063f17-17da-4cdf-946a-87ea6218477f","added_by":"auto","created_at":"2025-05-05 11:19:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":13557371,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple foci of the pancreatic intraepithelial neoplasms microdissected separately using the present method. (a) Histology of the serial H\u0026amp;E section, (b) the remaining H\u0026amp;E section after microdissection, (c) magnified histology, stereomicroscopic images, and electropherograms of each sampled area. Eleven foci were microdissected and analyzed for \u003cem\u003eKRAS\u003c/em\u003e. \u003cem\u003eKRAS\u003c/em\u003e c.34G\u0026gt;C (p.G12R) was detected in #1 and #2 lesions, and \u003cem\u003eKRAS\u003c/em\u003e c.35G\u0026gt;A (p.G12D) was detected in #3–#11 lesions. The serial H\u0026amp;E sections (upper left), post-microdissection histology (upper right), pictures under a stereomicroscope before (middle left) and after microdissection (middle right), and electropherograms (lower column) of \u003cem\u003eKRAS\u003c/em\u003e exon 2 using the \u003cem\u003eKRAS\u003c/em\u003ereverse prime are shown. The red arrow in the electropherogram of #1 and #2 indicates \u003cem\u003eKRAS\u003c/em\u003e c.34G\u0026gt;C, and the blue arrow in those of #3–#11 indicates \u003cem\u003eKRAS\u003c/em\u003e c.35G\u0026gt;A mutation.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/942d5849d9f5c5fa1d9b95e8.jpg"},{"id":81962430,"identity":"fdd54f8d-8c71-4e74-980f-cf39dee14115","added_by":"auto","created_at":"2025-05-05 11:11:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1528742,"visible":true,"origin":"","legend":"\u003cp\u003eA lesion of intraductal papillary mucinous neoplasm. (a) The serial H\u0026amp;E section and (b) post-microdissection H\u0026amp;E section implied the histology of the collected section. The pictures under stereomicroscopy (c) before and (d) after microdissection show that the structure of the tumor can be distinguished, and the appropriate area is microdissected. (e) The collected section in the tube cap is confirmed under a stereomicroscope. The electropherograms of (f) \u003cem\u003eKRAS\u003c/em\u003eexon 2 using the \u003cem\u003eKRAS\u003c/em\u003e reverse primer and (g) \u003cem\u003eGNAS\u003c/em\u003e exon 8 using the \u003cem\u003eGNAS\u003c/em\u003e forward primer. (f) The red arrow indicates \u003cem\u003eKRAS\u003c/em\u003ec.35G\u0026gt;A mutation. (g) The blue arrow indicates \u003cem\u003eGNAS\u003c/em\u003e c.601C\u0026gt;T mutation.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/75882ab9689cfb792939b02f.jpg"},{"id":81965074,"identity":"6d3dacaf-699a-43dc-8c34-231f3a8f1e17","added_by":"auto","created_at":"2025-05-05 11:28:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20610846,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/f6ea9d66-021d-4f6b-9210-185618eacc8c.pdf"},{"id":81962433,"identity":"1458e662-a7bf-409f-b952-09979b0a0ed6","added_by":"auto","created_at":"2025-05-05 11:11:53","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19604449,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymovie1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/d9b96bff3c3a944b06314b1f.mp4"},{"id":81962424,"identity":"5bc382eb-9c15-4890-a888-040406b9eb2f","added_by":"auto","created_at":"2025-05-05 11:11:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16737,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/ae735b4e041b87e765f8f809.docx"},{"id":81965073,"identity":"45ca9257-b6f3-4e3b-b9cf-ca6e5026fab3","added_by":"auto","created_at":"2025-05-05 11:27:52","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15232,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/866c6f039d38d8d41970e064.docx"},{"id":81962432,"identity":"e3b877f3-974d-49cb-8b0e-f844ac689c70","added_by":"auto","created_at":"2025-05-05 11:11:53","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":8139266,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/bb9389ab7558b8c85cc3b064.tif"},{"id":81962428,"identity":"55caa1e7-d38e-44a4-a9d0-796e70badc55","added_by":"auto","created_at":"2025-05-05 11:11:52","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14877,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5654096/v1/6e67c82ccad86780ae29825b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cost-Effective and Practical Manual Microdissection Method for Accurate High-Purity Tissue Sampling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRecent advances in molecular profiling have accelerated our understanding of many diseases. The improvements in high-throughput sequencing technologies for formalin-fixed paraffin-embedded (FFPE) tissues have enabled the analysis of clinical materials [1]. These advances have significantly affected many fields of biomedical research and clinical molecular diagnostics, including cancer. However, cancer tissues are composed of tumor and non-tumor cells, such as stromal, vascular, or inflammatory cells. The enrichment of the cancer cell ratio plays a vital role in molecular analysis [2]. Various kinds of dissection methods have been described, depending on the purpose of the investigation [3-5].\u003c/p\u003e\n\u003cp\u003eManual microdissection is a simple and inexpensive dissection method for FFPE and frozen sections using a sharp instrument under a microscope [3]. The surrounding non-neoplastic tissue is scraped from the section before the target tissue is collected (Fig. 1a). Manual microdissection allows the investigators to obtain an almost pure target tissue at minimal cost, for performing many molecular analyses, and thus has become the most common method of tissue enrichment in research fields. However, macro-dissection [5] provides enrichment of tumor purity in clinical diagnostics. This method uses serial hematoxylin and eosin (H\u0026amp;E) sections as a guide instead of a microscope. This allows for an easy rough dissection.\u003c/p\u003e\n\u003cp\u003eLaser capture microdissection (LCM) is a well-established cell separation technique that enables to obtain specific tissues with high purity [6, 7]. This method is most effective if the surrounding normal or stromal cells can affect the molecular analysis of the target tissue or if microscopic precursor or multifocal lesions need to be investigated separately. The samples obtained by LCM have been used in different types of research, including next-generation sequencing, expression arrays, and proteomic techniques [8]. In the field of clinical molecular diagnostics, the LCM method may be used for molecular tests on a special occasion [9].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere are several obstacles that hamper the routine application of LCM in clinical diagnostics, such as expensive equipment must be installed and the entire process is time-consuming and labor-intensive. We aimed to establish a low-cost method that could achieve precision close to that of LCM. Herein, we developed a modified manual microdissection technique for FFPE sections using equipment and instruments that most pathology laboratories have, with minimal additional costs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eProtocol of modified manual microdissection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scheme and workflow are illustrated in Figs. 1b and 2, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStep 1. Section the FFPE tissue into 5\u0026ndash;15\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026mu;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003em thick sections\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe FFPE tissues were sectioned using a microtome. Although the thickness of the sections depends on the required amount of nucleic acid, the sections that are too thin are easily broken to be picked up, and those that are too thick do not allow for the identification of target areas during microdissection. Approximately 10-\u0026mu;m thick sections are the best, although 5\u0026ndash;15 \u0026mu;m thickness can also be used for successful target identification and microdissection. The representative section in Fig. 2 was 10-\u0026mu;m thick, whereas the dissected section in Fig. 3 was 15-\u0026mu;m thick.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStep 2. Float the sections on warm water\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis step smoothens and stretches sections. When the section is not smooth, the dissection process in \u003cem\u003eStep 5\u003c/em\u003e can be difficult because of the shallow depth of focus under a stereomicroscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStep 3. Place the tissue onto a non-coating slide\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlass slides used in routine practice can be used in this method. The use of a new slide for each dissection can reduce the risk of contamination.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStep 4. Wipe the underside of the slide without drying the section\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is the most important step. Slides are typically air-dried using conventional manual microdissection. However, it is difficult to pick up the dissected section once the tissue firmly adheres to the glass slide. Therefore, in this method, dissection was performed without drying. The section was kept wet, creating a condition in which it slightly floated on a thin layer of water on a glass slide. The area of interest can be easily collected after sectioning. However, the underside of the slide must not be wet. The water between the slide and stage of the stereomicroscope interferes with the visualization of the FFPE section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStep 5. Dissect the section using a needle under a stereomicroscope and collect it into a tube or tube cap\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, dissect the non-dried section on the slide with a sharp instrument, such as using a disposable hypodermic needle, and a serial hematoxylin and eosin (H\u0026amp;E) section as a guide. Then, section the area of interest under a stereomicroscope (SZ-61; Olympus, Tokyo, Japan) with lighting from the side. The illumination from the side creates a contrast between the tissue structures in an FFPE section without any staining. While dissecting, another needle or finger of the other hand with a clean globe should hold the edge of the section. After cutting the entire circumference, the fragment should be collected in a sample tube. When the dissected section is small, it is better placed inside the tube cap, where a small amount of extraction reagent is applied. This manipulation may require skill, and we prepared a movie of Step 5, shown in Supplementary Movie 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepresentative cases\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA representative case of duodenal neoplasm is shown in Fig 3. To obtain tumor-rich material, the area indicated by yellow arrowheads was dissected (Fig. 3a, b). The non-stained FFPE sections were observed under a stereomicroscope before (Fig. 3c) and after (Fig. 3d) dissection, and DNA was extracted from the sections. Direct sequencing detected the \u003cem\u003eGNAS\u003c/em\u003e c.601C\u0026gt;T (p.R201C) mutation (Fig 3e).\u003c/p\u003e\n\u003cp\u003eWe demonstrated that multiple foci in a single section can be dissected separately using this method. The second case had multiple pancreatic intraepithelial neoplastic (PanIN) lesions (Fig. 4), and each PanIN lesion was separately dissected and analyzed by direct sequencing and/or cycleavePCR for \u003cem\u003eKRAS\u003c/em\u003e mutations. Although each lesion was minute (0.0667\u0026ndash;0.686 mm\u003csup\u003e2\u003c/sup\u003e in area) (Supplementary Table 2), we detected \u003cem\u003eKRAS\u003c/em\u003e c.34G\u0026gt;C (p.G12R) in two areas and \u003cem\u003eKRAS\u003c/em\u003e c.35G\u0026gt;A (p.G12D) in the remaining areas. The second lesion was collected from multiple serial sections (Supplementary Fig. 1). The amounts of extracted DNA are presented in Supplementary Table 2.\u003c/p\u003e\n\u003cp\u003eThe third case had intraductal papillary mucinous neoplasm (IPMN) (Fig. 5). A broader area could be dissected using this method (Fig. 5a\u0026ndash;e) and was analyzed for \u003cem\u003eKRAS\u003c/em\u003e and \u003cem\u003eGNAS\u003c/em\u003e mutations. Both \u003cem\u003eKRAS\u003c/em\u003e c.35G\u0026gt;A (p.G12D) (Supplementary Fig. 3f) and \u003cem\u003eGNAS\u003c/em\u003e c.601C\u0026gt;T (p.R201C) (Fig. 5g) mutations were present in IPMN.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTumor purity is a crucial factor that leads to appropriate results in molecular analyses. Although several dissecting methods have been used in research and clinical fields [3-9], precise dissection methods such as LCM require expensive equipment, and inexpensive methods such as macro-dissection make it difficult to achieve fine dissection. This study developed a simple and inexpensive modified manual microdissection method that enabled precise dissection.\u003c/p\u003e\n\u003cp\u003eMacrodissection is the easiest dissection method, wherein the FFPE section is dissected using instruments, such as a razor blade, without the aid of a microscope [5]. In this method, the target area is roughly dissected using serial HE sections as a guide. Unlike other microdissection methods, this method does not allow for precise dissection. In conventional manual microdissection [3], the tissue sections are attached to glass slides and non-tumor tissue is scraped away to obtain the targeted tumor tissue. In this method, deparaffinized and stained tissues are dissected under a microscope, allowing for fine separation. However, because the target tissue is collected after scraping off the surrounding tissue, it is possible that the tissue fragments may be mixed, which requires time and effort. In addition, it is difficult to confirm the sampled area retrospectively.\u003c/p\u003e\n\u003cp\u003eOur modified manual microdissection method was an improvement over the conventional manual microdissection method. The key feature of our method was to cut the FFPE sections under wet conditions before deparaffinization. Before drying, the sections were peeled off the glass slides. Therefore, this method enabled the removal and collection of the target tissue in one piece without scraping the surrounding tissue (Fig. 1a and b). This may have reduced the risk of contamination. Using our method, the section can be cut into any shape or size by LCM with a sharp needle (Supplementary Movie 1). It was also possible to sample a small area or different areas separately (Fig. 4).\u003c/p\u003e\n\u003cp\u003eFor small sections, static electricity may have caused the sample to be lost when collected in tubes. This can be avoided by cutting off the tube cap and applying a small amount of the extract solution before dissection, as shown in step 5. This enabled the collection of samples under a stereomicroscope (Supplementary Fig. 1 and Supplementary Movie 1). By performing H\u0026amp;E staining on the sections remaining after sample collection (Fig. 3b), the collected area could also be retrospectively confirmed by comparison with a serial H\u0026amp;E section (Fig. 3a).\u003c/p\u003e\n\u003cp\u003eOne disadvantage of this method, which is performed before deparaffinization, is that staining to identify tissue structures is impossible. However, it is possible to recognize tissue morphology even in unstained sections, like the presented case (Fig. 3c, 4, and 5c), by making appropriate preparations and observing under a stereomicroscope. Side lighting and semi-wet conditions make this possible.\u003c/p\u003e\n\u003cp\u003eMost equipment used in this method was inexpensive and readily available in ordinary laboratories. Although it was necessary to prepare a stereomicroscope, it was performed using instruments that are generally used in clinical practice, such as injection needles and glass slides, and did not require expensive equipment, such as an LCM [6]. If more amount of DNA is needed, a larger area (Fig. 5) or greater number of sections (Supplementary Fig. 1) can be dissected. This method can also be used for whole-exome sequencing, similar to LCM [10].\u003c/p\u003e\n\u003cp\u003eThe DNA quality obtained using this method is also suitable for molecular analysis. As shown in the present case (Fig. 3e), even small samples were successfully analyzed using Sanger sequencing, which is not highly sensitive. This was probably because the samples were collected before deparaffinization. This may prevent the denaturation and degradation of nucleic acids before extraction. In addition, the electropherogram showed that we could collect the samples with high tumor purity.\u003c/p\u003e\n\u003cp\u003eHowever, this method had some limitations. First, although this is an inexpensive and easy dissection method that can be performed in any laboratory, it requires precise handling and skill to precisely cut small areas of interest. Second, to collect samples without deparaffinization, deparaffinization must be performed at the time of extraction. However, if a dried specimen is deparaffinized on a slide, unnecessary areas can be removed using this method to increase tumor purity.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we described a modified method for manual microdissection of FFPE sections. This method allows investigators to perform microdissection under a stereomicroscope to collect highly enriched target tissues in a manner similar to LCM. In general, clinical specimens have low tumor purity as they contain mixed various tissues; hence, their molecular analyses may yield inaccurate results. However, our method is versatile because it can collect tumor-enriched specimens not only by isolating and obtaining tumor tissue but also by removing non-tumor tissue and gathering the remaining tumor tissue. In addition, our method can separate tissues from different parts of the same tumor in a single section, which allows for examining molecular changes in tumor progression and genetic differences between histologically different parts of the tumor. Therefore, our method is useful in both clinical and research settings.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCase selection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included three cases with duodenal or pancreatic neoplasia in which surgery was performed at Aichi Cancer Center between 2018 and 2021. The resected specimens were fixed in formalin and embedded in paraffin. A total of 16 lesions were microdissected by present method from FFPE sections and subjected to the molecular analyses described below. The uniqueness of this modified manual microdissection is summarized in two points: a sectioned tissue was floated on a layer of water on a glass slide, and the tissue was microdissected under wet conditions using a needle under microscopic observation. No special equipment was required, except for a stereomicroscope. The scheme and workflow are illustrated in Figs. 1b and 2, respectively. This study adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Aichi Cancer Center (Approval No. R011032).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDNA was extracted from the collected sections using a deparaffinization solution (Qiagen, Venlo, Netherlands) and QIAamp DNA FFPE Tissue Kit (Qiagen, Venlo, Netherlands), according to the manufacturer’s protocol. The amount of extracted DNA was measured using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, Massachusetts, U.S.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSanger sequencing and Cycleave polymerase chain reaction (PCR)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe tested whether the DNA extracted using this dissection protocol could be used for molecular analysis. We examined \u003cem\u003eGNAS\u003c/em\u003e exon 8 and \u003cem\u003eKRAS\u003c/em\u003e exon 2 using direct sequencing and/or cycleave-PCR. The methods for both the assays and primers have been described previously [11-13].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the technical staff members, Noriko Shibata and Motoko Nimura, of the Department of Pathology and Molecular Diagnostics, Aichi Cancer Center, for their technical assistance. This work was supported by JSPS KAKEN-HI (grant number 20K16179). We would like to thank Editage (www.editage.jp) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYasuko Fujita designed the study, collected samples, performed molecular analysis, wrote the manuscript, and prepared the figures and movies. Masataka Haneda contributed to the improvement of the method. Waki Hosoda organized the study and revised the manuscript. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMathieson W, Thomas GA. Why formalin-fixed, paraffin-embedded biospecimens must be used in genomic medicine: an evidence-based review and conclusion. J Histochem Cytochem 68:543\u0026ndash;552. https://doi.org/10.1369/0022155420945050 (2020)\u003c/li\u003e\n\u003cli\u003eKogo M, Fujimoto D, Hosoya K, Nagata K, Nakagawa A, Tachikawa R, Yamashita D, Kitamura Y, Imai Y, Tomii K. Tumour content ratio matters for detecting epidermal growth factor receptor mutation by cobas test in small biopsies; a retrospective study. BMC Cancer 20:104. https://doi.org/10.1186/s12885-020-6603-3 (2020)\u003c/li\u003e\n\u003cli\u003eHunt JL, Finkelstein SD. Microdissection techniques for molecular testing in surgical pathology. Arch Pathol Lab Med 128:1372\u0026ndash;1378. https://doi.org/10.5858/2004-128-1372-MTFMTI (2014)\u003c/li\u003e\n\u003cli\u003eWalsh EM, Halushka MK. A comparison of tissue dissection techniques for diagnostic, prognostic, and theragnostic analysis of human disease. Pathobiology 1\u0026ndash;10. https://doi.org/10.1159/000525979 (2022)\u003c/li\u003e\n\u003cli\u003eWisner L, Larsen B, Maguire A. Enhancing tumor content through tumor microdissection. J Vis Exp. https://doi.org/10.3791/62961 (2022)\u003c/li\u003e\n\u003cli\u003eEmmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA, Liotta LA. Laser capture microdissection. Science 274:998\u0026ndash;1001. https://doi.org/10.1126/science.274.5289.998 (1996)\u003c/li\u003e\n\u003cli\u003eSch\u0026uuml;tze K, Lahr G. Identification of expressed genes by laser-mediated manipulation of single cells. Nat Biotechnol 16:737\u0026ndash;742. https://doi.org/10.1038/nbt0898-737 (1998)\u003c/li\u003e\n\u003cli\u003eDatta S, Malhotra L, Dickerson R, Chaffee S, Sen CK, Roy S. Laser capture microdissection: Big data from small samples. Histol Histopathol. 30:1255\u0026ndash;1269. https://doi.org/10.14670/HH-11-622 (2015)\u003c/li\u003e\n\u003cli\u003eHiguchi R, Goto T, Nakagomi T, Hirotsu Y, Oyama T, Amemiya K, Mochizuki H, Omata M. Discrimination between primary lung cancer and lung metastases by genomic profiling. JTO Clin Res Rep 2:100255. https://doi.org/10.1016/j.jtocrr.2021.100255 (2021)\u003c/li\u003e\n\u003cli\u003eNo\u0026euml; M, Niknafs N, Fischer CG et al. Genomic characterization of malignant progression in neoplastic pancreatic cysts. Nat Commun 11:4085. https://doi.org/10.1038/s41467-020-17917-8 (2020)\u003c/li\u003e\n\u003cli\u003eYatabe Y, Hida T, Horio Y, Kosaka T, Takahashi T, Mitsudomi T. A rapid, sensitive assay to detect EGFR mutation in small biopsy specimens from lung cancer. J Mol Diagn 8:335\u0026ndash;341. https://doi.org/10.2353/jmoldx.2006.050104 (2006)\u003c/li\u003e\n\u003cli\u003eHosoda W, Sasaki E, Murakami Y, Yamao K, Shimizu Y, Yatabe Y. GNAS mutation is a frequent event in pancreatic intraductal papillary mucinous neoplasms and associated adenocarcinomas. Virchows Arch 466:665\u0026ndash;674. https://doi.org/10.1007/s00428-015-1751-6 (2015)\u003c/li\u003e\n\u003cli\u003eHosoda W, Takagi T, Mizuno N, Shimizu Y, Sano T, Yamao K, Yatabe Y. Diagnostic approach to pancreatic tumors with the specimens of endoscopic ultrasound-guided fine needle aspiration. 60:358\u0026ndash;364. https://doi.org/10.1111/j.1440-1827.2010.02527.x (2010)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microdissection, Manual microdissection, Formalin-fixed paraffin-embedded tissue, Laser microdissection, Tumor purity","lastPublishedDoi":"10.21203/rs.3.rs-5654096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5654096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Tissue-based molecular analyses have become a mainstay of biomedical research, including high-throughput DNA and RNA sequencing, and for appropriate results, microdissection is necessary to enrich the target cells. Many types of microdissection methods have been developed, among which laser capture microdissection (LCM) is the most precise. However, LCM is expensive and requires specialized equipment and membrane slides. Herein, we present a modified manual microdissection method that is simple, inexpensive, and does not require special equipment. On a wet condition after microtome sectioning of non-stained formalin-fixed paraffin-embedded tissue, dissection of targeted foci of as small as 0.02 square millimeters was possible using a needle under stereomicroscopic observation. Three representative cases with 16 foci were demonstrated, with successful detection of KRAS and/or GNAS mutations by direct sequencing and Cycleave polymerase chain reaction (PCR) assays. This modified manual microdissection method is simple and efficient, allowing us to obtain materials with a high tumor purity in any laboratory.","manuscriptTitle":"Cost-Effective and Practical Manual Microdissection Method for Accurate High-Purity Tissue Sampling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-05 11:11:48","doi":"10.21203/rs.3.rs-5654096/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e03fa80f-bb99-47ce-a090-56d92dd37149","owner":[],"postedDate":"May 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":42301479,"name":"Biological sciences/Cancer"},{"id":42301480,"name":"Health sciences/Medical research"},{"id":42301481,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-05-05T11:11:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-05 11:11:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5654096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5654096","identity":"rs-5654096","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}