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The present study investigated the influence of unbuffered formalin fixation and prolonged fixation on histomorphology, immunohistochemistry, polymerase chain reaction (PCR) analysis, and DNA quality in FFPE tissues, which are essential issues in veterinary diagnostic pathology. Fixation of tissues with 10% unbuffered formalin resulted in increased intercellular spaces and decreased color contrast in hematoxylin and eosin-stained sections, attenuated Ki-67 immunolabeling, advanced DNA fragmentation, and lower PCR amplification efficiency compared with 10% neutral-buffered formalin. Prolonged fixation resulted in attenuated immunolabeling for multiple antigens, advanced DNA fragmentation, and lower PCR amplification efficiency. Preserving formalin-fixed tissue in 70% ethanol prevented loss of immunolabeling by prolonged fixation. Regarding storing FFPE tissues, coating FFPE blocks with paraffin prevented chronological DNA fragmentation. Based on the results obtained in this study and previous publications, the authors propose a standard preanalytic tissue handling guideline for histology and molecular examinations using FFPE tissues in veterinary medicine. In particular, fixation with 10% neutral-buffered formalin within 72 hours is recommended to obtain high-quality FFPE samples widely applicable for precision molecular diagnostics in routine veterinary histological examinations.<\/jats:p>","DOI":"10.1177\/03009858251349126","type":"journal-article","created":{"date-parts":[[2025,6,27]],"date-time":"2025-06-27T01:51:13Z","timestamp":1750989073000},"page":"949-962","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":2,"title":["Standardization of tissue handling in veterinary pathology: How fixation affects morphological and molecular examination results"],"prefix":"10.1177","volume":"62","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5273-7221","authenticated-orcid":false,"given":"James K.","family":"Chambers","sequence":"first","affiliation":[{"name":"The University of Tokyo, Bunkyo-ku, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6963-578X","authenticated-orcid":false,"given":"Kenichi","family":"Watanabe","sequence":"additional","affiliation":[{"name":"Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masaki","family":"Michishita","sequence":"additional","affiliation":[{"name":"Nippon Veterinary and Life Science University, Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hitoshi","family":"Hatai","sequence":"additional","affiliation":[{"name":"Iwate University, Morioka, Iwate, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yusuke","family":"Tanaka","sequence":"additional","affiliation":[{"name":"Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"So","family":"Doge","sequence":"additional","affiliation":[{"name":"Nippon Veterinary and Life Science University, Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuhiko","family":"Ochiai","sequence":"additional","affiliation":[{"name":"Nippon Veterinary and Life Science University, Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shizuka","family":"Kato","sequence":"additional","affiliation":[{"name":"The University of Tokyo, Bunkyo-ku, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5228-7125","authenticated-orcid":false,"given":"Yuko","family":"Goto-Koshino","sequence":"additional","affiliation":[{"name":"The University of Tokyo, Bunkyo-ku, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-8237-260X","authenticated-orcid":false,"given":"Miyuu","family":"Tanaka","sequence":"additional","affiliation":[{"name":"Osaka Metropolitan University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3236-6050","authenticated-orcid":false,"given":"Takeshi","family":"Izawa","sequence":"additional","affiliation":[{"name":"Osaka Metropolitan University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"name":"Japanese College of Veterinary Pathologists Standardization of Animal Tissue Processing Committee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"179","published-online":{"date-parts":[[2025,6,26]]},"reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.7717\/peerj.1996"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1136\/jclinpath-2015-203174"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1177\/0300985821999831"},{"key":"e_1_3_3_5_2","first-page":"660","article-title":"Survey on actual condition of the fixation procedure for histopathological specimens in Japan: report on the quality control survey questionnaire of the Japanese Association of Medical Technologists","volume":"69","author":"Azuma M","year":"2020","unstructured":"Azuma M, Yamashita K, Ishida K, et al. 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