{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T03:03:04Z","timestamp":1787022184972,"version":"build-2736575974"},"reference-count":25,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T00:00:00Z","timestamp":1770249600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Pathology is a vital part of modern medicine, playing an essential role in disease diagnosis, prognosis, and therapy choice. The field of pathology depends on traditional tissue embedding techniques, still routinely using toxic and even carcinogenic substances to process tissues for diagnostics. Chronic exposure of humans to xylene and\/or formalin, two essential components in the process, may result in severe health problems. Accumulating insights resulted in a ban of formalin in Europe for multiple applications and tight restrictions for almost all other uses since its toxicity levels were upgraded to carcinogen grade 1B and mutagen grade 2. A remarkable exception to these regulations is the use of formalin in the field of pathology, because of the existing lack of formalin-free tissue embedding methods that produce high-quality pathology specimens. Here we show that a novel embedding technique employing supercritical Carbon dioxide (CO\n                    <jats:sub>2<\/jats:sub>\n                    ), used in a xylene- and formalin-free manner, results in high-quality tissue samples, suitable for the fundamental techniques used in pathology: histochemistry, immunohistochemistry, and immunofluorescence.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0340480","type":"journal-article","created":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T18:29:52Z","timestamp":1770316192000},"page":"e0340480","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["High-quality histochemistry, immunohistochemistry, and immunofluorescence on xylene- and formalin-free paraffin-embedded tissues"],"prefix":"10.1371","volume":"21","author":[{"given":"Maarten","family":"Niemantsverdriet","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne Mei","family":"Eshuis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naomi","family":"van der Horst","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rory","family":"Ogink","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pien","family":"van Smeerdijk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose","family":"van der Starre-Gaal","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7630-6039","authenticated-orcid":true,"given":"Agnes Marije","family":"Hoogland","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,2,5]]},"reference":[{"key":"pone.0340480.ref001","volume-title":"General and systematic pathology","author":"J Underwood","year":"2004","edition":"4"},{"key":"pone.0340480.ref002","volume-title":"Diagnostic criteria handbook in Histopathology","author":"PJ Tadrous","year":"2007"},{"key":"pone.0340480.ref003","doi-asserted-by":"crossref","DOI":"10.1017\/9781009106924","author":"T Nguyen","year":"2022","journal-title":"Immunohistochemistry"},{"key":"pone.0340480.ref004","author":"MSA Tuffaha","journal-title":"Immunohistochemistry in tumor diagnostics"},{"key":"pone.0340480.ref005","doi-asserted-by":"crossref","first-page":"1476685","DOI":"10.3389\/fmed.2025.1476685","article-title":"Mini review on skin biopsy: traditional and modern techniques","volume":"12","author":"N Alwahaibi","year":"2025","journal-title":"Front Med (Lausanne)"},{"issue":"1","key":"pone.0340480.ref006","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1159\/000542497","article-title":"Immunofluorescence Use and Techniques in Glomerular Diseases: A Review","volume":"4","author":"N Messias","year":"2024","journal-title":"Glomerular Dis"},{"key":"pone.0340480.ref007","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.semcancer.2021.06.009","article-title":"Analytical demands to use whole-genome sequencing in precision oncology","volume":"84","author":"M Meggendorfer","year":"2022","journal-title":"Semin Cancer Biol"},{"issue":"5","key":"pone.0340480.ref008","doi-asserted-by":"crossref","first-page":"1467","DOI":"10.1016\/S0002-9440(10)65461-2","article-title":"A high frequency of sequence alterations is due to formalin fixation of archival specimens","volume":"155","author":"C Williams","year":"1999","journal-title":"Am J Pathol"},{"issue":"3","key":"pone.0340480.ref009","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1007\/s40139-019-00194-6","article-title":"Using FFPE Tissue in Genomic Analyses: Advantages, Disadvantages and the Role of Biospecimen Science","volume":"7","author":"W Mathieson","year":"2019","journal-title":"Curr Pathobiol Rep"},{"key":"pone.0340480.ref010","first-page":"1167","article-title":"A review of environmental and occupational exposure to xylene and its health concerns","volume":"14","author":"K Niaz","year":"2015","journal-title":"EXCLI J"},{"key":"pone.0340480.ref011","unstructured":"Safety sheet: Formaldehyde__37%_w_w_in_aqueous_solution_MTR_CLP1_EN (Thermofisher)."},{"issue":"24","key":"pone.0340480.ref012","doi-asserted-by":"crossref","first-page":"13147","DOI":"10.3390\/ijerph182413147","article-title":"Volatile Organic Compounds (VOCs) as Environmental Pollutants: Occurrence and Mitigation Using Nanomaterials","volume":"18","author":"E David","year":"2021","journal-title":"Int J Environ Res Public Health"},{"key":"pone.0340480.ref013","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1186\/1755-8794-7-23","article-title":"Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing","volume":"7","author":"SQ Wong","year":"2014","journal-title":"BMC Med Genomics"},{"key":"pone.0340480.ref014","first-page":"305","article-title":"DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present","volume":"5","author":"C-Y Chen","year":"2014","journal-title":"Front Microbiol"},{"issue":"6","key":"pone.0340480.ref015","article-title":"The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when?","volume":"2","author":"MTP Gilbert","year":"2007","journal-title":"PLoS One"},{"key":"pone.0340480.ref016","unstructured":"Commission Regulation (EU) No 605\/2014 of 5 June 2014 amending, for the purposes of introducing hazard and precautionary statements in the Croatian language and its adaptation to technical and scientific progress, Regulation (EC) No 1272\/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures Text with EEA relevance."},{"key":"pone.0340480.ref017","unstructured":"Commission TE. Commission implementing regulation (EU) 2018\/183 of 7 February 2018 concerning the denial of authorisation of formaldehyde as a feed additive belonging to the functional groups of preservatives and hygiene condition enhancers. 2018."},{"key":"pone.0340480.ref018","unstructured":"Commission Regulation (EU) 2023\/1464 of 14 July 2023 amending Annex XVII to Regulation (EC) No 1907\/2006 of the European Parliament and of the Council as regards formaldehyde and formaldehyde releasers."},{"issue":"6","key":"pone.0340480.ref019","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1007\/s00418-018-1765-7","article-title":"Coping with formalin banning in pathology: under vacuum long-term tissue storage with no added formalin","volume":"151","author":"L Mastracci","year":"2019","journal-title":"Histochem Cell Biol"},{"key":"pone.0340480.ref020","unstructured":"Directive (EU) 2019\/983 of the European Parliament and of the Council of 5 June 2019 amending Directive 2004\/37\/EC on the protection of workers from the risks related to exposure to carcinogens or mutagens at work (Text with EEA relevance)."},{"issue":"6","key":"pone.0340480.ref021","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1369\/00221554211017859","article-title":"Do Tissues Fixed in a Non-crosslinking Fixative Require a Dedicated Formalin-free Processor?","volume":"69","author":"SG Frasquilho","year":"2021","journal-title":"J Histochem Cytochem"},{"issue":"6","key":"pone.0340480.ref022","doi-asserted-by":"crossref","first-page":"1198","DOI":"10.1111\/j.1365-2559.2012.04342.x","article-title":"Solvent-free tissue processing using supercritical carbon dioxide","volume":"61","author":"EP Bleuel","year":"2012","journal-title":"Histopathology"},{"issue":"12","key":"pone.0340480.ref023","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0316107","article-title":"Formalin-free tissue embedding is less hazardous and results in better DNA quality","volume":"19","author":"M Niemantsverdriet","year":"2024","journal-title":"PLoS One"},{"issue":"7","key":"pone.0340480.ref024","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1097\/PAI.0000000000001071","article-title":"Lessons Learned, Challenges Taken, and Actions Made for \u201cPrecision\u201d Immunohistochemistry. Analysis and Perspectives From the NordiQC Proficiency Testing Program","volume":"31","author":"S Nielsen","year":"2023","journal-title":"Appl Immunohistochem Mol Morphol"},{"issue":"10","key":"pone.0340480.ref025","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1136\/jclinpath-2015-203178","article-title":"Immunohistochemistry should undergo robust validation equivalent to that of molecular diagnostics","volume":"68","author":"K Elliott","year":"2015","journal-title":"J Clin Pathol"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0340480","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,5]],"date-time":"2026-02-05T18:29:58Z","timestamp":1770316198000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0340480"}},"subtitle":[],"editor":[{"given":"Jordan Robin","family":"Yaron","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,2,5]]},"references-count":25,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2,5]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0340480","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,5]]}}}