{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T03:49:20Z","timestamp":1785296960587,"version":"3.55.0"},"reference-count":48,"publisher":"Public Library of Science (PLoS)","issue":"10","license":[{"start":{"date-parts":[[2021,10,14]],"date-time":"2021-10-14T00:00:00Z","timestamp":1634169600000},"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>Modern histopathology is built on the cornerstone principle of tissue fixation, however there are currently no analytical methods of detecting fixation and as a result, in clinical practice fixation is highly variable and a persistent source of error. We have previously shown that immersion in cold formalin followed by heated formalin is beneficial for preservation of histomorphology and have combined two-temperature fixation with ultra-sensitive acoustic monitoring technology that can actively detect formalin diffusing into a tissue. Here we expand on our previous work by developing a predictive statistical model to determine when a tissue is properly diffused based on the real-time acoustic signal. We trained the model based on the morphology and characteristic diffusion curves of 30 tonsil cores. To test our model, a set of 87 different tonsil samples were fixed with four different protocols: dynamic fixation according to our predictive algorithm (C\/H:Dynamic, N = 18), gold-standard 24 hour room temperature (RT:24hr, N = 24), 6 hours in cold formalin followed by 1 hour in heated formalin (C\/H:6+1, N = 21), and 2 hours in cold formalin followed by 1 hour in heated formalin (C\/H:2+1, N = 24). Digital pathology analysis revealed that the C\/H:Dynamic samples had FOXP3 staining that was spatially uniform and statistically equivalent to RT:24hr and C\/H:6+1 fixation protocols. For comparison, the intentionally underfixed C\/H:2+1 samples had significantly suppressed FOXP3 staining (<jats:italic>p&lt;0<\/jats:italic>.<jats:italic>002<\/jats:italic>). Furthermore, our dynamic fixation protocol produced bcl-2 staining concordant with standard fixation techniques. The dynamically fixed samples were on average only submerged in cold formalin for 4.2 hours, representing a significant workflow improvement. We have successfully demonstrated a first-of-its-kind analytical method to assess the quality of fixation in real-time and have confirmed its performance with quantitative analysis of downstream staining. This innovative technology could be used to ensure high-quality and standardized staining as part of an expedited and fully documented preanalytical workflow.<\/jats:p>","DOI":"10.1371\/journal.pone.0258495","type":"journal-article","created":{"date-parts":[[2021,10,14]],"date-time":"2021-10-14T18:57:26Z","timestamp":1634237846000},"page":"e0258495","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["Making a science out of preanalytics: An analytical method to determine optimal tissue fixation in real-time"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2534-9666","authenticated-orcid":true,"given":"Daniel R.","family":"Bauer","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Torsten","family":"Leibold","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David R.","family":"Chafin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2021,10,14]]},"reference":[{"issue":"8","key":"pone.0258495.ref001","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1177\/33.8.3894502","article-title":"Formaldehyde fixation","volume":"33","author":"CH Fox","year":"1985","journal-title":"J Histochem Cytochem"},{"issue":"11","key":"pone.0258495.ref002","doi-asserted-by":"crossref","first-page":"1346","DOI":"10.5858\/arpa.2019-0009-SA","article-title":"Preanalytics and Precision Pathology: Pathology Practices to Ensure Molecular Integrity of Cancer Patient Biospecimens for Precision Medicine","volume":"143","author":"CC Compton","year":"2019","journal-title":"Arch Pathol Lab Med"},{"issue":"2","key":"pone.0258495.ref003","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/j.jneumeth.2009.06.002","article-title":"Advanced laboratory techniques for sample processing and immunolabeling using microwave radiation","volume":"182","author":"AM Ferris","year":"2009","journal-title":"J Neurosci Methods"},{"issue":"2","key":"pone.0258495.ref004","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1097\/01.paf.0000221050.64572.01","article-title":"Microwave processing and ethanol-based fixation in forensic pathology","volume":"27","author":"A Iesurum","year":"2006","journal-title":"Am J Forensic Med Pathol"},{"issue":"4","key":"pone.0258495.ref005","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1111\/j.1365-2559.2005.02269.x","article-title":"Possible role of tissue shrinkage in high-temperature antigen retrieval","volume":"48","author":"L Antunes","year":"2006","journal-title":"Histopathology"},{"issue":"1","key":"pone.0258495.ref006","first-page":"23","article-title":"Microwave-stimulated formaldehyde fixation of experimental renal biopsy tissues: computerised morphometric analysis of distortion artefacts","volume":"27","author":"LM Looi","year":"2005","journal-title":"Malays J Pathol"},{"issue":"4","key":"pone.0258495.ref007","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1080\/00313020410001722374","article-title":"Ultra-rapid microwave-stimulated tissue processing with a modified protocol incorporating microwave fixation","volume":"36","author":"ZA Hafajee","year":"2004","journal-title":"Pathology"},{"issue":"4","key":"pone.0258495.ref008","first-page":"612","article-title":"Microwave-assisted rapid tissue processing","volume":"122","author":"D Adams","year":"2004","journal-title":"Am J Clin Pathol"},{"issue":"5","key":"pone.0258495.ref009","doi-asserted-by":"crossref","first-page":"583","DOI":"10.5858\/2002-126-0583-CSFHTH","article-title":"Continuous-specimen-flow, high-throughput, 1-hour tissue processing. 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