{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T17:53:35Z","timestamp":1782496415151,"version":"3.54.5"},"reference-count":37,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T00:00:00Z","timestamp":1782432000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004484","name":"Tehran University of Medical Sciences and Health Services","doi-asserted-by":"publisher","award":["1400-1-410-52883"],"award-info":[{"award-number":["1400-1-410-52883"]}],"id":[{"id":"10.13039\/501100004484","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:sec id=\"sec001\">\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>To accurately diagnose cervical cancer, high-quality genetic material (DNA and RNA) from clinical samples is crucial. Current preservation methods often have limitations, including poor RNA stability and safety concerns. This study aimed to develop an optimized ethanol-based fixative to preserve DNA and RNA in cervical samples under ambient conditions.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec002\">\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>HeLa cells were fixed in ethanol-based fixatives containing polyhydric compounds (Sorbitol and polyethylene glycol [PEG]) and chelating agents. We used a central composite design (CCD) approach to evaluate the effects of pH, Sorbitol concentration, and PEG concentration on nucleic acid preservation. DNA and RNA quality were assessed using agarose gel electrophoresis, PCR, and real-time PCR. Cellular morphology was evaluated using Papanicolaou-stained slides. HPV genotyping of clinical samples was conducted using real-time PCR.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec003\">\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>The optimized fixative, developed in this study consisted of 40% ethanol, 4.3% Sorbitol, 1.2% PEG 8000, with a pH of 5.6. This new formula significantly improved DNA and RNA preservation compared to the commercial solution, PreservCyt. DNA showed high integrity and was successfully amplified in PCR test targeting HPV-18 oncogenes. RNA quality was confirmed through clear 28S and 18S rRNA bands and lower threshold cycle (Ct) values in qRT-PCR. HPV genotyping and morphological analysis revealed excellent preservation, enabling both molecular and cytological evaluations.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec004\">\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>The new ethanol-based fixative represents a promising cost-effective and environmentally friendly solution for preserving nucleic acids and cellular morphology in cervical samples. Its good performance under ambient conditions suggests it may serve as an option for Human Papillomavirus (HPV) testing and cervical cancer prevention, particularly in places with limited resources.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1371\/journal.pone.0349088","type":"journal-article","created":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T17:30:52Z","timestamp":1782495052000},"page":"e0349088","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimizing an ethanol-based fixative for enhanced nucleic acid preservation in cervical samples using a central composite design approach"],"prefix":"10.1371","volume":"21","author":[{"given":"Ghazal","family":"Sedaghat Shayegan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pouya","family":"Salehipour","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ali","family":"Najafi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2763-1964","authenticated-orcid":true,"given":"Mohammad Hossein","family":"Modarressi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,6,26]]},"reference":[{"issue":"2","key":"pone.0349088.ref001","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1097\/LGT.0000000000000525","article-title":"2019 ASCCP risk-based management consensus guidelines for abnormal cervical cancer screening tests and cancer precursors","volume":"24","author":"RB Perkins","year":"2020","journal-title":"J Low Genit Tract Dis"},{"issue":"3","key":"pone.0349088.ref002","doi-asserted-by":"crossref","first-page":"430","DOI":"10.1016\/j.ygyno.2010.11.013","article-title":"HPV mRNA tests for the detection of cervical intraepithelial neoplasia: a systematic review","volume":"120","author":"EA Burger","year":"2011","journal-title":"Gynecol Oncol"},{"key":"pone.0349088.ref003","doi-asserted-by":"crossref","first-page":"3116","DOI":"10.3389\/fmicb.2019.03116","article-title":"Human papillomavirus E6 and E7: the cervical cancer hallmarks and targets for therapy","volume":"10","author":"A Pal","year":"2020","journal-title":"Front Microbiol"},{"issue":"2","key":"pone.0349088.ref004","doi-asserted-by":"crossref","first-page":"69","DOI":"10.3390\/jmp4020008","article-title":"Performance of a 7-Type HPV mRNA test in triage of HPV DNA primary screen positive women compared to liquid-based cytology","volume":"4","author":"SW S\u00f8rbye","year":"2023","journal-title":"J Mol Pathol"},{"issue":"9","key":"pone.0349088.ref005","doi-asserted-by":"crossref","first-page":"1634","DOI":"10.3390\/diagnostics11091634","article-title":"Usefulness of E7 mRNA in HPV16-positive women to predict The risk of progression to HSIL\/CIN2","volume":"11","author":"C Mart\u00ed","year":"2021","journal-title":"Diagnostics"},{"key":"pone.0349088.ref006","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1186\/1743-422X-9-262","article-title":"A review of methods for detect human Papillomavirus infection","volume":"9","author":"ALP Abreu","year":"2012","journal-title":"Virol J"},{"issue":"7","key":"pone.0349088.ref007","doi-asserted-by":"crossref","first-page":"2643","DOI":"10.1128\/JCM.02570-10","article-title":"Sensitivity, specificity, and clinical value of human papillomavirus (HPV) E6\/E7 mRNA assay as a triage test for cervical cytology and HPV DNA test","volume":"49","author":"M Benevolo","year":"2011","journal-title":"J Clin Microbiol"},{"issue":"2","key":"pone.0349088.ref008","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1002\/cncr.11058","article-title":"Stability of archived liquid-based cervical cytologic specimens","volume":"99","author":"PE Castle","year":"2003","journal-title":"Cancer"},{"issue":"1","key":"pone.0349088.ref009","first-page":"1","article-title":"The effects of non-ideal temperature regimes on RNA quality from samples stored in RNAlater-like buffer: an attempt to replicate field conditions","volume":"2","author":"P Arnold","year":"2016","journal-title":"J Anal Mol Tech"},{"issue":"8","key":"pone.0349088.ref010","doi-asserted-by":"crossref","first-page":"734","DOI":"10.1002\/dc.22963","article-title":"Effective application of the methanol-based PreservCyt(\u2122) fixative and the Cellient(\u2122) automated cell block processor to diagnostic cytopathology, immunocytochemistry, and molecular biology","volume":"41","author":"BM van Hemel","year":"2013","journal-title":"Diagn Cytopathol"},{"issue":"2","key":"pone.0349088.ref011","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1002\/cncy.21355","article-title":"Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry","volume":"122","author":"A Kawahara","year":"2014","journal-title":"Cancer Cytopathol"},{"key":"pone.0349088.ref012","doi-asserted-by":"crossref","first-page":"393","DOI":"10.1159\/000489873","article-title":"Validation of a new low-cost, methanol-based fixative for cervical cytology and human papillomavirus detection","volume":"62","author":"KRM Leite","year":"2018","journal-title":"Acta Cytol"},{"issue":"1","key":"pone.0349088.ref013","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1136\/jcp.2007.047266","article-title":"Effects of fixation on RNA integrity in a liquid-based cervical cytology setting","volume":"61","author":"CAJ Horvath","year":"2008","journal-title":"J Clin Pathol"},{"issue":"5","key":"pone.0349088.ref014","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1159\/000524137","article-title":"Optimal preservations of cytological materials using liquid-based cytology fixatives for next-generation sequencing analysis","volume":"66","author":"R Tanaka","year":"2022","journal-title":"Acta Cytol"},{"key":"pone.0349088.ref015","article-title":"Sorbitol washing complex homogenate for improved DNA extractions","author":"AS Jones","year":"2020","journal-title":"PLoS ONE Lab Protocols"},{"issue":"1","key":"pone.0349088.ref016","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1186\/s13007-024-01175-6","article-title":"Assessing the effect of sample storage time on viral detection using a rapid and cost-effective CTAB-based extraction method","volume":"20","author":"D Mark","year":"2024","journal-title":"Plant Methods"},{"issue":"2","key":"pone.0349088.ref017","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1002\/elps.201100335","article-title":"Bleach gel: a simple agarose gel for analyzing RNA quality","volume":"33","author":"PS Aranda","year":"2012","journal-title":"Electrophoresis"},{"key":"pone.0349088.ref018","unstructured":"Chao YS, Clark M, Ford C. HPV self-sampling for primary cervical cancer screening: a review of diagnostic test accuracy and clinical evidence-an update. 2019."},{"issue":"1","key":"pone.0349088.ref019","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.sxmr.2019.09.005","article-title":"Cervical cancer screening: past, present, and future","volume":"8","author":"SL Bedell","year":"2020","journal-title":"Sex Med Rev"},{"key":"pone.0349088.ref020","unstructured":"Hologic Inc. Hologic women\u2019s health: products. 2025. [cited 2025 July 4]. https:\/\/hologicwomenshealth.com\/products\/aptimahpvassay\/"},{"key":"pone.0349088.ref021","unstructured":"Doorenweerd C, Beentjes K. Extensive guidelines for preserving specimen or tissue for later DNA work. https:\/\/www.yumpu.com\/en\/document\/view\/51104633\/extensive-guidelines-for-preserving-specimen-or-tissue-for-later-dna-"},{"issue":"7","key":"pone.0349088.ref022","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1002\/cncy.21276","article-title":"Preparation of DNA from cytological material: effects of fixation, staining, and mounting medium on DNA yield and quality","volume":"121","author":"A Dejmek","year":"2013","journal-title":"Cancer Cytopathol"},{"issue":"11","key":"pone.0349088.ref023","doi-asserted-by":"crossref","first-page":"1064","DOI":"10.1111\/apm.12082","article-title":"Stability of DNA, RNA, cytomorphology, and immunoantigenicity in Residual ThinPrep Specimens","volume":"121","author":"Y Kim","year":"2013","journal-title":"APMIS"},{"issue":"6","key":"pone.0349088.ref024","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1016\/j.anndiagpath.2008.07.004","article-title":"Histology without formalin?","volume":"12","author":"RJ Buesa","year":"2008","journal-title":"Ann Diagn Pathol"},{"issue":"1","key":"pone.0349088.ref025","doi-asserted-by":"crossref","first-page":"3","DOI":"10.5414\/NP300907","article-title":"KINFix--A formalin-free non-commercial fixative optimized for histological, immunohistochemical and molecular analyses of neurosurgical tissue specimens","volume":"35","author":"H Stefanits","year":"2016","journal-title":"Clin Neuropathol"},{"key":"pone.0349088.ref026","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1016\/j.cryobiol.2020.11.004","article-title":"Lipidomic changes in mouse oocytes vitrified in PEG 8000-supplemented vitrification solutions","volume":"99","author":"GT Jung","year":"2021","journal-title":"Cryobiology"},{"issue":"1","key":"pone.0349088.ref027","doi-asserted-by":"crossref","first-page":"4788","DOI":"10.1038\/s41467-023-40322-w","article-title":"spinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content","volume":"14","author":"J De Jonghe","year":"2023","journal-title":"Nat Commun"},{"issue":"2","key":"pone.0349088.ref028","first-page":"30","article-title":"Polyethylene glycol repairs damaged membrane; biophysical application of artificial planar bilayer to mimic biological membrane","volume":"2","author":"S Kouhzaei","year":"2013","journal-title":"Progr Biol Sci"},{"key":"pone.0349088.ref029","unstructured":"Daum GWFT. Biological specimen collection\/transport compositions and methods. 2011. https:\/\/doi.org\/US9416416B2"},{"issue":"3","key":"pone.0349088.ref030","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0017581","article-title":"DNase 2 is the main DNA-degrading enzyme of the stratum corneum","volume":"6","author":"H Fischer","year":"2011","journal-title":"PLoS One"},{"issue":"1","key":"pone.0349088.ref031","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1023\/A:1011903932645","article-title":"Binding citrate\/DNA in presence of divalent cations. Potential mimicry of acidic peptides\/DNA interactions","volume":"28","author":"I Calzuola","year":"2001","journal-title":"Mol Biol Rep"},{"issue":"13","key":"pone.0349088.ref032","doi-asserted-by":"crossref","first-page":"5951","DOI":"10.1016\/S0021-9258(18)50504-4","article-title":"Acid ribonuclease from HeLa cell lysosomes","volume":"254","author":"BK Saha","year":"1979","journal-title":"J Biol Chem"},{"issue":"1","key":"pone.0349088.ref033","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1038\/s42003-020-01260-1","article-title":"Polyol and sugar osmolytes can shorten protein hydrogen bonds to modulate function","volume":"3","author":"J Li","year":"2020","journal-title":"Commun Biol"},{"key":"pone.0349088.ref034","unstructured":"Ho D, Bruttig S. Preservation solutions for biologics and methods related there to. 2011. https:\/\/doi.org\/WO2011103114A1"},{"issue":"2","key":"pone.0349088.ref035","doi-asserted-by":"crossref","DOI":"10.1016\/j.cell.2024.10.048","article-title":"Mechano-dependent sorbitol accumulation supports biomolecular condensate","volume":"188","author":"S Torrino","year":"2025","journal-title":"Cell"},{"issue":"1","key":"pone.0349088.ref036","doi-asserted-by":"crossref","first-page":"12293","DOI":"10.1038\/s41598-018-29871-z","article-title":"Insight into the molecular mechanism behind PEG-mediated stabilization of biofluid lipases","volume":"8","author":"B P\u00e9rez","year":"2018","journal-title":"Sci Rep"},{"issue":"2","key":"pone.0349088.ref037","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1097\/PDM.0b013e3181b520f8","article-title":"Effects of formalin, methacarn, and fineFIX fixatives on RNA preservation","volume":"19","author":"I Dotti","year":"2010","journal-title":"Diagn Mol Pathol"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0349088","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T17:31:04Z","timestamp":1782495064000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0349088"}},"subtitle":[],"editor":[{"given":"Devesh U","family":"Kapoor","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,6,26]]},"references-count":37,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6,26]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0349088","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,26]]}}}