{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,7]],"date-time":"2025-12-07T21:39:54Z","timestamp":1765143594862,"version":"3.40.5"},"reference-count":54,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2021,7,6]],"date-time":"2021-07-06T00:00:00Z","timestamp":1625529600000},"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>The field of liquid biopsy has seen extensive growth in recent decades, making it one of the most promising areas in molecular diagnostics. Circulating cell-free DNA (ccfDNA) especially is used as an analyte in a growing number of diagnostic assays. These assays require specified preanalytical workflows delivering ccfDNA in qualities and quantities that facilitate correct and reliable results. As each step and component used in the preanalytical process has the potential to influence the assay sensitivity and other performance characteristics, it is key to find an unbiased experimental setup to test these factors in diagnostic or research laboratories. We defined one such setup by using blood from healthy subjects and commercially available products for blood collection, spike-in material, ccfDNA isolation, and qPCR assays. As the primary read-out, we calculated the probit model-based LOD95 (limit of detection of the 95<jats:sup>th<\/jats:sup>percentile) from the qPCR assay results. In a proof of principle study we tested two different but widely used blood ccfDNA profile stabilization technologies in blood collection tubes, the Cell-Free DNA BCT and the PAXgene Blood ccfDNA Tube. We tested assays for three different<jats:italic>EGFR<\/jats:italic>gene mutations and one<jats:italic>BRAF<\/jats:italic>gene mutation. The study design revealed differences in performance between the two tested technologies for all four mutations. In conclusion, we successfully established a blueprint for a test procedure capable of verifying and validating a liquid biopsy workflow from blood collection to the analytical result.<\/jats:p>","DOI":"10.1371\/journal.pone.0253401","type":"journal-article","created":{"date-parts":[[2021,7,6]],"date-time":"2021-07-06T20:37:37Z","timestamp":1625603857000},"page":"e0253401","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":3,"title":["Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0485-9501","authenticated-orcid":true,"given":"Thorsten","family":"Voss","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrea","family":"Ullius","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maike","family":"Sch\u00f6nborn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Uwe","family":"Oelm\u00fcller","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2021,7,6]]},"reference":[{"key":"pone.0253401.ref001","article-title":"Challenges and opportunities of cfDNA analysis implementation in clinical practice: Perspective of the International Society of Liquid Biopsy (ISLB)","author":"C Rolfo","year":"2020","journal-title":"Critical Reviews in Oncology\/Hematology"},{"issue":"9","key":"pone.0253401.ref002","doi-asserted-by":"crossref","first-page":"1448","DOI":"10.1093\/annonc\/mdz196","article-title":"Liquid biopsy in the era of immuno-oncology: is it ready for prime-time use for cancer patients?.","volume":"30","author":"P Hofman","year":"2019","journal-title":"Annals of Oncology"},{"issue":"5","key":"pone.0253401.ref003","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1002\/path.5048","article-title":"The value of cell-free DNA for molecular pathology","volume":"244","author":"C M Stewart","year":"2018","journal-title":"The Journal of Pathology"},{"issue":"1","key":"pone.0253401.ref004","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1373\/clinchem.2014.222679","article-title":"Circulating tumor DNA as a liquid biopsy for cancer","volume":"61","author":"E Heitzer","year":"2015","journal-title":"Clinical Chemistry"},{"issue":"6485","key":"pone.0253401.ref005","doi-asserted-by":"crossref","first-page":"1424","DOI":"10.1126\/science.abb0736","article-title":"Deciphering cancer clues from blood","volume":"367","author":"N Ma","year":"2020","journal-title":"Science"},{"issue":"2","key":"pone.0253401.ref006","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1038\/s41576-018-0071-5","article-title":"Current and future perspectives of liquid biopsies in genomics-driven oncology","volume":"20","author":"E Heitzer","year":"2019","journal-title":"Nature Reviews Genetics"},{"issue":"24","key":"pone.0253401.ref007","doi-asserted-by":"crossref","first-page":"6010","DOI":"10.1158\/1078-0432.CCR-16-0909","article-title":"A prospective evaluation of circulating tumor cells and cell-free DNA in EGFR-mutant non-small cell lung cancer patients treated with erlotinib on a phase II trial","volume":"22","author":"M Yanagita","year":"2016","journal-title":"Clinical Cancer Research"},{"issue":"5","key":"pone.0253401.ref008","doi-asserted-by":"crossref","first-page":"1103","DOI":"10.1158\/1078-0432.CCR-15-1031","article-title":"Detection of T790M, the acquired resistance EGFR mutation, by tumor biopsy versus noninvasive blood-based analyses","volume":"22","author":"T K Sundaresan","year":"2016","journal-title":"Clinical Cancer Research"},{"key":"pone.0253401.ref009","doi-asserted-by":"crossref","first-page":"1034","DOI":"10.15252\/emmm.201404913","article-title":"Serial monitoring of circulating tumor DNA inpatients with primary breast cancer for detectionof occult metastatic disease","volume":"7","author":"E Olsson","year":"2015","journal-title":"EMBO Molecular Medicine"},{"key":"pone.0253401.ref010","first-page":"241","article-title":"Les acides nucleiques du plasma sanguin chez 1 homme","volume":"142","author":"P. 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