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We aimed to develop an optimized protocol for single\u2010cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic\u2010activated cell sorting system, and CD45\u2010negative and cytokeratin\u2010positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole\u2010genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an <jats:italic>EGFR<\/jats:italic> exon 19 deletion in Case 1 (12\/19 cells, 63.2%), and <jats:italic>EML4\u2010ALK<\/jats:italic> fusion (17\/20 cells, 85%) with an alectinib\u2010resistant mutation of <jats:italic>ALK<\/jats:italic> (p.G1202R) in Case 2. To eliminate WGA\u2010associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single\u2010cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA\u2010associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.<\/jats:p>","DOI":"10.1111\/cas.14821","type":"journal-article","created":{"date-parts":[[2021,1,23]],"date-time":"2021-01-23T09:49:05Z","timestamp":1611395345000},"page":"2006-2019","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Development of an optimal protocol for molecular profiling of tumor cells in pleural effusions at single\u2010cell level"],"prefix":"10.1111","volume":"112","author":[{"given":"Ikuko Takeda","family":"Nakamura","sequence":"first","affiliation":[{"name":"Division of Cellular Signaling National Cancer Center Research Institute Tokyo Japan"},{"name":"Department of Respiratory Medicine Graduate School of Medicine Juntendo University Tokyo 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