BACKGROUND: The aim of this study was to determine the applicability of a sequential process using leukapheresis, elutriation, and fluorescence-activated cell sorting (FACS) to enrich and isolate circulating tumor cells from a large blood volume to allow further molecular analysis. METHODS: Mononuclear cells were collected from 10 L of blood by leukapheresis, to which carboxyfluorescein succinimidyl ester prelabeled CaOV-3 tumor cells were spiked at a ratio of 26 to 10⁶ leukocytes. Elutriation separated the spiked leukapheresates primarily by cell size into distinct fractions, and leukocytes and tumor cells, characterized as carboxyfluorescein succinimidyl ester positive, EpCAM positive and CD45 negative events, were quantified by flow cytometry. Tumor cells were isolated from the last fraction using FACS or anti-EpCAM coupled immunomagnetic beads, and their recovery and purity determined by fluorescent microscopy and real-time PCR. RESULTS: Leukapheresis collected 13.5 x 10⁹ mononuclear cells with 87% efficiency. In total, 53 to 78% of spiked tumor cells were pre-enriched in the last elutriation fraction among 1.6 x 10⁹ monocytes. Flow cytometry predicted a circulating tumor cell purity of ~90% giving an enrichment of 100,000-fold following leukapheresis, elutriation, and FACS, where CaOV-3 cells were identified as EpCAM positive and CD45 negative events. FACS confirmed this purity. Alternatively, immunomagnetic bead adsorption recovered 10% of tumor cells with a median purity of 3.5%. CONCLUSIONS: This proof of concept study demonstrated that elutriation and FACS following leukapheresis are able to enrich and isolate tumor cells from a large blood volume for molecular characterization.
BACKGROUND: The aim of this study was to determine the applicability of a sequential process using leukapheresis, elutriation, and fluorescence-activated cell sorting (FACS) to enrich and isolate circulating tumor cells from a large blood volume to allow further molecular analysis. METHODS: Mononuclear cells were collected from 10 L of blood by leukapheresis, to which carboxyfluorescein succinimidyl ester prelabeled CaOV-3 tumor cells were spiked at a ratio of 26 to 10⁶ leukocytes. Elutriation separated the spiked leukapheresates primarily by cell size into distinct fractions, and leukocytes and tumor cells, characterized as carboxyfluorescein succinimidyl ester positive, EpCAM positive and CD45 negative events, were quantified by flow cytometry. Tumor cells were isolated from the last fraction using FACS or anti-EpCAM coupled immunomagnetic beads, and their recovery and purity determined by fluorescent microscopy and real-time PCR. RESULTS: Leukapheresis collected 13.5 x 10⁹ mononuclear cells with 87% efficiency. In total, 53 to 78% of spiked tumor cells were pre-enriched in the last elutriation fraction among 1.6 x 10⁹ monocytes. Flow cytometry predicted a circulating tumor cell purity of ~90% giving an enrichment of 100,000-fold following leukapheresis, elutriation, and FACS, where CaOV-3 cells were identified as EpCAM positive and CD45 negative events. FACS confirmed this purity. Alternatively, immunomagnetic bead adsorption recovered 10% of tumor cells with a median purity of 3.5%. CONCLUSIONS: This proof of concept study demonstrated that elutriation and FACS following leukapheresis are able to enrich and isolate tumor cells from a large blood volume for molecular characterization.
Authors: Sean R R Hall; Yajuan Jiang; Elizabeth Leary; Greg Yavanian; Sarah Eminli; David W O'Neill; Wayne A Marasco Journal: Stem Cells Transl Med Date: 2013-07-11 Impact factor: 6.940
Authors: Jin-Woo Kim; Ekaterina I Galanzha; David A Zaharoff; Robert J Griffin; Vladimir P Zharov Journal: Mol Pharm Date: 2013-02-25 Impact factor: 4.939
Authors: Johannes C Fischer; Dieter Niederacher; Stefan A Topp; Ellen Honisch; Sarah Schumacher; Norma Schmitz; Luisa Zacarias Föhrding; Christian Vay; Imke Hoffmann; Nikola S Kasprowicz; Philip G Hepp; Svjetlana Mohrmann; Ulrike Nitz; Antje Stresemann; Thomas Krahn; Tanja Henze; Evelyn Griebsch; Katharina Raba; Jutta M Rox; Folker Wenzel; Christoph Sproll; Wolfgang Janni; Tanja Fehm; Christoph A Klein; Wolfram Trudo Knoefel; Nikolas H Stoecklein Journal: Proc Natl Acad Sci U S A Date: 2013-09-24 Impact factor: 11.205