Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: proof of concept.
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.
Eifler
Robert L
RL
Department of Clinical Medicine and Biotechnology, Danube University, Krems, Austria; Department of Blood Transfusion, Medical University of Vienna, Austria.
Lind
Judith
J
Falkenhagen
Dieter
D
Weber
Viktoria
V
Fischer
Michael B
MB
Zeillinger
Robert
R
D000328:Adult; D001810:Blood Volume; D005434:Flow Cytometry; D006801:Humans; D007937:Leukapheresis / Q000379:methods*; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured; D055815:Young Adult