Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: Proof of concept

Abstract

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 10 6 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 3 10 9 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 3 10 9 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.

Diagnostic leukapheresis enables reliable detection of circulating tumor cells of nonmetastatic cancer patients

“…Another factor that might contribute to a more reliable CTC detection by DLA is that the final LA products were generated from at least more than half of the whole circulating blood volume (cohort 2), thereby reducing the sampling error of a simple venipuncture. Eifler et al suggested by their proof-of-concept study using apheresis products spiked with cancer cell lines that an additional elutriation step might even further enhance CTC recovery after high-volume screening for CTCs by apheresis (21).…”

Challenges in the Enumeration and Phenotyping of CTC

“…This apparent conflict could be solved by in vivo detection methods, in which feasibility has been shown (48)(49)(50)(51). An alternative approach is to conduct leukapheresis for CTC isolation (52). Although this procedure is more cumbersome compared with a simple blood draw, it is more attractive than taking biopsies from the metastatic sites.…”

Circulating Tumor Cells: Liquid Biopsy of Cancer

“…An alternative approach is the development of leukapheresis, elutriation for subsequent ex vivo CTC analyses that use flow cytometry, and real-time PCR for molecular characterization (25 ). Eifler et al showed that leukapheresis collected 13.5 ϫ 10 9 mononuclear cells with 87% efficiency.…”