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.

Mechanical loading in the form of oscillatory fluid flow-induced shear stress was applied to meniscal cells while the biochemical response [intracellular calcium mobilization and sulfated glycosaminoglycan (GAG) production] was studied. Isolated rabbit meniscal cells were cultured onto microscope slides and placed in a parallel plate flow chamber. Cells were exposed to oscillating fluid flow-induced shear stresses of 4 Pa for sulfated GAG studies and 0-6.5 Pa for calcium studies. The calcium response was monitored using a fluorescent probe and imaging techniques, sulfated GAG production was measured using the modified 1,9-dimethylmethylene blue method, and thapsigargin was used to block intracellular calcium ([Ca 2þ ] i ) mobilization. A significant dosedependent relationship was found for the percentage of cells responding to oscillating fluid flow with an increase in [Ca 2þ ] i versus shear stress level. The percentage of cells responding decreased linearly from 72% AE 17% at 6.5 Pa to 28% AE 7% at 2.0 Pa to 2% AE 1% for baseline no-flow (0 Pa). No differences were found in the amplitude of the calcium response of responding cells for any shear stress level. Oscillating fluid flow-induced shear stresses of 4 Pa produced a significantly greater amount of sulfated GAGs (253 AE 95 ng GAG/mg cell protein) compared to the no-flow control (158 AE 86 ng/mg). The addition of thapsigargin to the media inhibited both the intracellular calcium response to oscillating fluid flow (less than 1.5% of the cells responded) and the increase in GAG production following oscillating fluid flow, which was returned to control levels (170 AE 72 ng/mg). These findings suggest that oscillatory fluid flow-induced shear stress increases intracellular calcium levels and sulfated GAG production. Furthermore, they suggests that calcium may modulate the biochemical pathway that leads to sulfated GAG production. ß