Abstract 2375: Rapid unbiased enrichment (negative selection) of circulating non-hematopoietic tumor cells directly from whole blood

There is increasing interest in analyzing circulating non-hematopoietic tumor cells (CTC) in peripheral blood to evaluate disease progression or response to treatment; however, CTC enrichment is required prior to most analytic procedures. The ideal enrichment method would be rapid, permit a high recovery of viable CTC, and would be independent of the expression of specific epithelial cell surface markers, since CTCs in the peripheral blood may be undergoing EMT (epithelial mesenchymal transition) and may not express epithelial markers. RosetteSep™ CD45 depletion of hematopoietic cells directly from whole blood meets these criteria. However, RosetteSep™ enrichment of CTC involves density gradient centrifugation, which entails careful layering of the sample over the density gradient medium and careful pipetting to remove the enriched cells after centrifugation. Centrifugation must be performed with the brake off to avoid disturbing the enriched cell layer, further lengthening the process. SepMate™, a centrifugation tube with a specialized insert, was developed to minimize mixing of the sample with the density gradient medium, thus allowing rapid layering of the sample on the density gradient medium and easy pouring off of the enriched cells after centrifugation. We compared CTC enrichment using RosetteSep™ and the standard tubes and protocol with RosetteSep™ using SepMate™ tubes and reduced cocktail incubation and spin times on 5 donor whole blood samples seeded with ∼1% CAMA cells. Purity of viable (PI negative) CTC obtained with SepMate™ with RosetteSep™ was 85 ± 7%; purity of CTC obtained with RosetteSep™ alone was 91 ± 5% (no significant difference, paired t test, p=0.050). Purity of CTC obtained with density gradient centrifugation only (no RosetteSep™), either with or without SepMate™, was 4 ± 2%. There was no significant difference in the recovery of enriched CTC under any of the conditions tested (RosetteSep™ ± SepMate™, SepMate™ alone, density gradient separation alone, Tukey-Kramer test, p>0.05). CTC enrichment was accomplished in