Abstract 511: Unbiased enrichment of circulating tumor cells directly from whole blood
Authors: Peters Carrie E. , Maestre-Battle Danay , Woodside Steven M. , Thomas Terry E. , Eaves Allen C. 
Published: July 2016

Abstract
Enrichment of rare circulating tumor cells (CTC) in peripheral blood is required prior to most analytic procedures. The ideal enrichment method would be rapid (&lt;30 minutes (min)), permit a high recovery of viable CTC, and would be independent of surface marker expression, since CTC in the peripheral blood may be undergoing epithelial-mesenchymal transition and may not express epithelial markers. We have developed a new immunomagnetic method (EasySep™ Direct) that fulfills these criteria and can be used directly with whole blood without any preparatory steps. The method was tested using whole blood samples seeded with the adenocarcinoma cell line CAMA to a final concentration of approximately 1%. The CAMA cells were then enriched using either EasySep™ Direct CTC Enrichment (immunomagnetic separation) or RosetteSep™ (immunodensity based control). For immunomagnetic enrichment, the samples were incubated with EasySep™ Direct Enrichment Cocktail for 5 min, then EasySep™ Direct RapidSpheres™ were added and the sample placed in a magnet for 10 min. Magnetically labeled cells were retained in the magnet and the unlabeled cells poured or pipetted off. Rapidspheres™ were added again and the magnetic incubation and pour-off steps repeated. To perform RosetteSep™ enrichment, the samples were incubated with either the CD45+ cell depletion cocktail or a more extensive hematopoietic cell depletion cocktail for 10 min. Samples were then pipetted onto Lymphoprep in SepMate™ tubes, centrifuged for 10 min at 1200 x g with the brake on, and poured off. The number of cells recovered with each procedure was counted and the depletion of hematopoietic cells (CD45+/EPCAM-), enrichment of CAMA cells (CD45-/EpCAM+), and depletion of red blood cells (RBCs, Glycophorin A+/CD45-) in each sample were evaluated by flow cytometry. EasySep™ Direct was evaluated on five different samples. Each sample was separated on five different EasySep™ magnet formats (5, 14, and 50 mL single tube magnets and 5 and 14 mL multi-tube magnets) with each condition in duplicate; the two RosetteSep™ cocktails were tested on three of the samples, each in duplicate. There was no significant difference in CD45 depletion, CAMA cell recovery, or residual RBC contamination between samples enriched using the different magnets (Least Squares Fit; Prob F&gt;0.05) or pooled and compared to the pooled RosetteSep™ controls (Least Squares Fit; Prob F&gt;0.05). Over all magnet separation conditions and samples, the mean log depletion of CD45+ cells was 2.9±0.4, the mean recovery of CAMA cells was 42±23%, and the residual RBC contamination was ∼9000 RBC/mL of start sample. Rare cells were enriched in 25 minutes using EasySep™ Direct and in less than 40 minutes using RosetteSep™ with SepMate™. CTC can be enriched directly from whole blood with either EasySep™ Direct or RosetteSep™. Since enriched cells are unlabeled there is nothing to interfere with subsequent further enrichment, culture, or evaluation.
Citation Format: Carrie E. Peters, Danay Maestre-Battle, Steven M. Woodside, Terry E. Thomas, Allen C. Eaves. Unbiased enrichment of circulating tumor cells directly from whole blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 511.