Abstract Circulating tumor cells (CTCs) are frequently present in the blood of cancer patients and can be captured by a variety of mechanisms. Here we describe a novel microfluidic chip that can capture CTCs by both cell size and affinity. Flat, plastic slides were embossed using silicone molds to produce two chip types. The T7 chip consists of posts with a fixed diameter arranged in a staggered pattern with a constant gap size between them (designed primarily for affinity capture), while the C5 chip consists of posts arranged in a gradient pattern with decreasing post diameters and gap distances across the chip surface (for size and affinity capture). Decreasing the distances between posts, to a minimum of 12 μ, creates a gap size gradient across the chip allowing smaller blood cells to flow through while capturing larger CTCs within the gaps. Both chips types were additionally functionalized with either an anti-EpCAM (epithelial cell adehsion molecule) monoclonal antibody (Ab) for specific affinity capture of epithelial cells or an irrelevant control IgG. High (H1650) and low (MDA-MB-231) EpCAM expressing cancer cells were spiked into buffer and pumped through the chips at a constant flow rate. Captured cells were then identified by on-chip immunofluorescence using an anti-cytokeratin Ab conjugated to phycoerythrin in combination with a nuclear specific fluorescent stain and imaged on an automated cell imaging platform at 5x magnification. Reproducibility of capture rate was acceptable for replicate measurements, with CV's of < 15% for within-day, day-to-day, and lot-to-lot precision. Affinity capture rate of H1650 cells, as demonstrated by EpCAM Ab T7 chips, was 37%. Size capture, as demonstrated using IgG C5 chips with H1650 spiked cells was 35%. EpCAM Ab coated C5 chips however, captured 97% of H1650 cells, verifying the advantage of both size and affinity capture. Control IgG T7 chips captured only 22% of H1650 cells. As further confirmation of the concept of dual capture, low EpCAM expressing MDA-MB-231 cells, were captured at 38% by size alone on IgG chips and showed no increase in capture rate on chips with EpCAM antibody. Heat maps of cell distribution showed that low EpCAM expressing cells captured by size were localized primarily in the small gap regions of the chip. Cells with high EpCAM, run on the EpCAM-specific antibody chip, were uniformly distributed across the entire surface of the chip, suggesting both capture mechanisms were operative. This microfluidic platform allows for affinity and size capture of heterogeneous CTCs on a novel, gradient EpCAM antibody chip. The platform can capture spiked cells with good precision. High efficiency cell capture is demonstrated by combining size and affinity, with almost 100% efficiency, regardless of EpCAM expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4898. doi:10.1158/1538-7445.AM2011-4898