Figure 1.

An overview of the most commonly utilized techniques for the process of CTC enrichment and detection. In general, four approaches currently exist for CTC enrichment: (1) size-based; (2) density-based; (3) immunomagnetic separation; and (4) microfluidic-based. Using size-based enrichment techniques, diluted whole blood is passed through a filtration device with specific sized pores (typically 8 µm). CTCs are captured based on differences in cell size between CTCs (typically >8 µm) and white blood cells (WBCs; typically <8 µm). Density-based enrichment utilizes Ficoll (or similar density gradient medium) to enrich for mononuclear cells (including CTCs) from other blood components. Immunomagnetic separation involves the use of iron-conjugated antibodies targeted toward CTCs (e.g., EpCAM; positive selection) or contaminating blood cells (e.g., CD45; negative selection) and incubation in a magnetic field. For microfluidic-based techniques, whole blood is slowly passed across a chip-based surface and isolated using either CTC targeted antibody-coated microposts (CTC Chip and iChip [21,22]), or dielectrophoresis (DEPArray [23,24]).Current CTC detection techniques use either a protein-based approach (i.e., immunofluorescence or flow cytometry) expressed by whole cells or secreted proteins (EPISPOT assay [25,26,27]), or nucleic acid-based approaches such as RT-PCR or RT-qPCR, applied at the level of single genes or using a multiplex approach.