2.?Techniques for CTCs IsolationIsolation of CTCs from whole blood could be done using biological (antigen expression) or physical features. Here, we summarise some of the most used methods for CTCs isolation from whole blood (Table 1).Table 1.Methods for CTCs isolation.2.1. Immunoaffinity/ImmunobindingAntibodies are extensively used to functionalise magnetic beads or nanostructured substrates (silicon nano/micropillars) that could be used to separate CTCs from blood cells [29,40,41,53�C55]. This approach is limited by antibody-antigen specificity and the complete process needs long interaction times. The antigen mostly used is EpCAM, an epithelial marker overexpressed in some carcinomas [22,23,40,56]. CTCs undergo changes in their epithelial signature during the metastatic process, avoiding the use of EpCAM as a universal marker [57�C59].

Therefore, all efforts are focused on characterisation and identification of additional markers able to distinguish CTCs from their counterparts in blood.Currently, CellSearch? (Veridex LLC, now Jenssen Diagnostics LLC, Raritan, NJ, USA) is the only technology approved by the US Food and Drug Administration for CTCs quantification in metastatic breast, prostate, and colon cancers. This technology uses magnetic beads coated with an anti-EpCAM antibody for CTCs isolation and the identification is mainly based on cytokeratins expression [8,9,24]. Although CellSearch? is an accepted platform with high value for cancer prognosis and monitoring, the low purity of the CTC-enriched samples, its low sensitivity, and its limitation to some cancer types [24] reinforce the need for more effective technologies for CTCs analysis.

In this sense, the combination of EpCAM-based CTCs immunoisolation with PCR quantitation methods represents an alternative to improve detection rates. Using this strategy, our group, among others, has obtained good results for the detection and characterisation of CTCs from metastatic colorectal cancer (mCRC) [60,61].This year, another Spanish group reported a novel system for CTCs counting in an in vitro model, using ImageStream (Amnis, Seattle, WA, USA). ImageStream is an imaging cytometry device that combines flow cytometry to select EpCAM-expressing cells and subsequent identification by fluorescence microscopy. The group compared this method with the CellSearch? system [27] and found an expected ratio of CTCs in most cancer patients [25].

Batimastat The use of flow cytometry in vivo was tested several years ago [62�C64]. This technology is now combined with current ones to achieve better results.Recently, an immune-filtration approach has also been developed and isolation of CTCs from lung cancer patients has been shown [29]. This device adds a magnetic sifter that has been re-engineered from a previous sifter [30].