Methods

The CTC Atlas presents images and data obtained with advanced molecular imaging techniques, including Imaging Flow Cytometry (ImFC), which combines the strengths of flow cytometry and fluorescence microscopy. The process begins with the collection of peripheral or tumor-draining blood samples into EDTA tubes (1), discarding the first 2 mL in the case of peripheral blood to avoid contamination with skin-derived cells. The fraction of mononuclear cells ((P)BMCs), which might also include circulating tumor cells (CTCs), is then isolated using density gradient centrifugation (Histopaque®-1077) (2).

The cells are subsequently fixed in 4% formaldehyde (3) and stored at −80 °C until further analysis (4). Prior to acquisition, samples are stained with an antibody cocktail tailored to the specific panel of selected markers (5), and nuclei are counterstained with DAPI. Prepared cells are analyzed using the Amnis® ImageStream® X Mk II platform, which allows for simultaneous acquisition of fluorescence and brightfield images (6). The resulting data enable detailed the identification of rare CTC populations, and their detailed phenotypic and morphological characterization (7).