Isolating circulating tumor cells via the cell attachment protocol:
Our protocol is an easy, cheap, and efficient way to screen patient samples for circulating tumor cells (CTCs). Many past studies have screened for CTCs in bone marrow aspirates, which can be both difficult and painful to obtain. Instead, we use a 10cc peripheral blood sample for CTC screening. Blood drawing is a less invasive procedure that can easily be incorporated into a potential routine screening test.
Our protocol has been designed to minimize cell loss during sample processing and to preserve the integrity of CTCs using a short red blood cell lysis and gentle centrifugation. Nucleated cells are attached to a specially coated adhesive slides and immunofluorescently labeled on the slide surface after fixation. The cells are labeled with a cytokeratin antibody cocktail that is directed against proteins abundant on intermediate filaments of the cytoskeleton in epithelial cells. As normal cells of the blood and bone marrow are mesenchymal in origin, they do not express cytokeratin proteins, making the disseminated epithelial cells readily distinguishable from normal blood cells. Additionally, CD45 is used as a negative marker to distinguish lymphocytes from CTCs.
Fiber-optic array scanning technology (FAST) for circulating tumor cell detection:
After patient slides are prepared, they are scanned by the FAST cytometer, developed for ultra-fast scanning of immunofluorescently labeled CTCs. This instrument can analyze 50 million nucleated cells and find rare cells in less than two minutes with a sensitivity of 98% and a specificity of ~4 x 10-6. The key innovation is a fiber-optic bundle with asymmetric ends. FAST enables an optical system with extraordinarily large field-of-view (50mm), a large effective numerical aperture (0.66), and a resolution appropriate to cell size (10 um). This large field-of-view minimizes slow mechanical motion resulting in the FAST cytometer’s large advantage in exposure rate over traditional optical scanning approaches. The large numerical aperture enables detection of dim objects.
A key aspect of FAST is relocating identified objects in a microscope for additional viewing or analysis. During a scan, the locations of FAST detected objects are collected. These locations are specified by a substrate coordinate system that is determined by alignment marks on the substrate. To relocate a FAST object with a microscope, these coordinates are transformed to a coordinate system on the microscope stage using the same alignment marks. Using object relocation, the FAST identified objects are imaged using automated digital microscopy (ADM). The combined specificity of FAST and ADM is ~ 10-6.
