Moreover, since EpCAM and other proteins (e.g. cytokeratins) are not expressed in some tumors and can be down regulated during the epithelial to mesenchymal transition (EMT), new enrichment strategies are required. First evidence indicates that CTC markers applied in human medicine are conserved in other species. Five of the more common markers including CK19 are also useful to detect CTC in the blood of dogs with malignant mammary tumors. Newer approaches are able to identify more cells out 7.5 ml of blood, like IsofFux or Maintrac. In very rare cases, CTCs are present in large enough quantities to be visible on routine blood smear examination. This is referred to as carcinocythemia or carcinoma cell leukemia and is associated with a poor prognosis. To date, a variety of research methods have been developed to isolate and enumerate CTCs. The only U.S. Food and Drug Administration (FDA) cleared methodology for enumeration of CTC in whole blood is the CellSearch system. Extensive clinical testing done using this method shows that presence of CTCs is a strong prognostic factor for overall survival in patients with metastatic breast, colorectal or prostate cancer. CTCs are pivotal to understanding the biology of metastasis and promise potential as a biomarker to noninvasively evaluate tumor progression and response to treatment. However, isolation and characterization of CTCs represent a major technological challenge, since CTCs make up a minute number of the total cells in circulating blood, 1–10 CTCs per mL of whole blood compared to a few million white blood cells and a billion red blood cells. Therefore, the major challenge for CTC researchers is the prevailing difficulty of CTC purification that allows the molecular characterization of CTCs. Several methods have been developed to isolate CTCs in the peripheral blood and essentially fall into two categories: biological methods and physical methods, as well as hybrid methods that combine both strategies. Techniques may also be classified based on whether they select CTCs for isolation (positive selection) or whether they exclude all blood cells (negative selection). Biological methods isolate cells based on highly specific antigen binding, most commonly by monoclonal antibodies for positive selection. Antibodies against tumor specific biomarkers including EpCAM, HER2 and PSA have been used. The most common technique is magnetic nanoparticle-based separation (immunomagnetic assay) as used in CellSearch or MACS.