**5. Unanswered questions in the field of CTCs, technically and clinically**

The unanswered question is that the optimal protocol or device has not been found or validated. Many investigators have clearly realized that the number of CTCs cannot easily compare with that counted by another system, but some are not. Recently, Fina et al. [307] compared two CTC isolation methods in a clinical trial. AdnaTest EMT‐1/ and EMT‐2/Stem CellSelect/Detect kits, and ScreenCell Cyto devices were both performed for all samples. Higher CTC detection rates were obtained with the AdnaTest approach when using for CTC‐ enrichment antibodies against ERBB2 and EGFR in addition to MUC1 and the classical epithelial surface marker EPCAM (13% versus 48%). When the physical properties of tumor cells were exploited, CTCs were detected at higher percentages than with positive‐selection‐ based methods. The results supported that different approaches for CTC detection probably identify distinct tumor cell subpopulations. Technical standardization before clinical validity would be the most urgent issue we have to solve.
