Molecular Characterization of Rare Single Tumor Cells

Rare cells can be defined as those of less than 0.1% frequency.Ultra-rare cells can be defined as those of less than 0.001% frequency. These are terms for ease of discussion and are not universally agreed upon. In terms of flow cytometry and cell sorting, rare cell applications not only push the limits of the technology but also require levels of staining specificity beyond those assumed by most biologists. It is important for engineers to understand that good technology can bemade irrelevant by bad cell staining and preparation. The greatest difficulty with rare cell applications is not the immediate technological constraints but the requirement that there be no weak links in the experimental methodology anywhere in the process from cell preparation, flow cytometry/cell sorting and data analysis or subsequent analysis of isolated cells. Each and every one of these steps in the methodology must be excellent and the entire process must be thought through to eliminate or deal with the weaker links of a given rare cell application. For reviews on “rare event” analysis techniques, see Refs. [1–3]. There are many rare cell applications of importance to basic or clinical research. The ones discussed in this paper help show the range not only of the applications but also of the technological challenges to engineers working in this field. Some basic research examples that are briefly discussed include: (1) isolation of rare cell clones with specific mutations or transfected genes, (2) isolation of clones with combinatorial libraries of inserted genes and (3) studies of environmentally inducedmutations in human cells.