Imaging multiple biomarkers in captured rare cells by sequential immunostaining and photobleaching

Several technologies recently have been developed for separating and counting circulating tumor cells (CTCs) in the human blood. CTCs play an important role in the metastasis of cancer. Most of the current applications are focused on the enumeration of CTCs; however, analysis of the enumerated CTCs has been proven to be increasingly important. Ensemble-decision aliquot ranking (eDAR) is a high-throughput method that allows the isolation of the CTCs from the whole blood with high recovery and a zero false-positive rate. Coupling a CTC separation and capturing method, such as eDAR, with a downstream immunostaining test provides information about the cell's expression of certain protein biomarkers. In this article, we introduce a semi-automated system for sequential immunolabeling and photobleaching on the eDAR platform. With our new technique, we were able to evaluate the expression of eight different biomarkers on isolated CTCs.