Highly efficient isolation and multistep analysis of tumor cells from whole blood

We present a microfluidic solution for improved tumor cell analysis based on selection-free isolation of nucleated cells from whole blood. It consists of a high-density silicon microcavity array combined with the novel fluidic strategy of microfluidic decanting. This enables multistep on-chip staining protocols comprising sample loading-blocking-extracellular staining-fixation-permeabilization and intracellular staining to quantify tumor cells. The performance of the workflow was investigated and proven by spiking colon cancer cell lines into whole blood for the detection of the epithelial tumor markers EpCAM and cytokeratin. Total cell recovery rates of ≥95% were achieved for different sample species. The method allows for rapid reagent exchange within 10 s each almost without cell loss compared to approximately 50% cell loss in reference centrifugal processing. The isolation of nucleated cells resulted in a high intra-assay precision with a CV of 2% and a single cell per well distribution of 90%, which is consistent with the theoretical estimate using Poisson statistics. The linearity of the method was demonstrated over three orders of magnitude with r² = 0.9998. These results demonstrate a highly efficient approach for the quantification of tumor cells from whole blood that could be integrated into automated point-of-care devices in the future.