By integrating multifunctional microfluidic chip with digital image analysis, here we report a fast-speed method for automatically and accurately enumerating CTCs (Circulating Tumor Cells). By eliminating the deviation induced by different criteria of diverse examiner and off-chip procedures, as well as significantly reducing the time cost of CTC examination, this study provides a strategy to narrow the gap between the existing chip-based CTC enumeration technologies and the commonly accepted clinical examinations.
We develop a multifunctional microfluidic chip which consists of three functional segments: blood filtering, cell isolating and cell positioning. There are three micro-valves to control the cell flow between segments. Firstly, the diluted blood was labelled by immune-magnetic beads which recognize EpCAM and pumped in to the filtering segment. 1500 micro-pillars were designed to eliminate all blood impurities which may jam the microchannel or interfere the fluorescent identification of CTC. EpCAM+ cells were isolated in next segment. Since the cells were forced to flow in a thin layer, the chip exhibits better isolation efficiency than traditional tube-based isolation. Also, the segment is gradually widened. The flow speed, as well as the dynamic force applied on cells, is corresponding weakened. Therefore, cells labeled with different amount of magnetic-beads would be captured in different areas of the segment, avoiding cell aggregation. Then 6000 V-shape micro-structures, whose dimension were specially design to single cell capture, were used for efficiently positioning single cells in the third segment. Finally, the whole segment was fluorescently imaged using a microscopy with automatic stage. We developed a piece of unique software to analysis all images with the same standard. The microchip benefits the image processing as follows: 1) cells were localized in limited areas, remarkably reducing the calculation workload; 2) the fixed size of V-shaped structures provides a reference to algorithm for excluding cell debris and other fluorescent spots by size comparison.
The chip performances were verified on 20 breast cancer patients. It was demonstrated that the proposed chip provided not only accurate CTC numbers, but also useful information for molecular diagnosis.