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ENSEMBLE-DECISION ALIQUOT RANKING (eDAR) FOR CTC ISOLATION AND ANALYSIS: A STUDY REPORT
Table of Contents
Introduction    3
Theories    3
Analysis    4
Conclusion    5
Bibliography    7
Introduction
The ideal method to isolate the rare cells such as circulating tumor cells (CTC) needs to fulfil four criteria. The method must be capable of high throughput so that milliliters of entire blood can be processed fast like within ten minutes. Method must be highly efficient and sensitive and the percentage of recovery must be higher than 90 per cent. Its limit of detection must be close to 1 CTC per blood sample. It must be capable of easily recovering live CTC with high purity. At last, it must be robust and economical to operate. To mitigate the problem, a new method called eDAR (ensemble-division aliquot ranking). This method is based on positive selection, in which blood samples are labelled with specific antibodies conjugated with fluorophores, then blood samples are divided in nanoliter aliquot that are optically checked and ranked for the absence or presence of the circulating tumor cells [1].
Theories
Ensemble-division aliquot ranking ensemble cells within each aliquot and ranked them in a single scan. It helps in selecting the aliquot of cells that must be investigated without compromising the throughput. Throughput is a key bottleneck in applying flow cytometry for CTC isolation. In traditional flow cytometry, the throughput is limited by the sequential analysis of individual cells. This took over 24 hours for 1 ml of blood containing 5 billion cells [11]. This throughput constraint is one of the main reasons that flow cytometry able to analyze only fractioned blood in CTC detection. This is done either by lysing red blood cell or by centrifuging the blood with density gradient buffer to isolate nucleated cells.
eDAR works like the flow cytometry yet there is significance difference. To manage the throughput, eDAR probes for rare cells in nanoliter aliquot of blood, so that each aliquot contains thousands of cells. High sensitivity of detection scheme able to detect one CTC per few nanoliter of blood in milliseconds. Aliquot containing CTC are automatically sorted and collected in small region of microfluid chip. In that region, purification method is applied. The isolated CTC can be imaged on microchip or restrained with biomarkers for further analysis [2].
eDAR provides three or four magnitude of improvement in throughput compared to other flow cytometry. Other methods spread the isolated the cell throughout the large area or on multiple slides, in contrast, eDAR trapped the cells in smaller area for rapid subsequent imaging. It isolated the rare cells from abundance of the cell. This method is elegant yet posed certain limitation to the process. Ensemble ranking process limits eDAR to the isolation of rare cells and prevent them from working well on target cells, which are, present in abundant. Despite the vast improvement in the method, eDAR is limited to the information from the fluorescence. Information using the light scattering data cannot be obtained due to large number of cells within the vicinity [3]. Flow cytometry detect in a single profile format allowed to obtain data from both fluorescence and light scattering. eDAR gives reduced hydrodynamic-induced cell stress. eDAR resultant into linear flow in cytometry, which minimizes shear stress on the cell. It preserves the viability of the rare...