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Beside detection of enriched or bound regions, an important question is to determine differences between conditions. While this is a common analysis for gene expression, for which a large number of computational approaches have been validated, the same question for ChIP-seq is particularly challenging owing to the complexity of ChIP-seq data in terms of noisiness and variability. Many different tools have been developed and published in recent years. However, a comprehensive comparison and review of these tools is still missing. Here, we have reviewed 14 tools, which have been developed to determine differential enrichment between two conditions.
However, these surface molecules have been shown to dynamically vary in expression during disease states e. This suggests that tumor antigen based positive selection approaches might not be able to isolate the entire population of CTCs. One strategy to overcome this pitfall is the use of size-based sorting technologies.
Early work used microfilters 31 while more recent studies involve the use of deterministic lateral displacement or DLD 32 , isolation by size of epithelial tumor cells or ISET 33 , and inertial focusing These technologies work on the presumption that CTCs are larger than typical blood cells, which is shown to be true for cancer cell lines but the limited amount of data with patient CTCs do not support this assumption 16 , Furthermore, the CTC size statistics are biased by the type of isolation technology used 35 , 36 , Another approach that does not rely on any single protein based enrichment of CTCs is the use of high-definition CTC analysis developed by Kuhn and colleagues, where all nucleated cells are panned onto slides for staining and subsequent multi-marker immunofluorescent imaging to identify CTCs Although nucleated cells including CTCs are attached onto a dozen or so specially developed large slides for imaging along with millions of contaminating WBCs, and the cells are not alive as they are fixed for processing, this technique clearly supports the unbiased isolation of CTCs and useful for central laboratory type settings.
To overcome the shortcomings of the existing approaches, we engineered an inertial focusing-enhanced microfluidic system, the CTC-iChip, which allows for high-efficiency negative depletion of normal blood cells, leaving CTCs in solution where they can be individually selected and analyzed as single cells 21 , The CTC-iChip combines hydrodynamic size-based separation of all nucleated cells leukocytes and CTCs away from red blood cells, platelets, and plasma, with subsequent inertial focusing of the nucleated cells onto a single streamline to achieve high-efficiency in-line magnetophoretic depletion of white blood cells WBCs that are tagged with magnetic beads in whole blood.
This antigen-independent isolation of CTCs enables the characterization of CTCs with both epithelial and mesenchymal characteristics. Furthermore, the high quality of RNA purified from viable, untagged CTCs is particularly well suited for detailed transcriptome analysis.
The CTC-iChip technology was successful but limited in its applicability due to long set up times, multiple manually interconnected chips and would have been difficult to implement within a clinical setting. Here we present the culmination of microfluidic and process engineering efforts to develop a high-throughput monolithic CTC-iChip, which uses deterministic lateral displacement, inertial focusing, and magnetophoresis to deplete blood cells at 15—20 million cells per second to sort CTCs.
The individual components previously manufactured using deep reactive ion silicon etching and PDMS polydimethylsiloxane soft lithography 21 , 38 have now been integrated on a single mass-produced plastic chip, vastly decreasing both technical requirements and hands on time and hence improving the accessibility of the technology.
The CTCs are unbiasedly collected in solution, rather than attached on a surface or with beads attached to their membranes and they spend less than 8 seconds within the monolithic chip based upon average flow speeds in each device stage and their connecting channels.
Additionally, there are already a few examples of research taking advantage of the improved purity and throughput of this technology 39 , This paper details the extensive characterization and rigorous testing of the limits of a technology that is already being utilized for clinical and scientific pursuits.
Results and Discussion The monolithic microfluidic device incorporates three different microfluidic technologies for rapid and high-throughput negative selection of circulating tumor cells that is completely controlled using on-chip fluidic resistors and a single pressure source.
The device consists of an injection molded fluidic layer that contains all the microfluidic features, which is then thermally bonded to an injection molded lid-layer containing molded through-holes for micro-macro interfacing. The microfluidic disks are made of medical-grade cyclic olefin copolymer COC and are manufactured using direct laser writing mastering in combination with variothermal localized time-dependent temperature control injection-compression molding.
A molded COC on-chip-cassette that is laser welded to the lid layer enables connectivity with the instrumentation and allows interfacing between the different microfluidic subcomponents.
The overall symmetrically parallelized chip architecture includes the integration of the following five microfluidic stages represented schematically in Fig.
An overall image of an integrated chip is shown in Fig.
Figure 1 Device and Process Flow - a Linearized schematic of the monolithic chip and how cells are processed. Blood pre-labeled with antibodies and magnetic beads targeting white blood cells and buffer enter the device and first go through a deterministic lateral displacement DLD device that separates nucleated cells from the red blood cells, platelets and unbound magnetics beads.
These cells are then accelerated through an inertial focusing device IF1 that causes the alignment of the cells without the addition of a sheath flow.
These aligned cells merge into a single channel and then pass through a magnetic field where the high gradient forces magnetically tagged cells towards the center of the channel MACS1.
This channel then splits collecting highly tagged cells in the center waste and all remaining cells are refocused in a second inertial focusing device IF2 and then enter a different region of the magnetic field where the gradient is higher leading to the removal of all labeled cells MACS2.
RBCs and smaller items pass straight through the array while the WBCs and CTCs are bumped across the array into a buffer co-flow and can be separated by splitting the flow streams at the outlet bottom. Full size image Tumor cell isolation is achieved by inputting whole blood that is pre-labeled with magnetic beads targeting white blood cells via CD45, CD16 and CD66b surface antigens, as well as a running buffer.
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