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Fractionation and Focusing of Leukocytes using Inertial Microfluidics
thesisposted on 2021-12-01, 00:00 authored by Prithviraj Mukherjee
Leukocytes or white blood cells (WBCs) account for less than 1% of all blood cells, yet have a significant impact on person’s health. Assessment of WBC subtypes, their numbers and status play a critical role in disease diagnostics. Traditionally, leukocyte isolation and counting is performed using Ficoll-Paque density gradient centrifugation or using immunofluorescence based cytometry (fluorescent activated cell sorting (FACS) and magnetic activated cell sorting (MACS)). While these systems offer high throughput, high separation efficiency, and high purity, they are expensive and rely on cell surface markers for target selection. Microfluidic systems have emerged as viable alternatives to these benchtop methods, offering small device size, reduced costs, rapid analysis, as well as high efficiency and high purity. In this work, a sheath-flow aided inertial microfluidic device is demonstrated for isolating subsets of leukocytes from human whole blood. Inertial microfluidics is a label-free approach that leverages hydrodynamic forces acting on cells suspended in flow and the inertia of the carrier fluid to sort cells based on their physical phenotype (primarily size, but also shape and deformability). Thus, it is suitable for sorting of leukocyte subsets due to their distinct differences in cell size. Inertial microfluidics was also used to demonstrate sheath-less focusing of cells in triangular cross-section microchannels. In these channels, due to asymmetry in the velocity profile, a size-dependent single stable equilibrium position near channel apex emerges. When coupled with a laser counting system, a sheathless flow cytometer with a throughput of >300/s can be demonstrated. Finally, this work also reports on a fabrication process for microfluidic devices that can be performed in a low-cost improvised fabrication room in ambient light, in place of a conventional yellow-light cleanroom environment. Ultimately, the approaches demonstrated in this work offer promising alternatives to isolating and sorting of leukocytes and their subpopulations.