Fractionation and Focusing of Leukocytes using Inertial Microfluidics

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

Single Stream Inertial Focusing in Low Aspect-Ratio Triangular Microchannels

A wide range of microfluidic devices for single stream focusing of cells and particles has emerged in recent years, based on both passive and active methods. Inertial microfluidics offers an attractive alternative to these methods, providing efficient and sheathless passive focusing of cells and beads. Nevertheless, in rectangular microchannels, presence of multiple equilibrium positions necessitates complicated solutions involving manipulation of 3D structure in order to achieve single stream flows. Here, we present a new approach to single-stream inertial focusing based on a triangular microchannel geometry. Changing channel cross-sectional shape leads to asymmetry in velocity profile, resulting in a size-dependent single stable equilibrium position near channel apex. We demonstrate that soft lithography masters for such microchannels can be fabricated in PMMA through micromilling, and 15 µm diameter beads can be efficiently focused in a single stream. Confocal microscopy was used to confirm focusing positions in the microchannel cross-section. We further integrate this device with a laser counting system to form a sheathless flow cytometer and demonstrated counting of beads with ~326 s -1 throughput. The use of triangular cross-section offers a number of benefits, including simplicity in fundamental principle and geometry, continence in design, small footprint, ease of integration, as well as high-precision single position focusing