Massively Parallel Inertial Ordering for High-Throughput Sheathless Imaging Flow Cytometry

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Massively Parallel Inertial Ordering for High-Throughput Sheathless Imaging Flow Cytometry

Soojung Claire Hur1, Henry Tat Kwong Tse2 and Dino Di Carlo2,3
University of California, Los Angeles
1Department of Mechanical and Aerospace Engineering
2Biomedical Engineering Interdepartmental Program
3Department of Bioengineering and California NanoSystems Institute

Rapid and accurate differentiation of cell types within a heterogeneous solution is a challenging but important task for various applications in biological research and medicine. Specifically, flow cytometry for blood analysis (i.e., complete blood counts (CBC)) is most often used to determine general patient health, blood diseases, and HIV or AIDs disease progression through quantitative measurement and analysis of cell populations in patients’ blood samples. However, modern clinical benchtop CBC flow cytometers are associated with high costs, lack of portability, and are incapable for point-of-care applications in resource limited settings. Previous efforts to miniaturize flow cytometry using microfluidic techniques employ various cell focusing, fluid pumping, and optical interrogation strategies. However, most miniaturized systems require logistically difficult and costly “sheath flows” to focus cells to a single optically interrogated volume, or throughputs are limited compared to commercially available flow cytometers’ 40,000 to 100,000 cells/s, thus necessitating the development of fundamentally new methods for high accuracy and throughput, cost efficiency, and ease of use.
Inertial lift forces in microchannels induce lateral migration of particles to produce ordered particle lattices downstream at precise focal planes defined by the channel geometry. In this study, we exploit inertial effects for label- and sheath-free flow cytometry. The particles or cells are focused to one uniform z-position to reduce the probability of overlap and out-of-focus blur and provide similar cell signature images for accurate detection and analysis. The device consists of 256 parallel channels with high-aspect ratio (W=16µm, H=37µm) with a sample rate as high as 1 mililion cells/s, limited by the field of view of our high-speed imaging optical interrogation method. Higher throughputs are possible with unique new wide-field-of-view imaging modalities. Microspheres (3%v/v) and diluted human blood samples (1 and 5%v/v) collected from healthy volunteers have been used to test the performance of the device for ordering and differentiation accuracy. Distribution of particle/cell focusing positions, travel velocity, and RBC/WBC differentiation were determined using high-speed microscopy and quantified using manual image analysis or custom- image analysis code.
It has been observed that precisely ordered particle/cell lattices travel with uniform velocity, Uave=0.208m/s. Moreover, using micro channels with inverted aspect ratio (W:H~2:1), uniform z-plane positions of particle lattices were at the centerline (σ=±7%). Furthermore, automated RBC and total leukocyte counts were performed, with accuracy of 83% and 81%, respectively, compared to manual counts.
We have developed a massively parallel inertial focusing microfluidic system that precisely positions particles/cells in both lateral and vertical directions, ensuring uniform cell travel velocity and focal positions. As no additional external forces (e.g. optical, magnetic, electrical) is required to create ordered streams of cells, this system has the potential toward point-of-care sheathless flow cytometry with extreme throughput, with potential future applications in portable hematology analysis.