Concurrent isolation of lymphocytes and granulocytes using prefocused free flow acoustophoresis

Microchip-based free flow acoustophoresis (FFA) in combination with two-dimensional cell prefocusing enables concurrent multiple target outlet fractionation of leukocytes into subpopulations (lymphocytes, monocytes, and granulocytes); we report on this method here. We also observed significantly increased accuracy in size-based fractionation of microbeads as compared to previously presented FFA multiple outlet systems. Fluorescence microscopy illustrates the importance of two-dimensional prefocusing where a sample mixture of 3, 7, and 10 μm beads are separated into well-confined particle streams and collected in their respective target outlets. Flow cytometry data for lymphocytes and granulocytes, respectively, in their corresponding outlets verify concurrent isolation of leukocyte subpopulations with high purity (95.2 ± 0.6% and 98.5 ± 0.7%) and high recovery (86.5 ± 10.9% and 68.4 ± 10.6%). A relatively low purity and high recovery of monocytes (25.2% ± 5.4% and 83.1 ± 4.3%) was obtained in the third target outlet. No subpopulation bias was observed. These data demonstrate an unprecedented separation of leukocyte subpopulations at flow rates of ∼100 μL/min and ∼1 M cells/mL sample concentrations, not previously reported in acoustofluidic systems. Two-dimensional prefocusing FFA with multiple target outlets is a viable alternative to current methods for particle fractionation and cell isolation, requiring a minimum of sample preparation and lowering analysis time and cost.