Label-free separation of leukocyte subpopulations using high throughput multiplex acoustophoresis

Multiplex separation of mixed cell samples is required in a variety of clinical and research applications. Herein, we present an acoustic microchip with multiple outlets and integrated pre-alignment channel to enable high performance and label-free separation of three different cell or particle fractions simultaneously at high sample throughput. By implementing a new cooling system for rigorous temperature control and minimal acoustic energy losses, we were able to operate the system isothermally and sort suspensions of 3, 5 and 7 m beads with high efficiencies (>95.4%) and purities (>96.3%) at flow rates up to 500 L min(-1) corresponding to a throughput of approximate to 2.5 x 10(6) beads per min. Also, human viable white blood cells were successfully fractionated into lymphocytes, monocytes and granulocytes with high purities of 96.5 +/- 1.6%, 71.8 +/- 10.1% and 98.8 +/- 0.5%, respectively, as well as high efficiencies (96.8 +/- 3.3%, 66.7 +/- 3.2% and 99.0 +/- 0.7%) at flow rates up to 100 L min(-1) (approximate to 100000 cells per min). By increasing the flow rate up to 300 L min(-1) (approximate to 300000 cells per min) both lymphocytes and granulocytes were still recovered with high purities (92.8 +/- 1.9%, 98.2 +/- 1 .0%), whereas the monocyte purity decreased to 20.9 +/- 10.3%. The proposed isothermal multiplex acoustophoresis platform offers efficient fractionation of complex samples in a label-free and continuous manner at thus far unreached high sample throughput rates.