Concurrent Isolation of Lymphocytes and Granulocytes Using Prefocused Free Flow Acoustophoresis

Abstract

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-micrometer 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 ~1M 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.

Clinical-Scale Cell-Surface-Marker Independent Acoustic Microfluidic Enrichment of Tumor Cells from Blood

“…One likely reason for this is that they are relatively large and dense, yielding an acoustophoretic migration velocity partly overlapping with that of some of the cancer cells. Granulocytes have previously been reported to have higher acoustophoretic migration rate than monocytes and lymphocytes 31 . This finding has further support in recent measurements of the acoustic impedances and sizes of neutrophils, lymphocytes and monocytes at single-cell level using iso-acoustic focusing 33 .…”

Acoustic Tweezing and Patterning of Concentration Fields in Microfluidics

“…The acoustic radiation force acting on particles in acoustic fields is used in these systems to manipulate particles and cells, thereby concentrating [14], trapping [15,16], separating [17], and sorting [18] bioparticles and cells based on their acoustomechanical properties. It would be of considerable interest if these methods could be extended to the manipulation of solute concentration fields in microfluidic systems.…”

Microfluidic Devices in the Fast‐Growing Domain of Single‐Cell Analysis

“…The later study only enabled the binary separation with a low throughput. To overcome these limitations, Grenvalle et al . used a 2D microchip with acoustic focusing zone for cell sorting (Figure d).…”

“…(d) Acoustophoresis based cell sorting, reprinted with the permission from Ref. . Copyright 2015 American Chemical Society.…”

“…The later study only enabled the binary separation with al ow throughput. To overcome these limitations, Grenvalle et al [47] used a2 Dm icrochip with acoustic focusing zone for cell sorting (Figure 3d). They separated leukocytes; howevert he throughput was not high enough.M ost of the studies exploit the positive acoustic contrast nature of the mammalian cells.…”