Separation of blood cells with differing deformability using deterministic lateral displacement(†)

Abstract

Determining cell mechanical properties is increasingly recognized as a marker-free way to characterize and separate biological cells. This emerging realization has led to the development of a plethora of appropriate measurement techniques. Here, we use a fairly novel approach, deterministic lateral displacement (DLD), to separate blood cells based on their mechanical phenotype with high throughput. Human red blood cells were treated chemically to alter their membrane deformability and the effect of this alteration on the hydrodynamic behaviour of the cells in a DLD device was investigated. Cells of defined stiffness (glutaraldehyde cross-linked erythrocytes) were used to test the performance of the DLD device across a range of cell stiffness and applied shear rates. Optical stretching was used as an independent method for quantifying the variation in stiffness of the cells. Lateral displacement of cells flowing within the device, and their subsequent exit position from the device were shown to correlate with cell stiffness. Data showing how the isolation of leucocytes from whole blood varies with applied shear rate are also presented. The ability to sort leucocyte sub-populations (T-lymphocytes and neutrophils), based on a combination of cell size and deformability, demonstrates the potential for using DLD devices to perform continuous fractionation and/or enrichment of leucocyte sub-populations from whole blood.

Keywords: blood separation; cell deformability; deterministic lateral displacement; digital holography; microfluidics; optical stretching.

Figures

Figure 1.

Schematic showing the fluid streamlines (at low Reynolds number) through an array of pillars. Each column is shifted vertically by ɛλ relative to the previous column, where λ is the inter-pillar distance, ɛ is the column shift fraction and g the gap between the pillars. The flow between the pillars is assumed to be parabolic with streamlines being divided by stall lines which begin and terminate on the pillars. The horizontal flow velocity is indicated by the colour intensity. (Online version in colour.)

Figure 2.

(a) Schematic of DLD separation device showing multiple sections each designed to achieve a characteristic critical diameter. (b) DRIE etched silicon master for casting of PDMS devices. (c) Section through a PDMS device showing the pillars. Depending on the cell type under study, the height of the pillars was varied: approximately 4 µm tall pillars for erythrocyte work and approximately 25 µm for experiments involving whole blood. (d) Micrographs showing the trajectories of a mixture of rigid polymer beads (i.e. non-deformable particles) at different positions along the DLD device. The numbers by each image correspond to the vertical column to column shift, and the associated critical diameter for that particular section of the DLD device. (Online version in colour.)

Figure 3.

(a) Schematic of the optical stretching set-up. (b) Microscope images of trapped erythrocytes held under 100 mW power (left) and stretched erythrocytes under 600 mW power (right). Each row of images shows cells having been exposed to different glutaraldehyde concentrations (concentration is noted in the right upper corner of each panel). (c) Average stretching curves for erythrocytes with various levels of glutaraldehyde cross-linking (n > 20 for each concentration). Solid lines represent power law fits to the data. Error bars show standard error of mean. (Online version in colour.)

Figure 4.

(a) Photograph of the outlet of the shallow DLD device showing separation of RBCs of different stiffness (mixture of 0 and 0.01% glutaraldehyde-fixed erythrocytes). Histogram shows cell distribution at the outlet for a mix of untreated and glutaraldehyde-treated RBCs (note that the displacement direction is reversed with respect to figures 1 and 2). The high-magnification images (top right) show deformation of compliant (0% glutaraldehyde) and stiff (0.01% glutaraldehyde) erythrocytes as they interact with the pillars within the DLD device. (b) Photographs of the outlet of the DLD device showing displacement of untreated erythrocytes at different flow rates (increasing flow rates are shown from left to right). (c) Variation in lateral displacement versus flow rate for erythrocytes of different stiffness. (Online version in colour.)

Figure 5.

(a) Microscope image showing separation of leucocytes (bright labelled cells) from whole blood using the deep DLD device. Leucocytes are labelled with CellTracker dye. Erythrocytes are not displaced (the greater depth of the device means they are free to orient along the direction of fluid flow) and run straight through to the outlet at the top left. (b) Lateral displacement of leucocytes (WBCs) is shown as a function of flow rate. Lateral displacement is seen to decrease as the flow rate is increased; this is due to cells deforming under shear. (Online version in colour.)

Figure 6.

(a) Images of fluorescently labelled leucocyte sub-populations (in diluted whole blood) flowing through the outlet of the deep DLD device. T-lymphocytes and neutrophils clearly exit the device with different displacement distributions. The bright vertical lines in the image are created from superimposition of multiple video frames; each image shows several hundred cells superimposed. (b) Histogram showing the lateral distribution of the T-lymphocyte and neutrophil populations at the outlet of the device.