Separation and Washing of Candida Cells from White Blood Cells Using Viscoelastic Microfluidics

Abstract

An early and accurate diagnosis of Candida albicans is critical for the rapid antifungal treatment of candidemia, a mortal bloodstream infection. This study demonstrates viscoelastic microfluidic techniques for continuous separation, concentration, and subsequent washing of Candida cells in the blood. The total sample preparation system contains two-step microfluidic devices: a closed-loop separation and concentration device and a co-flow cell-washing device. To determine the flow conditions of the closed-loop device, such as the flow rate factor, a mixture of 4 and 13 μm particles was used. Candida cells were successfully separated from the white blood cells (WBCs) and concentrated by 74.6-fold in the sample reservoir of the closed-loop system at 800 μL/min with a flow rate factor of 3.3. In addition, the collected Candida cells were washed with washing buffer (deionized water) in the microchannels with an aspect ratio of 2 at a total flow rate of 100 μL/min. Finally, Candida cells at extremely low concentrations (Ct > 35) became detectable after the removal of WBCs, the additional buffer solution in the closed-loop system (Ct = 30.3 ± 1.3), and further removal of blood lysate and washing (Ct = 23.3 ± 1.6).

Keywords: Candida; separation; viscoelastic fluid; washing; white blood cell.

Figure 1

Schematic of continuous separation, concentration, and purification of candida cells using viscoelastic fluid.

Figure 2

Viscoelastic separation of 4 and 13 μm particles at the (a) inlet and outlet depends on the flow rate factors of (b) 2.5, (c) 3.5, and (d) 4.5 at the inlet flow rate of 800 μL/min. Green and yellow triangles indicate 4 and 13 μm particles, respectively. (e) Concentration ratio and recovery rate of 4 μm particles at the center outlet (outlet A).

Figure 3

Stacked microscopic images showing viscoelastic closed-loop separation and concentration of Candida cells from white blood cells at a flow rate of 800 μL/min with FF = 3.3 at the (a) outlet at time T = 1 min, (b) outlet at time T = 5 min, and (c) outlet at time T = 18 min. Microscopic images of the 1:10 diluted sample before and after the closed-loop separation and concentration process at the (d) inlet, (e) outlet A, and (f) outlet B for manual counting. (g) Concentration of Candida cells and white blood cells at the inlet and outlet A and B.

Figure 4

Effect of the sample-to-sheath flow rate ratio (R) of (a) 9 and (b) 4 on the medium exchange of 4 μm particles from a 0.1% HA solution to deionized water (DW) in the co-flow device with AR = 2. The total flow rates were 100 μL/min. (c) Absorbances measured by the UV-VIS spectrophotometer at 254 nm wavelength using the negative sample (inlet A), the positive sample (inlet B), and collected samples at the outlets with a flow rate ratio of 4.

Figure 5

(a) Continuous purification of concentrated candida cells using a viscoelastic co-flow system. Stacked fluorescent microscopic images (left) and distribution of candida cells and 100 nm particles (right) at the outlet region. (b) The Ct values for the negative control (Ct = 38.6) represent the 0.1% (w/v) HA buffer solution. Before the separation process, candida cells could hardly be detected due to a large number of contaminants, including blood cells (Ct = 36.5). After the first-step separation, the Ct values for outlets A and B were 30.3 and 37.2, respectively. After the second-step washing process, the Ct value was measured to be 23.6. The dashed red line at Ct = 35 indicates the cutoff value for real-time PCR analysis. The error bars show the standard deviation from repeated measurements (n = 5).