Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift

Abstract

Background: Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated.

Methods: A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker.

Results: The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated.

Conclusion: The method allows for the enrichment of red blood cells early after the invasion by P. falciparum parasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.

Figure 1

Experimental setup. The sheath flow Q_sheath and the sample flow Q_sample are driven by two separate syringe pumps. They are connected to the PDMS microchannel via PTFE tubes. The microchannel has a width w = 91 μm in y-direction for the whole device. The sample and the sheath flow inlet have a height h = 102 μm (in z-direction). The separation process occurs in the 20 mm long separation channel (height h = 102 μm) before the microchannel gently widens over a distance x = 400 μm to h = 301 μm. The bifurcation between outlet 1 and outlet 2 is at z = 50 μm and outlet 2 is connected at an angle of 49°. Both outlets have a height of h = 250 μm. The injected cells are focused to the lower wall before they flow through the separation channel and experience the non-inertial lift effect. The expansion shown in the inset increases the absolute height differences which facilitates sorting. Finally, the cells are collected in the height reservoirs connected to outlet 1 (waste) and outlet 2 (enriched sample). We observe the process using an inverted video microscope.

Figure 2

Representative FACS analysis of the injected and collected samples. The histogram shows the relative counts of all three samples.

Figure 3

Schematic drawing of the different regimes of motion exhibited by RBCs in shear flow. (a) Tumbling at low shear stresses. The cell does not deform and rotates like a rigid body with a and b indicating the projection of the elliptical half axes in the paper plane. (b) At higher shear stress, the cell reorients itself and adopts the rolling motion. In this state of motion, the entire cell rotates. (c) At even higher shear stress, the RBCs reorient again and adopt the tank-treading state of motion. The cell is stretched and has a constant inclination angle θ with respect to the flow while the membrane rotates around the cytoplasm.