Density-Gradient Mediated Band Extraction of Leukocytes from Whole Blood Using Centrifugo-Pneumatic Siphon Valving on Centrifugal Microfluidic Discs - Fig 7

<p><b>Phase Switching Data and Quantitative Results</b> (a-b) highlight ‘phase-switching’. This trait occurs where the system switches from drawing one phase, the DGM, to the other phase, plasma, while leaving a significant number of PBMCs within the main sedimentation chamber (c) and image from the haemocytometer showing mononuclear leukocytes enumeration (d) comparison of mononuclear leukocytes extracted from the single pneumatic chamber (Disc A) to a whole blood count (hospital laboratory) and using a HemoCue™.</p

Projector mode operation.

<p>(i) Schematic of the reader in projector mode. A shadow of the read chamber can be projected onto a wall from a distance of ~1 m and can easily be discerned in a dim or dark room (ii) an image (acquired using a smartphone) of the read chamber shadow projected onto a wall. This is read as ‘5’ relative to the graduated markings. The projected image is approximately 50 times larger than the read chamber.</p

Schematic of Multi-layer discs Multi-layer discs are assembled from of 1.5-mm PMMA layers interspersed by 86-μm thin PSA films.

<p>Schematic of Multi-layer discs Multi-layer discs are assembled from of 1.5-mm PMMA layers interspersed by 86-μm thin PSA films.</p

WBC isolation using a dynamically CPSV (Disc D).

<p>(a) The disc loaded with DGM while the whole blood is introduced during disc acceleration. (b) RBCs sediment to the bottom. Note that the pneumatic chamber is extended by channel (lower level) indicated in a blue dash. This large pneumatic chamber is required to ensure that the valves function at the volumes processed. (c) Stratified blood in the chamber. (d) The spin rate is decreased and both siphons are simultaneously primed. Note that the siphon crests are located radially inwards of the bulk liquid and the liquid displaced radially inwards along the loading channel. (e) The spin rate is increased and both siphons empty. See ESI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155545#pone.0155545.s001" target="_blank">S1 Movie</a> showing blood processing in Disc D.</p

Comparison of blood centrifugation in configurations with continuous (Disc A) and split pneumatic chambers (Disc B).

<p>Note that only in the single chamber design (top) RBCs enter the pneumatic chamber due to the increased liquid displacement (c). (d) Note the different PBMC locations in the two designs above and below the siphon outlet. See ESI <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155545#pone.0155545.s001" target="_blank">S1 Movie</a> showing blood processing in Disc B.</p

Comparison of Hydrostatic, Dynamic and Hybrid centrifugo-pneumatic siphon valves (CPSVs).

<p>Gas pressure is indicated in subfigures through the intensity of colour. (a) Liquid is loaded to the disc. (b) Upon spinning, the liquid advances into the central chamber while seeking hydrostatic equilibrium. However, the centrifugal compression of the gas volumes in the compartments enclosed by the liquid creates a counter pressure. (c, d) In the hydrostatic mechanism, the air in the closed side chamber expands upon reduction of the spin rate, so the liquid level in the open central chamber rises above the crest point of the siphon to forward the liquid into the open receiving chamber. In the hybrid CPSV, air is compressed in the closed central chamber during fast spinning. After lowering the angular frequency, the resulting decompression of air and the reduction of the centrifugal field jointly lift the liquid levels in the side arms above the crest point to empty the liquid into the open outer chamber. The operation of the dynamic CPSV follows a similar mechanism. However, the crest point of the siphon is now located above the level of the hydrostatic equilibrium; the siphon valve thus only opens upon rapid change of the spin rate so inertia propels the flow until the meniscus in the outlet channel has protruded past the liquid level in the central chamber.</p

The optical reader for CD4 cell enumeration.

<p>(i) The low-cost device is 3D-printed from four separate parts. Additional parts are a threaded screw, objective lens and diffusion plate. The finger actuated chip is inserted into a moveable chip holder. Its chip location relative to the objective lens, and thus the focus, can be finely adjusted by turning the focusing screw. (ii) The chip is interrogated by looking through the objective. The packed height of CD4+ cells and bead conjugate can be estimated from graduated hatch marks. Alternatively, the hatching can be calibrated as ‘treat’ or ‘no treat’ based on clinical guidelines. (iii) Image of the optical reader. Also in the image is an alternative lens holder for larger objectives. Additionally, a ‘Projector insert’, a powerful, low cost LED powered by a 3V battery, can be placed into the reader in place of the diffusion plate. In this case, the shadow of the packed cells and graduated hatch marks can be projected against a wall or floor.</p

Comparison and characterisation of Discs A and B (Continuous Pneumatic Chamber vs Split Pneumatic Chamber).

<p>(a) Filling levels, relative to the datum radius (siphon crest at 25 mm), against the spin rate. Numerical modelling using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155545#pone.0155545.e004" target="_blank">Eq 1</a> (using volume data from 3D CAD models) is compared to experimental data (n = 4). Hydrostatic priming pressure curves are also shown (dashed lines). These curves indicate that both designs are optimally primed at about18 Hz. (b) Disc A at high (60 Hz) and low (2.5 Hz) spin rates. (c) Disc B at high (60 Hz) and low (2.5 Hz) spin rates. Note the dependence of the location of the liquid interfaces on the geometry of the pneumatic chamber. Liquid interfaces in Disc B (split pneumatic chamber) approach a stable configuration above 40 Hz. Note also that at 2.5 Hz the priming pressure is insufficient to overcome the capillary pressure in the slightly hydrophobic siphon channel.</p

The finger-actuated CD4 enumeration chip previously introduced by Glynn <i>et al</i>. [29].

<p>(i) The chip filled with dyed water to highlight its geometry. (ii) Schematic of chip and (iii) chip operation. To operate, the chip is first primed with buffer through degas driven flow. To load, the P1 chamber is depressed and sample is pipetted into sample input port. The P1 chamber is then released and, as the chamber relaxes to its earlier shape, the sample id drawn through the chip and past the capture chamber. Repeated pressing and release of the chamber reciprocally pumps the sample through the separation chamber. Figure is reproduced from Glynn <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189923#pone.0189923.ref029" target="_blank">29</a>] with permission of The Royal Society of Chemistry (RSC).</p

A portable optical reader and wall projector towards enumeration of bio-conjugated beads or cells

<div><p>Measurement of the height of a packed column of cells or beads, which can be direclty related to the number of cells or beads present in a chamber, is an important step in a number of diagnostic assays. For example, haematocrit measurements may rapidly identify anemia or polycthemia. Recently, user-friendly and cost-efficient Lab-on-a-Chip devices have been developed towards isolating and counting cell sub-populations for diagnostic purposes. In this work, we present a low-cost optical module for estimating the filling level of packed magnetic beads within a Lab-on-a-Chip device. The module is compatible with a previously introduced, disposable microfluidic chip for rapid determination of CD4+ cell counts. The device is a simple optical microscope module is manufactured by 3D printing. An objective lens directly interrogates the height of packed beads which are efficiently isolated on the finger-actuated chip. Optionally, an inexpensive, battery-powered Light Emitting Diode may project a shadow of the microfluidic chip at approximately 50-fold magnification onto a nearby surface. The reader is calibrated with the filling levels of known concentrations of paramagnetic beads within the finger actuated chip. Results in direct and projector mode are compared to measurements from a conventional, inverted white-light microscope. All three read-out methods indicate a maximum variation of 6.5% between methods.</p></div