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

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

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

WBC Isolation using a dual siphon, split pneumatic chamber CPSV (Disc C).

<p>(a) The disc is loaded with DGM as the whole blood is introduced during disc acceleration. (b) RBCs sediment. Note that the siphons have a number of capillary burst valves. The upper capillary valve on the lower siphon prevents the DGM pre-priming siphon while the disc is stopped for blood loading. (c) Stratified blood in the chamber. Note that the plasma remains below the siphon crest points. (d) The disc is decelerated and the bulk liquid is displaced radially inwards and the siphon prime. The siphon priming is halted by the capillary burst valves. The siphons must be primed at a lower frequency (~2.5 Hz) than the nominal frequency (~15 Hz) to prevent the capillary valves from bursting early or out of sequence. However, due to the low hydrostatic priming pressure at this spin rate, the crest of the lower siphon required treatment using a surfactant to achieve reliable priming. The upper siphon was not treated. (e) The spin rate is increased and the burst valve of the upper siphon capillary is opened, thus removing the plasma to the collection chamber. (f) The spin rate is increased further and the lower siphon valve opens for removing the WBCs (with some plasma and DGM) to the WBC collection chamber. 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 C.</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 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