Sickle cell vasoocclusion and rescue in a microfluidic device

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Original entry: Nan Niu, APPHY 226, Spring 2009

by Professor Mahadevan

Abstract

Sickle-cell

I find this article particularly interesting although it is not directly related to capillarity and wetting. I enjoyed the authencity and creativity of the authors in designing the set of experiments to carry out their research. Most importantly, this article interested me is because the disease or scientific phenomenon studied is a rare but has significant impact on human society. As an introduction, sickle cell disease, the first molecular disease to be identified more than a half century ago, has been studied extensively at the molecular, cellular, and organismal level. The pathophysiology of sickle cell disease is complicated by the multiscale processes that link the molecular genotype to the organismal phenotype: hemoglobin polymerization occurring in milliseconds, microscopic cellular sickling in a few seconds or less, and macroscopic vessel occlusion over a time scale of minutes, the last of which is necessary for a crisis. Using a minimal but robust artificial microfluidic environment, the authors shows that it is possible to evoke, control, and inhibit the collective vasoocclusive or jamming event in sickle cell disease. Moreover, the authors also shows that a key source of the heterogeneity in occlusion arises from the slow collective jamming of a confined, flowing suspension of soft cells that change their morphology and rheology relatively quickly.

Experimental Set-up

Fabrication and schematic of the device. The oxygen channels and vascular network were fabricated in separate steps, bonded via oxygen plasma activation, and attached to a glass slide. The widest crosssection in the vascular network on the left and right of the device is 4 mm * 12 um. The vascular network then bifurcates, maintaining a roughly equal total cross-sectional area. The gas channels were connected to two rotameters regulating the gas mixture that was fed into the device. The outlet of the gas network had an oxygen sensor to validate the oxygen concentration in the microchannels.

Experimental Set-up

The authors and researchers have used unconventional methods to achieve best experimental results. Their experimental set-up is as follows: the assembled microfluidic device was mounted on an inverted microscope, and the fluidic and gas sources were connected as shown in picture above. The microfluidic channels begin 4 mm wide, then split into roughly equal total cross-section areas until the smallest dimension, which then traverses 4 cm until the channels recombine sequentially at the outlet. The blood velocity was monitored most often in the 250-um channels, which were fed by four 60-um, eight 30-um, sixteen 15-um, or sixteen 7-um channels, depending on the device studied. Two rotameters controlled the gas mixture fed through the oxygen channels. The gas mixture diffused rapidly through PDMS to initiate occlusion or flow. The outlet gas concentration was monitored with a fluorescent oxygen probe to monitor the gas concentrations within the gas microchannels. Gravity-driven flow was used to inject blood into the vascular network and resulted inflowrates of up to 500um/sec. The authors and researchers performed more than 100 different such occlusion assays, capturing more than 1,000 videos with more than 100,000 total frames. Given a device with a particular minimal width, the authors flowed a patient blood specimen with a known HbS fraction and a known red blood cell concentration. The pressure difference is modulated by changing the height of the pressure head and the gas concentration in the fluid channel is also modulated by adjusting the gas mixture flowing through the adjacent gas channels. Videos were captured at intervals.

Findings

It is particularly interesting to see the finding of the experiment as discribed above. It is found that oxygen diffuses through the experimental device over time scales on the order of 10 second, which is roughly 10 times faster than occlusion and rescue events. The authors believe that it is important to consider the relative rates of ambient deoxygenation and hemoglobin oxygen unloading, especially when the collective chemical polymerization and collective hydrodynamics can act in concert. The authors insist that the diffusion times for water-filled fluid channels in the control experiment is similar to those for blood-filled channels because the fluid channel itself is 12 um high and represents only 10% of the total diffusion distance. Obviously, this is ideal because it closely simulates real time situation. The velocity profile measurements begin with measurable changes in velocity that will occur when intracellular oxygen concentration drops below 3% or rises above 1%. The authors also informed reader that in their experiments, rapid polymerization will occur when oxygen concentration reaches below 1–2%.

Result

Fig. 1
Fig. 2
Fig. 3

I have carefully scrutinized the results presented from the experiments. Because vasoocclusion fundamentally represents the inability of the blood to flow, the authors therefore measured the local velocity of the red blood cells in a microfluidic device with a selected minimal channel width. Also, they controlled the pressure difference driving the steady flow of blood using a constant hydrostatic head, and determined the time for occlusion as a function of ambient oxygen concentration. This experiment allows them to characterize the phase space of occlusion or jamming using three coordinates: the minimum channel width in the microfluidic device, the total hydrostatic pressure difference across the device, and the ambient oxygen concentration.

Talking about the two figures, I believe the results were indeed reflective and supported the initial claims of the authors well. Fig. 1 shows a phase space of vasoocclusion. For unaffected individuals with 100% hemoglobin A (HbA), all fixed-time isosurfaces are located very close to the origin because the time to occlusion becomes very large almost regardless of pressure, oxygen, and vessel width.

Fig. 2 shows that rescue occurs over a much shorter time scale than occlusion. The authors claimed that the evolution of the vasoocclusive event was highly stochastic with large variations. They believe this heterogeneity could arise from at least two sources: the highly cooperative nature of the HbS polymerization reaction whose onset is very slow relative to the subsequent explosive growth and the hydrodynamics of highly concentrated suspensions that are well known to jam.