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in the micro-channels, the cross-sectional area of the channel

the overall pressure drop across the channel or the overall flow

decreases, resulting in unequal flow through different sections of

rate through the channel based on an infusion pump.

the device. A method to estimate the local wall shear stress was

developed using erythrocytes as particles for tracking fluid

Rolling velocity versus wall shear stress

velocities. Particle image velocimetry (PIV) has been performed

A calibration plot showed that the average velocity of erythro-

using the erythrocytes to estimate the local flow environment.30,31

PIV is measured using specialized high-speed cameras, laser light

cytes increased with

the measured pressure

drop across

the

channels (Fig. 4A). The range of velocities in the calibration plot

sources, and image correlation methods to describe the velocity

covers

the velocities

observed

in experiments at a constant

field across the field of view. In this study, an average velocity

applied pressure. Reduced flow rates with higher pressure are due

estimation was used. The average velocity method uses conven-

to endothelial cells growing in the channels and reducing the

tional optics and cameras and is more appropriate for field use.

effective cross-sectional area. Erythrocyte velocities of 5–20 mm s 1

The average velocity method does not resolve the parabolic

have been observed in capillaries, which translates to wall shear

velocity field across the width of the micro-channel. Therefore,

stresses range of 0.1 to 0.7 kPa.32,33 These wall shear stresses are

this method is restricted in areas where flow is relatively laminar

in the same range observed in the mF-D described here.34–37 The

and unperturbed by obstructions. The method provides a useful

rolling velocity

of

individual

parasitized

erythrocytes

was

estimate of local velocities when particles are present in at least

observed

over

a

variety of wall shear

stresses (Fig. 4B). As

two consecutive images across the field of view. Over the velocity

expected,

the

rolling

velocity increased

with

the applied

wall

range measured, the average velocity appears to be linearly

shear stress and at higher shear stresses fewer parasitized eryth-

related to the pressure applied across the channel (Fig. 4A). This

rocytes tended to adhere. The relatively few number of parasit-

technique provides a reasonable estimate of the local flow rate

ized

erythrocytes observed

rolling

on

endothelial

cells

through the channels, and is superior to relying on a measure of

underscores the difficulty and unique characteristics of working

with fresh parasite field isolates. Normally for similar experi-

ments in non-endemic countries, parasite cultures are selected to

enrich for expression of adhesive characteristics before binding

experiments are performed. In an effort to keep the microfluidic

system as close to physiologic conditions as possible, a field

parasite isolate was chosen to directly demonstrate that the

techniques described capture parasite-endothelial cell interac-

tions over a variety of flow conditions. While a detailed investi-

gation across multiple parasite isolates and primary brain

endothelial cells is not presented here, the present work

demonstrates that these microfluidic technologies are ready for

field applications.

Conclusion

As the parasitized erythrocytes accumulate in the microcircula-

tion it is important to understand the conditions under which

they cytoadhere and how they migrate under various flow

conditions. The mF-CS described here was developed for field

experimentation to observe parasitized erythrocyte cytoadhesion

to primary endothelial cells. Quantifying the rolling behavior of

parasitized erythrocytes over a variety of shear stresses can help

describe the behavior of parasitized cells in micro-circulation.

These types of measurements could help future investigations

into interactions between endothelial cells and parasitized

erythrocytes. This report demonstrates that microfluidic systems

can be utilized to perform experiments in a malaria-endemic

area. Such a system can mimic the micro-circulatory conditions

in the deep capillary beds of organs and may improve our

understanding of malaria pathogenesis. The mF-CS and image

analysis tools described here provide a promising new resource

for investigating how cytoadhesion contributes to severe malarial

Fig. 4 The mean erythrocyte velocity increased linearly with the applied

infections.

pressure drop across the device (A). The wall shear stress was estimated

using the erythrocyte velocity and the depth of field of the objective lens.

Acknowledgements

The rolling velocity of parasitized erythrocytes’s increased as the esti-

mated wall shear stress increased (B). Each dot indicates an individual

This work was supported by the NIH under the following

parasitized erythrocyte.

grants R21 AI081234 (P.K.R.), K23AI079402 (K.B.S), and

This journal is ª The Royal Society of Chemistry 2011

Lab Chip, 2011, 11, 2994–3000 | 2999

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U19AI089688 (P.K.R.). We specifically thank Jason Stage, Dave

15

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Tucker and the late George Turner of Games4you LLC for

2007, 64, 1486–1498.

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D. J. Roberts, A. G. Craig, A. R. Berendt, R. Pinches, G. Nash,

developing software for the microcontroller pump.

K. Marsh and C. I. Newbold, Nature, 1992, 357, 689–692.

17

M. Antia, T. Herricks and P. K. Rathod, PLoS Pathog., 2007, 3, e99.

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