This work was supported by the Natural Sciences and Engineering Research Council of Canada. Microfabrication was performed with the support of the McGill Nanotools Microfab facility at McGill University and the Department of Electronics at Carleton University.

Blood is collected from healthy individuals with the approval of the ethics committee of the University of Ottawa (H11-13-06).
1. Microchannel Fabrication
The microchannels are fabricated based on the standard photolithography methods19.
<ol>
	<li>Design the microchannel geometry using a Computer Aided Design (CAD) software and print the configuration on a transparency photo-mask. These masks are crucial since they dictate the light path during the fabr...

<ol>
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J. 83 (5), 2482-2490 (2002).</li><li>Schmid-Sch&#246;nbein, H., Gaehtgens, P., Hirsch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+shear+rate+dependence+of+red+cell+aggregation+in+vitro.">On the shear rate dependence of red cell aggregation in vitro.</a> J. Clin. Invest. 47 (6), 1447-1454 (1968).</li><li>Pries, A. R., Secomb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+the+microcirculation.">Rheology of the microcirculation.</a> Clin. Hemorheol. Microcirc. 29 (3-4), 143-148 (2003).</li><li>Baskurt, O. K., Neu, B., Meiselman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Red Blood Cell Aggregation. , (2011).</li><li>Cho, Y. I., Mooney, M. P., Cho, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemorheological+disorders+in+diabetes+mellitus.">Hemorheological disorders in diabetes mellitus.</a> J. Diabetes Sci. Technol. 2 (6), 1130-1138 (2008).</li><li>Baskurt, O. K., Hardeman, M. R., Rampling, M. W., Meiselman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Hemorheology and Hemodynamics. , (2007).</li><li>Lindqvist, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viscosity+of+the+blood+in+narrow+capillary+tubes.">The viscosity of the blood in narrow capillary tubes.</a> Am. J. Physiol.-Legacy Content. 96, 562-568 (1931).</li><li>Goldsmith, H. L., Cokelet, G. R., Gaehtgens, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F&#229;hraeus:+Evolution+of+his+concepts+in+cardiovascular+physiology.">F&#229;hraeus: Evolution of his concepts in cardiovascular physiology.</a> Am. J. Physiol. 257 (3), H1005-H1015 (1989).</li><li>F&#229;hraeus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+suspension+stability+of+the+blood.">The suspension stability of the blood.</a> Physiol. Rev. 9 (2), 241-274 (1929).</li><li>Bauersachs, R. M., Wenby, R. B., Meiselman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+specific+red+blood+cell+aggregation+indices+via+an+automated+system.">Determination of specific red blood cell aggregation indices via an automated system.</a> Clin. Hemorheol. 9 (1), 1-25 (1989).</li><li>Hardeman, M. R., Dobbe, J. G., Ince, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laser-assisted+optical+rotational+cell+analyzer+(lorca)+as+red+blood+cell+aggregometer.">The laser-assisted optical rotational cell analyzer (lorca) as red blood cell aggregometer.</a> Clin. Hemorheol. Microcirc. 25 (1), 1-11 (2001).</li><li>Rampling, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+cell+aggregation+and+yield+stress.">Red cell aggregation and yield stress.</a> Clinical Blood Rheology. , (1988).</li><li>Dusting, J., Kaliviotis, E., Balabani, S., Yianneskis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coupled+human+erythrocyte+velocity+field+and+aggregation+measurements+at+physiological+haematocrit+levels.">Coupled human erythrocyte velocity field and aggregation measurements at physiological haematocrit levels.</a> J. Biomech. 42 (10), 1438-1443 (2009).</li><li>Kaliviotis, E., Dusting, J., Balabani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+variation+of+blood+viscosity:+modelling+using+shear+fields+measured+by+a+&#181;PIV+based+technique.">Spatial variation of blood viscosity: modelling using shear fields measured by a &#181;PIV based technique.</a> Med. Eng. Phys. 33 (7), 824-831 (2011).</li><li>Sherwood, J. M., Kaliviotis, E., Dusting, J., Balabani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+variation+of+blood+viscosity+and+velocity+distributions+of+aggregating+and+non-aggregating+blood+in+a+bifurcating+microchannel.">Spatial variation of blood viscosity and velocity distributions of aggregating and non-aggregating blood in a bifurcating microchannel.</a> Biomech. Model. Mechan. 13 (2), 259-273 (2014).</li><li>Chen, S., Barshtein, G., Gavish, B., Mahler, Y., Yedgar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+red+blood+cell+aggregability+in+a+flow+chamber+by+computerized+image+analysis.">Monitoring of red blood cell aggregability in a flow chamber by computerized image analysis.</a> Clin. Hemorhol. Microcirc. 14 (4), 497-508 (1994).</li><li>Xia, Y. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography.">Soft lithography.</a> Angewandte Chemie International Edition England. , 551-577 (1998).</li><li>Mehri, R., Mavriplis, C., Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+microfluidic+system+for+red+blood+cell+aggregation+investigation.">Design of a microfluidic system for red blood cell aggregation investigation.</a> J. Biomech. Eng. 136 (6), 064501-1-064501-5 (2014).</li><li>Pitts, K. L., Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-particle+image+velocimetry+for+velocity+profile+measurements+of+micro+blood+flows.">Micro-particle image velocimetry for velocity profile measurements of micro blood flows.</a> J. Vis. Exp. (74), e50314 (2013).</li><li>Bitsch, L., Oleson, L. H., Westergaard, C. H., Bruus, H., Klank, H., Kutter, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro+particle-image+velocimetry+of+bead+suspensions+and+blood+flows.">Micro particle-image velocimetry of bead suspensions and blood flows.</a> Exp. Fluids. 39 (3), 507-513 (2005).</li><li>Wereley, S. T., Gui, L., Meinhart, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+algorithms+for+microscale+particle+image+velocimetry.">Advanced algorithms for microscale particle image velocimetry.</a> AIAA J. 40 (6), 1047-1105 (2002).</li><li>Nguyen, C. V., Fouras, A., Carberry, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+measurement+accuracy+in+micro+PIV+by+image+overlapping.">Improvement of measurement accuracy in micro PIV by image overlapping.</a> Exp. Fluids. 49 (3), 701-712 (2010).</li><li>Pitts, K. L., Mehri, R., Mavriplis, C., Fenech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-particle+image+velocimetry+measurement+of+blood+flow:+validation+and+analysis+of+data+pre-processing+and+processing+methods.">Micro-particle image velocimetry measurement of blood flow: validation and analysis of data pre-processing and processing methods.</a> Meas. Sci. Technol. 23 (10), 105302 (2012).</li><li>Bhamare, M. G., Patil, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+blood+cell+analysis+by+using+digital+image+processing:+a+preliminary+study.">Automatic blood cell analysis by using digital image processing: a preliminary study.</a> Int. J. Eng. Res. Tech. 2 (9), 3135-3141 (2013).</li><li>Maitra, M., Gupta, R. 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Using the present methodology, it is possible to analyze qualitatively and quantitatively the RBC aggregates under different flow conditions and hematocrits. For successful testing and aggregate detection, it is crucial to determine the appropriate velocity ratio between the two fluids at the microchannel entry. This ratio is very important to obtain an optimal blood layer thickness where the velocity profile is quasi-linear20.
Another key factor for successful testing is a good ima...

An erratum was issue for <a href="http://www.jove.com/video/52719">Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation</a>. The introduction section was updated.

An erratum was issued for <a href="http://www.jove.com/video/52719">Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation</a>. The introduction section was updated.

Erratum: Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Red Blood Cells (RBCs) play a crucial role in determining the rheological behavior of blood. They are almost singularly responsible for the particular properties of blood in vitro and in vivo. Under physiological conditions, RBCs occupy 40% to 45% of the blood volume. In microcirculation, RBCs only occupy up to 20% of the blood volume due to smaller vessel diameters and the plasma skimming effect1. This phenomenon of plasma reduction in microcirculation is known as the F&#229;hr&#230;us effect. At low shear rates, RBCs are able to bridge together and form one dimensional or three dimensional structures called &#8220;rouleaux&#8221; or aggregates, hence contributing to the non-Newtonian behavior of blood. However, the mechanism of RBC aggregation is not completely understood. Two theories exist to model the aggregation of RBCs: the bridging of cells theory due to the cross-linking of the macromolecules2 and the force attraction theory caused by depletion of the molecules due to the osmotic gradient3.
Typically, for human blood, aggregates form at very low shear rates4 ranging from 1 to 10 sec-1. Above this range, RBCs tend to disaggregate and flow separately within the vessel.
Understanding the conditions of the aggregates formation is of a great importance to the hemorheology field in terms of defining blood&#8217;s rheological behavior. These aggregates are often seen at the macrocirculation level (&#62;300 &#181;m diameter)5. At this scale, blood is considered as a Newtonian fluid and a homogenous mixture. However, these aggregates are rarely seen in the capillary level (4-10 &#181;m in diameter) and are usually an indication of pathological conditions such as diabetes6 and obesity. Other pathological conditions that could change RBC aggregation include inflammatory or infectious conditions, cardiovascular diseases such as hypertension or atherosclerosis, genetic disorders and chronic diseases7. Therefore, understanding the RBC aggregation mechanism and analyzing these entities (by defining a relationship between the size of these aggregates and the flow conditions) could lead to the understanding of the microrheological behavior of blood and hence relate it to clinical applications.
RBC aggregates can be altered by several factors such as the hematocrit (volume of RBCs in blood), the shear rate, the vessel diameter, the RBC membrane stiffness and the suspending medium composition8-10. Therefore, controlled conditions are required in order to effectively analyze the RBC aggregates. Several methods are able to analyze aggregate formation by providing static aggregation measurements (aggregation index) that offers relevant information about blood behavior. These methods include, inter alia, the erythrocyte sedimentation rate method11, the light transmission method12, the light reflection method13 and the low shear viscosity method14.
Few studies have attempted to study RBC aggregation and determine the degree of aggregation in controlled flow conditions15-17. However, these studies indirectly investigate RBC aggregate sizes by determining the ratio of the occupied space in a shearing system measured based on microscopic blood images providing information on the degree of aggregation as well as the local viscosity. Chen et al.18 presented a direct measurement technique for RBC aggregate sizes and provided RBC aggregate size distribution for different shear stresses by varying the flowrate of the suspensions while monitoring the pressure drop across a flow chamber. The shear stresses are calculated based on the monitored pressure using Stokes equation18.
We therefore present a new procedure to directly quantify RBC aggregates in a controlled microfluidic environment, dynamically, under specific, constant and measurable shear rates. The blood flow in the shear system is directly observed (perpendicularly to the flow direction), providing a different angle on flow investigation compared to previous studies15,18 and a visualization of the full domain of interest. RBC-suspensions are entrained, in a double Y-microchannel (as illustrated in Figure 1), with a Phosphate Buffered Saline (PBS) solution hence creating a shear flow in the blood layer. Within this blood layer a constant shear rate can be obtained. The RBC-suspensions are tested at different hematocrit (H) levels (5%, 10% and 15%) and under different shear rates (2-11 sec-1). The blood velocity and shear rate are determined using a micro Particle Image Velocimetry (&#181;PIV) system while the flow is visualized using a high speed camera. The results obtained are then processed with a MATLAB code based on the image intensities in order to detect the RBCs and determine aggregate sizes.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>SU8-50 epoxy based negative Photo-resist</td>
			<td>MicroChem Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU8-50 developer</td>
			<td>MicroChem Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(dimethylsiloxane) (PDMS) Sylgard-184</td>
			<td>Dow-Corning</td>
			<td>3097358-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-50 series Plasma system&#160;</td>
			<td>Plasma Etch</td>
			<td>PE-50 series</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood collection tubes with K2 EDTA (ethylenediaminetetraacetic acid)</td>
			<td>FisherSci</td>
			<td>B367861</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge, i.e. Thermo Scientific CL2</td>
			<td>Thermo Scientific</td>
			<td>004260F</td>
			<td></td>
		</tr>
		<tr>
			<td>Poshpate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Tracer fluorescent particles solution (15 mL)</td>
			<td>FisherSci</td>
			<td>R800</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggregometer</td>
			<td>RheoMeditech</td>
			<td>Rheo Scan AnD300</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringes (50 &#181;L)</td>
			<td>Hamilton</td>
			<td>80965</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing (Tygon)</td>
			<td>FisherSci</td>
			<td>AAA00001</td>
			<td></td>
		</tr>
		<tr>
			<td>High speed camera (Basler)</td>
			<td>Graftek Imaging Inc.</td>
			<td>basler acA2000-340km</td>
			<td>A camera capable of recording 18 frames per seconds could be used.&#160;</td>
		</tr>
		<tr>
			<td>Double pulsed camera&#160;</td>
			<td>LaVision</td>
			<td>Imager Intense</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope MITAS</td>
			<td>LaVision</td>
			<td>MITAS</td>
			<td></td>
		</tr>
		<tr>
			<td>Nd:YAG laser</td>
			<td>New Wave Research</td>
			<td>Solo-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump (Nexus3000 and PicoPlus)</td>
			<td>Chemyx Inc. and Harvard Apparatus</td>
			<td>Nexus3000 and PicoPlus</td>
			<td></td>
		</tr>
		<tr>
			<td>DaVis software</td>
			<td>LaVision</td>
			<td>Davis</td>
			<td></td>
		</tr>
	</tbody>
</table>

controlled microfluidic environment for dynamic investigation of red blood cell aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

An example of the two-fluid flow in the double Y-microchannel is shown in Figure 2 for human RBCs suspended at 5%, 10% and 15% hematocrit and flowing at 10 &#181;l/hr. Figure 3 shows the difference in aggregate sizes when the flow in the channel is reduced from 10 &#181;l/hr to 5 &#181;l/hr for a hematocrit of 10%. This gives a qualitative notion of the sizes of the aggregates when varying the hematocrit and shear rate. Figure 5 follows the displacement of four human RBC...

Watch this Scientific Journal Video about Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation at JoVE.com

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

controlled-microfluidic-environment-for-dynamic-investigation-red

Research

JoVE Journal

Bioengineering

11.7K visualizações.  University of Ottawa.  O protocolo descrito detalhes um procedimento experimental para quantificar glóbulos vermelhos (RBC) agregados sob uma velocidade de corte constante e controlado, com base em técnicas de processamento de imagem. O objetivo deste protocolo é relacionar RBC tamanhos agregados à taxa de corte correspondente em um ambiente controlado microfluídico. 