Group Title: 7th International Conference on Multiphase Flow - ICMF 2010 Proceedings
Title: 6.7.3 - Visualization Study of Motion and Deformation of Red Blood Cells in Microchannel
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 Material Information
Title: 6.7.3 - Visualization Study of Motion and Deformation of Red Blood Cells in Microchannel Bio-Fluid Dynamics
Series Title: 7th International Conference on Multiphase Flow - ICMF 2010 Proceedings
Physical Description: Conference Papers
Creator: Guo, F.
Xiang, H.
Chen, B.
Publisher: International Conference on Multiphase Flow (ICMF)
Publication Date: June 4, 2010
 Subjects
Subject: red blood cell
microchannel
microfluidic chip
visualization
 Notes
Abstract: The size of red blood cell (RBC) is on the same order with the diameter of microvascular. Therefore, blood should be regarded as a two-phase flow system of RBCs suspended in plasma rather than a continuous medium of microcirculation. It is of great physiological and pathological significance to investigate the effects of deformation and aggregation of RBCs on microcirculation. In this study, a visualization experiment was conducted to study the microcirculatory behavior of RBCs suspension. Motion and deformation of RBCs in a microfluidic chip with straight, divergent and convergent sections of microchannel have been captured by microscope and high-speed camera. Meanwhile, deformation and movement of RBCs were investigated under different viscosity, hematocrit and flowrate in this system. For low velocity and viscosity, RBCs behave the normal biconcave disc shape and their motion was found as a flipping motion, not only deformed their shapes along the flow direction, but also rolled and rotated themselves. RBCs were also found aggregated to form rouleaux at very low flowrate and viscosity. However, for high velocity and viscosity, RBCs deformed obviously under the shear stress. They elongated along the flow direction and performed a tank-treading motion (TTM).
General Note: The International Conference on Multiphase Flow (ICMF) first was held in Tsukuba, Japan in 1991 and the second ICMF took place in Kyoto, Japan in 1995. During this conference, it was decided to establish an International Governing Board which oversees the major aspects of the conference and makes decisions about future conference locations. Due to the great importance of the field, it was furthermore decided to hold the conference every three years successively in Asia including Australia, Europe including Africa, Russia and the Near East and America. Hence, ICMF 1998 was held in Lyon, France, ICMF 2001 in New Orleans, USA, ICMF 2004 in Yokohama, Japan, and ICMF 2007 in Leipzig, Germany. ICMF-2010 is devoted to all aspects of Multiphase Flow. Researchers from all over the world gathered in order to introduce their recent advances in the field and thereby promote the exchange of new ideas, results and techniques. The conference is a key event in Multiphase Flow and supports the advancement of science in this very important field. The major research topics relevant for the conference are as follows: Bio-Fluid Dynamics; Boiling; Bubbly Flows; Cavitation; Colloidal and Suspension Dynamics; Collision, Agglomeration and Breakup; Computational Techniques for Multiphase Flows; Droplet Flows; Environmental and Geophysical Flows; Experimental Methods for Multiphase Flows; Fluidized and Circulating Fluidized Beds; Fluid Structure Interactions; Granular Media; Industrial Applications; Instabilities; Interfacial Flows; Micro and Nano-Scale Multiphase Flows; Microgravity in Two-Phase Flow; Multiphase Flows with Heat and Mass Transfer; Non-Newtonian Multiphase Flows; Particle-Laden Flows; Particle, Bubble and Drop Dynamics; Reactive Multiphase Flows
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Volume ID: VID00168
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Resource Identifier: 673-Guo-ICMF2010.pdf

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Paper No 7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


Visualization Study of Motion and Deformation of Red Blood Cells in Microchannel


F. Guo, H. Xiang and B. chen


State Key Laboratory of Multiphase Flow in Power Engineering, Xi'an Jiaotong University
Xi'an 710049, CHINA
chenbin@mail.xjtu.edu.cn


Keywords: Red blood cell, microchannel, microfluidic chip, visualization




Abstract

The size of red blood cell (RBC) is on the same order with the diameter of microvascular. Therefore, blood should be regarded
as a two-phase flow system of RBCs suspended in plasma rather than a continuous medium of microcirculation. It is of great
physiological and pathological significance to investigate the effects of deformation and aggregation of RBCs on
microcirculation. In this study, a visualization experiment was conducted to study the microcirculatory behavior of RBCs
suspension. Motion and deformation of RBCs in a microfluidic chip with straight, divergent and convergent sections of
microchannel have been captured by microscope and high-speed camera. Meanwhile, deformation and movement of RBCs
were investigated under different viscosity, hematocrit and flowrate in this system. For low velocity and viscosity, RBCs
behave the normal biconcave disc shape and their motion was found as a flipping motion, not only deformed their shapes
along the flow direction, but also rolled and rotated themselves. RBCs were also found aggregated to form rouleaux at very
low flowrate and viscosity. However, for high velocity and viscosity, RBCs deformed obviously under the shear stress. They
elongated along the flow direction and performed a tank-treading motion (TTM).


Introduction

Microcirculation refers to the blood flow in microvascular
network between arterioles and venules. In microcirculation,
oxygen and nutrient are delivered to living tissues, and
metabolic wastes are removed away. Therefore,
microcirculation plays an important role in the
environmental regulation of the living tissues and the
maintenance of normal physiological functions of cells,
tissues and organs.
Blood is a suspension comprised of plasma and blood cells.
The main content of blood cell is red blood cell (RBC,
erythrocyte), which accounts for 45% of blood volume,
95% of blood tangible composition, and more than 99% of
the particulate matter in blood. In large vessels, RBC is
negligibly small compared with vessel diameter and blood
can be treated as a homogeneous Newtonian fluid because
the flow rate of blood is high and whole blood viscosity
approaches an asymptotic value with the increase of shear
rate. However, blood should be regarded as a two-phase
flow system of RBCs suspended in plasma for
microcirculation since the size of RBC is on the same order
with capillary diameter in microcirculation and RBCs'
aggregation as well as deformability have great influences
on blood theological properties especially when blood flow
rate is low. Abnormal aggregation and deformability of
RBCs will lead to a decrease of blood flow rate and increase
of blood viscosity, thus improving the probability of
microcirculation disturbance. Long-term microcirculation
disturbance will cause many diseases such as coronary heart


disease, phlebitis, etc. In this sense, it is of great
physiological and pathological significance to investigate
the effects of deformation and aggregation of RBCs on
microcirculation.
Matured RBC is comprised of membrane and cytoplasm
without nucleus. The RBC membrane consists of 52%
protein, 40% lipid and 8% carbohydrate (Freitas, 1999),
which is responsible for many of the physiological functions
and mechanical properties of the cell. RBC membrane
consists of membrane skeleton and lipid bilayers. The RBC
membrane skeleton is a multi-protein complex formed by
structural proteins. It interacts with the lipid bilayer and
transmembrane proteins to provide the RBC withs strength
and plasticity (Pawlowski et al. 2006). The lipid bilayer is a
thin membrane made of two layers
ofphospholipids molecules. These membranes are flat
sheets that form a continuous barrier around cells. The lipid
molecules' good potential of lateral movement, rotating
motion, fatty acid chains swing and turnover movement
make RBC membrane having a strong fluidity, which
ensures RBCs can deform plastically with blood flow and
even pass though the microvascular with smaller diameter
than the size of itself. Normal RBC performs a biconcave
disc shape. In microcirculation, it can deform itself to
parachute, bullet, slipper shape, etc. The deformability of
RBC is an important factor affecting the high shear blood
viscosity. Depressed RBC deformability will cause the
increase of blood viscosity and flow resistance, which will
lead to many cardio-cerebrovascular diseases, such as
Myocardial Infarction and apoplexy (Mi et al. 2005).






Paper No


Common measurement methods of RBC deformability
include aspiration of cells into microtubule, filtration of
cells with microporous, viscometry of cell suspensions,
laser diffraction method, deformation of cells under fluid
shear stress, and so on (Avishay & Natane 2007). Recently,
microfluidics has been the subject of a vast body of
researchers with the rapid development of micro-electro
mechanical system (MEMS). Detections and analysis with
microfluidic technology have many advantages such as low
sample reagent cost, high processing control accuracy and
rapid response time. Based on the MEMS technology, micro
total analysis system (pTAS) can serve many useful
applications in biological field such as cytological test and
DNA analysis (Burns & Johnson 1998). For blood rheology,
many researchers have attempted to study the theological
properties of RBCs with silicon microchannels. Lima et al.
(2006) measured velocity distribution of RBC suspension
flow in a microchannel with a 0.1 x0.mm2 cross-section by
confocal micro-PIV They indicated that microchannels with
dimensions on the order of 100pm still obey macroscale
flow theory. Korin et al. (2007) studied the flow behavior of
RBC suspension in a straight microchannel with a 0.05
x1mm2 rectangular cross-section. The influence of shear
stress on RBC shape has been studied and good agreement
was obtained between experiment measurement and
numerical simulation. Their research revealed that RBCs'
deformability under high viscosity shear condition is
insensitive to the change of inner viscosity within the
physiological range and is highly affected by RBC shear
modulus. This conclusion indicated that the relationship
between shear rate and RBC deformability index (DI) could
be a useful tool to estimate membrane mechanical properties,
which will help diagnose of blood disorders. Shevkoplyas et
al. (2003) designed and fabricated a microfluidic chip with a
network of microchannels and visualized flow of red and
white blood cells in this network by high-speed camera.
Parachute and bullet shape as well as rouleaux formation of
red blood cells was observed in their experiment.

In present work, the flow behavior of RBC suspension was
visualized in a microfluidic chip, which consists of
microchannels with straight, divergent and convergent
sections. High quality images of RBCs motion and
deformation were recorded by microscope and high-speed
camera. RBCs' theological properties were investigated
under different viscosity, hematocrit and flowrate.

Experiment setup

In this work, a microfluidic experimental system with
functions of flow control, image capture and data
acquisition is established to obtain high-resolution images of
RBCs' motion and deformation.
The schematic of the experiment setup is shown in Figure 1.
RBCs suspension was injected into microchannel system as
pulseless steady flow by a Havard PHD2000 syringe pump,
with which flow rate can be controlled precisely (control
precision is 0.35%). after passing though the microchannel,
the suspension was discharged into a liquid waste. In the
microchannel, the motion and deformation of RBCs are
captured by a Leica DMI 500M microscope and recorded by
a Redlake HG-100k high speed camera with resolution of
1506x1506 pixels at 60-200 frames per second. Images was


7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

transferred into computer by Ethernet and then analyzed by
specified image-processing software.


Figure 1: schematic of experiment setup


Figure 2 illustrates the structure of the microfluidic chip,
which is fabricated by etching microchannel on the silicon
substrate and then bonded with a 7740 glass cover.
Compared to traditional machining processing method, this
MEMS technology can provide a smoother channel surface
(surface roughness is less than lpm). The image of the test
section photographed with 5x objective lens is shown in
figure 3. The shooting region consists of straight, divergent
and buffer microchannels, which have a rectangular
cross-section with a depth of 40pm. The width of the
straight channel was 200pm and the narrowest width was
40pm for the divergent and convergent sections. In our
experiment, we mainly focused RBCs' motion in straight
channel and RBCs' movement from straight to convergent
section. In this process, RBCs move from low to high speed
flow region and will deform remarkably.


Figure 2: microfluidic chip


Figure 3: test section of the microchannel


In our experiment, the narrowest part of the microchannel
was liable to be contaminative and blocked by RBCs
because of their aggregation and adhesion on the channel
wall. As shown in Figure 4a, once the channel was





7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


completely blocked up, the problem could be solved by
heating the microfluidic chip in high-temperature water bath
for half an hour, and then cleaning by high velocity absolute
ethanol, which could reduce the surface viscosity of the
channel wall and dissolve organic impurities.








(a) (b)
Figure 4: Microchannel blocking and its cleaning. (a)
channel blocking; (b) after cleaning

Before the experiment, we firstly washed channel surface by
strong acid and alkali such as aqua regia and sodium
hydroxide as well as organic solvent like trichloroethylene
to ensure there is no impurity adhering on the channel wall.
Then we flushed microchannel with deionized water and
phosphate buffer solution (PBS). At last the microchannel is
washed by 1% albumin solution which can precoat the
channel wall to reduce the adhesion of cells to silicon
substrate and cover glass (Wang et al. 2008).

Preparation of RBC suspension

Firstly, heparin was added into fresh blood obtained from
healthy adult volunteers for anticoagulation. Afterwards,
RBCs were separated from the sample by centrifugation and
aspiration of the plasma as well as buffy coat. Then the
RBCs were washed three times in PBS, which is an isotonic
salt solution to balance the osmotic pressure and maintain
the ionic strength as well as PH value so as to keep a healthy
physiological environment for RBCs. At last, we suspended
RBCs in PBS to get the hematocrit of 0.4%, 1% and 3%.
After preparation, RBCs suspension was stored at 4C and
the experiment was performed at room temperature. To
investigate the influence of viscosity on RBCs'
deformability, polyvinylpyrrolidone (PVP) was added to
individual samples by 8% (mass) to increase the viscosity of
RBCs suspension.

Results and discussions

Generally, plasma proteins in blood have the function of
bridge-link, which will make RBCs aggregate and show
rouleaux, reticular and branchlike formations. In our
experiment, RBC suspension (with PVP) was pumped into
the channel at flowrate of 0.0046ml/h after washing
microchannel with albumin solution. As shown in Figure 5,
RBCs' aggregation was observed at the right side of the
image. At the moment, the mean velocity in the channel was
0.16mm/s, which is lower than the normal blood flow
velocity in microvascular. Disaggregation of RBCs was
found by gradually increasing flow rate. When the flow rate
was higher than 0.lml/h, RBCs' aggregation completely
disappeared.


Figure 5: RBCs aggregation in the microchannel
(hematocrit: 1%, flow rate: o.(ll46b1nt h)

Deformation of RBC

With no nucleus, matured RBC is comprised of membrane
and cytoplasm. Membrane of RBC consists of membrane
skeleton and lipid bilayers. Membrane skeleton is a fibrous
network formed by the cross-link of protein molecules,
which provides RBC with strength and plasticity. While
lipid bilayer has a strong fluidity, which ensures RBC can
deform plastically with blood flow and even pass though
the microvascular with a smaller diameter than the size of
RBC
In the experiment, we firstly compared RBCs' motion
morphology under different shear stress. As shown in
Figure 6a, RBCs preserved a standard biconcave disc
shape under low flow rate and low viscosity (without PVP).
This shape has a high surface-to-volume ratio, which is
helpful to RBCs' plastic deformation and can provide a
large gas-exchange area. Figure 6b shows RBCs shape
under high flow rate and high viscosity (with PVP). Cells
were elongated by a higher shear stress and deformed into
a flattened ellipsoidal shape. This motion morphology of
RBC is usually described as tank treading motion (TTM),
which is caused by membrane's rotation around the interior
of the cell.


(b)
Figure 6: RBCs' deformation under different flow rate and


Paper No






Paper No


viscosity: (a) hematocrit 1%, flow rate 0.0046ml/h, without
PVP; (b) hematocrit 1%, flow rate 1.Oml/h, with PVP.

Figure 7 shows the snapshots when RBCs past though the
divergent section of the channel and a RBC near the
channel junction is marked out to analyze. It can be seen
that, flow velocity is very low near the wall in the straight
channel, so RBC can keep its biconcave disc shape (Figure
7a). When it is moving into the divergent section from the
straight channel, the flow velocity was greatly increased
because of the sudden convergence of the channel width.
So RBC shape was elongated gradually (Figure 7b) and
finally turned into a flattened ellipsoidal shape (Figure 7c)
in the divergent section.


7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

Common motion form of RBC

When suspended in a medium with lower viscosity than
cytoplasm, RBC will perform a flipping motion (Korin et
al. 2007). It not only deforms toward its moving direction,
but also rotates and rolls with its motion. Figure 8 is a
schematic diagram of RBC's rotation and rolling. RBC's
rotation refers to its rotating around its arbitrary symmetric
axis on the axisymmetric plane (Figure 8a). Meanwhile,
RBC also rolls around its axisymmetric axis, as shown in
Figure 8b.


4 Rotate








(a)flipping


(-4 Roll








(b) rolling


Figure 8: Schematic diagram of RBCs' rotation and
rolling

Figure 9 shows the process of RBCs' rotation in our
experiment. The two RBCs that marked out by red squares
rotated half cycle successively. The process of RBCs'
rolling is presented in Figure 10. Actually, RBCs always
flip and roll simultaneously.
A problem worth to be pointed out is that the direction of
the axis that RBCs rotate around are not unified through
the whole channel. In our experiment we found that the
flipping axes of most RBCs near the wall were toward the
normal direction of their moving direction (Figure 11). For
the RBCs near the centerline of the channel, the flipping
axes were toward their moving direction (Figure 12).
RBC's rotation can be explained by the parabolic velocity
distribution of RBC suspension. Velocity difference
exerted on RBCs adjacent to the wall was higher, so the
flipping of RBCs was more obvious. However, to the
present, no reasonable explanation was achieved for RBC
rolling (Wang et al. 2008).


ire 9: The rotation ol


Figure 10: The rolling motion of RBC


Figure 7: Deformation process of RBCs' passing though
the convergent section of the channel


v figure 11: KLus rotation near tue channel wall





Paper No


Fahraeus-Lindqvis effect and RBCs axis concentrate

In 1931, Fahraeus and Lindqvis found that in narrow
channels less than about 0.3 mm in diameter, the decrease
of apparent viscosity of blood occurs with the decrease in
channel diameter. This phenomenon is the so-called
Fahraeus-Lindqvis effect (Fahraeus and Lindqvist. 1931;
Goldsmith. 1971). Common explanation for this
phenomenon is tubular pinch effect (Hiroshi et al. 1979) or
plasma skimming effect (Pries et al. 1986; Schmid-
Schonbein et al. 1980). Tubular pinch effect is the
phenomenon that in laminar flow of suspensions through a
circular channel, particles will migrate into a concentric
annular region with the width about 60% of the channel.


7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

In the experiment, as shown in figure 12, this phenomenon
was observed at pumping velocity of Iml/h. When RBCs
moved into the straight channel from divergent section of
the channel, RBCs concentrated at the axis of the
microchannel, and a plasma layer with no RBC is formed
adjacent to the inner wall of the microchannel. This plasma
layer can decrease the apparent viscosity of blood and have
a lubricating effect on microcirculation. Figure 13 presents
the RBCs distribution at flow rate 0.15 ml/h. It can be seen
that with the reduction of RBCs flow rate, its aggregation
extent at the axis of the channel was weakened and the
plasma layer get thinner. When the flowrate reduced to
0.012ml/h (figure 14), the tubular pinch effect almost
disappeared. With the decrease of flow rate, the passing
velocity of RBCs decreased. More and more RBCs
squeezed at the narrowest part of the channel, leading to
the increase of local concentration and flow resistance at
divergent section and increase the possibility of channel to
be blocked.


(a) (b) (c)
Figure 12: tubular pinch effect of RBC at flow rate Iml/h: (a) hematocrit 8% (b) hematocrit 10% (c) hematocrit 15%







(a) (b) (c)
Figure 13: tubular pinch effect of RBC at flow rate 0.15ml/h: (a) hematocrit 8% (b) hematocrit 10% (c) hematocrit 15%







(a) (b) (c)
Figure 14: tubular pinch effect of RBC at flow rate 0.012ml/h: (a) hematocrit 8% (b) hematocrit 10% (c) hematocrit 15%


Conclusions

In this research a microfluidic experiment setup was setup
to observe the flow behavior of RBCs suspension in a
microchannel by capturing images of RBCs motion
morphology. RBCs aggregation and deformation were
studied under different flow rate, hematocrit and viscosity.
The conclusions are as follows:
1) The aggregation of RBCs happens under low flow rate
((.((l46111l h) and high viscosity (with PVP). With
increasing of flow velocity, RBCs will disaggregate under
high shear stress. When the flow rate was higher than
O.lml/h, RBCs' aggregation completely disappeared.


2) Under low flow rate and viscosity condition, RBCs will
maintain their biconcave disc shape, rotate and roll
simultaneously with their motion.
3) Under high flow rate and viscosity condition, RBCs will
perform flattened ellipsoidal shape and tank treading
motion.
4) Tubular pinch effect was observed: RBCs concentrated
at the axis of the microvessel, and a plasma layer with no
RBC is formed adjacent to the inner wall of microchannel.
This effect weakened with decreasing the flow velocity.
When the flowrate is lower than 0.012ml/h, the tubular
pinch effect almost disappeared.






Paper No 7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

References

Avishay, B. & K. Natane, The rheologic properties of
erythrocytes a study using an automated rheoscope. Rheol
Acta, Vol. 46, 621-627 (2007)
A .Hiroshi, K.Yasuo and A. Hiroshi, Study on the Tubular
Pinch Effect in a Pipe Flow: I. Lateral migration of a single
particle in laminar poiseuille flow, Bulletin of JSME,Vol.
22, 206-212 (1979)
Bums, M.A., Johnson, B.N., Brahmasandra, S.N., et al.,
An integrated nanoliter DNA analysis device. Science, Vol.
282(5388), 484-487 (1998)
Fahraeus, R., & T Lindqvist. The viscosity of blood in
narrow capillary tubes. Am. J. Physiol, Vol. 96, 562-568
(1931)
Freitas Jr., R.A., Cell membrane. In: Freitas, Jr., R.A.
(Ed.),Nanomedicine, Volume I: Basic Capabilities. Landes
Bioscience, Georgetown, TX Section 8.5.3.2. (2006)
Goldsmith, H. L. Red cell motions and wall interactions in
tube flow. Fed. Proc. Vol. 30, 1578-1590 (1971)
Lima, R., Shigeo, W, Tsubota, K., et al., Confocal
micro-PIV measurementsof three-dimensional profiles of
cell suspension flow in a square microchannel.
Measurement Science and Technology, Vol. 17, 797-808
(2006)
Pries, A. R., K. Ley, and P. Gaehtgens. Generalization of
the fahraeus principle for microvessel networks. Am. J.
Physiol, Vol. 251, 1324-1332 (1986)
Korin, N., A. Bransky and U. Dinnar, Theoretical model
and experimental study of red blood cell (RBC)
deformation in microchannels. Journal of Biomechanics,
Vol. 40, 2088-2095 (2007)
Mi, X.Q., Chen, Y, Zhou, Z.Y, Zhou, L.W and Chen, J.Y,
The in Vitro Effects of Low Power Laser Irradiation on
Animal Erythrocyte Rheology. Chinese Journal of Laser,
Vol. 31, 121-124 (211114
Pawlowski, PH., B. Burzyn and P. Zielenkiewicz,
Theoretical model of reticulocyte to erythrocyte shape
transformation. Journal of Theoretical Biology, Vol. 243,
24-35 (2006)
Schmid-Schonbein, G W., R. Skalak, S. Usami, and S.
Chien. Cell distribution in capillary networks. Microvasc.
Res, Vol. 19, 18-44 (1980)
Shevkoplyas SS, Gifford SC, Yoshida T, et al., Prototype of
an in vitro model of the microcirculation. Microvascular
Research, Vol. 65, 132-136 (2003)
Wang, C., X. Wang & P. Ye, The transport and deformation
of blood cells in microchannle, in 3rd IEEE Int. Conf. on
Nano/Micro Engineered and Molecular Systems. IEEE
Xplore: Sanya, China. pp. 116-119, 2008
Pawlowski, PH., B. Burzyn and P. Zielenkiewicz,
Theoretical model of reticulocyte to erythrocyte shape
transformation. Journal of Theoretical Biology, Vol. 243,
24-35(2006)




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