Applying a Microfluidic 'Deformability Cytometry' to
Measure Stiffness of Malaria-infected Red Blood Cells
at Body and Febrile Temperatures
MASsACHUSETTS INS
OF TECHNOLOGY
[JUN 17 2011
by
Sha Huang
LIBRARIES
Submitted to the Department of Electrical Engineering and Computer
Science in partial fulfillment of the requirements for the degree of
Masters of Science in Electrical Engineering and Computer Science
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY ARMRMS
June 2011
© Massachusetts Institute of Technology 2011. All rights reserved.
Signature of Author:
Department of Electrical Engineering and Computer Science
May 20, 2011
Certified by:
__
Jongyoon Han
Associate Professor of Electrical Engineering and Computer Science
Thesis Supervisor
Accepted by:
Leslie A. Kolodziejski
,'
-
Professor of Electrical Engineering and Computer Science
Chairman, Committee for Graduate Students
E
Applying a Microfluidic 'Deformability Cytometry' to
Measure Stiffness of Malaria-infected Red Blood Cells
at Body and Febrile Temperatures
by
Sha Huang
Submitted to the Department of Electrical Engineering and Computer
Science on May 20. 2011, in partial fulfillment of the
requirements for the degree of
Masters of Science in Electrical Engineering and Computer Science
Abstract
Red blood cells (RBCs) undergo repeated deformation as they traverse blood vessel,
capillaries and splenic cords; RBC deformability is therefore crucial in maintaining
normal blood circulation. During falciparum malaria, parasite proteins interact with the
spectrin network of host RBCs, moderately stiffening the ring stage infected cells (rings).
The subtle modification in the deformability of rings is however believed to be
significant enough to trigger their retention by human spleen. In addition, recent studies
demonstrated considerable stiffening of parasitized RBCs at febrile temperature,
highlighting the temperature-dependent physiological consequences in microcirculation.
A quantitative characterization of the dynamic process of RBC deformation at
physiologically relevant temperatures is therefore highly desirable. In this work, a
microfluidic device with bottleneck arrays is developed to mimic RBCs' travelling
through narrow in-vivo constrictions such as splenic cordal meshwork and blood
capillaries. For the first time, we report the dynamic mechanical responses of rings in a
large population of co-cultured uninfected cells at both body and febrile temperatures.
Experiments revealed that the deformability cytometer can differentiate parasitized RBCs
from normal RBCs most efficiently at febrile temperature, suggesting a potential role of
fever in facilitating splenic clearance. Similar dynamic deformability measurements were
also conducted on RBCs with anti-malarial drug treatment; the drug effect on the
deformability of both normal and parasitized cells is assessed.
Thesis Supervisor: Jongyoon Han
Title: Associate Professor of Electrical Engineering and Computer Science
and Biological Engineering
Acknowledgements
It has been almost two years since I first embarked on my journey as a graduate
student in MIT. Life has been even more enriching and exciting than I had envisaged.
The ample collaboration opportunities amongst dissimilar research groups and the state
of the art lab facilities have offered me the maximum research freedom one could
possibly have.
First of all, I would like to express my utmost gratitude to my research advisor
Professor Jongyoon Han, who introduced me into this amazing field of BioMEMS. I
would like to thank Prof. Han for his guidance, support and encouragement throughout.
His enthusiasm for science and his insights in conducting scientific research have
influenced me greatly and redefined my perspective towards research. In addition, I am
also deeply indebted to Prof. Han for the enormous research freedom he endowed me. He
encouraged me to explore areas of my own interests and he has always been accepting to
my new ideas, even though most of the time they did not work out well. I could not have
wished for a better advisor.
I would also like to thank all the Han group members who have been so
supportive and helpful. It has been a very comfortable and conductive environment to
work in. I greatly appreciate Dr. Pan Mao and Dr. Hansen Bow, both offered me a lot of
hands-on trainings and supervisions and helped me identify possible research directions.
Dr. Pan Mao's expertise in device design and fabrication helped me enormously when I
first entered Microsystems Technology Laboratories (MTL). Under his coach, I learnt
that thoughtfulness and patience are the two essential qualities for device fabrication and
for research in general. Dr. Hansen Bow was the person who initiated this malaria project
when he was a graduate student in Han group. He built the foundation for this project and
gave me a lot of guidance and advice during the initial phase of my research work. The
weekly project discussions with Hansen have been really educational and rewarding. The
other members in the group are also great people to work with and to spend time with
including Lih-Feng Cheow, Aniruddh Sarker, Lidan Wu, Leon Li, Rhokyun Kwak, Dr.
Chia-Hung Chen, Dr. Hiong Yap Gan, Dr. Sung Jae Kim, and Dr. Yong-Ak Song.
During my hard times at MIT, they offered me the warmest companionship.
Prof. Subra Suresh and his nanomechanics laboratory have also offered me a lot
of support without which this project could not have been accomplished. As current
director of national science foundation (NSF), Prof. Suresh still meet us whenever it is
possible and offered us a lot of invaluable advice on the general direction of the project.
His research group, currently led by Dr. Ming Dao, has been a pleasure to work with.
Special thanks to Dr. Monica Diez-Silva whose expertise in Malaria research has been a
great asset for this project.
During my summer internship at the National University of Singapore, the
constructive interactions I had with Prof. Chwee Teck Lim, Dr. Ali Asgar Bhagat and
graduate student Hanwei Hou have been also very valuable.
Last but not least, I would like to thank my dearest parents and my husband Yuan
Fang. I greatly appreciate their support and understanding. I would like to dedicate this
thesis to them to express my love and gratitude.
Contents
1. Introduction............................................................
7
Red Blood Cell (RBC) Deformability.......................................7
Plasmodiumfalciparum Malaria..........................................12
2. Existing Tools for RBC Deformability Measurement........16
RBC
RBC
RBC
RBC
deformability
deformability
deformability
deformability
measurement
measurement
measurement
measurement
by Optical Tweezers................16
by Micropipette Aspiration.............18
by ektacytometry........................19
in MicroFluidic devices.................19
3. Materials and Methods..............................................23
Device Fabrication..........................................................23
Parasite Culture.............................................................
25
Solution Preparation............................................................25
Experim ent Protocol............................................................25
4. Effect of Febrile Temperature on the Deformability.........28
of Malaria-infected Erythrocytes
Temperature-dependent iRBC deformability ..............................
Temperature-dependent uRBC deformability ...........................
Temperature-dependent hRBC deformability ...........................
Febrile condition enhances the separation resolution ...................
between iRBCs and uRBCs
Spleen as a mechanical filter...............................................36
28
32
33
34
5. Effect of Anti-malarial Drugs on the Deformability..........38
of Malaria-infected Erythrocytes
Artemisinin and its derivatives.............................................39
M alarone (Proguanil) ...........................................................
42
6. Effect of the Ring-infected Erythrocyte Surface..............44
Antigen (RESA) on the Deformability of
Malaria-infected Erythrocytes
7. Discussions........................................................47
Body temperature corresponds to the maximum...............................47
hRBC deformability
Fever in the pathogenesis of malaria...........................................49
Antim alarial drug effect......................................................
50
Reduced uRBC deformability may exacerbate malaria anemia............53
8. Conclusion and Future Plans...................................55
9. Bibliography.......................................................56
Chapter 1
Introduction
Human blood consists of approximately 55% plasma and 45% blood cells, of
which 99% are red blood cells (RBCs). One important function of RBC is to take up
oxygen in the lungs or gills and deliver it to all body tissues. During the 120-day lifespan of an average RBC, it circulates through the body for approximately 500,000 times
and undergoes repeated severe deformations while squeezing through blood capillaries
and splenic cords. RBC deformability is therefore crucial in maintaining normal blood
circulation. Decreased RBC deformability is both a cause of and biomarker for
potentially severe diseases such as diabetes, sickle cell anemia and malaria. Crossing
splenic interendothelial slits is probably the most stringent challenge on RBC
deformability because the slit size is estimated to be only 1pm. Spleen is therefore
perceived as a mechanical filter which facilitates the clearance of RBCs with reduced
deformability.
Red Blood Cell (RBC) Deformability
Red blood cells are also known as erythrocytes. Mature human erythrocytes are
non-nucleated discoid with average diameter and thickness of 7gm and 2pm respectively.
The biconcave shape evolves from the multilobulated reticulocyte during 48 hours of
maturation first in the bone marrow and then in blood circulation' (Figure 1). As the only
structural component of a mature RBC, the plasma membrane enclosing a large amount
of hemoglobin accounts for all of its diverse antigenic, transport and mechanical
characteristics. Essentially, RBC deformability comprises two major components:
membrane stiffness and cytosol (hemoglobin) fluidity.
Red blood cell membrane is currently viewed as a composite structure in which a
membrane envelope, composed of cholesterol and phospholipids, is anchored to an
elastic network of skeletal proteins via transmembrane proteins embedded in the lipid
bilayer' (Figure 2 and Figure 3). The principal skeletal proteins that form the spectrin
network are a- and P- spectrin, actin, protein 4.1 R, adducing, dematin, tropomyosin and
tropomodulin. Spectrin tetramer, the major structural component of the skeletal network,
is formed by the lateral interaction between a solitary helix of the a-chain from 1 dimer
(at the N-terminus) and 2 helices of the 0- chain from the other (at the C-terminus) to
create a stable triple-helical repeat 2. The shift in dimer-tetramer ratio could be induced by
shear-stress3, oxidative stress 4 or temperature5 and the shift in dimer-tetramer ratio is
believed to have a significant impact on the mechanical stability of RBCs 4. On the other
hand, the lipid bilayer composes both cholesterol and phospholipids (Figure 4). While the
cholesterol is believed to be equally disposed between the two leaflets, the four
phospholipids are asymmetrically distributed. Aminophospholipids including
phosphatidylserine (PS) and phosphatidylethanolamine are usually confined to the inner
leaflet, whereas cholinephospholipids, phosphatidylocholine (PC) and sphingomvelin are
predominately located in the outer leaflet. Several enzyme activities are involved in the
regulation of this membrane lipid asymmetry. "Flippase" moves membrane lipids from
outer to inner leaflet, and "floppases" does the opposite. Because macrophages recognize
and phagocytose RBCs which expose PS at their outer surface, the localization of PS to
the inner leaflet is essential for cells to survive their frequent encounter with
macrophages in the reticuloendothelial system. In fact, loss of lipid asymmetry leading to
PS exposure to the macrophages is believed to play a major role in premature destruction
of thalassemic and sickle cells" 6 .
Hemoglobin (Hgb) is the iron-containing oxygen-transport protein that is
involved in oxygen transportation. Abnormal Hgb concentration or altered Hgb structure
can lead to anemia and other genetic disease. In adult humans, Hgb protein typically
contains 4 subunit proteins non-convalently bound. Each subunit consists of a protein
chain associated with a non-protein heme group (Figure 5). The heme group consists of
an iron ion held in a heterocyclic ring, known as a porphyrin. Under homeostasis, heme is
controlled by its insertion into the "heme pockets" of hemoglobins. However, under
oxidative stress, some hemoglobin may release their heme prosthetic groups which are
highly cytotoxic. It is believed that the iron ion in the protoporphyrin IX ring undergoes
Fenton chemistry to catalyze in an unfettered manner and produces free radicals. This
deleterious effect could indeed play an important role in the pathogenesis malaria 7.
m
ROCS Now ftly
within blood vessel
W
w W
Figure 1: Normal Red Blood Cells in Blood Flow. Adapted from
National Heart Lung and Blood Institute Diseases and Conditions Index 8
Ankyrin
4.1R
Dernatin
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S SlasctTropomyosin
~~~elf
association
site
MmtbY
O
-
protohitament
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Ind dembft
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Figure 2: A schematic model of the red-cell membrane.
This figure was published in: Young NS, Gerson SL, High KA, eds. Clinical hematology. Mohandas
N, Reid ME, Erythrocyte structure, p 36-38.9
Membrane
Aprin
Glycophorin
Figure 3: Red Cell Spectrin Network. Adapted from Sigma Aldrich
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/structuralproteins/spectrin.html0
outer face
hydrophiic (polar} head
Sof phospholipId
s
hydrophobic
(nonpolar)
fatty acid tal
of phosphoipid
/
cholesterol/
inteal (intrinsic) protns
Inner face
peipherat (extrinsic) protein
@ 2007 Encyclopadia Britannica, Inc.
Figure 4: Schematic picture of the lipid bilayer. Adapted from Techno-Science
http://conquerordany.blogspot.com/2011/04/cell-structure-and-function.html"
Figure 5: Structure of human hemoglobin. The protein's a and P subunits are in red and blue, and
the iron-containing heme groups in green. Adapted from PDB IGZX ProteopediaHemoglobin2
Plasmodium falciparum Malaria
Malaria is the most deadly parasitic disease which affects 200 million people
worldwide and accounts for one million deaths annually 3 (Figure 6). The most virulent
malarial parasite Plasmodium falciparum can lead to severe complications and has the
highest mortality rate 4 . Cyclic febrile attack is a characteristic clinical feature of P.
faciparum malaria. The intermittent fever paroxysm corresponds to the release of
merozoites (free parasites) following schizont rupture. During intra-erythrocytic
development, the invasion of merozoites to other red blood cells (RBCs) reinitiates a 48
hour asexual reproduction cycle 5 . The infected cells (iRBCs) then undergo notable
morphological and rheological changes from the ring-form (rings) to trophozoite and
finally schizont (Figure 7). While rings are only moderately less deformable than
uninfected cells (uRBCs), trophozoites and schizonts can be 10 to 50 times stiffer 6 .
Malaria cases (per 100,000) by country, latest available data
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Figure 6: malaria cases. Adapted from
http://www.bu.edu/themovement/2010/10/14/th e-new-d rug-war-against-mal aria/"1
2004
Figure 7: Plasmodium falciparum malaria parasite life cycle. Adapted from
http://www.leidenuniv.nl/en/researcharchive/index.php3-c=217.htm' 8
In the pathophysiology of P. faciparum malaria, the alteration in the dynamic
deformability of iRBCs is critical for their clearance in the spleen. RBCs undergo
repeated deformations to traverse blood capillaries (3-5um) and splenic cordal meshwork.
The narrow splenic inter-endothelial slits (-lum) pose the most stringent mechanical
challenge on RBC deformability' 9. In vivo, only rings can be seen in the peripheral blood
whereas stiffer iRBCs such as trophozoites and schizonts are typically sequestered during
microcirculation. In fact, a substantial proportion of rings would also be retained by
human spleen as illustrated by recent studies on isolated-perfused human spleens20 . The
study suggests that even a subtle deformability shift in the rings could trigger their
clearance in the spleen.
Table 1: Asexual stages of plasmodium malaria
Asexual stages
Ring
Post-invasion time
0 - 24 hours
-
features
Only stage existing in host cell
-
Biconcave shape
-
-
Small "ring" observed on the surface of
host cell
RESA protein interact with spectrin
network
More pigmented regions
Knobs starting to appear on host cell
surface
KHARP protein may enhance the RBC
adhesion
Knobs on the surface of RBC
-
RBC becomes almost spherical
Trophozoite
24-36 hours
-
Schizont
36-48 hours
To better understand the dynamic deformation of RBCs in vivo, the nature of the
RBC deformability should be carefully characterized2 1 . Previously, the deformability of
individual RBCs has been measured by a number of ways, including micropipette
aspiration , atomic force microscopy , and optical tweezers" . Many of these
measurements apply quasi-static loads to attain notable deformation. The deformability
of the cells is hence characterized by the shear and bending moduli of the cell membrane.
However, when RBCs circulate in the blood capillaries and splenic cordal meshwork,
their ability to deform is also a time-dependent response which conventional static
measurements fail to correlate directly. The best and most direct way to study cells'
dynamic deformation under viscous fluid flow is to develop a microfluidic "deformability
cytometer" which simulates in vivo flow condition.
Another important aspect in simulating in vivo RBC deformation is the effect of
temperature. Almost every malarial patient displays intermittent fever, which
pathophysiologically corresponds to the onset of schizont rupture. Yet, how the febrile
conditions stimulate changes in the mechanical properties of both normal and parasitized
cells is still unclear. Recent studies shed light on the vital role of temperature in malaria
pathophysiology by demonstrating significant stiffening of rings at febrile conditions21 .
Fever could then be perceived as part of the body's protective mechanism which
facilitates more efficient parasite clearance in human spleen by stiffening the infected
cells. The attachment of parasite-derived protein, Ring-infected Erythrocyte Surface
Antigen (RESA), is believed to be responsible for the stiffening of infected cells at febrile
conditions . Compared to the iRBCs, the temperature effect on co-cultured uninfected
RBCs has often been disregarded, although the main cause of malarial anemia is the
massive loss of uninfected RBCs in patient' 9 . An important question posed is whether
this loss of uninfected RBCs has any biomechanical reason (e.g. stiffening of uninfected
RBCs), and if so, how we can understand the mechanism of such mechanical changes in
both iRBCs and uninfected RBCs.
Clinical studies revealed that patients treated with anti-malarial drug exhibit a
more rapid decline in parasitemia. However the accelerated parasite clearance is delayed
or even obscured in splenectomized patients2 5 . Therefore, active splenic retention is
believed to be the underlying mechanism facilitating rapid parasite clearance after antimalarial treatment' 9 . As spleen could mechanically filter stiffer cells from
microcirculation; the more efficient splenic clearance after drug treatment seems to
suggest that some of the tested anti-malarial drugs may be able to modify the mechanical
properties of healthy or parasitized cells. However, there is a lack of experimental proof
to confirm the exact role of anti-malarial drug on RBC deformability. In this project, we
would focus on two most popular anti-malarial drugs that have relatively fewer drug
resistance reported: Malarone (Proguanil) and Artemisinin with its derivatives. Two
questions are to be addressed in this thesis: 1) whether these two anti-malarial drugs have
similar mechanical impact on ring stage infected RBCs; 2) whether the drugs have any
adverse effect on healthy (uninfected) RBCs.
To answer these questions, our group previously introduced a microfabricated
'deformability cytometer' which measures dynamic mechanical responses of many
(-1000) RBCs in a population 2 6 . Distinct from conventional tools for single cell analysis,
our microfluidic device is able to process approximately 10 cells per second. The high
throughput enables us to measure a considerable proportion of cells from population,
permitting high sensitivity and low sampling error. In this thesis, we employ this tool to
investigate the subtle RBC deformability changes in response to temperature changes and
drug treatments, both for infected and normal (uninfected) RBCs. Enhanced
deformability difference between healthy and infected RBCs at febrile temperature, or
after drug treatment (but not in an additive manner) are shown from our experiments.
This observation suggests that temperature or drug may act on similar biological pathway
to stiffen the iRBCs in order to facilitate splenic clearance. Also from this study, we
demonstrated that this simple, high-throughput and inexpensive device could also be
potentially used as a malaria diagnostic tool in rural Africa where advanced imaging tools
are unavailable.
Chapter 2
Existing Tools for RBC Deformability
Measurement
As the red blood cell deformability plays a key role in blood circulation under
both physiological and pathophysiological conditions, searching for the right tool to
characterize RBC deformability has been an active research field over a few decades.
Several measurement techniques were developed which measure red cell "deformability"
either statically or dynamically. In this chapter, more commonly used measurement tools
are outlined and discussed. Some of these tools are able to produce single cell analysis
with low throughput whereas others are bulk measurements with high throughput but fail
to reflect individual trait.
RBC Deformability Measurement by Optical Tweezers (OT)
Optical tweezers (OT) generate force via a highly focused laser beam which could
trap micron-sized dielectric particles and manipulate sub-nanometer displacements. The
dielectric particle that is trapped by the laser beam experiences an attractive force
towards the laser beam focus and the restoring force is linear with respect to the
displacement between the particle and the focus. In this way, the force acting on the
particle can be modeled as a simple spring which follows Hooke's law:
F= -kU
where the constant k is the trap stiffness depending on the OT design and the particle size.
size. Experimentally, k is usually in the order of 50pN/pm, which corresponds to a
resolution of 0.5pN in force measurement.
Though the OT technique has been used to characterize micron particles for
almost 20 years, its application to measure cell deformability was fairly recent. In the
paper published by Mills et a 24 in 2007, OT was used to measure single RBC forcedisplacement response, from which membrane shear modulus was derived based on
computational modelings 2 7 . The schematic set up for a typical OT experiment is sketched
in Figure 8.
Figure 8A: picture of optical tweezers stretching a red cell. Adapted from
http://www.bioeng.nus.edu.sg/nanolab/galleryresearch.htm 2 1;
B: schematic diagrapm of optical tweezers. Adapted from Wikipedia:
http://en.wikipedia.org/wiki/Opticaltweezers#Physics of optical-tweezers 29
One distinct advantage of optical tweezers measuring RBC membrane stiffness is
its high precision and specificity. By stretching the beads that are strategically attached to
cell surface, optical tweezers can stretch RBCs in one or more directions 0 . Several
limitations are also associated to this method. For example, typically the maximum
optical force is limited to several hundred pico-Newton, which could be insufficient to
induce large deformation in the cells commonly encountered in vivo. Moreover, care
needs to be taken to avoid overheating the cells by laser light during operation.
RBC Deformability Measurement by Micropipette Aspiration (MP)
Micropipette aspiration is one of the most classic methods that measure RBC
membrane stiffness. During a typical MP experiment, RBCs are usually diluted by 1000
times from whole blood and suspended in a saline solution with comparable osmolarity.
A small micropipette with inner diameter between 1 to 3 pm is carefully inserted in the
RBC suspended solution and the target RBC is aspired into the mouth of the pipette
under a known suction pressure AP.
Compared to OT, MP is a more versatile method for measuring the mechanical
properties of living cells as the suction pressure could ranges from 0. 1pN/pm 2 to almost
atmospheric. That means this technique can probe soft, fluid-like cells, such as RBCs, as
well as very rigid cells. On the other hand, the high flexibility of red cells and their
biconcave shape often cause "buckling" problem while performing MP experiment. That
means, even at a low suction pressure, it is very easy for the entire cell to be sucked into
the micropipette. Additionally, as a tool aimed to measure RBC membrane property, a
considerable amount of hemoglobin would also be sucked into the pipette, depending on
the size of pipette. Though many complex models try to accommodate these variations in
pipette inner diameter, it remains a question whether measured membrane stiffness can
be consistent with pipettes of different sizes. Figure 9 illustrates a micropipette aspirating
a malaria infected red blood cell.
-
s---.
Figure 9: picture of micropipette aspiration of a malaria infected red blood cell. Adapted from
http://www.bioeng.nus.edu.sg/nanolab/galleryresearch.html 28
RBC Deformability Measurement by Ektacytometry (LORCA)
Ektacytometer is another method to measure cell deformation in Couette flow and
was first developed by Bessis and Mohandas in 1975 3. The basic idea is to suspend cells
in the narrow gap between two concentric cylinders and apply various shears on the cells
by rotating the outer cylinder at different speed. As a laser beam is passed through the
cell solution, the cells scatter the light to form a diffraction pattern which is circular at
low shear force and becomes ellipsoidal at higher shear. The ratio of the major and minor
axis of the ellipsoid indicates how deformable the cells are under a given shear force,
and the deformability index (DI) was properly defined as follows:
DI =
A- B
A +B
where A is the major axis of the ellipsoid and B is the minor axis of the ellipsoid.
Distinct from MP and OT measurement, Laser-assisted Optical Rotational Cell
Analyzer (LORCA) ektacytometry measures the average deformability of cell
populations. Therefore, as a bulk measurement tool, ektacytometry has a much higher
throughput and is capable of producing population-wide trait but it fails to reflect the
deformability of individual cells in a given population. This becomes an important
drawback when the target cells to be studied form a minority population in a given
sample, such as in malaria culture.
RBC Deformability Measurement by MicroFluidic devices (MF)
Most of the above mentioned measurement methods may be a good indicator of
how flexible a red blood cell is, but it is still difficult to correlate the calculated DI or
shear modulus directly with the in vivo blood flow. Hence, several microfuidic devices
were designed to mimic the RBC deformation in small capillaries.
The first realistic in vitro microfluidic realization of in vivo RBC deformation was
presented by Brody et al in 1995 3 (Figure 10). Though Brody et al. did not attempt to
measure malaria infected red blood cells, the pillar array structure proposed in that work
is the basis of the current malaria-deformability cytometry used in this project.
Comparing the single capillary system by Shelby et al. Brody's pillar array structure has
several distinct advantages: 1) the pillar array structure minimizes cell-cell interaction,
achieving single cell accuracy measurement; 2) clogging is less likely a problem as there
are several channels in parallel and the constriction is fairly short; 3) the pillar array
structure could be a better depiction of in vivo RBC deformation in microcirculation as
the cells have to undergo "repetitive" "severe" deformations while each deformation time
is not necessarily long.
Figure 10: Video frames of healthy RBCs passing through a 4pm constrictions. Picture adapted
from Brody et al. Biophy J 1995"
In 2003, Shelby et al developed a microfluidic model for single-cell capillary
obstruction34 . Red blood cells at different stages of malaria infection were passed through
the microchannel of 2, 4, 6, and 8pm size. It was found that while Ring-stage infected
erythrocytes were able to pass through all constricted channels, cells with later infection
stage exhibited decreased ability to squeeze through the microchannels and Schizont
stage infected erythrocytes were blocked even at 6 pm channel. At 8 pm channel, the
channel size is already comparable to average RBC size and little deformation is needed
to pass through the channel. (Figure 11). Though this experiment did not derive cell
deformability in a very rigorous manner, it demonstrated several in vivo concepts such as
"pitting" and "capillary blockage". However, this technique would probably suffer from
serious clogging issue and no quantitative data on cell deformability can be obtained
directly. Cell-cell interaction may play a significant role and it is difficult to interpret
single cell deformability based on this channel design.
aIm
Spm
6~±m
4iim
4Lum
Rings
Early
Trophozoites
Late
Trophozoites
Schizonts
Figure 11: Video images of four stages of malaria-infected RBCs (early ring stage, early trophozoite,
late trophozoite, and schizont) passing through microchannel with different size (2, 4, 6, and 8 sm).
Figure adapted from Shelby et al PNAS 2003 3
Based on the similar design principles first proposed by Brody et al, Bow et al.
optimized the pillar array structure for malaria diagnostic and related deformability
studies. The "microfluidic cytometry" could detect the minute deformability shift from
healthy to Ring stage malaria infected RBCs 26 . This optimized device is used for this
project and the Figure 12 depicts how single cells could pass through repeated 3 Prn
constrictions
Figure 12: Video frames of healthy RBCs passing through a 3sam constrictions.
Compare the aforementioned measurement tools which are commonly used to
probe RBC deformability, each has its own advantages and limitations in terms of
throughput, precision, specificity, and ease of operation. However, distinct from many
other material stability tests, the need for RBC deformation study is built on its
physiological importance. Therefore, when performing these deformation tests, it is
important to recreate a physiologically relevant environment, such as shear rate, pressure
gradient etc. At very low shear rate, the pseudo-static measurement could overlook the
inherent viscoelasticity of cell membrane and leads to misrepresentation. On the other
extreme when very high shear rate is applied, the red blood cells could be permanently
damaged. As RBCs pass through the splenic slit in vivo, shear rate is estimated to be -10
sec' and this number could be important potentially in interpreting the difference in
experimental results by different measurement tools3 5 . The table below summarizes
several key features of above mentioned measurement tools.
Table 2: Key features of different tools measuring RBC deformability
Tool
OT
(Mills et al24 )
MP
(Nash et a122)
LORCA
(Bessis
et
al
MF
(Bow et alf)
32
Dynamic
/Static
Static
Single cell /
Bulk
Single cell
Throughput
<1 cell / min
Shear Rate
(sec-)
7-50
Static/dynami
Single cell
<1 cell / min
<1
Static
Bulk
~ 1000 cell/min
50036
Dynamic
Single cell
~ 100 cell/min
10-100
C
)
Chapter 3
Materials and Methods
Device Fabrication
Layout program CleWin3.0 was used to design our microfluidic device, which
2
consists of a 500 x 500ptm 2 inlet reservoir, a 500 x 500 pLm
outlet reservoir, and parallel
capillary channels with triangular pillar arrays. Three different pore-size of 2.5, 3.0 and
4.0 pm were designed for the capillary channels to test optimum deformation condition
for the experiment (Figure 13). A silicon mold of the device was made using standard
silicon processing techniques. The photolithography step was done using a 5X reduction
step-and-repeat projection stepper (Nikon NSR2005i9, Nikon Prevision) and reactive-ion
etching (RIE) techniques were used to give the mold a final depth of 4.2pjm. Final device
was then casted from the silicon mold using polydimethylsiloxane (PDMS) and was
sealed by a glass slide using oxygen plasma.
a. Silicon Mold Fabrication
Silicon molds were fabricated at the Microsystems Technology Laboratories
(MTL) with the help of the staff members. The table below describes the process flow to
fabricate these glass devices.
Starti g material: 6" p test wafers, GREEN process in TRL/ICL
Step Action
Machine
Parameters
Cleaning
1_1
Piranha cleaning
Acid-hood
5 min; hydrogen peroxide:sulfuric acid
=
1:3
Pattern
Alignment
Marks
2_1
Resist coating
Coater 6
2 2
2 3
Exposure
Development
Etch
Stepper
Coater 6
ICL, ipm thickness,
4600rpm
ICL, 170ms
ICL, "PUDDLE2"
"T1HMDS",
Alignment Marks
3_1
Reactive Ion Etch
AME5000
ICL,
"ISABELLA LTO" :descum:20s, oxide
etch 10s
"pan Si etch" etch rate: 4-4.5nm/s. etch
35s
3 2
Resist removal
Pattern
Asher
3 min
Coater 6
ICL,
Microchannels
4_1
Resist coating
1p m
thickness,
"T1IHMDS",
4600rpm
4 2
4 3
Exposure
Development
Stepper
Coater 6
ICL, 170ms
ICL, "PUDDLE2"
TRL
"JBETCH" etch rate 2-2.5pm/min. Etch
Etch
Microchannels
5_1
Reactive Ion Etch
STS2
52
Resist removal
Asher
3 min
P1O
Measure 4 corners + centre point and
take average.
2 mi
Measurement
6_1
Measure final depth
b. PDMS-Glass Device Fabrication (Soft Lithography)
Polydimethylsiloxane (PDMS) is prepared by mixing 10:1 w/w base to curing
agent and degassing under vacuum for at least 1 hour. The degassed mixture is poured
onto the silicon master and postbake at 65'C over overnight. Cured PDMS is peeled from
the master following punching the 1.5mm holes at the reservoirs. Tubings will be
connected to the reservoir during subsequent experiment.
PDMS-glass bonding is made by plasma treatment of both PDMS and glass slide
for 1 minute. A strong bond forms instantaneously. Annealing on a hotplate at 95'C
for >1 hour significantly increase the bond strength. Detailed steps are outlined in the
table below:
1) Pre-clean the PDMS with tapes and preclean the glass slide with ethanol followed
by water rinsing.
2) Place the PDMS and glass in the vacuum chamber and turn on the pump.
3) Make sure the plasma is at the "off' position. Make sure the valve is fully closed.
4) Wait 60 second.
5) Turn the plasma on to HIGH. Fine tune the needle value counterclockwise until
the a bright purple color is observed.
6) Wait for 60 second.
7) Turn off the plasma and vacuum pump. Release the valve slowly.
8) Remove the PDMS and glass from the chamber and press them together.
Parasite Culture
P. falciparum 3D7A parasites (from Malaria Research and Reference Reagent
Source, American Type Culture Collection) were cultured in leukocyte-free human RBCs
(Research Blood Components, Brighton MA) in RPMI 1640 complete medium as
described 3 7 . Parasites cultures were synchronized by sorbitol lysis3 8 two hours after
merozoite invasion.
Solution Preparation
1x Phosphate buffered saline (PBS) was mixed with 1% w/v Bovine Serum
Albumin (BSA) (Sigma-Aldrich, St. Louis, MO) as a stock solution and was freshly
made on every experimental day. For experiments tracking fluid flow velocity, 200 nm
FluoSpheres europium luminescent microspheres (Molecular Probes, Eugene, OR) were
used at a final concentration of 5.0 x 10-4 percent solids. For experiments involving only
healthy RBCs, fresh whole blood (Research Blood Components, Brighton, MA) was used
at 100 times dilution (i.e. 1Il whole blood with 99 pl stock solution). For experiments
involving parasitized cells, Iml of cultured cells were spun down at 1,000rpm for 5
minute; 1 pl of the pellet was then aliquot to 200 pl stock solution.
1 pl of 50 pg/ml Cell Tracker Orange (Invitrogen, Carlsbad, CA), which stains the
membrane of the cell, was added to aforementioned 100 pl healthy RBC solution for
better imaging, 20 minutes before the experiment. To ensure no adverse effect on the cell
deformability was induced by the dye, a control experiment of same RBC solution
without staining was performed. No statistically significant difference in deformability
was found between the sample with and without staining.
10 l of 1 x 10-6 M thiazole orange dye (Invitrogen, Carlsbad, CA), which stains
the RNA of the cell, was added to the aforementioned 200 pl iRBC solution 20 minutes
before the experiment. The infected cells would appear bright under the green
fluorescence filter set (488nm excitation) whereas the uninfected cells were seen as dark
shadows.
Experimental Protocol
To accurately control the ambient temperature, the microscope surface was
replaced by a heating chamber (Olympus), which was preheated to a desired temperature
range (i.e. 30-40 0 C) for 30 minutes before the beginning of every experiment. Meanwhile,
the PBS-BSA stock solution was injected into the device to coat the PDMS walls to
prevent adhesion. This filling step need not be done inside the heating chamber, but the
PBS-BSA filled device needed to be placed into the heating chamber at least 5 minutes
before loading 10 pL of diluted blood sample in order to guarantee the temperature
stability. During temperature calibration phase, a thermocouple thermometer was used to
probe the exact temperature inside the heating chamber. When the temperature needed to
be adjusted to a different value, at least 5 minutes of waiting time was required to ensure
a new stable ambient temperature.
To apply a constant pressure gradient across the device, the pressure difference
between inlet and outlet reservoir was generated hydrostatically by fixing the difference
between inlet and outlet water column height. To ensure the pressure difference is
constant throughout the experiment, a large volume (60ml) syringe was selected to be
connected to the inlet reservoir, so that the water column height would not vary
significantly within several hours.
A
Heating chamber
Inleth
Outlet
-
.A..X.A.Tubing
\Glass slide
PDMS dev lce
Tbn
101pm
FHold
10 pm
F
3 pim
]"m
3 pmn
Figure 13: Experimental schematics. A heating chamber (Olympus) was mounted to the inverted
microscope stage. Four heaters for the inner water bath, microscope stage, chamber top, and lens
were designed to accurately control the ambient temperature inside the chamber. The PDMS-glass
bonded device consists of the inlet and outlet reservoirs and main channels with triangular pillar
arrays. It was placed inside the heating chamber with only the inlet reservoir connecting to an
external syringe via a 20cm-tubing. The reservoirs were 500x500 Pm2 squares with 20 sminterspacing cylindrical pillar arrays; these pillar arrays could pre-filter white blood cells from whole
blood, allowing only RBCs to pass through the main channels. Each of the parallel channels was 10
pillars wide and 200 pillars long. Along the flow direction, the inter-pillar spacing was 10 pm. This
spacing would allow deformed cells to recover and ready for subsequent deformations.
Perpendicular to the flow direction, the pore size varied from 2.5 to 4 pm for different channels. Cells
would experience different level of deformation when passing through these pores.
To capture the movement of RBCs inside the microchannels, a CCD camera
(Hamamatsu Photonics, C4742-80-12AG, Japan) was connected to the inverted
fluorescent microscope (Olympus IX71, Center Valley, PA). Images were automatically
acquired by IPLab (Scanalytics, Rockville, MD) at 100ms time interval and the postimaging analysis was done using ImageJ (NIH, http://rsb.info.nih.gov/ij/). The velocity of
individual RBCs was defined as the distance the cells moved divide by the time in
seconds.
The device constitutes a series of equally spaced triangular pillar arrays with pore
size ranging from 2.5 to 4um (Figure 13 of Chapter 1 illustrates the case of 3um pore
size). Compared to the diameter of an average RBC (~8um), the considerably smaller
pore sizes are designed to impose similar mechanical constrains on the cells as if they are
passing through blood capillaries and splenic meshwork. Driven by constant pressure
gradient in the sub-Pascal-per-micrometer range, RBCs are able to deform substantially
at each constriction and traverse along the channel 26. The dynamic deformability of
RBCs is then characterized by their velocity: the ability to deform repeatedly in order to
pass through multiple constrictions in series. Stiffer cells with longer entrance time at
each constriction would assume lower velocity and vice versa.
Chapter 4
Effect of Febrile Temperature on the
Deformability of Malaria-infected Erythrocytes
In this chapter, we study the temperature dependent deformability changes of
iRBCs and uninfected RBCs (uRBCs). One important note: Once the blood is extracted
from the body and cultured / maintained ex vivo for an extended period of time, RBCs'
chemical and mechanical properties shift significantly over time (storage lesion)39' 4 0
Therefore, as a proper control for the iRBCs (which underwent extended parasite culture
processes), 'uRBCs' in this thesis designates uninfected RBCs contained in the malaria
culture. On the other hand, healthy RBCs (hRBCs) in this thesis mean freshly drawn
(from less than a day old sample in vacutainer) RBCs.
Figure 14 presents microscopic screenshots illustrating both iRBCs and cocultured uRBCs moving in the microchannel at different temperature conditions. The
uninfected cells appear as dark shadows indicated by blue arrows, and the infected cells
with thiazole orange (TO) staining appear as bright dots indicated by white arrows. The
velocity of individual cells can then be derived by recording the time period for each cell
passing through 10 constrictions in series (i.e. equivalent to 200pjm distance travelled). In
this work, cell velocity is used to characterize the dynamic deformability of individual
RBCs. Larger cell velocity value corresponds to larger RBC deformability). The
temperature-dependent modifications on the dynamic deformability of both uRBCs and
iRBCs are investigated. The clinical relevance of these results is discussed on the basis
that human spleen mechanically filters stiffer RBCs19 .
Temperature-dependent iRBC deformability
Figure 15A demonstrates the temperature-dependent modification on iRBC deformability.
The velocity of infected cells exhibited a significant raise from 300C to 370C followed by
a notable drop at 40 0 C. The peak at 370 C marked the optimum temperature for maximum
iRBC deformability.
To investigate the deformability increase from 300C to 370C, several factors were
taken into consideration including cell membrane viscosity, intracellular fluid viscotiy,
buffer solution viscosity as well as possible confounding effects caused by the
modification of cytoskeletal structure and membrane proteins. It was noted that the PBS
buffer viscosity decreases by 33% from 19 C to 37 C and the blood viscosity decreases
by 2% for every degree C temperature increment (i.e. -31% decrease from 19 to 37'C).
At a given pressure gradient, elevated temperature would therefore increase the bulk fluid
flow speed, leading to a projected increase in cell velocity measurement. For better
comparison, normalized cell deformability was measured by performing the experiment
at equalized bulk fluid velocity over all temperatures. Assuming the combined viscosity
shift in iRBC and PBS buffer solution is linear, we computed the pressure gradient to be
applied at each temperature for constant fluid flow. This calibration was also
experimentally verified by measuring fluid velocity via 200nm non-deformable
polystyrene beads (Figure 15B). With constant beads speed of 226 ptm/s, the normalized
cell deformability (Figure 15C) inside 4pm-channel was found to be fairly constant from
300 C to 37'C. This result is consistent with similar measurements using other techniques,
such as micropipette aspiration and optical tweezers24 . Compare Figure 15A and C, we
concluded that observed -50% increase in cell deformability from room to body
temperature was predominantly caused by temperature related viscosity change in both
iRBCs and PBS buffer solution. Other factors such as the cytoskeletal structural
modification, membrane protein alternation played a minor role.
On the other hand, the significant drop in iRBC velocity between 37 C and 40 C
was preserved at constant local fluid velocity (Figure 15C). This remarkable stiffening
effect at febrile condition agrees with recent deformability measurement by optical
tweezers 24. The attachment of parasite-derived protein, Ring-infected Erythrocyte
Surface Antigen (RESA), is believed to be responsible for the notable drop in cell
deformability at febrile temperatures24 . While RESA would be necessary for the
parasitized cells to survive heat-induced damages, the protein related stiffening also
facilitates more efficient splenic clearance. Detailed discussion on RESA effect is
presented in a separate section.
Os
1s
2s
infected cel
<-> : distance moved by a uninfected cell over one second time period
: distance moved by an Infected cell over one second time period
Figure 14: Experimental images of iRBCs (white arrows) and uRBCs (blue arrows) in 3pm channels.
Driven by a constant pressure gradient of 0.36 Pa/sm, the cell motion was tracked at three different
temperatures: 30 oC, 37 oC, and 40 oC. While a uninfected cell appeared as a dark shadow, the GFPtransfected parasite inside an infected cell would be seen as a small fluorescent dot. The red and
black arrows indicated the distance moved by iRBCs and uRBCs respectively. With one second time
interval, the lengths of the arrows revealed the velocity of cells. The images on the top right corner
illustrated how a cell would pass through the pores.
uW T
50-
~CED
00
30
30
3500
10P
0
20
0
-
00
200
>
CO
100
80
3 00-
020
80
4
00
50
0
1
3C
Figre 305 ACostat
PesureGraieT
elvlc
0
3Csemperature
EI
pltfrinetdcll0asn
though 3pim channels. B 200nm microspheres were used to track fluid velocity at different
temperature and by adjusting the pressure gradient, equalized fluid velocity is achieved. C Constant
Fluid Velocity Cell velocity vs. temperature plot for infected cells passing through 4 pim channels.
Data was obtained at a constant local fluid velocity of 230 pim/s as calibrated by beads. Translated to
pressure gradients, the gradient applied was 0.36 Pa/sm at 30 *C, 0.312 Pa/sm at 37 "C, and 0.288
Pa/sm at 40 "C
Temperature-dependent uRBC deformability
Whereas iRBC deformability is commonly accepted as an important factor in the
pathogenesis of p. falciparum malaria, the temperature-dependent modification on cocultured uRBC deformability has often been overlooked. With the understanding that the
human spleen is capable of sequestering stiffer cell during microcirculation ,
deformability of uRBCs, as well as the deformability separation between iRBCs and cocultured uRBCs, are indeed important in order to better understand the mechanism of
splenic retention.
Figure 16 demonstrates the temperature-dependent modification on uRBC
deformability. Similar to that of iRBCs, the uRBC velocity increased significantly from
30*C to 37"C. From 37 "C to 40 "C, instead of a 40% drop displayed by iRBCs, the
decrease in uRBC deformability was subtle but still statistically significant (p <0.01).
Following the same viscosity argument as presented earlier; normalized uRBC velocity
was also measured at equalized bulk fluid velocity inside 4pm-channel. The result was
similar to that of normalized iRBC deformability: no significant change in the normalized
uRBC deformability was observed from 30"C to 37'C, and the velocity drop from 37"C to
404C was preserved.
The result again confirms the major role of viscosity in influencing the RBC
deformability from 30'C to 370C. Compared to the 50% drop in the average deformability
of iRBCs from body to febrile temperatures, the temperature-dependent effect on uRBC
deformability is subtle but still statistically meaningful (P<0.01). This result indirectly
confirmed that the significant drop in iRBC velocity at febrile condition is mainly due the
effect of the parasite-derived protein RESA, and is not something inherent in nonparasitized cells. This result is also consistent with similar measurement using other
techniques.
100
80
120
A
E60
100.
B
S80-
A
60400
0
Z
200
-
,20
30
37
Temperature (0)0
4b
40
2
030
0
C
0
37 C
40 OC
Figure 16 A Constant Pressure Gradient: Cell velocity vs. temperature plot for uninfected cells
passing though 3pm channels. B Constant Fluid Velocity Cell velocity vs. temperature plot for
uninfected cells passing through 4 pm channels.
Temperature-dependent healthy RBC deformability
The dynamic deformability of healthy RBCs (hRBCs) from room temperature (25
C) to febrile temperature (40 0C) was measured, in order to be compared with the result
for uRBCs (Figure 17). Similarly, under constant pressure-driven scheme, the hRBCs
became more deformable from room to body temperature, and the deformability peaked
at 370 C. The measured temperature-dependent RBC deformability was independent of
the thermal history of the sample, which was confirmed by changing the order in which
measurements are made at different temperature values. In addition, temperature-induced
deformability changes were reversible under the conditions tested (from 25 to 40 "C),
meaning that returning a sample to its original temperature restored the deformability
value measured at that temperature.
120-
"U
100*
4-pimchannel
Ave fluid flow =226 pm/s
1000
80-
80-
6020-
66
0
40-
240-
20-
27.5
30.0 32.5 35.0 37.5
Temperature (0C)
40.0
0.36OPa/pm
0.312Palpm
0.288Pa/pm
0
30
3mr7
TemfPerature (0C)
40
Figure 17 A Constant Pressure Gradient: Cell velocity vs. temperature plot for healthy cells passing
though 3pm channels. B Constant Fluid Velocity Cell velocity vs. temperature plot for healthy cells
passing through 4 pm channels. Data was obtained at a constant local fluid velocity of 230 pm/s as
calibrated by beads. Translated to pressure gradients, the gradient applied was 0.36 Pa/pm at 30 "C,
0.312 Pa/sm at 37 *C, and 0.288 Pa/pm at 40 *C
Febrile condition enhances the separation resolution between iRBCs
and uRBCs
The deformability separation resolution between normal and parasitized cells is
previously noted as the key parameter for efficient splenic clearance of infected RBCs.
The temperature-dependent cell deformabilities for both iRBCs and co-cultured uRBCs
were simultaneously measured at 30'C, 37*C, and 40'C (Figure 18). The deformability
separation resolution Rs between normal and infected cells was analyzed using the
formula below, where X1, X2 and o1, o-2 denote the mean and standard deviation of
normal and infected cell mobilities. A higher Rs value implies better separation.
SX2 - X1
2(o1 + q 2 )
While at all tested temperatures the infected RBCs displayed statistically
significant stiffening compared to uninfected cells (p < 0.001), the deformability
separation resolution between uRBCs and iRBCs enhanced substantially with increasing
temperature (Table 3). At 30'C, the separation resolution was only 0.28; the value
increased to 0.46 at body temperature, and to 0.94 at febrile condition. Furthermore, at
40 'C, the average iRBC velocity was 3.02a (i: standard deviation of uRBC velocity
distribution) away from the average uRBC velocity. This result implied a more sensitive
and specific deformability differentiation between normal and parasitized RBCs at febrile
2 32
condition The result is consistent with similar measurement using other techniques ,
and reconciles with our hypothesis that febrile condition facilitates more efficient splenic
retention. The velocity difference between normal and parasitized cells can be explained
by the RESA effect.
Table 3: temperature dependent resolution
Temperature ("C)
Resolution (R,)
30
0.28
37
0.46
40
0.94
120
120
30 0 C
100-
37 0 C
100-
80-
80-
60-
60
40-
40*
20-
20-
01
Uninfected RBCs
Infected RBCs
Uninfected RBCs
Infected RBCs
120
40 0 C
10080-
6040 -
20 0Uninfected RBCs
Infected RBCs
Figure 18 Cell velocity of both infected and co-cultured uninfected cells passing though 3sm
channels at different temperatures. Approximately 600 RBCs were tracked at a pressure gradient of
0.36 Pa/ sm. The velocity of uninfected RBCs was found to be statistically higher than parasitized
cells at all temperatures, and the cell velocity difference between uRBCs and iRBCs is maximised at
febrile condition (40 *C).
Spleen as a mechanical filter
Spleen is believed to work as a mechnical filter which removes stiffer cells from
circulation. The splenic retention model has been hypothesized by Buffet in his recent
review paper 22. To quantitative illustrate splenic clearance, Figure 18 is replotted into
Figure 19 at body (370C) and febrile (40C) temperatures. A hypothetical 'filtration
threshold' value can be assumed such that all the RBCs with velocity below that value
will be retained (filtered) at spleen. This threshold cannot be too high, because it will lead
to the loss of too many too many normal RBCs. If this splenic filtration threshold is set
too low, then many of iRBCs will pass and survive the splenic clearance mechanism.
Presumably, the splenic filtration threshold in vivo will be determined (by evolution)
based on this tradeoff.
Our result shows that splenic mechanical filtration process can be more 'specific'
at fever temperature, due to the greater separation between iRBCs and uRBCs. For
example, if the 'filtration threshold' is set to be 34ptm/s, which is 2a away from the
average uRBC velocity measured at 370C. At 370 C, only 12 out of 25 iRBCs traverse
below the threshold velocity, indicating the efficiency of splenic filtration of iRBC at
370C is only 48%; however at 40C, 24 out of 25 iRBCs has a velocity value lower than
34um/s, suggesting 96% of iRBCs will be cleared by spleen at febrile condition.
The significant improvement in splenic filtration efficiency (from 48% to 96%)
suggest a potentially important role of fever in the pathophysiology offalciparum malaria,
and in splenic clearance in general. On the other hand, as we compare the splenic
retention of uninfected RBCs at body and febrile temperatures, we found that while only
1.5% of uRBCs would be removed from blood stream at 37 0C, 9% of uRBCs are below
the threshold velocity at 40 0C. The exact mechanism why febrile condition would mildly
reduce uRBC deformability is still unclear, but the significantly increased amount of
uRBC removal might be the related to the pathology of malarial anemia.
10
37 OC
uninfected RBCs
EM52Infected RBCs
3
10-
:0
0
0
10
20
30
40
50
_
60
70
80
91
RBC Velocity (gm/s)
RBC Velocity (tim/s)
100-
80I
'I
I
/
I
I
I
I
/
10
20
30
40
50
60
70
80
RBC Velocity (gm/s)
Figure 19: A Cell velocity histogram with normal fit for both infected (red curve) and co-cultured
uninfected (black curve) RBCs at 37 "C. For the uninfected RBCs, their mean velocity and
standard deviation were (52.02, 9.41). For the infected RBCs, their mean velocity and standard
deviation were (33.04, 11.61). B Cell velocity histogram with normal fit for both infected (red
curve) and co-cultured uninfected (black curve) RBCs at 40 *C. For the uninfected RBCs, their
mean velocity and standard deviation were (44.82, 8.19). For the infected RBCs, their mean
velocity and standard deviation were (21.29, 5.87). The normal fit graphs at 37 "C and 40 "C were
overlaid in C.
Chapter 5
Effect of Anti-malaria Drugs on the
Deformability of Malaria-infected Erythrocytes
As one of the most deadly infectious disease, malaria is responsible for over 200
million clinical cases in 200941. Though a number of anti-malarial drugs were widely
used clinically, antimalarial drug resistance has increasingly emerged to be a major
public health problem which hinders the control of malaria. Figure 20 illustrates the dire
situation of chloroquine resistance in malaria endemic areas. While the urgent need for a
new malaria drug is clearly justified, it is also important to understand how these malaria
drugs are working. In fact, the exact drug mechanism of artemisinin - one of the most
commonly used malaria drug currently - is still an active area of research. In this report,
several existing malaria drugs including artemisinin and malarone are studied to examine
their effect on the deformability of both healthy and malaria-infected red blood cells. It is
hoped that by studying these existing drug, we could gain a better understanding of the
mechanisms of drug action in vivo.
Figure 20: Chloroquine resistant in Malaria-endemic countries in the Eastern Hemisphere. Adapted
from http://wwwne.cdc.gov/travel/yellowbook/2010/chapter-2/malaria.htm
Artemisinin and its derivatives
Figure 21: Chemical structure of artemisinin and its derivatives. Adapted from Wikipedia:
http://upload.wikimedia.org/wikipedia/commons/a/a5/Artemisininskeletal.svg 2 9
Artemisinin, also known as qinghaosu, was extracted from a traditional Chinese
herbal Qinghao for the treatment of fever 2000 years ago . Only in the late 1960s,
Chinese scientists discovered that it is also a natural candidate for malaria treatment: it
could clear malaria parasite from the body faster than any other drug known. The peak
plasma concentration is reached within 1-2 hours and the half-life of the drug is only 2-3
hours. The rapid absorption as well as fast parasite elimination makes Artemisinin an
efficient curative drug for the treatment of Plasmodium malaria.
Multidrug resistance has always been a serious problem in the treatment of
malaria. As a relatively new anti-malaria drug, artemisinin and its derivatives made a
remarkable impact on the reduction of P.falciparum malaria. However in recent years, an
increasing number of clinical cases report treatment failures with artemisinin
monotherapy.
a) Current Hypotheses on the Mechanisms of Artemisinin Drug Action
Though the exact mechanism of artemisinin drug action is still unclear, clinical
studies have shown two important characteristics of artemisinin derivatives: 1) The endoperoxide bridge is believed to be mainly responsible for the drug action.
Deoxyartemisinin, an artemisinin analog lacking endoperoxide bridge, is clinically
proven devoid of antimalarial activity, indicating the important role of peroxide in the
drug action43 . 2) Artemisinin is toxic to malaria parasite at nanomolar concentration and
has relatively benign toxicity to normal red blood cells even at micromolar
concentration 44 . It is very likely that artemisinin would have adverse impact on
uninfected red blood cells too, at much higher dosage. The malaria infected red blood
cells seem to have much higher drug uptake capability, which enhances the drug
selectivity. 4 5 Based on the endo-peroxide structure, Meshnick attempted to explain
artemisinin drug mechanism in terms of free radicals and his argument is summarized
below 44.
Table 4: Artemisinin drug mechanism from the perspective of free radicals
Claims
Free
radicals is
directly
responsible
for the
drug action
Experimental evidence
* Free radical scavengers antagonized the
in vitro antimalarial activity Free
radical generators promoted
antimalarial activity
* Lipid peroxidation observed after
artemisinin treatment
Free
radical
formation
via heme
*
Reported by
Krungkrai and Yuthavong,
198746
Scott et al. 198941
Wei and Sadzadeh, 199448
Berman and Adams, 199749
* Peroxide are source of reactive oxygen Halliwell and Gutteridge 5
species
e Oxidant and antioxidant confirmed free Levander et al 198951
radical effect in vivo and in vitro.
Meshnick et al 198943
Senok et al, 199710
e
Artemisinin
interacted
with Meshnick et al 1991"1
intraparasitic heme, which might be
able to activate artemisinin inside the
parasite into free radicals
Malaria parasite is rich in heme-iron, Rosenthal and Meshnick,
explaining the selectivity of artemisinin.
1996
b) Dynamic Deformability Measurement
In order to clarify any biomechanical consequence of artemisinin treatment on
RBCs, we measured dynamic deformability changes of drug treated red blood cells in
vitro.
In our experiment, artesunate, one artemisinin derivative that is used clinically,
was used to treat cultured P. falciparum malaria in vitro. The deformability of both
iRBCs and co-cultured uRBCs were measured at 2, 4 and 6 hours after artesunate drug
treatment. A synchronized culture of ring-stage iRBCs with -15% parasitemia was
resuspended at 0.1% hematocrit in malaria culture medium containing 0.01, 0.05 or 0.1
pg/ml of artesunate. (Clinically used dosage for artesunate treatment is 0.1 g/ml.) All
experiments were performed at 37'C.
Error! Reference source not found. demonstrates a pronounced stiffening effect
after artesunate treatment for both iRBCs and co-cultured uRBCs. After 6 hour artesunate
treatment, a 30%-50% velocity decrease is observed, while smaller and less pronounced
velocity decrease is found at both 2 and 4 hours after artesunate treatment. After 4 hour
drug treatment, within the effective dosage range of 0.01 to 0.1 pg/ml, no statistically
significant dose dependence is found in terms of velocity changes.
Black Circles: Parasite free
120- Red Squares: Infected
* Control:
free incubation
-
.
100-
Controli Drug:
0
2hr incubation
Drug:
4hr incubation
Drug:
6hr incubation
0
80E 60-
o
Jo
JD
nM
D0
400
0
:n013
::0 o
j
0
0
o00
o:r
aic
M
0
M0
a
*0
M0
0
0
0
0
0
0
20-
0
0l
Ej
Ill
0.00
0.01 0.05 0.10
0.01 0.05 0.10
Drug Concentration
0.01 0.05 0.10
(gg/ml)
Figure 22: Artesunate drug effect on the deformability of ring stage malaria cells. The black circles
represent co-cultured uninfected RBCs and the red circles represent the ring stage infected RBCs.
Malarone (Proguanil)
At present, artemisinin is not used as a preventive drug for travelers going to
malaria-endemic countries possibly due to its relatively short half time (2~3 hours).
Malarone, a combination of two anti-malaria agents (atovaquone and proguanil
hydrochloride) is commonly prescribed as both preventive and curative drug. The
chemical structure of proguanil is shown in Figure 23.
H
N
CI
IO
H
H
NYWN
NH
NH
CH3
CH3
Figure 23: Chemical structure of proguanil. Adopted from
http://en.wikipedia.org/wik/Proguanil5 2
a) Current Hypotheses on the Mechanisms of Proguanil Drug Action
Proguanil acts as a dihydrofolate reductase inhibitor which prevents the
formation of tetrahydrofolate. As a type of antifolate, proguanil acts mainly during
DNA and RNA synthesis and is cytotoxic during the S-phase of the cell cycle. It has a
greater toxic effect on rapidly dividing cells which replicate DNA more frequently. In
the context of plasmodium malaria, it is hypothesized that proguanil is able to exert
toxic effect on the infected cells and selectively kill malaria parasite. On the other
hand, the chemical structure of proguanil also suggests its possible role as an oxidant.
In fact, proguanil oxidation has been studied both in vivo" and in vitro .
b) Dynamic Deformability Measurement
In our experiment, the deformability of both iRBCs and co-cultured uRBCs
were measured at 2, 4 and 6 hours after proguanil drug treatment. A synchronized
culture of rings with -15% parasitemia was resuspended at 0.1% hematocrit in
malaria culture medium containing 0.1, 0.4 or 0.8 pg/ml of proguanil. (Clinical
dosage for proguanil 0.1 pg/ml as a preventive treatment and is 0.4 ig/ml as a
curative treatment). All experiments were performed at 37 C.
Figure 24 demonstrates a evident stiffening effect after proguanil treatment for
both iRBCs and co-cultured uRBCs. After 6 hour proguanil treatment, a 30%-50%
velocity decrease is observed, while smaller and less pronounced velocity decrease is
found at 4 hours after proguanil treatment while no significant stiffening is observed
after 2 hour proguanil drug treatment. For 6 hour drug treatment, within the effective
dosage range of 0.1 to 0.8 pg/ml, no statistically significant dose dependence is found
in terms of velocity changes.
Control: 6 hour
70 1
.
_.
.
...
60
50
E 40
=L
%-o
30
O
*
20
10
0
2 hour
6 hour
Control 400 800
100
4 hour
400
800
100
6 hour
400
800
concentration (ng/ml)
Figure 24: Proguanil drug effect on the deformability of ring stage malaria cells. The black circles
represent co-cultured uninfected RBCs and the red circles represent the ring stage infected RBCs.
Chapter 5
Effect of the Ring-infected Erythrocyte Surface
Antigen (RESA) on the Deformability of Malariainfected Erythrocytes
RESA, also known as Pfl55, is a 155-kDa protein which contains two repetitive
sequences. Between the two repeat regions, RESA has a segment of 70 residues with
similarity to the human DnaJ chaperone proteins, suggesting a possible chaperone-like
property of RESA 55 . Synthesized in mature-stage parasites, RESA is stored in organelles
known as dense granules. Upon invasion, it is released into the host cell cytosol, and
subsequently binds to repeat 16 of p-spectrin (pR16) and stabilizes the spectrin tetramer
relative to the dimer". Studies demonstrate several biomechanical consequence of RESA
binding to host cells including increased membrane stiffness 24, reduced susceptibility to
heat shock 2 7, and increased resistance to multiple malaria invasion.
In this study, a transgenic resal gene knock-out P. falciparum clone and its resal
242
revertant clone were generated by transfection of P. falciparum FUP/CB (resal+)24,27
This technique has been patented by the Institut Pasteur France and the parasites are
prepared by research scientist Monica Diez-Silva.
Previously, the effect of RESA on the membrane stiffness of ring stage iRBCs has
been reported in vitro by optical tweezers experiment2 4 . In this section, we investigate the
effect of RESA on the dynamic deformability of iRBCs and co-cultured uRBCs. Figure
25 plots RBC velocity for three parasite cultures: wild-type RESA1+, resal-KO, and
resal-rev parasites. Measurement was done at 37'C with constant bulk fluid flow in 3pm
channels.
In Figure 25A, the average velocity of wild-type iRBCs was 14.1 pm/s,
significantly lower than that of co-cultured uRBCs (25.91 pm/s). However, after
knocking out RESA (Figure 25B), the iRBCs harboring resal-KO parasites (22.6 pim/s)
displayed similar deformability as co-cultured uRBCs (24.5 pm/s). No statistical
significance in the stiffness was detected between resal-KO iRBCs and co-cultured
uRBCs. Compare Figure 25A and B, the infected cells with RESA knocked out is
considerably less stiff than wild type resal+ iRBCs.
To ensure the observed stiffness difference between resa-KO iRBCs and wild
type iRBC are indeed due to the absence of RESA, we knocked in RESA and tested the
stiffness of resal-rev iRBCs (Figure 25C). The resal-rev iRBCs (16.832pm/s) display
similar stiffness as that of wild type resal+ iRBCs and are significantly stiffer than cocultured uRBCs (28.167pm/s). In terms of the deformability of uRBCs, no significant
difference was detected among wild-type resal+, resal-KO and resal-rev parasite
cultures.
Significant stiffening of parasitized cells was observed at febrile condition,
demonstrating a physiologically important role of fever in the pathogenesis of
Plasmodium falciparum malaria. The effect of RESA was also investigated at elevated
temperature. Figure 26 compares the wild-type iRBC stiffness at body and febrile
temperatures. The average cell velocity at 40 C was 10.23pm/s, significantly stiffer than
that at 37 "C (14.145pm/s, p<0.00 5 ). Similar trend was observed for resal-rev parasite
cultures. In contrast, the average cell velocity for resal-KO iRBCs at 40 C is 22.6pm/s
and no statistical significance can be concluded when comparing with resa l -KO iRBCs at
37 "C (24.56pim/s). This observation confirms that RESA is responsible for the feverrelated stiffening of infected cells.
A
40
U)
E
40-
o
30-
30
20-
10-~
0
4
20-
Q
-
10100
nI
parasite-free RBCs
parasite-free RBCs
resal+
50
C
40-
E
300
0
20-
100
10-p-r
parasite-free RBCs
resa1-rev
resal-KO
Figure 25 Cell velocity of both parasite-free
RBCs and Ring-stage malaria infected RBC
at body temperature A) Wild type; B) with
RESA knock out; and C) with resa knock in.
The black circles represent parasite-free
RBCs, the red circles represent wild-type
infected RBCs, the blue circles represent
infected RBCs with resa knocked out, and
the green circles represent infected RBCs
with resa knocked back in.
resa -KO
resa 1+
Ap=0.00256
E
" 30-
E430-
0
0
* 20-
0
0
00
V.
73 20-
I
37 *C
10-
00
0--
40 *C
37 *C
40 *C
resa -rev
C
p=0.00953
00
37 "C
400 C
Figure 26 Cell velocity of Ring-stage malaria infected RBC at body and febrile temperature A) Wild
type; B) with RESA knock out; and C) with resa knock in. The black circles represent parasite-free
RBCs, the red circles represent infected RBCs
Chapter 6
Discussion
The temperature- or drug- dependent deformability study has been performed for
healthy (hRBC), co-cultured but unparasitized (uRBC), and parasitized RBCs (iRBC).
Several observations (listed below) can be made when we reconcile all the results, which
will be discussed in depth here. These observations provide novel and valuable insights
on the pathology of malaria and RBC's structure / function in general.
(1) 37C (normal body temperature) seems to be the 'optimum' temperature for
maximum deformability for all cell types.
(2) Whereas the decrease in defornability from 370C to 40 0 C is subtle for healthy and
uninfected RBCs, a 50% fall in iRBC velocity was observed, indicating a probable
role of fever in facilitating more efficient parasite clearance by human spleen. RESA
is identified as the parasite protein which is responsible for the iRBC stiffening
especially at febrile temperature.
(3) Mechanically stiffen the infected red blood cells may be one consequence shared by
all antimalarial angents, which possibly enhances splenic filtration of parasites in the
similar way as febrile temperature.
(4) The splenic retention model suggests a possible the connection between malarial
anemia and uRBC clearance.
Body temperature corresponds to the maximum hRBC deformability
Temperature-dependent deformability for all cell types including iRBCs, uRBCs
and hRBCs were investigated (Figure 15, 16 and 17). For all cell types, body temperature
seems to (37 C) correspond to the maximum RBC deformability and different levels of
cell stiffening were observed at febrile temperature. Whereas the stiffening trend in
iRBCs was well documented by other measurement methods 24 and can be explained by
the enhanced expression of RESA at febrile temperature, it is unclear why hRBCs and
uRBCs become less deformable at febrile temperatures. One possible explanation could
be the heat-induced expression of heat shock proteins. Hsp70 proteins are made up of two
regions: the amino terminus is the ATPase domain and the carboxyl terminus is the
substrate binding region5 6. The Hsp70 chaperones help to fold many proteins and Hsp70
assisted folding involves repeated cycles of substrate binding and release. The observed
subtle drop in cell velocity at febrile temperature could possibly due to heat-induced
expression of Hsp7O which regulates the spectrin chain 57
From the measurement perspective, the fairly constant normalized deformability
from room to body temperature (Figure 17) is consistent with prior measurements by
micropipette aspiration 58, optical tweezers24, magnetic twisting cytometry , and
ektacytometry 2 . In contrast, the previously noted abrupt increase in RBC deformability
at the transition temperature near body temperature 59 was not observed. This discrepancy
is possibly due to the considerably much larger deformation force required for the 1.3pim
micropipette experiment5 9 . On the other hand, the subtle RBC stiffening trend from body
to febrile temperature was not uniformly observed in earlier experimental measurements:
similar observation was reported in magnetic twisting cytometry measurement ; general
stiffening in the average shear modulus was illustrated by optical tweezers but no
statistical significance was reported 24; and no stiffening effect was detected by
micropipette aspiration experiments 58. The slight inconsistency among different
measurement techniques could be explained from several factors, including the
magnitude and duration of shear stress, shear rate, and competing effect of viscosity vs.
elasticity. The RBC deformability measurement by rotating disc demonstrates that
different magnitude and duration of shear stress would lead to different temperaturedependent deformability trend60 . Additionally, magnetic twisting cytometry as well as our
microfluidic cytometer measure the coupled effect of shear, bending moduli and the
relaxation time, while other measurement techniques may be dominated by a subset of
these factors. For example, the viscous effect of cells is inherently dynamic, and depends
on several factors such as temperature, spectrin network fluctuation , and the relaxation
time. Its impact on cell deformation in our experiment will be clearly different from
many stiffness measurements which apply quasi-static force for large deformations3o 6 1 6, 2
To resolve the cofounding effect caused by viscosity and to simulate the quasi-static
loading conditions in micropipette aspiration and optical tweezers, we repeated the
experiment at much lower pressure-gradient of 0.072 Pa/pm and 0.120 Pa/ptm. (Figure 27)
No statistical significance in these measurements can be found.
A
B
0.072 Pa/pm
120'
E o-
.
00
40-
30
80 -
0
40
0
C
0
00
0
0
o
-
20"
60 -
20-
0.120 Pa/pm
50 -
-
0.072 0.120
0.240
Pressure gradient (Palpm)
0.360
0
I
3
0
37 C
=.424
p=0.18
0
40C
3fC
40 C
Figure 27: Cell velocity measurement repeated at different pressure gradient (a) and at both body
and febrile condition (b). Neglegible effect from TO staining is seen on the velocity measurement
Fever in the pathogenesis of malaria
Unlike a very subtle stiffening in hRBCs and uRBCs, the ring stage malaria
infected cells display nearly 50% drop in their average velocity in the device at febrile
temperature. This remarkable stiffening of iRBCs at febrile temperature was also reported
in recent studies by optical tweezers 24 and magnetic twisting cytometry 1 . The ring-stage
parasite-derived protein RESA was previously identified to be responsible to alter the
deformability of infected cells 55 . By binding to PR16, RESA constrains the cytoskeletal
structure of infected RBCs by preventing spectrin from undergoing heat-induced
conformational changes, thereby increasing infected RBC survival at febrile temperatures.
Yet, this stiffening may facilitate splenic clearance of infected RBCs from the circulation.
The stiffening role of RESA has been confirmed in our dynamic deformability
measurement as shown in Figure 25 and 26. At febrile temperature, the wild-type
infected RBCs are significantly stiffened (p<0.005) whereas the resa-ko iRBCs do not
display significant temperature-induced stiffening (p=0.186).
One important biomechanical consequences of iRBC stiffening could be an
enhanced splenic retention of infected RBCs. The human spleen filters RBCs based on
rigidity, among other factors. By reducing the deformability of parasitized cells, fever
increases the separation resolution between normal and infected RBCs, facilitating more
efficient splenic filtration of parasitized RBCs. The uRBC population is inherently
diverse, due to age and other factors6 3 and the wide velocity distribution uRBCs are likely
attributed by this diversity. The separation resolution between uRBCs and iRBCs can
potentially serve as a non-chemical biomarker in the malarial diagnosis.
Antimalarial Drug Effect
The impact of anti-malarial drugs on the dynamic deformability of ring stage
RBCs as well as co-cultured uninfected RBCs is investigated in vitro. Two antimalarials
were tested independently: Artesunate and Proguanil. Though the two drugs are believed
to have different mechanisms of action, the change in cell deformability after drug
treatment is seen to be similar. For infected RBCs, this drug-induced stiffening could lead
to increased parasite splenic retention, and may even constitute a part of drug mechanism.
This result is consistent with in vivo experiments, which demonstrate a markedly
longerparasite clearance time in splenectomized patients, regardless the antimalarial drug
that has been administered19.
In the experiment assessing artesunate effect on RBC deformability, the
separation resolution between iRBCs and uRBCs was doubled after 6 hour artesunate
treatment, suggesting that artesunate may be responsible for enhanced specificity and
efficiency in splenic parasite clearance. Clinical studies performed in patients with and
without a spleen also confirmed that artemisinin or one of its derivatives is indeed
actively involved in process of splenic parasite clearance25 . For the purpose of this project,
two specific questions are to be addressed on the mechanism of artesunate action: 1)
whether the mechanical stiffening caused by artesunate is drug specific, i.e. other
antimalarial agents do not induce mechanical stiffening; 2) whether our result provide
additional information to current theory on artesunate drug mechanism.
To answer the first question, another common anti-malaria drug, proguanil, is
tested in vitro. Proguanil is well known to act as antifolate which inhibits the forrmation
of tetrahydrofolate . Though acting in a different way compared to artesunate, proguanil
exhibits a similar stiffening trend on both uRBCs and iRBCs (Figure 24). This result
brings us to ponder the similarity between the two common antimalarial agents. One key
feature in artemisinin and its derivatives is its endoperoxide bridge chemical structure.
Deoxyartemisinin, an artemisinin analog lacking endoperoxide bridge, has been clinically
proven to be devoid of antimalarial activity 4 4 . As we discussed briefly in the previous
chapter, the endoperoxide bridge is capable of generating oxygen free radicals and
exerting oxidative stress on the red blood cells. In short, artesunate-mediated oxidation 47
could be an important attribute for the mechanism of drug action. On the other hand,
though there is a lack of in vivo studies on the oxidative stress exerted by proguanil in the
context of malaria, proguanil oxidation has also been reported both in vivo 5 3 and in
vitro54.
By looking at the chemical structures of other common antimalarials, we could
see almost all the antimalarials, such as mefloquine, chloroquine, and pyrimethamine
display similar chemical structure that can characterized as oxidant. That observation
makes us to hypothesize that most antimalarials could share the same chemical attribute
of imposing some level of oxidative stress on RBCs, leading to mechanical cell stiffening.
The stiffened cells are more prompted to be filtered out from human spleen.
To verify the hypothesis that the drug induced oxidative stress would reduce the
deformability of RBCs, the effect of the third drug, pentoxifylline, on RBC deformability
is studied in vitro. Pentoxifylline is well known as a radical scavenger 4 and its
antioxidant properties have been studied in vitro65 . Our microfluidic experiment with
pentoxifylline indicates a significant increase in hRBC deformability (Figure 28), which
is consistent with our hypothesis that oxidative stress could modulate RBC deformability.
25-
Healthy 1day old blood
000
20-
E
00
0
00
15-
%ow
0L 10-0
5
0
0
0
0
0
00000
00
0
0
0
0
oo
000
0
0O
0
oooo
00
control
2hr 20
4hr 100
Figure 28: Pentoxifylline induced RBC softening. Red circles represent healthy RBCs without
pentoxifylline treatment. Green circles present healthy RBCs with 2 hour pentoxifylline treatment at
the dosage of 20pg/ml. Blue circles present healthy RBCs with 4 hour pentoxifylline treatment at the
dosage of 100pg/ml
To address the second question raised, an immediate question would be the
selectivity and specificity of drugs induced cell stiffening. While the drugs are capable of
stiffen the infected RBCs, would the uninfected RBCs be stiffened inadvertently? Indeed,
our microfluidic experiments find a subtle but significant (p<0.005) drop even in uRBC
velocity after antimalarial drug treatment, indicating the drugs may have adverse effect
(oxidative stress) on uninfected cells as well. However, the efficiency of these drugs
evolves as they induce much greater stiffening in iRBCs. This stiffening will occur for
both uRBCs and iRBCs, but more precipitously for iRBCs, leading to a filtration
'separation' between uRBCs and iRBCs at the spleen.
It is interesting to note that drug-induced RBC deformability changes are similar
to that by febrile temperature. While the stiffening of iRBCs at febrile temperature is
probably due to RESA protein, the molecular mechanism for drug-induced stiffening of
both iRBCs and uRBCs are not yet fully known. Still, it is well known that oxidative
stresses act on cells in a similar manner as heat shocks, and the two processes are
significantly related66 . One hypothesis we have is the oxidative stresses may enhance the
expression RESA or other heat-shock proteins just in the similar way as febrile
temperature does. While the oxidative stress affects the heat shock proteins in hRBCs
only slightly (as we have discussed in earlier section), the expression of RESA is
responsible mainly for the increased stiffness on iRBCs under temperature or oxidative
stress. But the effect is unlikely to be additive as both mechanism may have similar
pathway by binding to similar location of the P-spectrin4 24 ,ss
On the other hand, drug-induced "pitting" (i.e. removing intraerythrocytic parasite
without destroying the host RBC) was previously noted by Chotivanich et al 7 as an
alternative mechanism of the artesunate drug action. Though this "pitting" effect is not
the main interest in this report, it could be potentially investigated carefully by optimizing
the device structure and flow conditions.
Reduced uRBC deformability may exacerbate malaria anemia
Either under febrile temperature or after antimalarial drug treatment, two
interesting but previously unappreciated observations were made in the alternation of
uRBC mechanical properties: (1) the unparasitized but malaria parasite co-cultured RBCs
(uRBC) are less deformable than healthy RBCs (hRBC); (2) the minute but statistically
significant drop in uRBC and hRBC deformability from body to febrile temperature
(Figure 16, 17, 23 and 24). These subtle modifications on uRBC stiffness may render
additional support on the splenic retention model which was hypothesized by Buffet et
al.19 .
Several factors could account for the first observation such as the source of the
cells and sample preparation methods. The healthy RBCs were obtained from fresh blood
cells within 2-days old whereas the uninfected RBCs analyzed were parasite co-cultured
RBCs which in average are much older than fresh cells3 9' 4 0. This additional ex-vivo
culture conditions may render the average uRBCs stiffer. An alternate explanation could
also be that healthy RBCs (hRBCs, which has not seen parasites and infected RBCs) are
inherently more deformable than uninfected RBCs (uRBCs, which was co-cultured with
iRBCs), possibly caused by parasite-derived proteins or other factors. Some clinical
studies suggested that a proportion of the co-cultured but non-parasitized cells are
invaded by parasite molecules21,22,68,69 which may consequently stiffen the cells. To
verify whether the observed stiffening of uRBCs compared to hRBCs is indeed due to
exposure to parsite molecules, we compared the in vitro deformability of uRBCs and
hRBCs drawn from the same patient and perform the same sample preparation method.
Figure 29A shows that after 2 day culture condition, hRBCs and uRBCs exhibit the same
120
120
m
[m~] hRBC with TO staining
100
100 uRBC, with TO staining
10
- 80 .
-0
[
hRBC with TO staining
] hRBC, without TO staining
100-
4
EV
60
80
60-
>40-
>
20-
8
40 20-
0
hRBC, with TO
uRBC, with TO
0hRBC, with TO
hRBC, w/o TO
Figure 29 A. Compare the in vitro deformability of uRBCs and hRBCs. B Compare the effect of TO
staining on the hRBC deformability measurement.
velocity as measured in vitro. This indicates that the experimentally observed uRBC
stiffening is largely due to incubation condition and cell aging process39
The second observation - a minute but still statistically significant drop in
uRBC/hRBC deformability under febrile temperature or after drug treatment - is very
interesting. The molecular mechanism for this is still unclear: it could be due to the
expression of heat shock proteins (triggered either by heat or by oxidative stress), which
lead to a slight modification of the spectrin networks6 , or regulate the membrane lipid
polymorphism6'70 . Notwithstanding, the physiological implication of uRBC / hRBC
stiffening is significant. Malarial anemia is the most frequent but severe manifestations of
malaria7 1 . Massive loss of red blood cells, which causes malarial anemia, cannot be
entirely attributed to the destruction of infected RBCs (iRBCs), which usually constitute
only a small fraction of total RBCs in patients. Instead, the major cause of malarial
anemia is that many uninfected RBCs (uRBCs) are lost in patients' blood, mostly in the
spleen and / or the liver19 . Malaria-related dyserythropoiesis is likely a minor factor,
because a complete removal of erythropoiesis brings about only a minor decrease in RBC
population7 2 . The temperature or drug related uRBC/hRBC stiffening might indeed be
one of the causes behind malarial anemia.
Chapter 9
Conclusion and Future Plans
In conclusion, our result has the important implication that the efficiency with
which diseased RBCs is cleared by the spleen may be directly dependent on elevated
body temperature. Speculatively, our findings suggest an important role of fever in
enhancing splenic clearance of less deformable parasite-infected RBCs from the
circulation at febrile temperatures. On the other hand, fever may aggravate the loss of
uninfected RBCs which in the worst case may inadvertently lead to malarial anemia. The
studies on the effect of antimalarial drug treatment suggest mechanical stiffening of
infected red blood cells may be one trait shared by different drugs with dissimilar
mechanism of action.
Future plans of this project could involve several directions. From an engineering
aspect, the deformability cytometry modified and optimized to apply to different cell
lines in which mechanical properties are of an important biomarker. Preliminary
experiments have been performed on MCF7 breast cancer cells and mesenchymal stem
cells confirming the feasibility. On the other direction, more in-depth studies are desired
for the red cell deformability under different environmental and biochemical stresses. For
example, most antimalarials are oxidant; whether the oxidation stress is a key in
influencing the red cell deformability is still unclear. It is also be interesting to
investigate whether the oxidative stress can indeed simulate temperature stress and
activate related heat shock proteins in the red blood cells and hence modify RBC
deformability.
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