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Mechanical Characterization of Red Blood Cells Using Microfluidic Devices
by
Zhensong Xu
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Department of Mechanical and Industrial Engineering
University of Toronto
© Copyright by Zhensong Xu 2018
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Abstract
Mechanical Characterization of Red Blood Cells Using Microfluidic Devices
Zhensong Xu
Doctor of Philosophy
Department of Mechanical and Industrial Engineering
University of Toronto
2018
The mechanical properties of red blood cells (RBCs) have been proven to play important roles in
the regulation of various biological activities and have shown clinical significance. During the
past few decades, a number of research studies have been conducted to understand the
relationship between RBC mechanical properties and diseases. The work described in this thesis
focuses on characterizing mechanical properties of RBCs from sickle cell trait (SCT) carriers and
the degradation/recovery of stored RBCs in transfusion medicine.
The mechanical properties and changes of SCT RBCs under deoxygenated and acidic
environments, two typical conditions present in the circulation of athletes undertaking strenuous
exercise, were measured. The results revealed that SCT RBCs are inherently stiffer than RBCs
from non-SCT healthy subjects, and a lower pH further stiffens the SCT cells. Furthermore, at
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both normal and low pH levels, deoxygenation was found to cause no change of SCT RBCs
stiffness.
In transfusion medicine, compromised RBC deformability can lower the transfusion efficiency or
intensify transfusion complications. This thesis reports microfluidic mechanical measurement of
stored RBCs under the physiological deformation mode. The results revealed that the effective
stiffness of RBCs increases over the storage process. RBCs stored for one week already started
to show significantly higher stiffness than fresh RBCs, and stored RBC stiffness degraded faster
in the last three weeks than in the first three weeks.
Although the stiffness of stored RBCs degrades over the storage process, whether the stiffness of
stored RBCs can be reversed after transfusion remains unknown. A microfluidic platform was
used in this thesis to measure the evolution of RBC stiffness recovery under in vivo-like
conditions. RBCs stored up to 6 weeks (42 days) in the blood bank were measured, revealing that
the degraded stiffness of RBCs over the storage process can be recovered in human serum within
120 minutes. However, the recovered stiffness of older RBCs (stored for 4-6 weeks) is still 1.6 –
2.1 times higher than that of fresh RBCs. Furthermore, ATP, which provides energy to keep
RBC membrane mechanically functional, was also measured. The ATP concentration in stored
RBCs was found to increase in human serum.
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Acknowledgements
The past four years I spent in my Ph.D. study have been the most rewarding years in my life. I
would like to express my appreciation to all those who provided me the possibility to complete
this degree. My deep gratitude goes first to my advisor, Prof. Yu Sun, for all his advice and
guidance throughout the past four years at the University of Toronto. I have learned a great deal
from Prof. Sun’s energy and critical thinking, which helped me shape this thesis. I often
encountered difficulties to address some of the comments from Prof. Sun during my research,
however, it was those comments that helped me learn fast and significantly improved my
following work. Thinking thoroughly is not only applied in my research, but also expands out of
academia, which allows me to efficiently create solutions for questions in different aspects. Prof.
Sun’s support is also what I am much appreciated. His supportive and knowledgeable comments
always recharge my energy in research, and he encouraged me to attend international
conferences where my presenting skills were improved and my knowledge was broadened. Prof.
Sun’s constant encouragement and tremendous help during my PhD study always inspired and
motivated me to be passionate for research.
My thesis is challenging, and I am glad to have the support from my great collaborators. I would
like to express my great appreciation to Dr. Chen Wang for his close collaboration and valuable
guidance and discussions. Whenever I needed samples or discussions, Dr. Wang was always
willing to help. I thank Prof. Axel Guenther for his knowledgeable advice in my committee
meetings which greatly improved my research. I also thank Prof. Xinyu Liu and Prof. Francis
Lin for serving on my defense committees.
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I am glad to be surrounded by many talented people during my graduate study. They did not only
provide me enormous help with my graduate study, they are also my valuable friends who I feel
fortunate to have. I am especially grateful to Yi Zheng, Yuan Wei, Xian Wang and Wenkun who
spent endless hours working with me. I thank Jun Wen for being a fabulous lab mate and friend
for years, who showed me the beauty of Toronto when I first came to this lovable city, and I
enjoyed the fun conversations we had during the past years; Devin Luu for being a reliable friend,
who gave me a lot of support at sin & redemption and helped me edit this thesis; Changhong for
working out together and giving me advice in research. I would also like to thank all past and
present members of the Advanced Micro and Nanosystems Laboratory for all their helpful
discussions and encouragements throughout the years.
Finally but not least, I wish to thank my parents for their encouragement and love over the years.
They always respect my interest and support every decision I make in my life, which gives me
the courage to move forward without hesitation. They are also my best friends who share my joy
and sorrow. I am far away from home but never far away from my parents.
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Table of Contents
Acknowledgements ...................................................................................................................................... iv
Table of Contents ......................................................................................................................................... vi
List of Figures ............................................................................................................................................ viii
1. Introduction ........................................................................................................................................... 1
1.1. RBC introduction .......................................................................................................................... 1
1.1.1. Membrane lipids ................................................................................................................... 3
1.1.2. Membrane proteins ............................................................................................................... 4
1.1.3. Skeleton proteins ................................................................................................................... 5
1.2. RBC stiffness measurement .......................................................................................................... 7
1.2.1. Filtration ................................................................................................................................ 7
1.2.2. Micropipette aspiration ....................................................................................................... 10
1.2.3. Atomic force microscopy .................................................................................................... 12
1.2.4. Optical tweezers .................................................................................................................. 12
1.2.5. Magnetic twisting ................................................................................................................ 15
1.2.6. Quantitative phase imaging ................................................................................................. 17
1.2.7. Microfluidic measurement .................................................................................................. 18
1.3. Pathophysiological conditions and RBC stiffness ...................................................................... 23
1.3.1. Diseases ............................................................................................................................... 23
1.3.2. Malaria ................................................................................................................................ 24
1.3.3. Sickle cell disease ............................................................................................................... 25
1.3.4. Sickle cell trait .................................................................................................................... 27
1.3.5. Stored RBCs ........................................................................................................................ 28
1.4. Research objectives ..................................................................................................................... 31
2. Stiffening of sickle cell trait red blood cells under simulated strenuous exercise conditions ............. 32
2.1. Introduction ................................................................................................................................. 32
2.2. Methods ....................................................................................................................................... 33
2.2.1. Blood specimens ................................................................................................................. 33
2.2.2. Device and measurement .................................................................................................... 34
2.3. Results and discussion ................................................................................................................ 37
2.3.1. Determination of RBC shear modulus ................................................................................ 37
2.3.2. SCT RBCs become stiffened under lower pH .................................................................... 38
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2.3.3. Hypoxia does not induce SCT RBC stiffening ................................................................... 42
2.4. Discussion ................................................................................................................................... 45
2.5. Conclusion .................................................................................................................................. 46
3. Stiffness increase of red blood cells during storage ............................................................................ 48
3.1. Introduction ................................................................................................................................. 48
3.2. Device fabrication ....................................................................................................................... 50
3.3. Methods ....................................................................................................................................... 52
3.4. Experimental Results .................................................................................................................. 57
3.4.1. Effective stiffness is not velocity dependent under low velocities. .................................... 61
3.4.2. Temperature effect on RBC effective stiffness ................................................................... 63
3.4.3. RBC effective stiffness increases during storage. ............................................................... 65
3.5. Discussion ................................................................................................................................... 67
3.6. Conclusion: ................................................................................................................................. 71
4. Stiffness and ATP Recovery of Stored Red Blood Cells in Human Serum ........................................ 73
4.1. Introduction ................................................................................................................................. 73
4.2. Methods ....................................................................................................................................... 75
4.3. Results ......................................................................................................................................... 78
4.3.1. Stiffness recovery of stored RBCs in human serum ........................................................... 78
4.3.2. Fresher RBCs reaching steady-state shear modulus faster .................................................. 81
4.3.3. Temperature effect on RBC stiffness recovery ................................................................... 83
4.3.4. Shape recovery of stored RBCs in human serum ................................................................ 85
4.3.5. ATP concentration recovery of stored RBCs in human serum ........................................... 87
4.4. Discussion ................................................................................................................................... 90
4.5. Conclusion .................................................................................................................................. 95
5. Conclusions ......................................................................................................................................... 96
5.1. Contributions ............................................................................................................................... 96
5.2. Future directions ......................................................................................................................... 99
6. Bibliography: .................................................................................................................................... 102
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List of Figures Figure 1.1 (a) RBC takes oxygen from lungs and releases the oxygen to tissues. (b) RBC’s thickness at
the thickest point is 2 – 2.5 µm and the minimum thickness in the center is 0.8 – 1 µm. RBC has a
dimeter of 6 – 8 µm. .............................................................................................................................. 2
Figure 1.2 Current model of the RBC membrane. A phospholipid bilayer is tethered to the spectrin
network (spectrin α and spectrin β proteins) via a number of proteins such as band 3, ankyrin, protein
4.1, and Glycophorin. Spectrin α and spectrin β form the structure underneath cell membrane and
provide mechanical support. Most of the known proteins are shown [26]. .......................................... 6
Figure 1.3 (a) An array of parallel micro channels. (b-d) Artificial microvascular networks mimicking
vessels can be used to measure the deformability of red blood cells. ................................................... 9
Figure 1.4 (a) Different micropipette aspiration methods used to measure the mechanical properties of red
blood cell (RBC) membranes. (b)Microfluidic pipette aspiration of RBCs using a funnel channel
chain. ................................................................................................................................................... 11
Figure 1.5 (a) Optical tweezers measuring the RBC deformability with focused laser beams that transfer
linear momentum or angular momentum of light. To measure the shear modulus of the RBCs, two
microbeads are attached to the opposite sides of a RBC and force was applied by the optical tweezers.
(b) Experimental observations of RBC deformability measurement using optical tweezers, and
simulation results. (c) 3D image of an RBC measure by AFM. ......................................................... 14
Figure 1.6 RBCs with magnetic beads bound to the surface. SEM image of an RBC with a bead on the
surface. C) Magnetic field is applied onto the bead to generate a torque to deform the RBC. d)
Mechanical simulation to predict the deformation by the force applied by the magnetic bead.
Permission is not required (thesis/dissertation)................................................................................... 16
Figure 1.7 A network of microfluidic channels to measure RBCs deformability. The transit time of the
individual cells are recorded using a high-speed camera. (b) A microfluidic system for mechanical
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characterization of RBCs. The transit time is obtained from electrical impedance signal captured
when RBCs are passing through the constriction channel. ................................................................. 19
Figure 1.8 (a)Hydrodynamic RBC deforming microfluidic device. Cells are focused to the center lines of
the channels and deformed by fluid. Cell deformation is measured by analyzing images recorded by
a high-speed camera. (b)Laser diffraction technique incorporating microfluidic rheometer to measure
RBC deformability with elongation index defined. ............................................................................ 22
Figure 2.1 (a) Schematic of the microfluidic device for RBC mechanical property measurement. RBCs in
solution of different pH levels are loaded into the middle channel and adhere onto the glass channel
bottom. Oxygen level is accurately varied by pumping air or nitrogen into the two side channels.
Schematic illustration of the deformation of a cell element. (b) RBCs are deformed under shear stress
(1.8 kPa). After the release of shear stress, RBCs recover to their original shape .............................. 36
Figure 2.2 (a) Shear modulus of SCT RBCs under physiological pH level 7.35 (blue) and acid pH level of
6.85 (red). Error bars represent standard error of the mean value. All samples (n=30-70 for each
sample) show a significant difference between different pH levels (*p < 0.05). (b) Shear modulus of
normal RBCs does not reveal significant difference under different pH levels (p value > 0.05; n=30-
70 for each sample). Error bars represent standard deviation. c) Box plot showing the summarized
shear modulus of RBCs from the 7 tested SCT samples and the 7 tested normal samples under
different pH conditions(**p=5.5 × 10-15,***p=2.2 × 10-29,****p=1.9 × 10-82; n=200-300). ........ 42
Figure 2.3 At physiological pH 7.35 (a) and low pH 6.85 (b), normal RBCs elastic modulus did not
change significantly when the channel was deoxygenated (*p>0.75). (c)(d) For SCT RBCs,
although shear modulus became higher at pH 6.85 than at pH 7.35, deoxygenation did not induce
further increase (*p>0.75). (e) Summarized shear modulus of SCT RBCs under different oxygen
levels, and no difference was observed. Error bars represent standard deviation. .............................. 44
x
Figure 3.1(a) Microfluidic device with a constriction channel where RBCs are bent by drag force. (b)
RBC’s shape change over storage showing that up to 12% cells change their shapes from discocytes
to echinocytes. ..................................................................................................................................... 54
Figure 3.2 (a) Simulation shows that the highest stresses occur in RBC membrane regions closest to the
channel walls. (b) At a flow velocity of 0.02 m/s, shear stress becomes significantly higher than 300
Pa for a channel width smaller than 10 µm, which can lead to RBC lysis. When channel width is
larger than 14 µm, shear stress is too low to deform RBCs. ............................................................... 56
Figure 3.3 Schematic force diagram. AFM experiment and force displacement curve. Contact width
estimation by applying a force of 150 pN. .......................................................................................... 60
Figure 3.4 Experimentally measured deformation index DI =L/d and effective stiffness values of RBCs
from a fresh sample. (a) DI significantly decreases at higher flow velocities (*p < 0.001). (b) In
comparison, effective stiffness of RBCs is not flow velocity dependent. Error bars represent the
standard deviation. .............................................................................................................................. 63
Figure 3.5 Effective stiffness of RBCs at different temperatures. RBCs under 4°C show higher effective
stiffness. RBC stiffness measured at 25°C shows no significant difference from that measured at
37°C (*p < 0.001). Error bars represent the standard deviation. ......................................................... 65
Figure 3.6 Effective stiffness of RBCs at different storage time points. RBCs stored for one week already
started to show significantly higher stiffness than fresh RBCs. Stored RBC stiffness degraded faster
in the last three weeks than in the first three weeks(*p < 0.001 and **p < 0.0001). Error bars
represent the standard deviation. ......................................................................................................... 66
Figure 3.7 (a) RBC membrane skeletal network. The affinities and interactions of key proteins are
regulated by biochemical parameters such as SNO and ATP, leading to RBC membrane stiffness
changes. (b) SNO (blue) and ATP (red) degradation during RBC storage. Data shown here are from
[30][31][32]. Approximately 90% SNO is depleted during the first week of storage, and ATP
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remains unchanged during the first three weeks of storage and starts to decrease significantly in
Week 4. ............................................................................................................................................... 69
Figure 4.1 (a) RBCs strongly adhered to glass substrate after settling for 15 minutes. RBCs in
microchannel were perfused with 37 °C human serum for 120 minutes. (b) RBCs were deformed by
shear flow (flow rate of 10 µL/min). Images of RBC deformation were recorded, and the shear
modulus of each RBC was quantified. ................................................................................................ 77
Figure 4.2 Stiffness of RBCs perfused for 120 minutes in human serum vs. end-point measured stiffness
of RBCs incubated in human serum in an incubator without perfusion (n=1300-3000 RBCs for each
condition). No difference was observed between the perfusion group and incubating group (*p>0.05),
showing that shear stress from perfusion did not induce bias in RBC stiffness recovery. ................. 78
Figure 4.3 (a) Shear modulus evolution of two-week old RBCs. PBS perfusion (blue line) did not cause
RBCs to recover their shear modulus. With perfusion of 37 °C human serum (red line), RBCs shear
modulus remained unchanged at 4.3 µN/m for 60 minutes; however, between 70-80 minutes, the
shear modulus value continuously decreased. By 90 minutes, stead-state was reached, and shear
modulus became 2.7 µN/m. (b) Steady-state shear modulus values after 120-min perfusion. With
human serum perfusion, RBCs stored for one week or two weeks were able to recover their shear
modulus close to the level of fresh RBCs. Older RBCs (four-six weeks) revealed limited capability
of stiffness recovery. *p <0.05&**p<0.001. Error bars represent the standard deviation. For each
condition, n= 1800-3000 RBCs. ......................................................................................................... 80
Figure 4.4 Stiffness of RBCs perfused up to 120 minutes in human serum vs. perfused up to 8 hours
(n=790-3000 RBCs for each condition). No difference was observed (*p>0.05), showing that longer
perfusion did not induce further recovery of shear modulus. ............................................................. 82
Figure 4.5 Steady-state shear modulus of stored RBCs that were PBS or serum perfused at 25 °C or 37 °C
(n=1260-3000 RBCs for each condition). The results show that 25 °C and 37 °C did not cause a
statistically significant difference in the stiffness of PBS-perfused or serum-perfused RBCs. .......... 84
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Figure 4.6 (a) Higher percentages of one-week and three-week old RBCs, compared to five-week old
RBCs, reached their steady-state shear modulus by 80 minutes. b-d) Compared to 25 °C, 37 °C
accelerated the stiffness recovery process for stored RBCs in human serum. Blue and orange lines
are the second degree polynomial fitting trend lines. Error bars represent the standard deviation. .... 85
Figure 4.7 Before incubation in human serum (blue line), a higher percentage of echinocytes existed in
old RBC samples. After 120-min incubation in human serum, the percentage of echinocytes
decreased from 3.8 % to 2.1 % for one-week old RBCs, from 4.3% to 3.3% for two-week old RBCs,
from 4.5% to 3.3% for three-week old RBCs, from 6.2% to 4.3% for four-week old RBCs, from 8.6%
to 6.2% for five-week old RBCs, and from 9.3% to 7.3% for six-week old RBCs. For each condition,
n=2200-2800 RBCs. ........................................................................................................................... 86
Figure 4.8 (a) Lack of ATP increases the spectrin-membrane affinity, leading to higher RBC stiffness.
When ATP is re-synthesized, spectrin-membrane binding becomes dynamic, causing the RBC to be
more deformable. (b) ATP concentration in RBCs that were stored from one week to six weeks. For
each data point, n=3 samples with each sample containing about 8000 RBCs. Older RBCs showed
lower ATP concentration than fresh RBCs, and their ATP concentrations increased during the
serum-incubation process. Steady-state values were reached by ~30 minutes. .................................. 89
Figure 4.9 RBCs were cultured in human serum for 8 hours. For each sample, the intracellular ATP
concentration by 120 minutes and at the end of the 8-hr incubation was compared. For each
condition, n=3 samples with each sample containing about 8000 RBCs. For all stored RBCs (one-
week to six-week old), no difference was observed, indicating that longer incubation (>120 minutes)
did not induce further re-synthesize of ATP. ...................................................................................... 90
1
Chapter 1
1. Introduction
1.1. RBC introduction
Human body is composed of about 37 trillion cells [1]. They provide structure for the body and
carry out specialized functions. Cells need energy to carry out their functions, which consumes
oxygen, and this requires the oxygen to be delivered to every part of the human body after the
oxygen is breathed in. The transportation of oxygen is carried out by the circulatory system
where the heart pumps oxygenated blood to the body and deoxygenated blood to the lungs. The
most abundant cells in blood are red blood cells (RBCs) which are the principle entities for
delivering oxygen, as shown in Figure 1.1(a). Among all the 37 trillion cells, 30 trillion of them
are RBCs, and approximately 2.4 million new RBCs are produced per second in human adults.
RBCs develop in the bone marrow and circulate for about 100–120 days in the body before their
components are recycled by macrophages.
Although RBCs are the most abundant cells in human body, they are unique in that unlike other
cells, mature RBCs lack a cell nucleus and most organelles. RBCs are produced in the bone
marrow with a nucleus and when they almost reach maturity, they then undergo enucleation
process when RBC’s nucleus is removed. The enucleation process cuts off a segment of the cell
containing the nucleus, which is then extruded from the RBC and further swallowed by a
macrophage [2]. The absence of a nucleus allows the RBCs to provide the maximum space for
hemoglobin to carry more oxygen. It also allows the cell to have its distinctive bi-concave shape
and be flexible, as shown in Figure 1.1(b). Such distinguishing feature provides RBCs the ability
to undergo large passive deformations during repeated passage through the narrow capillaries of
2
the microvasculature to fully deliver oxygen. Due to RBC’s lack of nucleus and most organelles,
RBC’s membrane, its only structural component, accounts for most of cell’s antigenic, transport,
and mechanical characteristics [3]. Abnormal RBCs with compromised deformability accounts
for decreased RBC life span, and leads to anemia in different RBC disorders.
Figure 1.1 (a) RBC takes oxygen from lungs and releases the oxygen to tissues. (b) RBC’s thickness at the thickest point is 2 – 2.5 µm and the minimum thickness in the center is 0.8 – 1 µm. RBC has a dimeter of 6 – 8 µm.
The RBC membrane plays a great role in keeping deformable. There are two major components
of RBC membrane: 1) lipid bilayer formed by its lipid main constituents with many
transmembrane proteins embedded; and 2) a spectrin-based membrane skeleton located on the
inner surface of the lipid bilayer [3].
3
1.1.1. Membrane lipids
The lipid bilayer composition participates in defining physical properties such as fluidity and
deformability of a cell. The lipid bilayer is composed of cholesterol and phospholipids in equal
proportions by weight. Cholesterol is evenly distributed between the inner and outer leaflets.
Five major phospholipids are asymmetrically disposed. Phosphatidylcholine and sphingomyelin
are located in the outer leaflet, while phosphatidylethanolamine and all phosphatidylserine,
together with the phosphoinositol, are confined in the inner layer [4][5]. The phospholipid
asymmetry is maintained by a group of both energy-dependent and energy-independent transport
activities. Energy-dependent activities include “flippases” which move phospholipids from the
outer layer to the inner layer, and “floppases” that do the opposite. Both “flippases” and
“floppases” describe the movement of phospholipids against a concentration gradient.
“Scramblases”, on the other hand, move the phospholipids bi-directionally down their
concentration gradients, which is an energy-independent activity [6][7]. These phospholipids
movement activities involve few proteins such as P-type ATPases, ATP-binding cassette
transporters and a putative scramblase [8][9].
The maintenance of the asymmetric distribution of phospholipids, plays a great role in regulating
biological function, especially the localization of phosphatidylserine to the inner layer.
Phosphatidylserine can act as an apoptosis signal when they are flipped to the outer layer of the
membrane, then macrophages can recognize the exposed phosphatidylserine and engulf the
RBCs [10]. Normally phosphatidylserine is actively held in the inner side of the cell membrane
by “flippase”, and the confinement of this lipid in the inner layer is essential for the RBC to
survive its frequent encounter with macrophages, especially in the spleen where the pores of the
4
red pulp meshwork in spleen are quite small (5 µm), which give RBCs more chance to interact
with macrophages. However, when the “flippase” activity is lost, which could happen when an
RBC undergoes apoptosis or with some disease, phosphatidylserine is no longer restricted in the
inner layer of the membrane. “Scramblase” will take place and phosphatidylserine will exchange
between the two sides of the cell membrane, resulting more of phosphatidylserine exposed on the
outer layer. This expose of phosphatidylserine has been suggested to play a role in destruction of
thalassemia and sickle cell disease RBCs [11][12][13]. Furthermore, phosphatidylserine shows
higher adhesion, and the restriction of phosphatidylserine to the inner layer of membrane also
inhibits the adhesion of RBCs to vascular endothelial cells [14]. When phosphatidylserine is kept
in the inner layer of the membrane, the interaction between phosphatidylserine and inner spectrin
network can also regulate membrane mechanical property [15][16].
1.1.2. Membrane proteins
There are more than 50 types of transmembrane proteins existing on RBC membrane, as shown
in Figure 1.2. A large fraction of them define the various blood group antigens, and other
proteins serve as transport proteins, signalling receptors, and etc. [17][18]. Among all
transmembrane proteins, two macromolecular complexes of membrane proteins are the most
relevant to structural integrity of RBC membrane, one Ankyrin-based, and the other 4.1R
protein-based. The structural integrity related proteins are usually responsible for the linking of
membrane to the skeleton network underneath. For example, Band 3 and RhAG link the lipid
5
bilayer to the skeleton through the interaction with Ankyrin. Glycophorin C, Rh, and other
proteins, on the other hand, build their connection to the skeleton through protein 4.1R
[19][20][21]. The membrane protein linkages with skeletal proteins play an important role in
regulating the interaction between the lipid bilayer and the membrane skeleton, and thus keep the
RBCs to maintain the favorable membrane surface area for the maximum oxygen exchange.
Meanwhile, the lodging and dislodging between the membrane proteins to the skeleton ensure
the RBC membrane to be deformable enough so they could squeeze through the capillaries
which are only few micro meters wide [22]. In addition, Band 3 also plays a key role in
regulating RBC metabolism in ion and gas transport functions [23].
1.1.3. Skeleton proteins
RBC membrane skeleton is composed majorly of spectrin (mostly spectrin α and spectrin β),
actin and some associated proteins including protein 4.1R, Ankyrin, adducin, and etc. (Figure
1.2)[24][25]. Spectrin is a long, flexible, helix like protein composed of two chains (spectrin α
and spectrin β) orientated in opposite directions. Each chain contains multiple spectrin repeats
with a “head” end and a “tail” end [26]. The functional domains at the “head” end are for
spectrin dimer-tetramer association and for Ankyrin binding. The domains at the “tail” end are
for binding to protein 4.1R, protein 4.2R and actin [27][28]. Isolated spectrin α or spectrin β
chains bind to each other at a pair of nucleation sites by repeats near the spectrin tail, while the
binding between spectrin α and spectrin β is through the association of two proteins called dimer
and tetramer [29]. The tetramers dissociate and reform when the membrane is deformed by shear
forces caused by the blood shear flow [30]. This allows the RBCs to sustain great distortions
when RBCs are passing through the micro vessels. The skeleton network is tethered to the RBC
6
membrane at two sites: one mediated by Ankyrin that couples spectrin to Band 3 and the other
mediated by protein 4.1R that couples to Glycophorin C [31]. The affinity of the spectrin
skeleton and membrane can be regulated by the proteins connecting between them, and the
continuous de- and reattachment of the spectrin-membrane binding results in a deformable RBC
membrane.
Figure 1.2 Current model of the RBC membrane. A phospholipid bilayer is tethered to the spectrin network (spectrin α and spectrin β proteins) via a number of proteins such as band 3, ankyrin, protein 4.1, and Glycophorin. Spectrin α and spectrin β form the structure underneath cell membrane and provide mechanical support. Most of the known proteins are shown(reproduced by permission of [26]).
7
1.2. RBC stiffness measurement
1.2.1. Filtration
Stiffer RBCs can enhance the chance of being trapped and cleared in the spleen, which has been
shown in animal in vivo studies [32]. The spleen consists of the following parts: 1) the white pull,
a lymphoid tissue that contains the most of the immune effector cells, 2) the red pulp, a reticular
meshwork where aberrant RBCs are trapped and destructed, 3) and a marginal zone between the
white pulp and red pulp [33]. Healthy RBCs, which are quite deformable, are able to pass
through the meshwork in the spleen easily and continue travelling in the human body. However,
due to the small pore size (~5 µm) of the red pulp meshwork [34], less deformable RBCs can be
trapped and further cleared by macrophages. This mechanism is usually used for aged RBCs to
be cleared, since after about 120 days of circulation, aged RBCs become stiff and recycled by the
spleen. Inspired by the mechanism of spleen, the filtration method was introduced as a technique
for measuring RBCs deformability. This method examines the ability of multiple RBCs to pass
through membrane with small sized pores as the filter [35]. During filtration, whole blood (or
partially diluted blood) is passed through the holes in a membrane filter by applying a certain
positive or negative pressure. Blood containing more stiff RBCs takes longer time or requires
higher pressure to pass through membrane filter.
The filtration technique is easy to implement for RBC deformability measurement. However, to
compare the deformability between samples, it requires the testing samples to have the same
concentration of RBCs [36]. Additionally, if whole blood sample is used, white blood cells
contained in the sample are stiffer than RBCs, which could also potentially induce the clogging
of the pores, result in a longer passing time, and require a higher pressure [37][38]. Besides the
8
stiffness of RBCs or white blood cells, other factors could also influence the filtration results.
For example, diabetic patients may have a higher blood plasma viscosity [39], resulting in the
high viscosity of whole blood, which could cause a longer passing time in filtration measurement.
Thus, measuring RBCs deformability by the filtration method could result in significant
variations across experiments.
Microfluidic filtration was designed to solve the issues of traditional filtration techniques by
fabricating multiple micro channels and observing RBCs under microscopy imaging. Whole
blood or PBS-diluted RBCs can be introduced into the microchannels with a certain pressure or
flow rate to flow RBCs through the microchannels. The behaviors (e.g., deformation and rate of
shape recovery) of RBCs when passing through the microchannels are recorded by camera, and
RBC stiffness is analyzed by data processing and mechanics modeling. Stiffer RBCs take longer
time to pass through the microchannels or get trapped in the channels [40][41]. By using reversal
flow, stiffer RBCs can also be trapped at a certain area [42] and the number of stiff RBCs can be
counted and used to represent the percentage of stiff RBCs in the sample. By using microfluidic
channels, only a small amount of blood is required, and high-throughput deformability
measurement of RBCs can be realized. Microfluidic devices have also been used to mimic in
vivo capillary blood flow system (Figure 1.3) [43][44]. Deformability is determined by
quantifying the threshold pressure required to traverse the constrictions. Microfluidic filtration
devices have been used to measure the deformability of malaria-infected RBCs [45] or sickle cell
disease RBCs [46]. This method could provide deformability assessment of an RBC population,
and only phenomenological metrics of RBCs deformability instead of inherent stiffness or
modulus of RBCs membrane can be measured.
9
Figure 1.3 (a) An array of parallel micro channels. (b-d) Artificial microvascular networks mimicking vessels can be used to measure the deformability of red blood cells (reproduced by permission of [47]).
10
1.2.2. Micropipette aspiration
Population-based measurements are able to assess the relative deformability change of RBCs;
however, they also mask the behavior of minority subpopulations and the differences between
cells. Single RBC deformability measurement is required to understand the heterogeneity in an
RBC sample. Micropipette aspiration is one of the first single cell measurement techniques and
has been used to measure the mechanical properties of RBC membrane [48]. A micropipette
aspiration measurement system consists of a glass micropipette with an inner diameter of 1-3 µm.
A negative pressure is applied through the micropipette, as shown in Figure 1.4 (a). The
deformation of the RBC membrane aspirated into the pipette is observed under a microscope.
Several RBC membrane stiffness measurement methods have been introduced [49]: 1) With a
given pressure, to measure the ratio between the length of RBC membrane aspirated and the
radius of the micropipette radius; 2) To measure the pressure required to aspirate the RBC
membrane to a certain length into the micropipette; and 3) To measure the pressure necessary for
the whole RBC to be aspirated into the micropipette. By measuring the amount of RBC
membrane aspirated with different negative pressure, Evans determined that the shear modulus
of the RBC membrane to be ~9 µN/m [50][51]. By using microfabrication techniques, an array
of microfluidics-based micropipette aspiration channels were constructed for micropipette
aspiration measurement of RBC deformability with a higher throughput, as shown in Figure 1.4
(b). The pressure applied on each RBC was calculated through hydrodynamic equivalent circuit,
and the deformation of each RBC in the channel was measured by microscope. Stiffness of
RBCs parasitized by Plasmodium falciparum was measured along with other types of cells
11
including RT4 human bladder cancer cells and L1210 mouse lymphoma cells [52][53].
Figure 1.4 (a) Different micropipette aspiration methods used to measure the mechanical properties of red blood cell (RBC) membranes. (b)Microfluidic pipette aspiration of RBCs using a funnel channel chain (reproduced by permission of [54]).
12
1.2.3. Atomic force microscopy
Atomic force microscopy (AFM), which was invented to achieve high resolution topography
imaging of an object, was adapted to perform RBC deformability measurement [55]. It uses a
cantilever with a tip of various shapes (triangular, parabolic or cylindrical) as a probe [56]. The
tip is placed on the sample surface and as the tip raster scans across the sample, the cantilever is
bent according to the surface topography, as shown in Figure 1.5. The minor vertical
deformation of the cantilever could cause position change of the laser beam reflected by the tip.
The reflected laser dot can be precisely detected by photodiodes, providing information of the
vertical deflection change of the cantilever [57].
Soon after the invention of AFM, it was also used to measure the mechanical properties of a
sample with a nanometric resolution, for example RBC membrane stiffness [58][59][60]. The
measurement was conducted by using AFM as an indenter and by monitoring the cantilever
deflection during indentation. By using this method, Young’s modulus of RBC membrane under
different physiological conditions was measured. The Young’s modulus of a healthy RBC’s
membrane Young’s modulus was found to be around 4.4 kPa [61]. Stiffness of RBCs from
thalassemia and diabetes mellitus was also measured by using AFM, and the effect of the disease
on RBC membrane stiffness was understood [61][62].
1.2.4. Optical tweezers
The stiffness of RBCs can be measured by using optical tweezers. Optical tweezers can be used
to manipulate micro objects that could be as small as a single molecule. Optical tweezers use
highly focused laser beams to create an optical trap which could hold a micrometer or
13
nanometer-sized dielectric particle at its centre [63]. When the particles are near to the center of
the trap, the focused laser beam applies a force on particles in the beam along the direction of
beam propagation, which is caused by the conservation of momentum. Photons that are scattered
by the tiny dielectric particles impart momentum to the dielectric particle, and the force
generated is also referred as scattering force. The scattering force can be determined by the
refractive indices of sample, surrounding medium, laser power, and sample size [64][65]. By
adjusting the power applied by the optical tweezers, a force with an order of pN can be achieved,
which is enough to deform the RBC. By measuring the deformation of the RBC membrane at the
given force, the mechanical properties of the RBCs could be determined (Figure 1.5).
The most common way of using optical tweezers is to use the scattering force to deform beads
which are attached to the RBC membrane. Two microbeads are first attached to RBCs from
opposite directions. The laser beam is then applied to generate a scattering force [66]. The
deformation of the RBC membrane is recorded by a microscope and the shear modulus can be
determined by using mathematical models. The shear modulus values of RBCs measured by
using this method vary from 10 to 30 µN/m [67][68].
14
Figure 1.5 (a) Optical tweezers measuring the RBC deformability with focused laser beams that transfer linear momentum or angular momentum of light. To measure the shear modulus of the RBCs, two microbeads are attached to the opposite sides of a RBC and force was applied by the optical tweezers. (b) Experimental observations of RBC deformability measurement using optical tweezers, and simulation results. (c) 3D image of an RBC measure by AFM (reproduced by permission of [47]).
15
1.2.5. Magnetic twisting
Magnetic twisting cytometry is able to apply direct mechanical loading onto the membrane of a
single RBC. A ferromagnetic microbead is first tightly bound to the RBC surface, and then
magnetic field is generated to precisely control the torque applied to the bead. RBC membrane
deforms in response to the torque applied (Figure 1.6). By controlling the magnetic field, the
microbeads exhibit wide ranges of forcing time scale and forcing amplitude to RBC membrane.
The motion of the beads and the deformation of the RBC membrane are recorded by a camera.
Then the shear and loss moduli of single RBCs can be determined by varying oscillating
frequency from 0.1 Hz to 100 Hz and applied magnetic field from 1 Gauss to 10 Gauss [69]. By
using magnetic twisting cytometry, healthy RBC membrane shear modulus is measured to be
around 6-12 µN/m, while the loss modulus increases as the frequency increases, which is around
0.2-0.8 pNꞏµm. Magnetic twisting cytometry has also been used to determine the stiffness
change of diseased RBCs, for example Plasmodium falciparum parasitized RBCs. Stiffness
change of RBCs infected with Plasmodium falciparum has been measured over all the stages of
parasite maturation. Temperature range from room temperature to febrile condition (41 °C) was
also studied. The results showed a dramatic increase in the stiffness of Plasmodium falciparum-
infected RBCs at a temperature of 41 °C [70].
16
Figure 1.6 RBCs with magnetic beads bound to the surface. SEM image of an RBC with a bead on the surface. c) Magnetic field is applied onto the bead to generate a torque to deform the RBC. d) Mechanical simulation to predict the deformation by the force applied by the magnetic bead. (Permission of [69] is not required for thesis/dissertation use).
17
1.2.6. Quantitative phase imaging
RBC membrane fluctuation is closely correlated with its mechanical property [71]. RBC can be
clearly seen under microscope and the biconcave shape can be clearly observed. RBC samples
are optically transparent in visible light, and only the light intensity can be measured by
conventional bright field microscopy, which does not provide enough information to determine
RBC membrane fluctuation. However, quantitative phage imaging techniques can measure the
amplitude and also phase information [72]. Since optical information is related to the physical
property of a sample, and there is a significant optical phase delay through the transparent
samples, quantitative phase imaging could provide high contrast images of the measured samples.
By using quantitative phase imaging, dynamic fluctuations of RBCs can be determined [73][74].
Fluctuations of RBCs membrane are used to characterize the membrane stiffness by determining
the in-plane shear modulus, and the stiffness difference between healthy RBCs and Plasmodium
falciparum-infected RBCs was analyzed [75].
18
1.2.7. Microfluidic measurement
Microfluidic devices have emerged as a promising tool to precisely control fluid and manipulate
cells. There has been growing evidence that cell stiffness can be used to provide a label-free
biomarker for determining cells states [76][77][78]. Thus, with the ability to manipulate a small
volume of samples and provide the heterogeneity information by measuring hundreds of
thousands single cells, microfluidic channels have been widely used to study cells stiffness as an
important biomarker. A real-time deformability cytometry was designed by flowing cells through
a microfluidic channel [79]. The deformability information was captured by a CMOS camera and
analyzed real time. This method was able to detect the cytoskeletal alteration, identify cell-cycle
phases and track stem cell differentiation into distinct lineages.
Since RBCs pass through micrometer-sized capillaries in vivo, microfluidic channels with a size
of micrometers can be used to mimic capillaries for measuring the stiffness of RBCs.
Constriction channels that are smaller than RBCs provide an efficient method to deform RBCs
for stiffness measurement. RBC’s deformability affects several parameters including elongation
and transit time when RBCs pass through the channels, and these parameters can be recorded by
high-speed imaging. A constriction channel device was first used to study the stiffness difference
between healthy RBC samples and malaria parasite-infected samples [80]. The results showed
that healthy RBCs showed higher elongation in the constriction channels and could pass through
small channels more easily than malaria parasite infected RBCs. RBC’s size can cause a
difference in the elongation of RBCs in the constriction channel. A two-stage microchannel was
designed to measure the size and the deformability of RBCs separately [81]. Besides RBC
deformation in the constriction channel, transit time can also be used to evaluate RBC
19
deformability (Figure 1.7). This is usually applied when RBCs are driven into the channel with a
certain pressure. Due to the resistance from the constriction channel, stiffer RBCs take longer
time than softer RBCs to pass through the channels (transit time). Transit time is usually
measured by analyzing images taken by a high-speed camera [82]. By applying electric voltage
on two ends of the constriction channel, the impedance difference caused by the RBCs passing
through the channels can also be measured to determine the transit time and further the
deformability of the RBCs [54].
As mentioned before, RBC deformability measured by using constriction channels is affected by
the cell size. Along with cell size, adhesion between cell membrane and channel walls is also
coupled with RBC deformability. Thus, transit time does not necessarily reveal RBC’s
deformability since larger RBCs or more adhesive RBCs also show longer transit time. Since the
constriction channel provides mechanical stimulation by physically contacting RBCs and is
normally smaller than RBCs, the channel is susceptible to clogging.
Figure 1.7 A network of microfluidic channels to measure RBCs deformability. The transit time of the individual cells are recorded using a high-speed camera. (b) A microfluidic system for
20
mechanical characterization of RBCs. The transit time is obtained from electrical impedance signal captured when RBCs are passing through the constriction channel (reproduced by permission of [54]).
21
Besides the force provided by the direct contact of the microfluidic channel walls, the shear
stress generated by the flow can provide mechanical stimulation for the RBCs to deform [83].
Ektacytometry, for instance, is one of the primary methods for measuring RBC deformability.
Suspension of RBCs is subjected to the flow stress generated by different shearing geometries
(Couette flow, plate-plate, Poiseullie flow). RBCs are elongated by the shear stress, and the
deformation of RBCs can be captured by a technique termed laser diffractometry. When a laser
beam is applied on an RBC suspension, light is scattered by the RBCs and forms a single image,
referred to as a diffraction pattern. The shape of this diffraction pattern reflects the average shape
of a high number of cells (e.g., thousands). When determining deformability of RBCs, the
pattern is fitted to an elliptical shape with a long axis L and short axis W. RBC deformability is
described by the elongation index, EI=(L-W)/(L+W).
RBC deformability change due to glutaraldehyde treatment, which stiffens RBC membrane by
crosslinking membrane lipids and proteins, or heat treatment has been well studied by using the
Ektacytometry method. Based on this method, LORCA as a commercial product has been
manufactured. LORCA consists of two concentric cylinders with a gap of 0.3 mm which can
hold 1 mL whole blood for RBC measurement. The outer cylinder can rotate with varying speeds,
and the fixed inner cylinder is integrated with a laser light source along with a light sensor and
temperature control unit. Not only RBC deformability in terms of EI, but also RBC aggregation
behaviour has been studied by using this instrument, as shown in Figure 1.8. One disadvantage
of using EI to quantify deformability is that EI is phenomenologically defined and is strongly
dependent on flow velocity.
22
Figure 1.8 (a)Hydrodynamic RBC deforming microfluidic device. Cells are focused to the center lines of the channels and deformed by fluid. Cell deformation is measured by analyzing images recorded by a high-speed camera. (b)Laser diffraction technique incorporating microfluidic rheometer to measure RBC deformability with elongation index defined (reproduced by permission of [47][54]).
23
1.3. Pathophysiological conditions and RBC stiffness
1.3.1. Diseases
RBC stiffness has been associated to many blood related diseases such as sepsis, diabetes, and
malaria infection. Sepsis is usually caused by immune response triggered by an infection due to
the injury or disease. It has been associated with hemodynamic alternations and microcirculatory
disturbances [84]. Although cardiac output has been observed to increase in sepsis patients,
nutrient blood flow to the liver, kidneys, and muscles is markedly reduced [85]. Thus, sepsis can
lead to poor organ function and insufficient blood flow which could further cause septic shock.
Deformability of RBCs of sepsis patients has been well studied, and the results show that
compared to healthy RBCs, a significant decrease in deformability of sepsis patients’ RBCs was
observed [86][87]. As discussed before, RBC membrane is a composite of a lipid bilayer and an
underlying spectrin skeleton. The connection between the skeleton and lipid bilayer determines
the RBC’s response to external force when passing through vessels. Adducin, an RBC membrane
protein, is a target for the calcium-dependant regulating protein which could promote spectrin-
actin interactions. Increased calcium cytosolic concentrations in sepsis is suspected to lower the
spectrin-actin interaction, which could further lower deformability of the RBCs in sepsis
[86][88].
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia and usually caused by
low insulin levels. It is one of the most common causes of renal failure or cardiovascular
diseases [89][90]. It has been shown that diabetes is associated with higher blood viscosity and
reduced RBC deformability [91][92]. Lower RBC deformability can result in impaired perfusion
at the tissue level, which is a major complication of diabetes mellitus [93][94]. The abnormal
24
RBC deformability of diabetes mellitus is believed to be caused by the lipid-protein interactions
and increased glycosylation [95][96]. Filtration technique has been used to study the
deformability of diabetes RBCs, where a constant negative pressure was given to diluted
suspension of RBCs to pass through membranes with straight channels. The passing rate was
used to indicate the RBCs deformability, and a compromised RBC deformability was observed
in diabetes RBCs [97]. Ektacytometry, as discussed before, has also been used to investigate
diabetes RBC deformability, and impaired diabetes RBC deformability was observed in terms of
EI [98][99].
1.3.2. Malaria
Malaria causes the death of over 1 million people each year globally[100]. Malaria is a disease
caused by Plasmodium parasites. When affected blood enters the human body, sporozoites (a
motile infective form) enters the bloodstream and migrate to the liver where they infect liver
cells. During the potential dormant period in the liver, they multiply and differentiate to
thousands of merozoites, which will escape into blood and infect RBCs. The parasites wrap
themselves in the cell membrane to avoid being detected by immune system [101]. Within the
RBCs, the parasites further reproduce themselves, periodically breaking out of the host cells to
invade more fresh RBCs [102].
Although the parasites protect themselves from immunes system’s attack by hiding themselves
within liver and blood cells, parasites in the RBCs will lead to a more vulnerable RBC
membrane. Vulnerable RBCs will be easily destroyed by the spleen during the circulating. The
malaria parasites cleverly secrete some adhesive proteins on the RBC membrane, helping RBCs
stick to the blood vessels, preventing infected RBCs from entering the spleen [103]. However,
25
the sticky RBCs will clog the vessels and the blockage of the micro vessels can cause symptoms
such as Placental Malaria [104][105].
Malaria infected RBCs’ membrane skeleton is strongly interrupted by parasite protein, which
could lead to abnormal RBC membrane stiffness [106]. As discussed before, RBC membrane
lipid bilayer is connected to the spectrin skeleton through proteins. When the RBCs are invaded
by malaria parasites, parasite-encoded proteins such as KAHRP (knob-associated histidine-rich
protein), PfEMP3 (P. falciparum Erythrocyte Membrane Protein 3) and RESA (ring-infected
erythrocyte surface antigen) will form some knob structures underneath the membrane
[107][108]. The knob structures modify RBC membrane and lower RBCs’ deformability.
The mechanical properties changes caused by malaria have been well studied through multiple
techniques including the methods introduced previously like Ektacytometry [109][110] and
micropipette aspiration [111][112]. Results have shown that malaria infected RBCs have much
lower deformability than healthy RBCs, and the compromised deformability contributed by
RESA is significantly more severe at febrile temperature condition (41 °C) [113].
1.3.3. Sickle cell disease
Malaria-resistant genes have been identified in tropical populations at risk for the disease,
suggesting that naturally occurring mutations are an evolutionary response. Genetic factors
including sickle cell disease, thalassaemia, and etc. have provided different levels of malaria
resistance. Sickle cell disease, which describes a condition when RBCs can turn into rigid and
sickle-like shape, illustrates some trade-offs that have occurred due to the malaria [114]. Sickle
26
cell disease RBCs are more rigid and vulnerable than healthy RBCs, which provides little
protection for the malaria parasites and easily exposes the parasites to immune system. Sickle
cell disease RBCs also have shorter life span (~40 days) than healthy RBCs (~120 days), which
leaves malaria parasites no time to fully grow and reproduce themselves to infect more cells.
Without endemic malaria, however, the sickle cell mutation is a total disadvantage. Sickle cell
disease describes the inheritance of two mutated β1-globin genes, the sickle hemoglobin gene
(HbS), from both parents. The mutated genes will encode abnormal hemoglobin S instead
normal hemoglobin A. Hemoglobin S causes no apparent effects in conditions of normal oxygen
concentration. However low oxygen concentration will cause hemoglobin S to aggregate and
form long chain of fibrous precipitates [115], known as polymerization. The presence of the long
chain polymers distorts the RBC from bi-concave shape to sickle-like shape, which is known as
sickling. Although sickled RBCs recover to bi-concave shape when the oxygen concentration is
rebuilt, cycling between sickled and unsickled causes RBCs to lose their ability to recover and
stay permanently sickled. Besides the shape change, sickled RBCs membrane is also modified
due to the higher concentration of Ca+2 , which leads to a higher RBC stiffness [116]. Sickled
RBCs possess abnormal shape and more rigid membrane, preventing themselves from passing
through narrow capillaries smoothly, leading to vessel occlusion and ischaemia [116][117].
Microfluidic experiments have been used to understand sickle cell disease RBCs morphological
and rheological changes [118]. Channels with size of capillaries were fabricated where sickle cell
disease RBCs were infused into the channels with pressure controlled, when the oxygen level
was controlled to investigate the change of RBCs under different oxygen concentration. The
27
results show that sickle cell disease RBCs pass through the channels smoothly under normal
oxygen conditions, while blockage was observed when the oxygen concentration was lowered.
1.3.4. Sickle cell trait
As described in the previous section, sickle cell disease patient inherits two mutated β1-globin
genes, the sickle hemoglobin gene (HbS), from both parents. Sickle cell trait carrier, on the other
hand, inherits one abnormal gene from one parent along with one normal hemoglobin gene (HbA)
from the other parent. Since individuals with sickle cell trait carry one normal hemoglobin gene,
they can generate normal hemoglobin, so they do not display the severe symptoms of sickle cell
disease patients. Thus SCT is typically considered to be benign and harmless [119], and they can
protect the carriers from malaria. However, exercise-related collapses within SCT athletes is a
serious complication. SCT as a risk factor for sudden death in physical training was first
comprehensively studied in 1987 [120]. Since then, there have been numerous reports
associating SCT and sudden death in young athletes engaged in vigorous exertion [121][122].
Autopsy observations showed vascular occlusion in brain, heart, lungs and livers in SCT sudden
death cases, indicating that the cause of death was vaso-occlusion due to SCT RBCs change
during physical exertion [123]. RBCs from SCD patients are known to become stiffer and even
sickled under deoxygenated or acidic conditions, causing the blockage of blood vessels
[116][124]. However, whether SCT RBCs also become stiffer under deoxygenated or acidic
conditions remain unknown.
28
1.3.5. Stored RBCs
Sickle cell disease or sickle cell trait related RBCs abnormality is the result of natural selection;
while storage-induced RBC degradation, referred to as “storage lesion”, occurs in RBC
preservation for transfusion. After major blood groups (O, A, and B) were discovered, which
made blood transfusion much safer, the preservation of blood donation was put onto studies to
realize non-direct transfusion [125]. The purpose of RBC storage system is to allow longer
storage with more RBCs circulating 24 hours after transfusion [126], and nowadays the “gold
standard” issued by Food and Drug Administration requires a minimum level of post-transfusion
RBC survival of 75 % after 24 hours [127].
During storage, RBCs undergo biophysical changes, referred to as storage lesions. Storage-
associated mechanical changes (e.g., decrease in deformability) can cause a decrease in
transfusion efficacy and an increase in harmful effects [128][129]. Stiff RBCs result in a higher
clearance by the spleen and are known to contribute to respiratory distress and systemic sepsis
[130][131]. Tissue ischemia, for instance, is believed to be contributed by microcirculatory
occlusion caused by poorly deformable RBCs [132]. Furthermore, among the critical ill patients
with sepsis who had older RBCs transfused, sepsis can be aggravated [133][134]. Since septic
patients have constricted vessels, poorly deformable RBCs can be trapped in the microcirculation,
leading to tissue hypoxia and exacerbating patients’ health conditions [135][136]. Thus,
mechanical changes of RBCs over the storage process are important to understand.
It is known that the storage process induces RBCs mechanical property changes and more
significant storage lesions have been observed in longer stored RBCs. However the clinical
outcomes between patients transfused with shorter stored RBCs and longer stored RBCs remain
29
controversial in terms of higher rate of mortality or longer ICU staying time. More than 40
observational studies have concluded that use of older stored RBCs is associated with a
significantly increased risk of adverse complications after transfusion [137][138]. These studies
observed patients with patient number from 52 to 387,130 and examined the effect of RBC
storage on various clinical outcomes including mortality, rates of infection, and length of ICU
Stay in the hospital [139][140][141]. The time point to distinguish younger and older RBCs was
mostly chosen to be 14 days or 21 days [142][143]. In these studies, patients transfused with
older RBCs showed higher rate of mortality and infection, or longer ICU staying time than
patients transfused with younger RBCs, which seems to conclude that younger RBCs are used
exclusively might save lives.
Results from more recent clinical studies, on the other hand, reported no significant difference in
clinical consequences between fresher and older RBCs [144]. Five clinical studies detected no
important clinical consequences of older RBCs [145][146][147][148][149]. In two larger trials,
transfusion of younger RBCs, as compared with older RBCs, did not significantly reduce the
complications of prematurity in very-low-birth-weight infants (patient number 377) or reduce the
rates of organ failure or adverse events among 1098 patients undergoing cardiac surgical
procedures [150][151].
The storage of RBCs is known to cause cell degradation (e.g., RBC stiffness and ATP), referred
to as “storage lesion”. For instance, mechanical stiffness changes of stored RBCs have been
widely studied, and the results consistently revealed that the stiffness of RBCs increases over the
storage process [152][153][154][155]. However, the controversy in clinical outcome of
30
transfusing fresher and older RBCs suggests that the degradation of stored RBCs can possibly be
reversed. Whether in vivo condition helps stored RBCs recover stiffness remains unknown.
31
1.4. Research objectives
The overarching goal of the thesis is to develop microfluidic systems for characterizing
mechanical properties of RBCs and understand RBC property changes in pathological and
storage conditions. Specific objectives include:
To design a microfluidic system for sickle cell trait RBC mechanical characterization
under typical exercise-induced conditions. To measure stiffness change of RBCs over
blood storage using human-capillary like microchannels.
To quantify the stiffness recovery evolution of stored RBCs under in vivo-like conditions
(i.e., in human serum).
32
Chapter 2
2. Stiffening of sickle cell trait red blood cells under simulated strenuous exercise conditions
2.1. Introduction
Athletes are often seen as the healthiest entities in the human population; hence, the occurrence
of sudden deaths in this group [156] can be shocking and have a devastating impact on
communities and the public [157][158]. According to an analysis conducted by the National
Collegiate Athletic Association (NCAA) from 2004 to 2008 [156], 36 out of 80 medical causes
of athletes’ death (45%) were identified to be exertional sudden deaths [159]. Among all the
exertional sudden deaths, sickle cell trait (SCT)-related cases caused most controversies
[160][161][162].
SCT describes the inheritance of one normal hemoglobin gene (HbA) from one parent along with
one mutated β1-globin gene, the sickle hemoglobin gene (HbS), from the other parent [163]. By
the end of 2009, there were approximately 300 million people worldwide with SCT.
SCT is typically considered to be benign and harmless [119]. However, as more cases of SCT
athletes’ sudden deaths were reported [159][164], heated debates over whether SCT should be
considered as a death cause during exercise and whether athletes should be screened for SCT
arose. Rationally analyzing these issues requires clear understanding of the properties of SCT
RBCs [159][165][166].
Vaso-occlusion, usually caused by the block of blood vessels, is one of the most fatal and
common symptoms in sickle cell disease (SCD). It also appears to be a crucial contributor to
33
sudden deaths in SCT individuals [166]. RBCs from SCD patients are known to become stiffer
and even sickled under deoxygenated or acidic conditions, causing the blockage of blood vessels
[124][116]. However, whether SCT RBCs also become stiffer under deoxygenated or acidic
conditions is not known.
During strenuous exercise, athletes’ muscles are under maximal oxygen consuming condition,
which lowers the oxygen level in circulation. Furthermore, although human blood pH is
normally 7.35, in strenuous exercise the higher concentration of hydrogen ions in human body
makes blood pH drop below 7.0 and even to 6.8 under extreme conditions for a short time period
[167][168]. Along with deoxygenation, the acidic blood condition has also been confirmed to
trigger the sickling of SCD RBCs and is hypothesized to stiffen SCT RBCs [169][170].
In this chapter, we focused on determining whether lowered pH and deoxygenation conditions
can trigger the stiffening of SCT RBCs which could be associated with higher vaso-occlusion
risks. The measurements were made on both normal RBCs and SCT RBCs, using a microfluidic
system that is capable of controlling oxygen and pH levels.
2.2. Methods
2.2.1. Blood specimens
The study was performed in accordance with the institutional guidelines for using human tissue
samples. Blood samples were collected for routine tests and used for study only after they were
completed for clinical tests and would be otherwise discarded. The study protocol was approved
by the Mount Sinai Hospital Research Ethics Board, in which the informed consent was not
required because the samples were selected retrospectively and no patient identification
34
disclosed to the study, and the study had no effect on the clinical test or patient management.
SCT was confirmed by sickle cell test and standard hemoglobin electrophoresis in the clinical
laboratory. Blood samples including seven normal blood samples and seven SCT blood samples
were stored with ethylenediaminetetraacetic acid (EDTA, 1.5 mg ml−1) and used within 48
hours. Before introduced into the device under room temperature, blood samples tested under
normal pH level were diluted 200 times in phosphate-buffered saline (PBS, pH= 7.35± 0.05),
while blood samples tested under acid conditions were diluted 200 times in acid-adjusted PBS
(pH=7.10±0.05 and pH=6.85±0.05).
2.2.2. Device and measurement
As shown in Figure 2.1(a), the microfluidic device consists of three parallel channels. The
middle channel is used for loading and testing RBCs, and the other two channels are used to
control the oxygen level in the middle channel. Diluted blood sample is introduced to the middle
channel and left settling for 15 minutes. Due to the presence of the carboxyl group of sialic acids
in the cell membrane, RBCs show negative charge, while glass slide has positive charge, so
RBCs strongly adhere to the glass substrate due to the difference in electrical charge on the cell
membrane and glass surface [171]. In experiments, when the pressure was varied from 0 Pa to 9
Pa, none of the RBCs was detached or revealed noticeable displacements. Two water tanks
containing PBS are connected to the inlet and outlet of the middle channel to maintain the
osmolality and pH level. Pumping either air or nitrogen into the two side channels controls the
oxygen level in the device due to the gas permeability characteristic of polydimethylsiloxane
(PDMS) [172]. The cross-sectional area of the three channels is 60 µm 300 µm. The gap
35
between neighboring channels is 100 µm for facilitated gas exchange between the gas channel
and the cell testing channel. Details of fabrication and design of the microfluidic device have
been described in our previous work [173]. In addition, pH level is controlled by adding
hydrochloric acid and confirmed before and after each experiment by using pH measurement
instrument (Hanna FC 240B pH electrode). RBCs are deformed under shear stress (0.9 Pa)
generated by a regulated vacuum source (pressure difference is 1.8 kPa) and this shear stress is
comparable to in vivo condition [174]. After the release of the shear stress, the RBCs recover to
their original shape. The dynamic recovery process is captured using a CCD camera connected to
a microscope. Mechanical models are developed to extract the shear modulus of each RBC.
36
Figure 2.1 (a) Schematic of the microfluidic device for RBC mechanical property measurement. RBCs in solution of different pH levels are loaded into the middle channel and adhere onto the glass channel bottom. Oxygen level is accurately varied by pumping air or nitrogen into the two side channels. Schematic illustration of the deformation of a cell element. (b) RBCs are deformed under shear stress (1.8 kPa). After the release of shear stress, RBCs recover to their original shape
37
2.3. Results and discussion
2.3.1. Determination of RBC shear modulus
When an RBC is in a shear flow, its membrane undergoes deformation by shear stress.
According to the Kelvin-Voigt (KV) model [175][176],
T λ (1)
where T (µN/m) is the average tension force acting on RBC membrane, μ (µN/m) is the elastic
shear modulus expressed in force per unit length, and λ is the extension ratio of RBC membrane.
λ where l is the RBC’s length when deformed under shear stress, and l is the RBC’s
original length [177].
The flow in microfluidic channel in this work is driven by a pressure difference (ΔP). The
velocity profile of pressure-driven flow is
ν 1 (2)
where η is dynamic viscosity of the fluid. Since the microchannel has a rectangular cross-section
and its width (w) is much larger than the channel height (h), hydraulic radius R is approximately
equal to h. Since w>>h, the error caused by the approximation is minor and doesn’t affect the
conclusion in this work. Shear stress on the microchannel bottom where RBCs are located is
τ η │ ∆ (3)
38
To calculate tension force T in the direction of extension, a small element (dA) is taken for force
equilibrium analysis, as shown in Figure 2.1. Since only the steady-state behavior is considered,
tension force and shear force on each element are balanced.
T x 2τ Y x (4)
where Y x is the half-width of the element dA. Thus, tension force T is
T (5)
where A is the surface area of the RBC. Shear modulus μ is determined by substituting Equation
(5) into Equation (1).
2.3.2. SCT RBCs become stiffened under lower pH
RBCs from healthy donors (control) and SCT individuals were first tested using the microsystem
without adjusting the pH level. Figure 2.2 shows that under normal pH levels, SCT RBCs are
significantly stiffer than normal RBCs, which is in agreement with previous results [173][178].
The higher stiffness of SCT RBCs could lead to a higher blood viscosity in the vascular system
[179]. In large blood vessels, lower RBC deformability limits cell orientation in flow and thus
increase blood viscosity [180]. In small blood vessels, stiffer RBCs lead to a lower Fahraeus-
Lindqvist effect, which increases the flow resistance and blood viscosity [181]. In healthy non-
SCT carriers, endothelial cells lining the blood vessels can generate vasodilators (e.g., Nitric
Oxide) to mediate increased blood viscosity. Differently, SCT carriers are known to develop
impaired vascular functions and generate less vasodilators [166][182].
39
We next measured the shear modulus of both SCT and normal RBCs at lower pH levels. As
shown in Figure 2.2(a), the shear modulus of all the seven tested SCT RBC samples consistently
became higher under a low pH level of 6.85 (p < 0.05). This low pH level was chosen because it
is known that in strenuous exercise the higher concentration of hydrogen ions in human body can
make pH drop to approximately 6.8 [167][168]. We then aimed to investigate whether moderate
exercise could induce SCT RBC stiffening. Hence, we conducted experiments under an
intermediate pH value (pH 7.10) which mimics the moderate exercise condition and no
significant difference in shear modulus was observed, as shown in Figure 2.3 (c).
In contrast, RBCs from normal subjects did not respond significantly to low pH levels. When pH
was reduced to 6.85, as shown in Figure 2.2(b) including seven samples’ data, the shear modulus
of normal RBCs increased slightly. However, the difference was not statistically significant (p≥
0.05). In our experiments, normal RBCs only started to reveal stiffness changes with statistical
significance at pH levels lower than 6.0 (data not shown) which is a physiologically irrelevant
condition [183][184][185].
As can be seen in Figure 2.2(c) where all of the seven samples are summarized together and
reported in box plots, when pH was reduced from 7.35 to 6.85, SCT RBCs were stiffened
significantly while control RBCs’ stiffness only increased slightly. The average shear modulus of
normal RBCs increased from 2.09 ± 0.67 µN/m to 2.27 ± 0.83 µN/m while the average shear
modulus of RBCs from SCT individuals increased significantly from 2.81 ± 0.7 µN/m to 4.05 ±
1.08 µN/m. The results indicate that SCT RBCs are inherently stiffer than control/normal RBCs,
and SCT RBCs are more sensitive to lowered pH levels than normal RBCs. Statistical analysis
confirms a significant difference of the shear moduli of normal RBCs and SCT RBCs under the
40
acidic condition (2.27 ± 0.83 µN/m vs. 4.05 ± 1.08 µN/m) (p=1.9 × 10-82). The significant
stiffening of SCT RBCs under the acidic condition could cause difficulties for the SCT RBCs to
pass through minuscule vessels and capillaries. Along with the increased blood viscosity, this
could lead to a higher chance of vessel blockage. The resulting vaso-occlusion events could
result in acute ventricular failure and contribute to sudden death in SCT carriers.
41
42
Figure 2.2 (a) Shear modulus of SCT RBCs under physiological pH level 7.35 (blue) and acid pH level of 6.85 (red). Error bars represent standard error of the mean value. All samples (n=30-70 for each sample) show a significant difference between different pH levels (*p < 0.05). (b) Shear modulus of normal RBCs does not reveal significant difference under different pH levels (p value > 0.05; n=30-70 for each sample). Error bars represent standard deviation. c) Box plot showing the summarized shear modulus of RBCs from the 7 tested SCT samples and the 7 tested normal samples under different pH conditions(**p=5.5 × 10-15,***p=2.2 × 10-29,****p=1.9 × 10-82; n=200-300).
2.3.3. Hypoxia does not induce SCT RBC stiffening
We then tested the oxygen effect on SCT and normal RBCs. As shown in Figure 2.3, the
stiffness of SCT RBCs was not found to increase by deoxygenation under physiological pH
(7.35), which is in agreement with previously reported result [173]. We speculated that under
acidic conditions deoxygenation might cause SCT RBCs to become even stiffer compared to the
condition of low pH only. However, the measurement results revealed that deoxygenation is not
capable of stiffening SCT RBCs further. In the deoxygenation experiments, we infused Nitrogen
into the two side channels for 20 minutes to reduce the oxygen concentration from 20% (air) to 0%
(pure Nitrogen). The validation of Oxygen depletion was described in our previous work [173].
As discussed in the previous section, the shear modulus of SCT RBCs increased from 2.81 ± 0.7
µN/m to 4.05 ± 1.08 µN/m under pH 7.35 and 6.85. These shear modulus values largely
remained the same when the channel was deoxygenated (for pH 7.35, oxygenated: 2.81 ± 0.7
µN/m vs. deoxygenated: 2.82 ± 0.7 µN/m; for pH 6.85, oxygenated: 4.05 ± 1.08 µN/m vs.
deoxygenated: 4.02 ± 1.04 µN/m). The differences were confirmed by using Mann-Whitney
nonparametric analysis to be insignificant (p>0.75). In order to decouple the effect of
43
deoxygenation and pH levels, the RBC samples were diluted 200 times, and it was confirmed
after each experiment that there was no significant difference in pH after deoxygenation.
44
Figure 2.3 At physiological pH 7.35 (a) and low pH 6.85 (b), normal RBCs elastic modulus did not change significantly when the channel was deoxygenated (*p>0.75). (c)(d) For SCT RBCs, although shear modulus became higher at pH 6.85 than at pH 7.35, deoxygenation did not induce further increase (*p>0.75). (e) Summarized shear modulus of SCT RBCs under different oxygen levels, and no difference was observed. Error bars represent standard deviation.
45
2.4. Discussion
SCT was recently reported to be associated with strenuous exercise-related mortality
[159][160][164]. Existing results on mechanical properties of RBCs from SCT individuals are
limited. Here we examined SCT RBCs’ stiffness change under controlled oxygen and pH levels.
The results reveal that SCT RBCs are significantly stiffer than RBCs from non-SCT healthy
subjects. Lower pH resulted in 28% increase in SCT RBCs’ shear modulus (Figure 2.2).
The stiffness increase of SCT RBCs could be due to several physiological alterations. It was
reported that SCT RBCs contain a higher concentration of Ca+2, which can enhance the binding
of cytoplasmic domain of band 3 (CDB3) to the cytoskeleton bound Ankyrin [178][186]. This
stronger binding caused by increased Ca+2 can possibly contribute to the higher rigidity of the
membrane of SCT RBCs. At lower pH, CDB3 becomes even more compact [183][187], which
can further enhance the binding and stiffening of SCT RBCs. In addition, Monocarboxylate
transporter 1 (MCT-1) activity has been speculated to impact RBC stiffness [188][189][190].
Since MCT-1 activity is inherently stronger in SCT RBCs than normal RBCs at low pH, the
higher concentration of hydrogen ions leads to even stronger MCT-1 activity, the enhancement
of MCT-1 activity could also be responsible for the significant increase of shear modulus
measured on SCT RBCs at pH 6.85. However, since no significant difference was observed
when pH was lowered to 7.10, we speculate that at pH 7.10, cytoplasmic domain of band 3
(CDB3) is not sufficiently compact to enhance the binding to the cytoskeleton bound Ankyrin,
leading to insignificant stiffening of RBCs. The results also suggest that under normal exercise
conditions where pH only slightly decreases, the stiffening of SCT RBCs would not be
significantly evident.
46
Experimental results also show that deoxygenation did not induce further stiffening of SCT
RBCs (Figure 2.3). During strenuous exercise, both blood oxygen and pH levels become lower.
Although deoxygenation did not directly impact the stiffness of SCT RBCs, low oxygen levels
during exercise can contribute to the lowering of blood pH [191]. Our data measured in the
simulated strenuous exercise condition indicate that low pH rather than hypoxia is effective in
triggering SCT RBC stiffening. In addition, although SCD RBCs are known to sickle under
acidic and/or hypoxia conditions, no sickling of SCT RBCs was observed under these conditions
in this work. Besides the stiffening of SCT RBCs, other exercise-induced physiological changes
can also possibly lead to a higher risk. For example, higher epinephrine during exercise has an
effect on SCT RBCs’ adhesion [192][193]. Increased adhesion can contribute to stronger
interactions between RBCs and epithelial cells and thus, trigger inflammatory pathways.
Dehydration, a common condition occurring during exercise, has also been reported to affect
RBCs’ physical properties [194]. The stiffening of SCT RBCs, inflammation, and dehydration
individually and together can be associated with a higher risk among SCT individuals during
strenuous exercise.
2.5. Conclusion
This study aims to address the question whether RBCs of SCT individuals become stiffened
during strenuous exercise. RBCs from SCT individuals and non-SCT subjects were tested under
simulated strenuous exercise conditions (i.e., low oxygen and low pH). The results show that
RBCs from SCT individuals are inherently stiffer and are sensitive to low pH which induces
significant stiffness increase in SCT RBCs, implying that the stiffening of RBCs could occur in
SCT individuals during strenuous exercise. Furthermore, the experimental results revealed that
47
deoxygenation alone did not cause SCT RBCs to increase their stiffness. However, since low
oxygen levels contribute to the lowering of blood pH, the stiffening of SCT RBCs in vivo could
result from a combined effect of low oxygen and low pH.
48
Chapter 3
3. Stiffness increase of red blood cells during storage
3.1. Introduction
More than 108 million blood donations are collected globally every year. Regulations in many
countries specify 42 days (6 weeks) as the shelf life for stored RBCs, and a first-in-first-out
inventory management approach is standard. Large-scale clinical studies involving 200-1,800
patients indicated that patients transfused with older RBCs tend to have a higher risk of mortality
than those receiving fresher RBCs [195][196][197][198].
During storage, RBCs undergo several biochemical and biophysical changes, referred to as
storage lesions. Storage-associated biomechanical changes (e.g., decrease in deformability) can
cause a decrease in transfusion efficacy and an increase in harmful effects [128][129]. Poorly
deformable RBCs result in a higher clearance by the spleen and are known to contribute to
respiratory distress and systemic sepsis [130][131]. Clinical research has also identified a
number of disease conditions such as splanchnic ischemia developed in patients who had been
transfused with older RBCs. Tissue ischemia, for example, is believed to be contributed by
microcirculatory occlusion which is caused by poorly deformable RBCs [132]. Furthermore,
among the critical ill patients with sepsis who had older RBCs transfused, sepsis can be
aggravated [133][134]. Since septic patients have constricted vessels, poorly deformable RBCs
can be trapped in the microcirculation, leading to tissue hypoxia and exacerbating patients’
health conditions [135][136]. Thus, mechanical changes of RBCs over the storage process are
important to understand.
49
Micropipette aspiration was first used to investigate the deformability change during RBC
storage [199]. The negative pressure required to aspirate an RBC into the micropipette was used
as an indicator of RBCs’ deformability. The result showed that the longer stored RBCs required
a higher negative pressure to be aspirated into the micropipette, suggesting a poorer
deformability. Optical tweezers were used to stretch an RBC, revealing that 35-day RBCs were
more difficult to stretch than fresh RBCs [200]. More recently, ektacytometry was used to
characterize the deformability of stored RBCs by stretching RBCs with shear stress induced by a
rotating plate. The extent of RBC elongation, defined as elongation index (EI), was used to
indicate the deformability under a certain shear stress [152][153]. Microfluidic measurement was
also reported for investigating stored RBCs’ deformability, wherein the deformation index (DI)
did not show significant differences at different storage time points [201].
The deformation of an RBC contains three modes: area expansion, shear, and bending of the cell.
Area expansion describes the isotropic area dilation or compression of the membrane surface
under a force. Shear describes the extension of the in-plane extension of the membrane surface
with the same membrane area. Bending characterizes the deforming behavior of a membrane
under an out–of-plane force. Micropipette aspiration is suitable for measuring the area expansion
stiffness, while shear stiffness (named as shear modulus in many cases, although with a unit of
N/m) can be measured by using optical tweezers or shear flow. Both area expansion stiffness and
shear stiffness reflect an RBC’s in-plane properties. When an RBC is deformed under an in vivo-
like flow condition, bending must also be considered in the deformation. In this case, RBC
deformation results from the collective effects of area expansion, shear, and bending. Although
an in vivo-like flow condition was created on a microfluidic device, the defined deformation
index (DI) is a phenomenological parameter which strongly depends on flow velocities, which is
50
unsuitable to use as a metric for evaluating RBC mechanical degradation during storage. More
detailed discussion on DI’s dependence on flow velocities is provided in the Experimental
Results section.
This chapter reports microfluidic measurement of stored RBCs, wherein RBCs are deformed in
the folding mode and velocity-independent effective stiffness is used to evaluate RBCs’
mechanical degradation during storage. The results reveal that RBCs’ effective stiffness already
becomes significantly higher in the first week of storage and consistently increases over the 6-
week storage period. Interestingly, the time points of effective stiffness increase were found to
coincide well with the degradation patterns of S-nitrosothiols (SNO) and adenosine triphosphate
(ATP) in RBC storage lesion.
3.2. Device fabrication
The PDMS microfluidic device was fabricated by using standard lithographic techniques. The
masks for the device were designed in AutoCAD (Autodesk, Inc., USA) and were printed as
transparencies by CAD/Art Services, Inc.. Table 3.1 shows the details of the fabrication of
alignment marker. Alignment of features on the photoresist layers was accomplished by first
patterning a chromium layer on a glass slide. The SU-8 negative photoresist (MicroChem,
Newton, MA, USA) was used both as a seeding layer and feature master with two different
heights (20 µm and 60 µm). Details on fabricating the SU-8 layers are shown in Table 3.2.
PDMS was made with the standard 10:1 mixing ratio of base to curing agent. PDMS was then
poured onto the mold and cured in an oven at 80 °C for 40 minutes. The entire PDMS structure
was peeled off the SU-8 master. The structure was washed in acetone, methanol, and DI water,
dehydrated on a hotplate at 150 °C for 10 min, and then O2 plasma bonded to a clean glass slide.
51
Table: 3.1: Fabrication of alignment markers
Step Procedure
1 Starting with chromium-coated slides, clean with acetone, methanol, and DI water
2 Dehydrate glass slides on hotplate at 150 °C for 30 min
3 Pour positive photoresist S1811 onto the slide
4 Spin: step 1: 500 rpm, 5 s, 1 acl; step 2: 3000 rpm, 30 s, 8 acl
5 Pre-bake slide on hotplate at 95 °C for 2 min to remove solvent
6 Place slide and mask in mask aligner. Soft contact UV exposure for 6 s
7 Develop slide in MF-321 for 2 min. Rinse in DI water and dry with N2 gun
8 Hard bake slide on hotplate at 95 °C for 1 min
9 Etch chromium layer in CR-2 for 1 to 2 min. Rinse in DI water and dry with N2 gun
10 Develop in AZ-300T for about 5 min. Rinse in DI water and dry with N2 gun
Table 3.2: Fabrication of SU-8 seeding, 20 µm and 60 µm feature layers
Step Procedure
1 Dehydrate glass slides on hotplate at 150 °C for 30 min
2 Pour SU-8-5 on entire slide (seeding layer)
3 Spin: step 1: 500 rpm, 5 s; 1 acl, step 2: 3000 rpm, 30 s, 3 acl
4 Pre-exposure bake: 65 °C for 1 minute, 95 °C for 3 min
5 Place slide in mask aligner. Flood exposure for 6 s
52
6 Post-exposure bake: 65 °C for 1 minute, 95 °C for 1 min
7 Hard bake at 175 °C for 2 hours
8 Pour SU-8-25 on entire slide (20 µm layer)
9 Spin: step 1: 500 rpm, 5 s; 1 acl, step 2: 4000 rpm, 30 s, 3 acl
10 Pre-exposure bake: 65 °C for 1 min, 95 °C for 5 min
11 Place slide and mask in mask aligner. Soft contact exposure for 10 s
12 Post-exposure bake: 65 °C for 1 min, 95 °C for 5 min
13 Develop in SU-8 developer for several min
14 Pour SU-8-25 on entire slide (60 µm layer)
15 Spin: step 1: 500 rpm, 5 s; 1 acl, step 2: 1000 rpm, 30 s, 3 acl
16 Pre-exposure bake: 65 °C for 5 min, 95 °C for 15 min
17 Place slide and mask in mask aligner. Soft contact exposure for 12 s
18 Post-exposure bake: 65 °C for 1 min, 95 °C for 4 min
19 Develop in SU-8 developer for several min
20 Hard bake at 175 °C for 2 hours
3.3. Methods
The device (Figure 3.1) consists of wide channels (500 µm × 60 µm) for introducing cells and a
constriction channel (12 µm × 20 µm) for inducing shear force to deform RBCs. Two focusing
channels were used to center and reorient RBCs to ensure that most of the cells were deformed
symmetrically in the center of the constriction channel [201]. Before introduced into the device,
fresh RBCs and RBCs (from 5 subjects) stored for one week to six weeks were diluted 200 times
53
in phosphate-buffered saline (PBS) to minimize the coincidence occurrences of multiple cells in
the channel. Although pH of RBC storage medium decreases to 6.5 by the sixth week [202],
stored RBCs were tested in PBS (pH: 7.35) since pH 7.35 is more physiologically relevant [203].
Before the RBCs were introduced into the microfluidic device, their shape at rest was first
evaluated as shown in Figure 3.1(b). During storage, RBCs progressively change their shape
from smooth discs called discocytes to bumpy discs called echinocytes, and the percentage of
echinocytes over the storage process increases to around 12% by the end of six-week storage.
The deformation of the bent RBCs in the channel was recorded by a camera with a frequency of
5,000 frames per second and shutter time of 30 µs (HiSpec 1, Fastec Imaging Corp., U.S.).
54
Figure 3.1(a) Microfluidic device with a constriction channel where RBCs are bent by drag force. (b) RBC’s shape change over storage showing that up to 12% cells change their shapes from discocytes to echinocytes.
The size of the constriction channel was chosen via finite element simulation and experimental
validation. Figure 3.2 shows that the highest shear stress occurs on the RBC membrane closest to
the channel wall. The flow velocity used in this study was 0.02 m/s, which is limited by the
55
shutter time of the camera. When the channel width is smaller than 10 µm, the highest shear
stress increases significantly to 300 Pa, sufficiently high for causing RBC lysis [204][205].
When the channel width is larger than 14 µm, the shear stress is not sufficient to bend the RBCs.
Thus, the channel width was chosen to be 12 µm in this study.
After an RBC enters the constriction channel, the cell reaches its steady-state shape before
approaching the end of the channel which is 160 µm long. The shear stress acts on the cell and
imposes a drag force to deform the RBC. The drag force can be approximated analytically
according to [206]
𝐹 3𝜋𝜇𝑑𝑣𝑓 (1)
where µ is medium’s viscosity; 𝑣 is the flow velocity; d is the diameter of the deformed RBC
(see Figure 3.1); wall factor 𝑓 [207] with the ratio λ=d/D, where D is the size of the
constriction channel on the microfluidic device; then shear force acting on the end of the cell
𝐹 𝐹. Finite element simulation in COMSOL, using a model of a rectangular microchannel
and deformed biconcave RBC shape, confirmed that the error in drag force quantification with
Eq. (1) is consistently within 10% for all flow velocities. The RBC’s deformed diameter d and
the deflection L were both measured from 5,000 Hz imaging. The effective stiffness of the RBC,
reflecting the collective effects of area expansion, shear, and bending, is hence
𝑘 (2)
56
Figure 3.2(a) Simulation shows that the highest stresses occur in RBC membrane regions closest to the channel walls. (b) At a flow velocity of 0.02 m/s, shear stress becomes significantly higher than 300 Pa for a channel width smaller than 10 µm, which can lead to RBC lysis. When channel width is larger than 14 µm, shear stress is too low to deform RBCs.
57
3.4. Experimental Results
Microfluidic measurement of effective stiffness was first validated by AFM indentation. In
microfluidic measurement, RBC is located in the center of the microchannel, and shear-induced
force is symmetrically applied to the cell. As shown in Figure 3.3, the distributed shear stress 𝜏
is equivalent to a force 𝐹 acting on a position, y=y’ such that the two force systems are
equivalent with the same resultant force and the same resultant moment. The AFM experiment
was designed accordingly, and individual RBCs with half adhered on a substrate and the other
half suspended were measured by AFM indentation. Comparable effective stiffness values from
microfluidic measurement and AFM measurement of 6-week old RBCs were obtained (95 ± 7
µN/m, n = 220 vs. 108 ± 18 µN/m, n = 11).
3.4.1. AFM validation
To mimic the situation of an RBC experiencing shear force in the microfluidic channel, in the
validation experiments, we fixed the center of the RBC and used an AFM (Bioscope Catalyst,
Bruker) tip to deform an RBC edge.
In microfluidic measurement, RBC is located in the center of the microchannel, and shear-
induced force is symmetrically applied to the cell. As shown in Figure 3.3, the distributed shear
stress (𝜏 ) is equivalent to a force (𝐹 ) acting on a position, 𝑦 𝑦′ such that the two force
systems are equivalent with the same resultant force and the same resultant moment.
τ y y′ dy τ y′ y dy (3)
τ at each position y was quantified from finite-element simulation and satisfies
58
τ dy F (4)
Combining Eq. (3) (4) gives
y'=0.7385r
where r is the radius of the RBC, which is within the range of 3-4 µm but varies from one cell to
another. In experiments, each RBC’s radius was measured via imaging. These results mean that
the microfluidic shear situation is equivalent to the application of F , along a line, on the position
y' of the RBC body.
Correspondingly, in experiments, each RBC was accurately positioned with a micromanipulator
to have half of the cell adhered on a glass slide while the other half suspended (Figure 3.3) and a
rectangular AFM cantilever tip (MSNL-10 B: spring constant 0.023 N/m, length 210 µm, width
20 µm, thickness 0.5 µm) was used to deform the free end. The cantilever tip applied F on the
position y' of each RBC. An example force-displacement curve from AFM measurement of an
RBC is shown in Figure 3.3. The same RBC sample was tested in our microfluidic device. For
direct comparison, effective stiffness for the AFM measured RBCs was calculated as Fs/L.
The results from both groups are summarized in Table 1. It can be seen that the effective
stiffness measured by AFM is slightly higher than the microfluidic device-measured results.
Several error sources could have contributed to the difference. (1) The AFM cantilever’s spring
constant was carefully calibrated with the thermal tune method integrated in the AFM by Bruker.
The error of the calibrated spring constant is within 5%, directly reflected in the AFM applied
force. (2) The exact position of AFM cantilever tip for applying the force Fs also contains errors,
although the best experimental care was used. The position error is approximately one pixel
59
(0.1613 µm/pixel), and for a 4 µm RBC, this position error y' = 2.9 ± 0.1613 µm causes an error
of 3.8% in the quantified effective stiffness of the RBC. (3) Finally, in the derivation of
equivalent loading position (Eq. (3) (4)), the force is supposed to be applied along a line (dy →
0). However, in experiments, when the cantilever tip contacts the RBC, the width of the
contacting area is small but not zero. Finite element simulation of the experimental situation
reveals that the contact width is approximately 0.13 µm, as shown in Figure 3.3. Putting this
width back to the theoretical calculation (dy = 0.13 µm), it caused an error of 3.5% in the
quantified effective stiffness of the RBC. In summary, the validation experiments contained
identifiable error sources as any measurement; however, the results support the validity of our
microfluidic measurement.
Table 3.3: Microfluidic device and AFM measured effective stiffness of RBCs (6 weeks)
Technique Microfluidic (n=220 RBCs) AFM (n=11 RBCs)
Effective stiffness 95 ± 7 µN/m 108 ± 18 µN/m
60
Figure 3.3 Schematic force diagram. AFM experiment and force displacement curve. Contact width estimation by applying a force of 150 pN.
61
3.4.2. Effective stiffness is not velocity dependent under low velocities.
To understand the dependence of deformation index (DI, defined as L/d [201]) and effective
stiffness on flow velocity, measurements were made on RBCs from a fresh sample at ambient
temperature. Figure 3.4 shows the RBCs’ DI and effective stiffness data measured at different
flow velocities. DI significantly changed (0.79 ± 0.09 vs. 1.01 ± 0.11, *p < 0.001) when the flow
velocity was increased from 0.01 m/s to 0.02 m/s. Similarly, in an ektacytometry study [208]
where RBCs were deformed in the stretching mode, the extent of RBC elongation defined as
elongation index (EI) was also found to increase at higher flow velocities. These results confirm
that the indices, DI and EI, strongly depend on flow velocity, and the use of different flow
velocities in experiments can lead to different conclusions in DI or EI change during RBC
storage. The drag force quantification in Eq. (1) takes into account the effect of flow velocity, v.
At low flow velocities when the parachute shape is sustained, both the drag force F and RBC
deformation/deflection L increase as flow velocity increases. Experimental measurement on
fresh RBCs confirmed that their effective stiffness remained unchanged under different flow
velocities (28.5 ± 8.4 µN/m; p value > 0.1) as shown in Figure 3.4(b), indicating that effective
stiffness is largely flow velocity independent under low velocities and is a more appropriate
metric for characterizing RBCs’ mechanical degradation than the phenomenological parameters
of DI and EI.
To further investigate the effect of flow velocity on RBC’s effective stiffness, 3D finite element
simulation (COMSOL) was conducted. RBC was modeled as a shell (i.e., cell membrane;
thickness: 10 nm, Young’s modulus: 1 kPa [26]) encapsulating fluids (i.e., hemoglobin, as
incompressible [27]). It was modelled as a biconcave disk (diameter: 8 µm) with a thickness at
the thickest point of 2 µm and a minimum thickness in the center of 1 µm. In simulation, flow
62
velocities were varied from 0.01 m/s to 0.1 m/s in the constriction channel (12 µm × 20 µm in
cross section, the same as in experiments). Experimentally varying drag force and measuring
RBC deflection is difficult because of the practical limitation of the high-speed camera’s shutter
time for clearly measuring RBC deformation, limiting the flow velocity to < 0.03 m/s. The
simulation results reveal that the drag force increases linearly with the flow velocity; however,
RBC deflection increases linearly only when the flow velocity is lower than 0.06 m/s, after
which nonlinearity occurs. This is because the stress induced by a flow velocity higher than 0.06
m/s exceeds RBC’s yield stress (250 Pa to 300 Pa) [204][205][209]. Thus, the flow velocity <
0.03 m/s used in experiments was not considered to be sufficiently high to induce nonlinearity in
RBC effective stiffness.
Finite element simulation was also conducted to investigate the effect of microfluidic channel
width on RBC effective stiffness (flow velocity: 0.02 m/s, as used in experiments). The results
reveal that when the channel width is larger than 11 µm, the effect of channel width on RBC
effective stiffness becomes negligible. Simulation also shows (data not shown here) that when
the microfluidic channel width is larger than 14 µm, RBC deformation is only approximately 0.1
µm for 6-week old RBCs, due to the small flow-induced force, making RBC deformations
difficult to measure via imaging. Thus, a channel width of 12 µm was chosen in our microfluidic
device.
63
Figure 3.4 Experimentally measured deformation index DI =L/d and effective stiffness values of RBCs from a fresh sample. (a) DI significantly decreases at higher flow velocities (*p < 0.001). (b) In comparison, effective stiffness of RBCs is not flow velocity dependent. Error bars represent the standard deviation.
3.4.3. Temperature effect on RBC effective stiffness
In blood banks, RBCs are stored at 4°C. We next measured the effective stiffness of stored RBCs
at different temperatures to understand temperature effect on the stiffness of stored RBCs. In the
fresh RBC group (Figure 3.5), freshly collected RBCs were stored at 4°C for 5 hours and then
diluted in PBS that had been stored at 4°C; or diluted in PBS that had been incubated at 37°C for
microfluidic measurement. After the 37°C measurements, RBCs were cooled down to ambient
temperature of 25°C and then measured. The same protocol was used for collecting data in the ‘2
weeks’ and ‘5 weeks’ groups (Figure 3.5) for RBCs stored at 4°C for two weeks and five weeks,
respectively. In all measurements, pH was maintained consistently at 7.35 ± 0.05.
As shown in Figure 3.5, within each of the three groups (fresh, ‘2 weeks’, and ‘5 weeks’, n>
2,000 RBCs), the effective stiffness of RBCs measured at 4°C was always significantly higher
64
than at 25°C and 37°C. However, the effective stiffness of RBCs showed no difference when
measured at 25°C and 37°C. It is known that as temperature is increased from 4°C, the lipid tails
in the RBC membrane become unsaturated, resulting in extra free space within the lipid bilayer
[210][211]. Furthermore, as temperature increases, phosphatidylcholine lipids in the RBC
membrane turn more from a crystal-like arrangement to a liquid-like state. This transition is
largely completed at 22°C when lipid tails are fully unsaturated [210][212][213]. Our results, for
the first time, quantitatively reveal how RBCs’ effective stiffness decreases from 4°C to 25°C
and 37°C. The stiffness data supports previous findings of RBC membrane lipid packing changes
and the transition of saturation states at low to high temperatures. Additionally, data in all three
groups (fresh, ‘2 weeks’, and ‘5 weeks’) also indicated no significant difference in measuring
RBCs’ effective stiffness at 25°C and at 37°C.
65
Figure 3.5 Effective stiffness of RBCs at different temperatures. RBCs under 4°C show higher effective stiffness. RBC stiffness measured at 25°C shows no significant difference from that measured at 37°C (*p < 0.001). Error bars represent the standard deviation.
3.4.4. RBC effective stiffness increases during storage.
Fresh RBCs and RBCs stored up to 6 weeks were tested under ambient temperature. Based on
the measurement of over 5,000 RBCs from five different samples, the effective stiffness of RBCs
at different storage points was quantified and is summarized in Figure 3.6. RBCs stored for one
week already started to show significantly higher stiffness than fresh RBCs (one week: 37.2 ±
8.6 µN/m vs. fresh: 26.5 ± 8.3 µN/m; **p < 0.0001). No significant difference for RBCs stored
from 1 week to 3 weeks was observed until the fourth week (60.5 ± 12.3 µN/m) when the
stiffness of stored RBCs increased drastically. RBC stiffness then further increased to 79.9 ±
17.2 µN/m (5 weeks) and 86.2 ± 17.5 µN/m (6 weeks). These results indicate that stored RBC
66
stiffness degrades faster in the last three weeks than in the first three weeks, and 6-week old
RBCs have an effective stiffness almost four times that of fresh RBCs (86.2 ± 17.5 µN/m vs.
26.5 ± 8.3 µN/m).
Figure 3.6 Effective stiffness of RBCs at different storage time points. RBCs stored for one week already started to show significantly higher stiffness than fresh RBCs. Stored RBC stiffness degraded faster in the last three weeks than in the first three weeks(*p < 0.001 and **p < 0.0001). Error bars represent the standard deviation.
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3.5. Discussion
RBC stiffness is determined by the membrane skeletal network of RBCs. In the RBC membrane,
a phospholipid bilayer is tethered to the spectrin network (mainly the spectrin α and spectrin β
proteins) via a number of proteins such as band 3, ankyrin, protein 4.1, and Glycophorin, as
shown in Figure 3.7(a). Spectrin α and spectrin β form the structure underneath cell membrane
and provide mechanical support [214][215]. Ankyrin is responsible for the bridging of spectrin to
band 3, which is one of the major RBC membrane-spanning proteins in the lipid bilayer
[216][217]. The affinity between ankyrin and spectrin is modulated by S-nitrosylation, a process
involving post-translational protein modifications [218][219]. Another protein, protein 4.1, also
plays a key role in regulating membrane stiffness by interacting with spectrin and Glycophorin,
and their interactions are modulated by phosphorylation, a process that involves the addition of
phosphate to an organic compound by consuming adenosine triphosphate (ATP) [220]. The
structure and the interactions between these proteins are changed during RBC storage due to the
degradation of several biochemical parameters such as S-nitrosothiols (SNO) and ATP
[221][222][223]. The degradation of these biochemical parameters over the RBC storage process
has been widely reported, and the depletion of SNO and ATP has been speculated to play
important roles in regulating RBCs’ mechanical properties [220][224][225][226].
During RBC storage, the SNO level becomes lower, leading to lower activity of S-nitrosylation
[221]. The detachment between spectrin and ankryin, which is induced by S-nitrosylation,
becomes less frequent, and the detached end of spectrin thermally diffuses back to ankyrin and
reattach [219][227]. Higher affinity between the spectrin and ankyrin proteins can contribute to a
higher stiffness of the RBC membrane. In the meanwhile, gradual ATP depletion is known to
68
occur during RBC storage [222]. Due to insufficient energy provided by ATP for the detachment
of Glycophorin from the spectrin network [220], Glycophorin tends to re-attach to spectrin by
protein 4.1 [225], and the higher affinity between Glycophorin and spectrin can also contribute to
the increase of RBC membrane stiffness.
Our data show that the stiffness of stored RBCs increased significantly during the first week of
storage. Interestingly, this timing well matches the degradation pattern of SNO. It is known that
the SNO level decreases by 70% after only one day of RBC storage, and by the end of the first
week of storage, up to 90% SNO is depleted [221], as shown in Figure 3.7(b). Thus, we reason
that the significant depletion of SNO in the first week of RBC storage has caused the significant
RBC stiffness increase by Week 1 as our data revealed (one week: 37.2 ± 8.6 µN/m vs. fresh:
26.5 ± 8.3 µN/m). Since little SNO is left at the end of first week of storage, the effect of SNO
depletion on RBC stiffness becomes less obvious in Week 2-Week 6. Compared to Week 1-
Week 3, RBC stiffness increase was significantly more rapid in the last three weeks (Week 4-
Week 6). This faster increase in stiffness during the last three weeks’ storage can attribute to the
degradation timings of ATP. The ATP level has been shown to remain largely unchanged during
the first three weeks’ storage but start to drastically decrease in Week 4 [222][223], as shown in
Figure 3.7. It is thus likely that the increase of stored RBCs’ stiffness in Week 4-Week 6 is
mostly caused by the depletion of ATP.
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Figure 3.7 (a) RBC membrane skeletal network. The affinities and interactions of key proteins are regulated by biochemical parameters such as SNO and ATP, leading to RBC membrane stiffness changes. (b) SNO (blue) and ATP (red) degradation during RBC storage. Data shown here are from [30][31][32]. Approximately 90% SNO is depleted during the first week of storage, and ATP remains unchanged during the first three weeks of storage and starts to decrease significantly in Week 4.
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Whether and how RBCs change their stiffness during storage has critical relevance to patient
safety and treatment effectiveness in transfusion medicine [129][228]. RBCs over the storage
process have been mechanically characterized in both stretching and folding modes, and
different metrics for indicating RBC deformability were used [153][201][229]. Elongation index
(EI) and deformation index (DI) were phenomenologically defined and are strongly dependent
on flow velocity. Instead of measuring EI or DI, shear modulus of RBCs was measured using the
optical tweezers method to characterize stored RBC deformability changes [154]. Quantifying
the laser tweezers-applied forces was obtained by comparing the experimental data with finite
element simulation. Older RBCs were found to be stiffer than fresh ones. Unfortunately, no shear
modulus change data were presented beyond 21-day storage in [154] although 42 days is
specified as the shelf life in most countries. Recently, a deformability-based microfluidic device
was reported for sorting stiff and less stiff RBCs [230]. It was observed that significant
difference in the sorting results existed between RBCs stored less than and longer than 28 days,
indicating that RBCs became stiffer after 28-day storage. This result agrees well with our
quantified effective stiffness changes that the effective stiffness of RBCs starts to increase
drastically from Week 4 (28 days). Note that the sorting device is not capable of quantitating the
mechanical property changes of RBCs; instead, it leverages RBC stiffness changes for RBC
sorting.
Our work, for the first time, measured RBCs’ inherent stiffness change over the storage process
by deforming RBCs in the bending mode (an in vivo-like deformation mode). Effective stiffness,
a flow velocity independent parameter, was defined and used to quantify the mechanical
degradation of stored RBCs. Effective stiffness reflects the resistance of an RBC against bending
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deformation under external forces, for instance, induced by blood flow in vivo or fluidic flow in
microfluidic channels. The results reveal that RBCs stored for one week already started to show
significantly higher stiffness than fresh RBCs, and stored RBC stiffness degraded faster in the
last three weeks than in the first three weeks. These results and the interesting coincidence
between the time points of effective stiffness increase and the degradation patterns of SNO and
ATP in stored RBCs motivate us to pursue a systematic correlation between biomechanical and
biochemical parameters in RBC storage lesion; they can also likely trigger deeper analyses of
patient data gathered from previous and present large-scale clinical studies in transfusion
medicine to better understand RBC storage age and clinical results, for instance, do 1-week and
3-week old RBCs cause a mortality difference in transfused patients? There also exists some
technical issue that deserves more investigation. For instance, to better understand the effect of
biochemical degradation on RBCs’ stiffness changes, different from existing studies on RBC
populations [221][222][223], performing biochemical measurements on single RBCs and
stiffness measurements on the same RBCs would permit a more precise correlation.
3.6. Conclusion:
This chapter presented microfluidic measurement of effective stiffness changes of RBCs over the
storage process. Instead of using phenomenological metrics such as deformation index (DI) and
elongation index (EI) as in existing studies, effective stiffness that reflects RBC’s inherent
mechanical property and is flow velocity independent is used to quantitatively describe the
mechanical degradation of stored RBCs. Fresh RBCs and RBCs stored up to 6 weeks were
measured on a microfluidic device in the bending mode mimicking their deformation when they
72
pass through microcapillaries in vivo. The results revealed the pattern of effective stiffness
increase and the time points where drastic stiffness increase occurred. The coincidence with the
degradation patterns of SNO and ATP was also discussed.
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Chapter 4
4. Stiffness and ATP Recovery of Stored Red Blood Cells in Human Serum
4.1. Introduction
According to the World Health Organization, 92 million units of blood are collected annually in
164 countries [231]. More than 21 million units of red blood cells (RBCs) as the main type of
blood product are transfused every year in the United States [232]. Regulations stipulated by the
US Food and Drug Administration (FDA) and many other countries specify 42 days as the shelf
life for stored RBCs [127]. This “gold standard” was established based on the criteria of post-
transfusion RBC survival of 75 % or above after 24 hours [233], where post-transfusion RBC
survival was measured by monitoring transfused RBCs labeled with 51Cr [234]. However, 25%
RBC clearance is higher by orders of magnitude than the normal daily RBC clearing ratio
(~0.8%) in the human body [235].
In transfusion medicine, there has been a decades-long debate about whether older RBCs (i.e.,
RBCs that are stored longer) can cause worse transfusion outcome than fresher ones
[236][237][238]. More than 40 clinical studies reported that the use of older RBCs was
associated with a significantly increased risk of adverse complications after transfusion
[137][138][129]. These studies examined the effect of RBC storage on several clinical outcomes
including mortality, rates of infection, and length of ICU stay in hospital [139][140][141]. The
time point to distinguish fresher and older RBCs was mostly chosen to be 14 days or 21 days
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[129][142]. In these studies, patients transfused with older RBCs showed higher rates of
mortality and infection, or longer ICU staying time than patients transfused with fresher RBCs.
Results from more recent clinical studies, on the other hand, reported no significant difference in
clinical consequences between fresher and older RBCs [144][145][146][147][148][149]. In two
larger-scale clinical trials, transfusion of fresher RBCs, as compared with older RBCs, did not
significantly reduce the complications of prematurity in very-low-birth-weight infants (patient
number 377) or reduce the rates of organ failure or adverse events among 1,098 patients
undergoing cardiac surgical procedures [150][151].
In the human body, RBCs must be transported to every tissue to deliver oxygen; otherwise,
tissue hypoxia occurs, which can result in pulmonary hypertension, stroke, and cardiovascular
dysfunction [239][240][241]. A proper stiffness must also be maintained by RBCs to allow them
to pass through capillaries in microcirculation [242]. Poorly deformable RBCs (i.e., higher
stiffness) result in a higher clearance by the spleen and are known to contribute to respiratory
distress and systemic sepsis [130][131]. Clinical research has also identified other disease
conditions such as splanchnic ischemia developed in patients who had been transfused with older
RBCs that are known to have higher stiffness than fresher ones [132]. Furthermore, among the
critically ill patients with sepsis who had older RBCs transfused, sepsis became aggravated
[134][133]. Since septic patients have constricted vessels, poorly deformable RBCs can be
trapped in microcirculation, leading to tissue hypoxia and exacerbating patients’ health
conditions [135][136].
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The storage of RBCs is known to cause cell degradation (e.g., RBC stiffness and ATP), referred
to as “storage lesion” [243][244]. For instance, mechanical stiffness changes of stored RBCs
have been widely studied [199][200][155], and the results consistently revealed that the stiffness
of RBCs increases over the storage process. However, the controversy in clinical outcome of
transfusing fresher and older RBCs suggests that the degradation of stored RBCs can possibly be
reversed, and parameters such as RBC stiffness and ATP can recover after transfusion.
Therefore, the questions that motivated the present study are as follows. Can the stiffness of
stored RBCs recover in vivo? To what extent? How does storage duration cause differences in
the recovery of stiffness? Furthermore, since ATP concentration in RBCs plays an important role
in regulating the mechanical stiffness of RBCs [244][220][222], how well and quickly can the
ATP level recover after RBC transfusion? This chapter reports microfluidic measurement of the
evolution of stiffness and ATP recovery of stored RBCs under in vivo-like conditions (i.e., in 37
C human serum) and provides quantitative evidence to these questions.
4.2. Methods
The microfluidic device, constructed via standard PDMS soft lithography, had a microchannel of
60 µm in height and 1,000 µm in width. With the flow rate of 10 µL/min used in our
experiments, the generated shear stress of 0.5 Pa is comparable to in vivo condition [174], is
sufficiently high to deform RBCs (Figure 4.1), and is far below the yield stress of RBCs (~100
Pa) [205]. RBCs (from 7 different subjects) stored for different periods of time were first diluted
in PBS by 200 times and then introduced into the microfluidic channel of the device. They
strongly adhered to the glass substrate after settling for 15 minutes [245]. The RBCs were heated
by a heating plate (37 °C, HWPT-384S) throughout experiments. Human serum (type AB, male,
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Sigma-Aldrich) of 37 °C was continuously perfused in the microchannel for 120 minutes, and
images of RBC deformation [Figure 4.1(b)] were recorded at 1 Hz. The shape of each individual
RBC was measured via image processing in Matlab, and the shear modulus of each RBC was
quantified, according to our previously reported method [203]. Briefly, according to the Kelvin–
Voigt (KV) model, 𝑇 𝜆 , where T (µN/m) is the average tension force acting on the
RBC membrane, which was calculated from the shear stress applied on the RBC and the area of
the RBC [246][176]; and λ is the extension ratio of the RBC membrane, 𝜆 , where 𝑙 is the
RBC’s length when deformed under shear stress, and 𝑙 is the RBC’s original length. RBC’s
elastic shear modulus (𝜇) expressed in force per unit length (µN/m) was determined, and higher
shear modulus indicates a higher RBC membrane stiffness. Non-stored fresh RBCs were also
tested in the experiment as a benchmark to evaluate how stored RBCs recover their properties in
37 C human serum.
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Figure 4.1 (a) RBCs strongly adhered to glass substrate after settling for 15 minutes. RBCs in microchannel were perfused with 37 °C human serum for 120 minutes. (b) RBCs were deformed by shear flow (flow rate of 10 µL/min). Images of RBC deformation were recorded, and the shear modulus of each RBC was quantified.
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4.3. Results
4.3.1. Stiffness recovery of stored RBCs in human serum
PBS and human serum were both used to perfuse into the micro channel, and the stiffness change
of individual RBCs was monitored. Figure 4.3 shows the shear modulus evolution of two-week
old RBCs (i.e., stored for two weeks). During 120 minutes of perfusion, no stiffness change was
observed in the PBS-perfusion group. In the serum-perfusion group, the shear modulus of RBCs
remained unchanged at 4.3 µN/m for approximately 60 minutes and then started to decrease.
When the shear modulus decreased to approximately 2.7 µN/m, steady-state was reached, and no
further change was observed. These results confirmed that stored RBCs are capable of
recovering their shear modulus in human serum, and the recovery process was in the time scale
of tens of minutes.
Figure 4.2 Stiffness of RBCs perfused for 120 minutes in human serum vs. end-point measured stiffness of RBCs incubated in human serum in an incubator without perfusion (n=1300-3000
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RBCs for each condition). No difference was observed between the perfusion group and incubating group (*p>0.05), showing that shear stress from perfusion did not induce bias in RBC stiffness recovery.
One-week to six-week old RBCs (3,000 RBCs from 7 different subjects) were tested, and their
stiffness recovery after 120-min perfusion with PBS and human serum are summarized in Figure
4.3 (b). The RBCs stored for one week and two weeks recovered their shear modulus from 4.1 ±
0.5 µN/m and 4.3 ± 0.5 µN/m to 2.9 ± 0.3 µN/m and 3.2 ± 0.4 µN/m, which are about 1.1 and
1.2 times the shear modulus of fresh RBCs (2.6 ± 0.3 µN/m). However, RBCs stored for four
weeks and longer were only able to recover from 5.5 ± 0.6 µN/m to 4.2 ± 0.9 µN/m (four
weeks), from 6.2 ± 0.7 µN/m to 5.1 ± 1.1 µN/m (five weeks), and from 6.9 ± 0.6 µN/m to 5.5 ±
1.1 µN/m (six weeks). These results indicate that RBCs stored longer than three weeks have
limited capability of stiffness recovery. For instance, six-week old RBCs after recovery have a
shear modulus twice that of fresh RBCs (5.5 ± 1.1 µN/m vs. 2.6 ± 0.3 µN/m), i.e., very poor
deformability. Note that for stored RBCs of all ages (one week to six weeks), stiffness recovery
was not biased by the shear stress from perfusion (Figure 4.2).
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Figure 4.3 (a) Shear modulus evolution of two-week old RBCs. PBS perfusion (blue line) did not cause RBCs to recover their shear modulus. With perfusion of 37 °C human serum (red line), RBCs shear modulus remained unchanged at 4.3 µN/m for 60 minutes; however, between 70-80 minutes, the shear modulus value continuously decreased. By 90 minutes, stead-state was reached, and shear modulus became 2.7 µN/m. (b) Steady-state shear modulus values after 120-min perfusion. With human serum perfusion, RBCs stored for one week or two weeks were able to recover their shear modulus close to the level of fresh RBCs. Older RBCs (four-six weeks)
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revealed limited capability of stiffness recovery. *p <0.05&**p<0.001. Error bars represent the standard deviation. For each condition, n= 1800-3000 RBCs.
4.3.2. Fresher RBCs reaching steady-state shear modulus faster
The time fresher and older RBCs took to reach their steady-state shear modulus was also
different. For instance, by 80 minutes, 85% of one-week old RBCs and 80% of three-week old
RBCs reached their steady-state shear modulus. In contrast, the percentage for five-week old
RBCs was only 47% [Figure 4.6 (a)]. Although the measurement was made continuously for 120
minutes, most of the stored RBCs reached their steady-state shear modulus between 60 minutes
and 90 minutes. Between 90 minutes and 120 minutes, for all the stored RBC samples (one-week
to six-week), almost no RBC revealed a change in its shear modulus. As shown in Figure 4.6 (a),
by the end of the 120-min human serum perfusion, there were still ~30% of the five-week old
RBCs showing no recovery at all. We also conducted additional experiments to continuously
perfuse stored RBCs for 8 hours (Figure 4.4), finding no further recovery of the RBCs and the
results were consistent with those obtained from the 120-min perfusion experiments. These data
show that older RBCs require longer time to reach steady-state shear modulus, and a higher
percentage of older RBCs cannot recover their stiffness in human serum.
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Figure 4.4 Stiffness of RBCs perfused up to 120 minutes in human serum vs. perfused up to 8 hours (n=790-3000 RBCs for each condition). No difference was observed (*p>0.05), showing that longer perfusion did not induce further recovery of shear modulus.
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4.3.3. Temperature effect on RBC stiffness recovery
We tested the shear modulus of PBS-perfused RBCs and serum-perfused RBCs at both 25 C
(room temperature) and 37 C. As shown in Figure 4.5, when the stored RBCs were perfused
with PBS, after two hours of perfusion, no significant difference in the steady-state shear
modulus values was found between the 25 C group and the 37 C group, indicating that 37 C
alone cannot induce RBC stiffness recovery. The lipid tails in the RBC membrane become more
unsaturated as temperature increases, resulting in more free space within the lipid bilayer and
thus, a more deformable membrane [210]. At 22 C, the stiffness of the RBC membrane has
reached the steady state because the lipid tails are fully unsaturated [212]. This phenomenon is
consistent with our finding that 25 C and 37 C did not cause a statistically significant
difference in the stiffness of PBS-perfused RBCs.
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Figure 4.5 Steady-state shear modulus of stored RBCs that were PBS or serum perfused at 25 °C or 37 °C (n=1260-3000 RBCs for each condition). The results show that 25 °C and 37 °C did not cause a statistically significant difference in the stiffness of PBS-perfused or serum-perfused RBCs.
For serum-perfused RBCs, we also found no difference in the steady-state shear modulus
between the 25 C group and the 37 C group. However, the RBCs perfused at 37 C showed a
faster recovery process than at 25 C. Figure 3(b-d) shows the data collected on one-week, three-
week and five-week old RBCs. Higher percentages of 37 C RBCs than 25 C RBCs reached
their steady-state values by 80 minutes. This can be attributed to higher temperature-caused
faster ATP-regulated unbinding of RBC membrane proteins [247]. Our results suggest that,
compared to 25 C, 37 C does not produce an additional effect on the stiffness recovery of
stored RBCs; however, it accelerates the stiffness recovery process in the human serum
environment.
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Figure 4.6 (a) Higher percentages of one-week and three-week old RBCs, compared to five-week old RBCs, reached their steady-state shear modulus by 80 minutes. b-d) Compared to 25 °C, 37 °C accelerated the stiffness recovery process for stored RBCs in human serum. Blue and orange lines are the second degree polynomial fitting trend lines. Error bars represent the standard deviation.
4.3.4. Shape recovery of stored RBCs in human serum
During storage, RBCs change their shape from biconcave to echinocyte [248]. To investigate the
shape recovery of stored RBCs (i.e., reversal from echinocytes to biconcave), RBCs were
cultured in 37 C human serum for 120 minutes. The percentage of echinocytes before and after
human serum incubation was measured, as shown in Figure 4.7. Before incubation in human
serum, the percentage of echinocytes was approximately 4% for the one-week to three-week old
RBCs. The percentage increased to 6% for the four-week RBCs, and by the end of the storage
86
(i.e., six weeks) the percentage increased to about 9%, agreeing well with previously reported
results of storage lesion [248][249]. After 120 minutes of incubation in human serum, the
percentage of echinocytes for one-week to three-week old RBCs decreased to around 2% while a
higher percentage of older RBCs remained echinocytes (4%, 6%, and 7% for four-week, five-
week, and six-week old RBCs, respectively), indicating that RBCs stored for a shorter time
period are more capable of recovering their shape from echinocytes back to biconcave.
Figure 4.7 Before incubation in human serum (blue line), a higher percentage of echinocytes existed in old RBC samples. After 120-min incubation in human serum, the percentage of echinocytes decreased from 3.8 % to 2.1 % for one-week old RBCs, from 4.3% to 3.3% for two-week old RBCs, from 4.5% to 3.3% for three-week old RBCs, from 6.2% to 4.3% for four-week old RBCs, from 8.6% to 6.2% for five-week old RBCs, and from 9.3% to 7.3% for six-week old RBCs. For each condition, n=2200-2800 RBCs.
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4.3.5. ATP concentration recovery of stored RBCs in human serum
RBC membrane is composed of a phospholipid bilayer tethered to the underneath spectrin
network (spectrin and spectrin ), as shown in Figure 4.8(a). RBC stiffness is dependent on the
interactions between the phospholipid bilayer and the underlying spectrin cytoskeleton [244].
The interactions are regulated by the binding between spectrin and 4.1R, a protein embedded in
the phospholipid bilayer [250][215]. The spectrin/4.1R binding is modulated by phosphorylation
which consumes intracellular ATP [220]. Luciferin technique (ATP Bioluminescent Assay kit;
Promega, Madison, Wisconsin, United States) was used to measure RBC intracellular ATP
concentration. For each sample, about 8000 RBCs (containing enough total amount of ATP to
emit light for measurement) were first washed by PBS at least three times. Then the RBCs were
lysed by 5% trichloroacetic acid (TCA) to extract the ATP in the RBCs. Tris-acetate, as a buffer
solution, was then added to neutralize the TCA. The rL/L reagent from the assay kit was added
into the ATP exacted solution and react with ATP to emit light with a wavelength of 560 nm
measured by a fluorescent camera. The light intensity was used to quantify the amount of ATP.
The assay kit contains a vial of ATP Standard (10-7 M) which was used as the reference to
quantify measured ATP in each experiment following the assay kit’s protocol. Then the average
ATP concentration with a unit of M (mol/L) was calculated from the measured amount of ATP
(with a unit of mol) and the total volume of the RBCs (each RBC has a volume of around 90 pL).
Fresh RBCs and stored RBCs (one-week to six-week) were incubated in human serum at 37 C.
For each time point during serum incubation, ~8,000 RBCs were used for quantifying their
average ATP concentration. We also measured the intracellular ATP concentration of fresh
RBCs (3.50 ± 0.09 mM) as the control group. As shown in Figure (b), one-week storage did not
88
induce significant reduction of ATP concentration (3.50 ± 0.08 mM). However, for the RBCs
stored for four weeks, the ATP concentration was only 3.10 ± 0.08 mM; and for six-week old
RBCs, it was as low as 2.35 ± 0.04 mM. These results of ATP degradation are consistent with
the values previously reported on RBC storage lesion [222]. Mitochondria are the organelles that
use oxygen to synthesize ATP in most types of cells. RBCs, which do not contain mitochondria,
synthesize ATP by glycolysis, an oxygen-independent metabolic pathway that converts glucose
into lactate. Glycolysis contains ten enzyme-catalyzed reactions, and in two of these reactions
(converting 1, 3-Bisphosphoglycerate to 3-Phosphoglycerate, and converting
Phosphoenolpyruvate to Pyruvate) ATP is synthesized in the cytoplasm. It is known that RBC
storage causes a consistent decrease of glucose over the storage duration [222], resulting in
reduced glycolysis activities and thus the reduction of intracellular ATP concentration.
In human serum incubation, the increase of ATP concentration of stored RBCs, especially the
older RBCs (e.g., four to six weeks), was already apparent within the first 10 minutes of serum
incubation, and by 30 minutes their ATP concertation started to plateau. Longer incubation (8
hours) was also conducted, and it was verified that the steady-state ATP concentration of the
stored RBCs was indeed already reached by 120 minutes after which no further change occurred
(Figure S4). Our data reveal that the ATP concentration of stored RBCs increased in human
serum but cannot fully recover for older RBCs. For instance, for two-week old RBCs, serum
incubation increased their ATP concentration to full recovery, compared to that of fresh RBCs,
from 3.37 ± 0.05 mM to 3.50 ± 0.05 mM by 120 minutes. However, the steady-state ATP
concentration of six-week old RBCs after 120-min serum incubation only increased from 2.35 ±
0.04 mM to 2.74 ± 0.05 mM (vs. 3.50 ± 0.09 mM for fresh RBCs).
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Figure 4.8 (a) Lack of ATP increases the spectrin-membrane affinity, leading to higher RBC stiffness. When ATP is re-synthesized, spectrin-membrane binding becomes dynamic, causing the RBC to be more deformable. (b) ATP concentration in RBCs that were stored from one week to six weeks. For each data point, n=3 samples with each sample containing about 8000 RBCs. Older RBCs showed lower ATP concentration than fresh RBCs, and their ATP concentrations
90
increased during the serum-incubation process. Steady-state values were reached by ~30 minutes.
Figure 4.9 RBCs were cultured in human serum for 8 hours. For each sample, the intracellular ATP concentration by 120 minutes and at the end of the 8-hr incubation was compared. For each condition, n=3 samples with each sample containing about 8000 RBCs. For all stored RBCs (one-week to six-week old), no difference was observed, indicating that longer incubation (>120 minutes) did not induce further re-synthesize of ATP.
4.4. Discussion
Despite the many clinical studies of RBC transfusion that investigated whether older RBCs cause
worse clinical outcomes, these clinical trials provided conflicting information [129][228] with
some showing that older blood is less effective [139][140][142] but with others showing no such
difference [149][150]. Up to today, there still exist strong controversies regarding whether the
age of stored RBCs is a factor in transfusion efficacy. Intuitively, fresher RBCs may function
better than older RBCs because RBC storage is known to induce biomechanical and biochemical
91
degradations to RBCs, and longer storage leads to more severe degradations [199][200][155].
The controversial clinical trial results could have been biased by the vastly different conditions
of the patients involved in the clinical studies; however, the elusiveness is also due to the lack of
understanding of how well and quickly stored RBCs after transfusion can recover their key
parameters, such as stiffness and ATP concentration.
After blood donation and processing, RBCs are placed in the preservation medium for storage at
4 C. The low temperature is to keep the rate of glycolysis at a lower limit and minimize the
proliferation of bacteria that might have entered the blood unit [129]. In the preservation
medium, acid citrate functions as anticoagulant, mannitol is for preventing RBCs from
swelling/hemolysis, and a strictly protocoled amount of glucose is also added into the
preservation medium to help maintain the metabolism of RBCs [251]. Over the storage duration,
the glucose concentration significantly decreases due to glycolysis. This leads to a lower ATP
concentration in stored RBCs [222] and causes RBCs’ stiffness to increase, as shown in Figure
2(b) and Figure 5(b). It should be noted that RBC preservation protocols do not allow a high
amount of glucose to be added into the preservation medium since adding a high amount of
glucose can cause impairing complications such as the glycosylation of RBC membrane and
skeletal proteins [252].
Storage-induced degradations of RBCs can lead to the clearance of transfused RBCs in vivo. In a
healthy human being, only ~0.8% RBCs are cleared within 24 hours [253]; in contrast, a
significantly higher percentage of stored RBCs after transfusion are cleared (e.g., 25% for six-
week old RBCs) [253]. The clearance of RBCs mainly occurs in the spleen [254] which consists
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of a meshwork with many tiny pores (e.g., 5 µm) [34]. RBCs with a higher than normal stiffness
have a higher chance to be trapped within the meshwork and cleared [255]. A higher stiffness
itself has been shown to serve as a signal for macrophages to induce phagocytosis for RBC
clearance [256][257].
When stored RBCs are transfused into the human body, the major microenvironment they
encounter is the human serum. In this work, we quantitatively studied the stiffness and ATP
recovery of stored RBCs in 37 C human serum. The results showed that in 37 C human serum,
stored RBCs are able to recover their stiffness and ATP concentration to varying extents
depending on their age of storage. As summarized in Table I, one can see that for one-week old
RBCs, although the shear modulus before recovery was 1.6 times that of fresh RBCs, 97% of the
cells had their stiffness recovered in human serum to be 1.1 times that of fresh RBCs; and the
ATP concentration of one-week old RBCs after recovery showed no difference from that of fresh
RBCs. For three-week old RBCs, 89% RBCs recovered their stiffness to be 1.3 times that of
fresh RBCs; and the recovered ATP concentration was only 5% lower than that of fresh RBCs.
However, for six-week old RBCs, only about 70% of the RBCs showed stiffness recovery in
human serum; their shear modulus after recovery was still 2.1 times that of fresh RBCs; and their
ATP concentration after recovery was 25% lower than that of fresh RBCs. Overall, the results
indicate that fresher RBCs (one-week to three-week) have significantly higher capability of
stiffness and ATP recovery in human serum than older RBCs (four-week to six-week).
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Table 4.1: RBC shear modulus and ATP concentration before and after recovery
Age of the
stored RBCs
Shear modulus before
recovery (µN/m)
Shear modulus after
recovery (µN/m)
Recovered RBCs
percentage (%)
ATP concentration
before recovery (mM)
ATP concentration
after recovery (mM)
Fresh (benchmark) 2.6 ± 0.3 2.6 ± 0.3 N/A 3.50 ± 0.09 3.50 ± 0.09
One week 4.1 ± 0.5 2.9 ± 0.3 97 ± 2.2 3.50 ± 0.08 3.53 ± 0.02
Two weeks 4.3 ± 0.5 3.2 ± 0.4 95 ± 1.7 3.37 ± 0.05 3.50 ± 0.06
Three weeks 4.5 ± 0.5 3.3 ± 0.4 89 ± 8.3 3.28 ± 0.06 3.39 ± 0.02
Four weeks 5.5 ± 0.6 4.2 ± 0.9 85 ± 5.7 3.10 ± 0.08 3.30 ± 0.06
Five weeks 6.2 ± 0.7 5.1 ± 1.1 72 ± 3.8 2.76 ± 0.05 3.15 ± 0.04
Six weeks 6.9 ± 0.6 5.5 ± 1.1 70 ± 3.2 2.35 ± 0.04 2.79 ± 0.05
Our experiments showed that neither stiffness nor ATP concentration recovered when the stored
RBCs were incubated in PBS. Compared to PBS, the compositions of human serum are more
diverse [222]. For instance, human serum contains much glucose and pyruvate while these
components are absent in PBS [258]. Glucose is converted into lactate by the metabolic pathway
of glycolysis. Among the ten reactions involved in glycolysis, 1, 3-bisphosphoglycerate is
converted to 3-phosphoglycerate, and phosphoenolpyruvate is converted to pyruvate. The
subsequent conversion of pyruvate to lactate provides nicotinamide adenine dinucleotide (NAD),
which promotes one of the reactions in glycolysis, i.e., glyceraldehyde 3-phosphate conversion to
1,3-bisphosphoglyceric acid, and the molecules produced in this reaction are utilized by RBCs to
synthesize ATP [259]. Our data quantitatively revealed the recovery of ATP concentration in
stored RBCs when they were incubated in human serum. Since it is known that ATP regulates
the tension of the spectrin-membrane connection and thus RBC stiffness [244][220], it is likely
that the stiffness recovery of stored RBCs in human serum is proceeded by the recovery of ATP
concentration.
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Our experiments also revealed that the process of stiffness recovery and ATP recovery were on
the scale of tens of minutes. For instance, for one-week old RBCs, the recovered steady-state
ATP concentration and stiffness value were reached by 10 minutes and 80 minutes, respectively.
For six-week old RBCs, the recovery of ATP concentration and stiffness respectively took 30
minutes and 90 minutes. To put these numbers in context, an RBC completes the circulation for
one cycle in the human body within a minute [260]. The long recovery time (tens of minutes)
could increase the chance for stored RBCs to be cleared by the spleen. This might imply that
when feasible in RBC transfusion, pre-treatment of stored RBCs in the target patient’s serum
might help achieve a higher transfusion efficacy. Our results also show that, comparing six-week
old RBCs and three-week old RBCs, about 20% less RBCs can recover in stiffness. The
recovered six-week old RBCs had a stiffness that is 1.7 times that of recovered three-week old
RBCs, and the recovered ATP concentration was 20% lower than in three-week old RBCs. This
significantly poorer recovery capability might alert a revisit of the policy for the 42-day shelf life
of RBC storage. Admittedly, we note that this study used human serum to mimic the in vivo
environment, but the environment in the human body is more than what serum provides. For
instance, can other factors in the human body, such as adrenaline, which is absent in the serum
used in our study, speed up the recovery of stored RBCs? It is our hope that this study could
trigger the next steps of more comprehensive characterization of the recovery behaviors of stored
RBCs (e.g., 2,3DPG and SNO) and the quantitation of the in vivo recovery of stored RBCs in
transfusion medicine.
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4.5. Conclusion
This chapter reports microfluidic measurement of the evolution of stiffness and ATP recovery of
stored RBCs under in vivo-like conditions (i.e., in 37 C human serum). The results provide
quantitative evidence to the following questions. Can the stiffness and ATP of stored RBCs
recover in human serum? To what extent? How does storage duration cause differences in the
recovery of stiffness and ATP concentration? It was found that despite the degradation induced
by storage, stored RBCs were able to recover their stiffness and ATP concentration in human
serum. RBCs stored for one to three weeks were capable of recovering their stiffness and ATP
concentration close to fresh RBCs, while after the recovery older RBCs (four to six weeks) had a
significantly higher stiffness and lower ATP concentration than that of fresher RBCs. The results
also revealed that the process of stiffness recovery and ATP recovery were on the scale of tens of
minutes. These findings bring new insight to how well and quickly stored RBCs recover after
transfusion.
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Chapter 5
5. Conclusions
RBC’s membrane accounts for most of RBC’s antigenic, transport, and mechanical
characteristics. Abnormal RBCs with compromised deformability accounts for decreased RBC
life span, and leads to anemia in different RBC disorders or RBC products. In this thesis, a
microfluidic device that allows for measuring RBC mechanical property with in vivo-like
conditions applied and adjusted, was developed and applied to characterize mechanical
properties of RBCs from sickle cell trait (SCT) carriers and the degradation/recovery of stored
RBCs in transfusion medicine. The main findings and contributions of the thesis are summarized
as following:
5.1. Contributions
1. Developed microfluidic systems to address the question whether RBCs of SCT
individuals become stiffened during strenuous exercise. RBCs from SCT individuals and
non-SCT subjects were tested under simulated strenuous exercise conditions (i.e., low
oxygen and low pH). The results indicate that RBCs from SCT individuals are sensitive
to low pH which induces significant stiffness increase in SCT RBCs, implying that the
stiffening of RBCs could occur in SCT individuals during strenuous exercise.
2. Designed a microfluidic device for measurement of effective stiffness changes of RBCs
over the storage process. Compared to existing techniques using phenomenological
97
metrics such as deformation index (DI) and elongation index (EI), effective stiffness that
reflects RBC’s inherent mechanical property and is flow velocity independent is used to
quantitatively describe the mechanical degradation of RBCs during the blood storage.
The results show that stored RBC stiffness degraded faster in the last three weeks than in
the first three weeks.
3. Characterized stored RBCs stiffness recovery by using a microfluidic device. Stiffness
evolution of RBCs stored up to 6 weeks was measured under an in vivo-like condition
(37 °C human serum perfusion). The results revealed the storage time’s effect on stored
RBCs’ steady-state stiffness. The time required for RBCs to recover also differs between
RBC samples stored for various weeks. ATP, as a crucial organic chemical that provides
energy to keep RBC membrane mechanically functional, was also measured with RBCs
treated by human serum.
In conclusion, this thesis introduced the mechanical characterization of RBCs using microfluidic
devices, and also provided knowledge to understand RBC property changes in pathological
condition such as sickle cell trait and storage conditions. The results showed that although sickle
cell trait RBCs do not sickle or become stiffer under low oxygen condition, lower pH during
strenuous exercise could lead to the stiffening of RBCs. Blood storage condition was also studied
and the results revealed that longer stored RBCs have higher stiffness, which could be partially
recovered in in vivo-like condition, and the findings bring new insight to how well and quickly
stored RBCs recover after transfusion.
The devices used in this thesis are also easy to be implemented in a clinical setting. After the SU-
8 layer is fabricated, as discussed in the device fabrication section, the PDMS curing and
98
bonding can be easily done within few hours. It takes only one microscope and image processing
software to test the stiffness of RBCs. This can be used to perform a quick screening of RBCs’
mechanical properties before the blood transfusion. Meanwhile, the devices used in this thesis
allow the monitoring of single RBCs under different micro environment, which could be
implemented in clinical setting for drug testing.
99
5.2. Future directions
5.2.1. RBC modulus measurement in bending mode
In this thesis, we measured the effective stiffness of RBCs which reflects the resistance of an
RBC against bending deformation under external forces. However, studies are still limited
where systematic approaches are taken to investigate the cell membrane modulus along with
viscosity when the RBCs are deformed in bending mode. To further extract the modulus and
viscosity information, a thorough model of the dynamic process of an RBC passing through a
capillary-like channel needs to be built and applied to the experiments. In future studies,
elastic spring model can be used to represent the RBC membrane [261]. In this model, the
skeleton structure of an RBC membrane can be simulated by 80 to 120 (depending on the
experiment sensitivity) mass particles, which are interconnected by springs. I expect by
correlating simulation and experimental result, an RBC membrane modulus can be extracted.
Furthermore, by monitoring the dynamic shape changes when an RBC enters/leaves the
channel, we can also use the spring model to determine RBC’s viscosity [262].
5.2.2. Single RBC biochemical properties measurement
In this thesis, we measured the ATP changes of RBCs during the storage as well as during
the recovery. Although population-based ATP measurements are able to assess the relative
ATP change of RBCs and show the recovery of ATP, they also mask the behavior of
minority subpopulations and the differences between single cells. Single RBC ATP
measurement is required to understand the heterogeneity in an RBC sample. What is needed
100
now are effective ways to isolate and process large numbers of individual RBCs for ATP
measurements. This requires cell isolation under uniform conditions. One possible strategy is
droplet microfluidics, which is an effective method to isolate and analyze thousands of
individual cells for ATP measurement [263][264]. 2,3-DPG and SNOs are also crucial
biochemical properties for RBC to perform functionally in human bodies, for example
delivering oxygen [265], and single RBC measurement of 2,3-DPG and SNOs can also be
used to assess the quality of stored RBCs. 2,3-DPG concentration of RBCs can be
determined by using the detection kit from Roche Diagnostics (Mannheim, Germany), and
the SNOs can be selectively converted to NO which can be detected as the chemiluminescent
product of its reaction with ozone [266].
5.2.3. In vivo study of stored RBC recovery
We have studied the stored RBCs recovery under in vivo-like conditions (i.e., in 37 C
serum) in this thesis; however, we note that this study used serum to mimic the in vivo
environment, but the body environment is more complicated than serum [267]. To study the
recovery of stored RBCs in an in vivo condition, animal tests should be considered in the
future studies. Both swine and sheep have proven to act as good test subjects for RBC studies
in transfusion medicine [268][269]. To track the RBCs in animal subjects, stored RBCs can
be labeled with 51Cr [234] before the transfusion. When the blood sample is collected after a
certain time of circulation in animal subjects, labeled RBCs can be sorted by flow cytometry
[270] for further biophysical and biochemical properties study.
101
5.2.4. In vitro production of RBCs
RBC donation and transfusion have been used for various medical conditions to replace patients’
lost RBCs. However, a number of problems persist, including insufficiency of supply and the
debate about whether older RBCs (i.e., RBCs that are stored longer) are inferior than fresher
ones, as discussed in this thesis. It would be valuable if mature RBCs can be produced in vitro
rather than collected from donors. Immature hematopoietic progenitor cells have shown to be
able to develop into mature RBCs [271]. However, present in vitro produced RBCs still contain
large number of nucleated RBCs which can cause harm to human body. In future work, adjusting
the enucleation medium or co-culturing with macrophages [255][272] may have the potential to
further promote the enucleation of RBCs in in vitro production studies.
102
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