EFFECTS OF SUSPENSION OSMOLARITY ON RBC MECHANICAL PROPERTIES AND SHEAR
INDUCED HEMOLYSIS
Matthew Grzywinski, Amanda Daly, Marina Kameneva PhD
Hemorheology, Hemodynamics, and Artificial Blood Research Laboratory McGowan Institute for Regenerative Medicine
INTRODUCTION
Storage of human red blood cells (RBCs) began around
1910 when the anticoagulant properties of citrate were
discovered. Over the last century, RBC storage techniques
were improved so patients can receive healthy RBCs after
longer storage periods. Today, about 80 million transfusions
per year are performed worldwide [1,2]. Many parameters,
such as temperature and nutrient concentrations, must be
controlled for RBC storage to be successfully. To control these
parameters, RBCs are stored in preservative solutions, such as
CPDA-1 or AS-5, which have osmolarities higher than
physiological osmolarity. The solutions currently in use allow
RBCs to be stored up to 42 days [3].
Despite these preservation techniques, RBC properties
change during storage. Previous studies have shown that RBCs
are less deformable, have a lower gas carrying capacity, and do
not survive long in circulation after a period of storage [1,4,5].
Zehnder et al. demonstrate that RBCs stored in hypertonic
conditions had higher hemolysis, osmotic fragility, and
viscosity in addition to having lower deformability and
aggregability [6]. These results suggest that the osmolarity of
the RBC suspension may contribute to the changes in RBC
properties during storage. However, none of these studies have
examined how RBCs in high osmolarity respond to exposure to
shear stress. Patients who receive older RBC units are more
likely to experience serious complications such as renal failure,
respiratory insufficiency, multi-organ failure, and in-hospital
death [2].
RBCs’ short lifetime after transfusions suggests that stored
RBCs are more susceptible to shear stress than normal cells.
This can have serious consequences in a patient with a device
such as a left ventricle assist device (LVAD). These devices
can apply shear stresses on RBCs that are higher than
physiological shear stresses [7].
OBJECTIVES / SUCCESS CRITERIA
This study hopes to determine the effects of suspension
osmolarity on RBC susceptibility to shear stress, mechanical
fragility, and deformability.
The authors will conduct experiments on six units of blood.
P values of less than 0.05 will suggest that there is a significant
difference in the shear inducted hemolysis and RBC
mechanical fragility in normal and high osmolarity. A p value
of less than 0.05 will also represent a significant difference in
average RBC deformability in normal and high osmolarity and
average RBC deformability before and after exposure to shear
stress. Finally, the authors hope to obtain a correlation
coefficient of at least 0.60 between all hemolysis and
mechanical fragility results.
METHODS
One-day-old bovine blood was filtered and washed three
times with PBS. The washed cells were then suspended in
normal osmolarity PBS (285 mOsm) and high osmolarity PBS
(350, 450, and 530 mOsm) so that the hematocrit was 40%.
Each preparation consisted of a normal and a high osmolarity
suspension.
To expose the RBCs to shear stress, the suspensions were
circulated through the flow loop pictured in figure 1 by a
Levitronix PediMag pump for 60 minutes. This centrifugal
pump was used because it generates negligible hemolysis in
one hour. During testing, the pressure difference across the
microchannel, which was responsible for applying the shear
stress to the cells, was kept constant around 500 mmHg.
Baseline, 0, 30, and 60 minute suspensions samples were
collected through the outflow stop cock. The amount of
hemolysis was determined by measuring the concentration of
free hemoglobin in the supernatant of the samples. The
concentration of free hemoglobin was determined by measuring
the absorbance of the supernatant at 540 nm on a Spectronic
Genesys 5 spectrometer.
Figure 1. Schematic of flow loop showing important
components.
To analyze the mechanical fragility of the RBCs, Six test
tubes were prepared for both the normal osmolarity suspension
and the high osmolarity suspension. Three tubes were
experimental samples and received five stainless steel ball
bearings, two tubes were controls, and one tube was be a no
rock control. The experimental tubes and control tubes were
rocked on a platform rocker with a constant frequency (18
cycles/min) and amplitude (±17°) for 60 minutes. After
rocking, the concentration of free hemoglobin in the
experimental (Hbexp), control (Hbcontrol), and no rock control
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samples were determined by measuring the absorbance of the
supernatants of the samples of 540 nm. The total hemoglobin
concentration (Hbtotal) was also measured using a hemoximeter.
These concentrations were used to calculate the mechanical
fragility index (MFI).
The viscosity of the baseline RBC suspensions were
measured using a Brookfield cone-and-plate viscometer with a
CP40 cone at 25 °C. Viscosity was used to calculate shear
stress, shear rate, and Reynold’s number.
Baseline and 60 minute RBCs from the normal and
high osmolarity suspensions were suspended in 5.6% PVP
solution and sheared at 100 s-1, 500 s-1, and 1000 s-1 (shear
stress of approximately 3 Pa,15 Pa, and 30 Pa respectively) by a
Linkam CSS450 shear device. ImageJ was used to measure the
major axis (M) and minor axis (m) of the 100 cells per shear
rate. From these dimensions the elongation index (EI) of the
cells were calculated.
Paired t-tests were used to compare the hemolysis,
mechanical fragility, and deformability results from the 12
trails conducted. Values are reported as average± standard
error.
Free Hemeglobin
(mg/dL)
RESULTS
In the shear-induced hemolysis test, the concentration of
free hemoglobin in the high osmolarity suspension (85.5 ± 67.1
mg/dL) was significantly higher than the free hemoglobin
concentration in normal osmolarity suspension (41.1± 18.8
mg/dL) (p=0.020). RBCs high osmolarity suspensions (1.82 ±
0.50) were also more fragile than RBCs in normal osmolarity
suspensions (1.31 ± 0.28) (p=0.003).
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150
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DISCUSSION
This study suggests that mechanical properties of RBCs
change when they are in a high osmolarity environment. The
results met the success criteria for sample size, and
demonstrating a significant difference in shear induced
hemolysis and RBC mechanical fragility. Further more, a
satisfactory correlation coefficient between mechanical fragility
and hemolysis data was obtained. However, the design criteria
for demonstrating a difference in RBC deformability in normal
and high osmolarity suspensions, and RBC deformability
before and after exposure to shear stress were not met.
This study had several limitations. First, no test was
done to determine if the changes in RBCs properties caused by
high osmolarity were permanent. It is possible that if these
cells were returned to a physiological osmolarity environment,
they would return to their normal state. Also, the PVP
suspension the RBCs were suspended in for the deformability
test had a normal osmolarity of 285 mOsm.
This study can be expanded on in future research. For
example, the study could be repeated after a storage period.
CONCLUSION
The mechanical properties of RBCs in high osmolarity
suspensions are different than the mechanical properties on
normal osmolarity suspension. The high osmolarity of RBC
storage solution may contribute to RBCs short lifespan after
transfusion and the other changes that occur to the cells during
storage. If these consequences are avoided, transfusion patients
may be at a decrease risk for serious complications such as
organ failure and death.
ACKNOWLEDGMENTS
The Author would like to thank Dr. Kameneva and the
Kameneva laboratory graduate students.
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RBC deformability decreases after exposure to shear stress.
However, in some histograms comparing baseline and 60
minute deformability, there is a leftward shift in the elongation
index of the cells. These shifts suggest that the number of
highly deformable cells decreased after exposure to shear
stress.
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1.00
2.00
3.00
MFI
Figure 2. Graph of concentraion of free hemeglobin vs. MFI.
From figure 2, MFI and free hemoglobin seem to be
exponentially related. The following equation was obtained
from an exponential regression (R=0.67, R2=0.45):
There was no significant difference in the average
deformability for RBCs in normal and high osmolarity
suspensions. There is also no evidence to suggest that average
REFERENCES
[1]: Hess, Red Cell Changes During Storage, Transfusion, 2010
[2]: Dzik, Fresh Blood for Everyone? Balancing Availability
and Quality of Stored RBCs, Transfusion Medicine, 2008
[3]:
AABB,
Circular
of
Information,
2009,
http://www.aabb.org/resources/bct/Documents/coi0809r.pdf
[4]: Gilson et al., A Novel Mouse Model of Red Blood Cell
Storage and Posttransfusion in vivo survival, Blood
Components, 2009
[5]:Henkelman et al., Is Red Blood Cell Rheology Preserved
During Routine Blood Bank Storage, Transfusion, 2010
[6]: Zehnder et al., Erythrocyte Storage in Hypertonic and
Isotonic Conservation Medium, Vox Sanguinis, 2008
[7]: Selgrade et al., Computational Fluid Dynamics Analysis to
Determine Shear Stresses and Rates in Centrifugal Left
Ventricular Assist Device, Artificial Organs, 2012
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