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Rheologic changes of hypothermic preserved red blood cells
Henkelman, Sandra
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Rheologic changes of hypothermic
preserved red blood cells
Sandra Henkelman
Rheologic changes of hypothermic preserved red blood cells.
By Sandra Henkelman
Department of BioMedical Engineering
University Medical Center Groningen, University of Groningen
Groningen, the Netherlands
ISBN (book)
: 978-90-367-5399-9
ISBN (digital document)
: 978-90-367-5400-2
Copyright © 2012 by S. Henkelman
All rights are reserved. No part of this publication may be reproduced or transmitted in any
form or by any means, without permission of the author.
Cover design:
In Zicht grafisch ontwerp
Lay-out:
Ingrid Engelsman
Printed by:
GVO drukkers & vormgevers B.V.
Rheologic changes of hypothermic
preserved red blood cells
Proefschrift
ter verkrijging van het doctoraat in de
Medische Wetenschappen
aan de Rijksuniversiteit Groningen
op gezag van de
Rector Magnificus, dr. E. Sterken,
in het openbaar te verdedigen op
woensdag 14 maart 2012
om 14.30 uur
door
Sandra Henkelman
geboren op 26 mei 1976
te Groningen
Promotores:
Prof. dr. G. Rakhorst
Prof. dr. ir. H.J. Busscher
Copromotor:
Dr. W. van Oeveren
Beoordelingscommissie:
Prof.dr. A.R. Absalom
Prof.dr. R.A.W. van Lier
Prof.dr. G.J. Verkerke
Paranimfen:
A.J. Munnike
MD. O.C. Varnav
Ir. G.E. Engels
Ir. W. van der Houwen
The world is your Oyster
Voor Opa en Oma…
Contents
Chapter 1.
Introduction
Chapter 2.
Use of hydroxyethyl starch for inducing red blood cell
aggregation (Clin.Hemorheol.Micro. 2012)
19
Chapter 3.
Red blood cell aggregation; an important phenomena in
damage control resuscitation?
33
Chapter 4.
Is red blood cell rheology preserved during routinely blood
bank storage? (Transfusion 2010, 50: 941-948)
39
Chapter 5.
Utilization of cryopreserved red blood cells in transfusion
medicine.
57
Chapter 6.
The effects of cryopreservation on red blood cell rheologic
properties (Transfusion 2010, 50: 2393-2401)
77
Chapter 7.
A new freezing technique allows storage of RBCs with 20%
glycerol at - 80°C.
97
Chapter 8.
General discussion
115
Summary
127
Samenvatting
133
Acknowledgements
139
1
List of Abbreviations
AABB
AI
ATP
AS-3
CPD
2,3-DPG
DEHP
ECs
EI
EPM
FFP
FWB
Hb
Hct
HES
HGM
LGM
LORCA
MCV
MCHC
MODS
OD
PS
PVC
PVP
RBCs
SAGM
SIRS
SD
T½
T1DM
TRALI
American association of blood banks
aggregation index
adenine triphosphate
additive solution 3
citrate-phosphate-dextrose
2,3-diphosphoglycerate
di(2-ethylhexyl)phthalate
endothelial cells
elongation index
electrophoretic mobility
fresh frozen plasma
fresh whole blood
hemoglobin
hematocrit
hydroxyethyl starch
high-glycerol method
low-glycerol method
laser-assisted optical rotational cell analyzer
mean cell volume
mean cell hemoglobin concentration
multiple organ dysfunction syndrome
optical density
phosphatidyl serine
polyvinylchloride
polyvinylpyrrolidone
red blood cells
saline-adenine-glucose-mannitol additive solution
systemic inflammatory response syndrome
standard deviation
aggregation half-time
type-1 diabetes mellitus
transfusion related acute lung injury
Chapter 1
Introduction and aim of the thesis
Chapter 1
1.1 Introduction
Blood transfusion experiments were practiced as early in the 17th century. However, it was
the discovery of the ABO blood groups in the early 1900 that boosted transfusion
medicine.1 Blood transfusion practice has considerably improved since then and nowadays
refrigerated stored red blood cells (RBCs) are routinely infused to compensate for excessive
blood loss or to correct for abnormal hemoglobin content. Yet, during refrigerated storage
the RBCs undergo structural and biochemical alterations, collectively referred to as the
storage lesion, which could compromise their function after infusion. Particularly,
alterations in the RBC flow properties (i.e. rheologic properties) may impede the RBCs to
properly function in the microcirculation. This chapter describes, from a rheologic
perspective, the RBC characteristics and the cellular changes that are observed during
refrigerated blood bank storage. In addition, cryopreservation of RBCs will be discussed as
an alternative approach to counteract storage induced lesions and thus to extend the shelf
life of RBCs.
Characteristics of RBCs
RBCs are the most abundant cells in the blood. Adult humans have approximately 20-30
trillion circulating RBCs and on an average 2.6 million human RBCs are produced every
second by the red bone marrow and released into the blood via marrow sinusoids.2 RBCs
have unique structures and properties which enable them to distribute oxygen and collect
carbon dioxide in the body. Mature RBCs are shaped as biconcave discs (Figure 1.1) that
lack nuclei, mitochondria and other cell organelles. Yet, the RBCs are rich in hemoglobin
(Hb), an iron-containing protein that is able to reversible bind oxygen and carbon dioxide.
Figure 1.1. Human red blood cells under physiological conditions.
2
Introduction
In the RBC, energy is provided via the anaerobic glycolysis pathway. In this pathway
glucose is broken down in pyruvate with among others adenine-triphosphate (ATP) and
2,3-diphosphoglycerate (2,3-DPG) as intermediate metabolites. Subsequently, pyruvate is
converted to lactate and transported out of the cell. ATP as an energy source is essential for
the overall functioning of the RBC. Loss of ATP has been associated with shape changes,
enhanced cation permeability, rigid cell membranes, altered surface expression of
phospholipids, microvesiculation and decreased RBC viability.3-10 Furthermore, ATP
released by the RBC is a potent vasodilatation signaling molecule that adapts the blood
flow according to local needs.11,12 Recently, the ATP content was also correlated with the
oxygenation capacity of the RBC.13,14 In the RBC binding of 2,3-DPG to the Hb induces a
conformation state which will release oxygen from the Hb. Due to the high binding affinity
of 2,3-DPG for deoxygenated Hb, oxygen is released in regions with low oxygen tension.
Loss of 2,3-DPG will increase the oxygen affinity to the Hb which may hamper the oxygen
delivery to the tissues.2
The RBC membrane is composed of a lipid bi-layer and an underlying viscoelastic
cytoskeleton. This bi-layer consists of a layer of proteins which, due to the sialic acid
groups, bears a net negative charge. The viscoelastic cytoskeleton enables the RBC to adapt
their shape to maintain the flow behavior in the microvessels.
RBCs in the microcirculation
The microcirculation consists of a network of arterioles, capillaries and venules with a
diameter of less than 100 µm.15 In the capillaries exchange of oxygen and carbon dioxide
between circulating RBCs and the surrounding tissues takes place. Most microcirculatory
vessels are embedded within an organ. Adequate flow in the microcirculation is thus a
requisite for normal organ perfusion and function. The RBC rheologic properties (i.e. the
ability of RBC to aggregate, deform and adhere to endothelial cells) are important
determinants of the flow behavior of blood and subsequently the oxygenation of the microvascular environment.16,17
The ability of RBCs to form linear aggregates (Rouleaux: Figure 1.2) or more complex
three dimensional aggregates in regions with low shear rate is a phenomenon that has been
studied for decades. RBC aggregation is depending on opposing shear forces. On the one
hand RBC aggregation is counteracted by the repulsive force between negatively charged
3
Chapter 1
cells as well as the force generated by the blood flow. On the other hand, RBC aggregation
is promoted by the presence of plasma proteins, most importantly fibrinogen, or by high
molecular weight polymers.18,19 At present there are two theories proposed to explain the
RBC aggregation mechanism: a bridging and a depletion theory.20 In the bridging theory, it
is hypothesized that macromolecules adsorbed onto the RBC surface form bridges between
adjacent RBCs, whereas in the depletion theory RBC aggregation is induced due to an
osmotic gradient difference between the polymer concentration near the RBC surface
versus the suspending medium. Although RBC aggregation markedly enhances the blood
viscosity at low shear rate,21 the physiological role of this process is still elusive. Under
normal physiological conditions, RBC aggregates are easily dispersed by the rise in blood
flow rate. However, in certain pathologic states such as cardiovascular disorders, sepsis,
pre-eclampsia, diabetes mellitus, chronic renal failure, rheumatoid arthritis, inflammation,
hypertension, thalassemia and sickle cell disease,18,22-26 stronger and or larger aggregates
are formed which are more resistant to dispersion by the blood flow. Essentially, enhanced
RBC aggregation has the potential to impair the blood flow in the microcirculation and
contribute to the occlusion of micro-vessels.26,27
Flow
O2
CO2
O2
Artery
Capillaries
Figure 1.2. RBC Rouleaux formation in the microcirculation.
4
Vein
Introduction
The ability of RBCs to deform due to applied shear stress enables these cells to adapt their
size to squeeze through narrow capillaries. It also allows RBCs to reduce the blood
viscosity at high shear rate, so that blood remains fluid even at a high hematocrit. The RBC
deformability is depending on the geometry of the cell, the viscosity of the cytoplasm as
well as on the viscoelastic properties of the cytoskeleton.28 Alterations in these properties
can make the RBC more rigid and impair or block the blood flow in the microcirculation.
Ultimately, rigid RBCs will be sequestered by the spleen and destroyed. The RBC
deformability is thus essential for adequate tissue perfusion and cell survival.28,29 Reduced
RBC deformability has been observed in sepsis, diabetes mellitus, malaria, hypertension,
thalassemia and sickle cell disease.23-25
The adherence of RBCs to the vascular endothelium is, under physiological conditions,
negligible. However, structural changes in the RBC membrane may promote adherence to
endothelial cells (ECs) and impair the microcirculatory blood flow.26,30Adherence of RBCs
to ECs is mediated by expression of phosphatidylserine (PS) on the RBC surface.31,32 PS
expression also triggers recognition by macrophages which clears the RBCs from the
circulation.33-35 Enhanced RBC-EC interaction has been observed in malaria, diabetes
mellitus, thalassemia, sickle cell disease.26
In summary, changes in the rheologic properties of RBCs may form a circulatory risk by
hindering adequate tissue perfusion and contributing to ischemia or even infarction.26,36,37 It
is therefore not surprisingly that flow disturbances in the microcirculation are closely
associated with morbidity and mortality.38,39 These findings highlight the importance of
RBC rheologic properties as functionality parameters in transfusion medicine.
Usage of the Laser-assisted Optical Rotational Cell Analyzer
The laser-assisted optical rotational red cell analyzer (LORCA; R&R Mechatronics, Zwaag
The Netherlands)40,41 is a useful device to determine the aggregation and deformation
behavior of RBCs ex vivo.42,43 In general, a laser beam is directed through the RBC
solution and RBC aggregation is determined by means of backscattered light. Initially, the
RBC suspension is sheared to disperse pre-existing RBC aggregates. After several seconds
the shear is halted and the light reflection from the suspension is recorded over a certain
timeframe and subsequently analyzed by a computer program. As RBCs start to form side
to side aggregates (Rouleaux) the intensity of the backscattered light decreases
5
Chapter 1
exponentially. The aggregation behavior of RBCs is described by the aggregation index
(AI), which depends both on the kinetics and extent of aggregation.
The RBC deformability can be determined at various fluid shear stresses by means of laser
diffraction analysis. In general, a laser beam is directed through a sheared diluted RBC
solution and the diffraction pattern produced by the deformed cells is analyzed by a
computer program. Under the influence of shear stress, RBCs will gradual deform from a
biconcave to ellipsoid shape. Based upon the geometry of the diffraction pattern the
elongation index (EI) is calculated, in which an increased EI at any given shear stress
indicates greater cell deformation.
RBC Senescence
Human RBCs have an in vivo life span of approximately 120 days after which they are
selectively removed from the circulation by macrophages.35,44 This mechanism is essential
since it prevents Hb release from aged RBCs directly in the circulation. Aged RBCs are
characterized by cell shrinkage, microvesiculation and PS exposure at the cell surface.
Aged RBCs also have a decreased ability to deform and an enhanced tendency to
aggregate.19,45-47 RBC injury due to energy depletion, osmotic shock or oxidative stress
reduces the life span of the RBC.45 Refrigerated storage also induces cellular changes in the
RBC that diminish the life span and possibly the functionality of RBCs.
1.2. Refrigerated storage of RBCs
Worldwide RBCs are routinely infused in order to compensate for excessive blood loss as
well as to improve the oxygen carrying capacity. Annually, developed countries transfuse
approximately one RBC storage unit for every 20 inhabitants.48 Yet, recently RBC
transfusions has been under intensive evaluation. This is mainly because infusion of
prolonged refrigerated stored RBCs have been linked to adverse outcome in terms of
postoperative infections, length of hospital stay, multiple organ dysfunction syndrome
(MODS), transfusion related acute lung injury (TRALI), cancer progression and even
mortality.49-56
6
Introduction
Requirements of refrigerated stored RBCs
To date RBCs are routinely stored at 2-6°C for a maximum of 5 to 6 weeks, depending on
the preservation solution used. Notably, in the Netherlands RBCs are refrigerated and
stored in saline-adenine-glucose-mannitol (SAGM) solution for a maximum of 5 weeks.
International guidelines require that the hemolysis in a refrigerated RBC storage unit must
remain below allowable levels (i.e., 0.8% in Europe and 1% in The United States) and that
at least 75% of the infused RBCs must still circulate 24 hours after infusion.57,58
Interestingly, these guidelines do not specifically reflect the RBC ability to function after
infusion.
Quality of refrigerated stored RBCs
Although storage at 4°C slows down the biochemical processes in the RBC, cellular
metabolism is not completely suppressed at these temperatures. During refrigerated storage
a variety of changes have been observed that could compromise the RBCs to function after
infusion. These changes include decreased concentrations of 2,3-DPG, ATP and membrane
sialic acid content, translocation of PS to the cell surface, oxidative injury to membrane
lipids and proteins, shape change to spheroechinocytes, membrane blebbing and
accumulation of potassium, free HB, cytokines, bioactive lipids and (pro-coagulant)
microvesicles in the RBC storage unit.59-63
The RBCs rheologic properties also become impaired during refrigerated storage. RBCs
demonstrate an increased tendency to aggregate, diminished deformability and augmented
adhesiveness to ECs starting already from the second week of storage.64-68 These alterations
could hamper the RBCs to properly function in the microcirculation. Especially, since the
majority of the RBC units that are infused have a lifespan that exceeds 2 weeks of
storage.69-71 Yet, studies which propose a detrimental association with the infusion of longterm refrigerated stored RBCs are often biased and not adequately corrected for known
confounding factors.72-76 These factors include among others the number of RBC units
transfused, mixture of RBCs of different durations, severity of illness and the utilization of
leukoreduction.
7
Chapter 1
Leukofiltration of RBCs
In most developed countries, leukoreduction of RBC units prior to storage is a mandatory
practice. Initially leukoreduction was implemented to prevent HLA alloimmunization,
however by reducing the leukocyte count from a mean of 109 to 106 cells per RBC unit, less
detrimental substances such as cytokines, proteolytic enzymes and oxygen free radicals
accumulate in the RBC storage unit.77-80 Notably, leukoreduction reduces the risk of HLA
alloimmunization, febrile non-hemolytic transfusion reactions, immunomodulation and
transmission of infectious agents.81-85 It has been reported that leukoreduction prior to RBC
storage diminish the extent of storage induced RBC lesions.86,87 Hence, a lower degree of
hemolysis, microvesiculation, potassium leakage and osmotic fragility was observed during
refrigerated RBC storage as compared to their non-leukoreduced counterparts.80,88,89 In
addition, leukoreduced refrigerated stored RBCs showed negligible PS exposure and
adhesiveness to ECs with storage time.89-91 Interestingly, it was also observed that oxygen
free radicals and enzymes derived from activated leukocytes can compromise the
aggregation and deformation behavior of RBCs.86,92,93 Despite these findings, the
aggregability and deformability (i.e. rheologic features) of leukoreduced refrigerated stored
RBCs are yet to be determined.
In general, leukoreduction has been widely regarded as a beneficial practice to transfusion
medicine.81,94,95 However, whether leukoreduced refrigerated stored RBCs are functional in
the microcirculation or might be deleterious to transfusion outcome is still unclear.15,96-99 As
a result concerns regarding the infusion of prolonged stored RBCs are ongoing and a more
restrictive transfusion policy is currently being favored.100,101 Alternatively, storage of RBC
at ultra-low subzero temperatures halts the biochemical processes in the cell and could
therefore represent a promising approach to circumvent storage induced lesions and
subsequently to extend the shelf life of preserved RBCs.
1.3. Cryopreservation of RBCs
Since the discovery in the 1950s that glycerol protects RBCs from freezing injury,
cryopreserved RBCs have been under intensive investigation.48 Currently cryopreserved
RBCs (commonly known as frozen RBCs) are primarily used for controlling an inventory
in situations where the RBC availability is limited or unpredictable. Such as is the case for
8
Introduction
storage of RBC with rare blood groups or for usage in military settings.102,103 Occasionally
cryopreservation is used to preserve autologous RBCs.
Freezing methods
To date, there are two freezing methods approved for the preservation of RBCs.57,58 On the
one hand, RBCs can be frozen rapidly using a low-glycerol method (LGM) with a final
concentration of approximately 20% glycerol (wt/vol) at temperatures below -140°C. On
the other hand, RBCs can be frozen slowly using a high-glycerol method (HGM), allowing
storage of RBC units with a final concentration of approximately 40% (wt/vol) glycerol at
temperatures between -65°C and -80°C. Cryopreserved RBCs can be preserved up to at
least ten years if the correct storage temperature is guaranteed. The HGM cryopreserved
RBCs tolerate wide fluctuations in temperature during freezing and are more stable during
post-thaw storage.104,105 In addition, HGM cryopreserved RBCs did not require liquid
nitrogen which eased storage and transportation conditions. Therefore, the HGM is
currently the most applicable RBC freezing method in Europe and the United States.
Deglycerolization of thawed RBCs
Usage of glycerol as a cryoprotectant requires an intensive deglycerolization washing
procedure post-thaw in order to reduce the glycerol concentration within the cell. This is
necessary, since incomplete deglycerolized RBCs will cause hemolytic transfusion
reactions and renal failure after infusion.106,107 Washing also considerably reduces the
amount of detrimental substances such as bioactive lipids, microparticles, cytokines,
potassium and free Hb as well as leukocytes from the RBC unit.62,63,77,78,108-110 As a result,
transfusion of cryopreserved RBCs are associated with less febrile transfusion reactions,
alloimmunization as well as occurrences of TRALI and systemic inflammatory response
syndrome (SIRS).48,111
Requirements of cryopreserved RBCs
Preservation of RBCs at ultra-low subzero temperatures ceases the biological activity of the
cells which enables them to be preserved for years.112,113 However, once thawed the shelf
life of RBCs is limited. Deglycerolized RBCs are primarily stored in SAGM preservation
9
Chapter 1
solution for up to 48 hours or in AS-3 preservation solution for up to 14 days.
Cryopreserved RBCs need to be deglycerolized to reduce the residual glycerol content to
below 1%.104 Furthermore, international guidelines require that the hemolysis in the RBC
units must remain below allowable levels (i.e. 0.8% in Europe and 1% in The United
States) and that the RBC post-thaw recovery after deglycerolization (i.e. freeze-thaw-wash
recovery) must exceed 80%. Also, at least 75% of cryopreserved RBCs must still circulate
24-hours after infusion.57,58 However, these guidelines also do not reflect the ability of
RBCs to function after infusion. Hence, frozen storage subjects RBC to a range of
chemical, thermal and mechanical forces, which might affect their oxygen delivering
capacity after infusion.
Quality of thawed RBCs
The quality of HGM cryopreserved RBCs is dependent on the pre-freeze and post-thaw
storage time, the anticoagulant and additive solution used as well as on the biological
variation among RBC units. The cooling process per se only minimally induces cellular
damage when high glycerol concentrations were used.112,114 In general, RBCs intended for
frozen storage are refrigerated stored prior to processing. The 2,3-DPG content of
refrigerated stored RBCs rapidly declines due to the low pH of the storage solution. By
limiting this pre-freezing storage time, higher 2,3-DPG values could be obtained postthaw.114 Yet, the 2,3 DPG content continue to diminish during postthaw storage.115,116
Cryopreserved RBCs maintain high ATP content following post-thaw storage in SAGM
and AS-3. However, during postthaw storage also the ATP content gradually declines. This
decline is more prominent in AS-3 due to the lower pH of this storage solution.117 When the
pre-freezing storage time was limited to three days no significant changes in PS exposure,
CD-47 expression and membrane microvesiculation was observed with HGM
cryopreserved RBCs that were post-thaw stored in SAGM solution.118 In the past the
rheologic properties of cryopreserved RBCs have only briefly been addressed. It was
demonstrated that the aggregability and deformability of LGM cryopreserved RBCs were
diminished.119-121 Yet, the rheologic properties of HGM cryopreserved RBCs are still to be
determined.
10
Introduction
Implementation of new freezing technology
For routine clinical usage, cryopreserved RBCs must be safe, effective and reasonably
priced. Cryopreserved RBCs are more expensive, inefficient and time consuming than
refrigerated stored RBCs.48 However, ongoing technological advances could potentially
expand the utilization of cryopreserved RBCs in transfusion medicine. A promising
approach is the usage of a liquid Bio-freezer (Supachill, Lubbock, USA) with a high heat
absorption capacity. This Bio-freezer consists of a reservoir of fluid in which samples are
immersed. Consequently, the heat is rapidly dissipated from the sample and less freezing
damage is observed. This Bio-freezer has been successfully used to freeze bovine sperm
cells with a high post-thaw quality.122,123 The rate of heat release during freezing is also an
important contributor to the post-thaw quality of RBCs.124 Traditional freezing of RBCs at
–25°C in a mechanical freezer, normally requires high glycerol solutions to minimize ice
formation.125 However, usage of high concentrations of cryoprotectants also exert
detrimental effects on the cell.126 The rapid heat exchange of the liquid Bio-freezer, might
allow RBCs to be frozen with lower glycerol concentrations at higher temperatures. The
latter could reduce the osmotic stress and subsequently the freeze-thaw-wash related cell
loss. This characteristic could make the Bio-freezer particular interesting for
cryopreservation of RBCs.
1.4. Aim of this thesis
The aim of this thesis is to gain a better understanding of the RBC quality from a rheologic
perspective in transfusion medicine. Alternatively, the utilization of cryopreservation for
long-term storage of RBCs will be explored.
11
Chapter 1
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15
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Phelan HA, Gonzalez RP, Patel HD, Caudill JB, Traylor RK, Yancey LR, Sperry JL, Friese RS, Nakonezny
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by leukodepletion filters and washing. Transfus.Apher.Sci. 2008, vol.38: 141-147.
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16
Introduction
93.
Srour, Bilto YY, Juma M, Irhimeh MR. Exposure of human erythrocytes to oxygen radicals causes loss of
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96. Tsai AG, Cabrales P, Intaglietta M. Microvascular perfusion upon exchange transfusion with stored red
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103. Hess JR, Thomas MJ. Blood use in war and disaster: lessons from the past century. Transfusion. 2003,
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A, Verhoeven AJ. Stability after thawing of RBCs frozen with the high- and low-glycerol method.
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17
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110. Arnaud FG, Meryman HT. WBC reduction in cryopreserved RBC units. Transfusion. 2003, vol.43: 517-525.
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18
Chapter 2
Use of hydroxyethyl starch for inducing
red blood cell aggregation
Sandra Henkelman
Gerhard Rakhorst
Henny C. van der Mei
Henk J. Busscher
Accepted for publication in the journal of:
Clinical Hemorheology and Microcirculation
(Reproduced with permission of IOS press)
Chapter 2
Abstract
The aggregation of human RBC remains of biological and clinical interest. Replacement of plasma
proteins by polymers to induce RBC aggregation may help to unravel the fundamentals of the
aggregation process. Two theories exist to explain the RBC aggregation mechanism: a depletion and a
bridging theory. RBC aggregation induced by hydroxyethyl starch (HES) polymers increases with
polymer size, which suggests that aggregation is induced via the bridging theory. In the present study,
the electrophoretic mobility (EPM) was measured to investigate RBC aggregation induced by 200kDa HES polymers. In addition, we evaluated if these polymers were useful for demonstrating
aggregation differences between RBCs from healthy and type-1 diabetes mellitus (T1DM) subjects.
Our results demonstrate that the EPM values of RBCs in 200-kDa HES solutions were less negative
than could be predicted by the viscosity of the suspension, supporting the bridging theory.
Furthermore, aggregation analysis using the LORCA demonstrated that 200-kDa HES solution
enhanced RBC aggregation of healthy and diabetic subjects in a similar manner as standard 500-kDa
dextran solutions. In conclusion: our data supports the bridging mechanism underlying 200-kDa HES
induced RBC aggregation. In addition, both polymers are useful for demonstrating cellular induced
aggregation differences between RBCs from healthy and T1DM subjects.
20
Polymer induced RBC aggregation
2.1. Introduction
RBC aggregation is an important determinant of the flow behavior of blood. In regions with
low shear rate, human RBCs form linear stacks of cells and multi-cellular aggregates that
markedly enhance the blood viscosity and further reduce the blood flow rate.1 Under
physiological conditions RBC aggregates are easily dispersed when the blood flow rate
increases. However, under pathologic conditions stronger and larger aggregates may form
that are more resistant to dispersion by the blood flow, therewith contributing to the
occlusion of microvessels and hindering adequate tissue perfusion.2,3 Despite numerous
studies, the physiological role of RBC aggregation still remains unclear. It has been
demonstrated that different RBC aggregation tendencies can exist between healthy
individuals.4 In addition, enhanced RBC aggregation is observed in a variety of diseases
such as cardiovascular disease, diabetes mellitus, renal failure, sepsis, thalassemia and
sickle cell disease.3,5,6
The RBC surface consists of a layer of proteins, called the glycocalyx, which bears a net
negative charge. The ability of RBCs to aggregate is a balance between repulsive and
attractive forces. On one hand RBC aggregates are dispersed by repulsive electrostatic
forces between negatively charged RBCs as well as the prevailing shear forces generated by
the blood flow. On the other hand the promotion of aggregation is induced by the presence
of plasma proteins, especially fibrinogen, or by high molecular weight polymers.5,7
Usage of high molecular weight polymers to induce RBC aggregation gives more insight in
the aggregation process because it will exclude aggregation differences that are induced by
variations in plasma protein composition. Non-ionic polymers like dextran, polyethylene
glycol and to a lesser extent hydroxyethyl starch (HES) have been investigated for their
RBC aggregation inducing properties.8-14 Like dextran, HES polymers are used in plasma
substitutes to treat for example hypovolemia during surgery.15,16 HES polymers are
available in a broad range of molecular weights all with different tendencies to promote
RBC aggregation. In this regard, only HES polymers with low aggregation tendency, such
as the 130-kDa HES solutions, are currently preferred for infusion, whereas HES polymers
with hyper-aggregating tendencies, such as 450-kDa HES solutions, are less favored in
clinical practice.11,17,18 However, the 200-kDa HES polymer, which has intermediate RBC
aggregation tendencies, could be useful as a pro-aggregant in RBC rheologic studies.
21
Chapter 2
At present there are two theories that explain the RBC aggregation mechanism induced by
high molecular weight polymers.19 In the bridging theory, it is hypothesized that
macromolecules adsorbed onto the RBC membrane form bridges between adjacent cell
surfaces, which help overcome repulsive electrostatic forces between the RBCs. In the
depletion theory, RBC aggregation occurs due to a lower concentration of macromolecules
near the RBC surface compared to the suspending medium. This depletion of
macromolecules near the cell surface leads to an osmotic gradient that causes fluid to move
away from adjacent RBCs, which subsequently favors RBC aggregation.
Previous data support the depletion theory as the mechanism of dextran induced RBC
aggregation.13,20 The mechanism through which HES induces RBC aggregation has not yet
been established. However, since stronger and larger RBC aggregates are formed with HES
solutions of increasing molecular weight,18 an adsorption mediated aggregation mechanism
is expected to exist for this polymer. This is in contrast with the depletion mechanism,
where RBC aggregation has a nonlinear dependency on the molecular weight of the
polymer.8
Electrophoresis has shown to be a useful tool for studying the aggregation behavior of
RBCs.9,13,21,22 By measuring the electrophoretic mobility (EPM) of RBCs in polymer
solutions, a better understanding of the underlying aggregation mechanism can be obtained.
The EPM of RBCs in saline solutions demonstrates a negative value. In this regard a less
negative EPM of RBCs in polymer solutions (i.e. a lower mobility of RBCs) suggests
bridging mediated aggregation due to interactions of polymers with the RBC surface. In
contrast, a more negative EPM value (i.e. a higher mobility of RBCs) is explained by
depletion of polymers near the RBC surface.23
Differentiating between plasmatic and cellular factors leading to altered RBC aggregation is
important from both a biological and clinical point of view. Usage of high molecular weight
polymers will manifest RBC aggregation differences which are induced by cellular factors
such as RBC shape, deformability and membrane surface properties. This intrinsic tendency
of RBC to aggregate (i.e. RBC aggregability) is dependent on the type and the molecular
weight of polymers in solution.4
In the past standard dextran polymers have been useful pro-aggregants in RBC rheologic
studies.5,24 Like the standard 500-kDa dextran polymers, the 200-kDa HES polymers could
be useful to manifest cellular induced aggregation differences between RBCs from healthy
individuals and patients with a disease. Blood from type-1 diabetes mellitus (T1DM)
22
Polymer induced RBC aggregation
patients is especially suitable for aggregation testing. Particularly, because in these patients
alterations in both plasma and RBC cellular properties are responsible for enhanced RBC
aggregation tendencies.25 Nowadays, RBC aggregation can be studied using the LORCA.
This technique allows RBC aggregation to be studied ex vivo either in whole blood or in
polymer solutions.
In this study, the EPM of RBCs from healthy volunteers was explored in 200-kDa HES and
standard 500-kDa dextran solutions in order to elucidate the underlying aggregation
mechanism. Furthermore, the LORCA was used to investigate if these polymers could
discriminate cellular induced aggregation differences between RBCs from healthy
volunteers and T1DM patients.
2.2. Materials and methods
Sample preparation
Blood was collected from healthy volunteers and T1DM patients after informed consent
and in accordance with University Medical Center Groningen protocols. Briefly, whole
blood was anticoagulated with EDTA (1.5 mg/ml) and part of it was used in aggregation
experiments within 1 hour after collection. To obtain RBCs, whole blood was centrifuged at
1100 x g for 12 min to remove the buffycoat and supernatant. The concentrated RBCs were
washed twice with PBS (pH 7.4), and resuspended in 0.9% sodium chloride solution (NaCl;
pH 4.5 or pH 7.4) to a final hematocrit (Hct) of 40 ± 1%. Aggregation experiments and
EPM measurements with washed RBCs were performed within 4 h after collection. A 10%
HES solution with a molecular weight of 200-kDa and a molar substitution of 0.5 was
obtained from Fresenius, 's-Hertogenbosch, the Netherlands. Dextran fractions with a
molecular weight of 500-kDa were obtained from Sigma-Aldrich, Steinheim, Germany. A
2% dextran solution (wt/vol) was prepared by dissolving this polymer into 0.9% NaCl
solution with pH 7.4.
Electrophoretic mobility and viscosity measurements
The speed of RBCs in an electrical field was determined in suspension using a Laser Zee
Meter 501 equipped with image analysis options (PenKem, Bedford Hills, N.Y.), as has
been described previously.26 Briefly, RBCs were suspended in either 0.9% NaCl pH 7.4,
23
Chapter 2
0.9% NaCl pH 4.5, 10% 200-kDa HES pH 4.5 or 2% 500-kDa dextran pH 7.4 solutions at a
Hct of 0.05%. The electrophoresis chamber was filled with the RBC suspension and a
voltage difference of 150V was applied over the chamber. EPM values were measured at
20°C in duplicate with RBCs obtained from four different healthy volunteers. EPM values
were obtained from samples that contained a minimum of 35 RBCs per suspending
solution. The viscosities of the suspending solutions were measured in duplicate at 20°C
using a viscometer (DV-II+ Pro, Brookfield, USA). All EPM values are reported as
measured and are not corrected for differences in viscosity between the various solutions.
Aggregation measurements
RBC aggregation was assessed in vitro, by a laser-assisted optical rotational cell analyzer
(LORCA; R&R Mechatronics, Zwaag, the Netherlands).27 Aggregation was tested with 1
ml of either whole blood or with RBCs suspended in 200-kDa HES or 500-kDa dextran
solutions. In short, RBC suspensions were centrifuged for 1 min at 3500 x g and the
supernatant was discarded. RBCs were resuspended in either 10% 200-kDa HES or 2%
500-kDa dextran solutions, to obtain a Hct between 35 ± 5%. The aggregation of the RBCs
was monitored after complete disaggregation under increased shear stresses. Both the
aggregation measuring procedure and the subsequent analyses were computer controlled.
Aggregation of RBCs was expressed by the aggregation index (AI), where a larger AI
reflects an increased ability to aggregate. The AI depends on the kinetics and extent of
aggregation. The AI was determined after correcting the Hct in all the samples to a constant
value of 45%. The kinetics of aggregation (T1/2) was expressed by the time necessary to
induce 50% aggregation. All measurements were performed at 37°C.
Statistical analysis
Statistical analysis was performed using statistical software (SPSS, version 16.0, SPSS Inc.,
Chicago, IL). Data were tested for normality with the Kolmgorov-Smirnov goodness-of-fit
test. The EPM values were analyzed by using a repeated-measure ANOVA. Post hoc
comparisons were performed to quantify differences between suspension solutions by using
paired t-tests.
Aggregation differences between RBCs from healthy and T1DM subjects were determined
by using unpaired t-tests. Within subjects, paired t-tests were performed to show
24
Polymer induced RBC aggregation
differences with regard to whole blood. A Bonferroni correction was applied to correct for
multiple comparisons. Differences are considered to be significant with a two-tailed p-value
of less than 0.05. Results are presented as means ± SD.
2.3. Results
RBC electrophoretic mobility and viscosity
The EPM of RBCs suspended in polymer-free saline solutions was not significantly
affected by the pH. RBCs suspended in 200-kDa HES solutions showed significantly less
negative EPM values as compared to RBCs suspended in polymer-free solutions (-0.04 vs
-0.58 10-8m2V-1s-1). However, the EPM values of RBCs suspended in 200-kDa HES
solutions were less negative, than would be expected based upon the inverse relation
between suspension medium viscosity and EPM values found (Figure 2.1A).28
In contrast, RBCs suspended in 500-kDa dextran solutions demonstrated significantly more
negative EPM values as compared to RBCs suspended in polymer-free solutions (-1.40 vs
–0.74 10-8m2V-1s-1). However, these observed EPM values were considerable more negative
than would be expected based upon the inverse relation between the suspending medium
viscosity and the EPM values found (Figure 2.1B).
RBC aggregation
Whole blood from T1DM subjects showed an increased tendency to aggregate as compared
to that of healthy subjects (p < 0.05: Figure 2A). Moreover, the AI induced by 200-kDa
HES solution is higher with RBCs from T1DM subjects than that of healthy subjects (p <
0.05). However, it was notable that the AI induced by 200-kDa HES polymers was similar
to the AI induced by the 500-kDa dextran polymers.
No significant differences in the aggregation half-time between whole blood from healthy
and T1DM subjects (Figure 2B) were observed. The 200-kDa HES solution markedly
lowered the T1/2 as compared to that observed with whole blood of healthy and T1DM
subjects (p < 0.05), indicating a faster aggregation process with the use of this polymer.
However, both polymers did not induce significant differences in T1/2 between RBCs from
healthy and T1DM subjects.
25
Chapter 2
A B Figure 2.1. The electrophoretic mobility of human RBCs suspended in 200-kDa HES (A) or standard 500-kDa
dextran (B) solutions as a function of their viscosity. The dashed line is the predicted relation between EPM and
suspension viscosity assuming an inverse relation between these variables. Values are expressed as mean of eight
experiments.
26
Polymer induced RBC aggregation
A B
Figure 2.2. Aggregation indexes (A) and aggregation half-time (B) for RBCs obtained from healthy and T1DM
human subjects in either autologous plasma or RBCs resuspended in polymer solutions. Values are expressed as
the mean ± SD of ten different subjects. Significant changes are illustrated in the figure (p < 0.05); * significantly
different from whole blood, † significantly different from its healthy counterpart.
27
Chapter 2
2.4. Discussion
The found EPM values of healthy RBCs suspended in 200-kDa HES solution supports the
bridging theory as the mechanism underlying HES induced RBC aggregation. Although
200-kDa HES and 500-kDa dextran polymers induce RBC aggregation via different
mechanisms, we demonstrated that both polymers are useful pro-aggregants for manifesting
cellular induced aggregation differences between RBCs from healthy and T1DM subjects.
Aggregation of RBCs is a physiological phenomenon that has been studied for decades.5
Although RBC aggregation is an important determinant of blood viscosity in regions of low
shear rate, the physiological role of this process still remains elusive. Nevertheless,
enhanced RBC aggregation has been observed in various diseases.3,5,6 The use of standard
polymer suspensions to induce RBC aggregation will give more insight in the fundamentals
of the aggregation process. Currently, HES polymers are primarily used as plasma
expanders.15,16 However, studies demonstrating that 200-kDa HES polymers could be
useful pro-aggregants in rheologic studies were lacking.
In the present study, the EPM values of RBCs suspended in 200-kDa HES solution were
considerable less negative than the linear reciprocal viscosity change that is predicted by
the Helmholtz–Smoluchowski equation.28 The EPM of RBCs in an electrical field is
determined by the viscosity of the suspending fluid as well as the RBC surface charge
density and the glycocalyx thickness.5,23,29 Given that HES polymers are nonionic, it is
unlikely that these EPM changes are associated with alterations in RBC surface charge
density. Instead, absorption of nonionic HES polymers onto the RBC surface may have
attributed to structural changes within and near the RBC glycocalyx, explaining the less
negative EPM values.30 Clearly, our EPM measurements underline the presence of a
bridging mechanism between RBCs when using 200-kDa HES polymers.
In contrast, dextran induced aggregation is characterized by an optimal aggregation
tendency at a molecular weight of approximately 500-kDa.8 In this study, the EPM values
of RBC in 500-kDa dextran solutions became more negative despite the higher viscosity of
the bulk suspending medium. These findings are in agreement with a depletion interaction
in which a lower viscosity near the RBC surface compared to the bulk suspending medium,
enhances the EPM of the RBCs.19 A drawback with current depletion theories is that
polymer absorption onto RBC membranes are often not taken into account due to
experimental limitations.5,8,13 Nevertheless, a depletion theory does not require an absolute
28
Polymer induced RBC aggregation
lack of absorption on RBCs in order to initiate aggregation, but merely that the absorbed
polymer level is less than the polymers present in the bulk suspending medium.5,31
Recently, Liu et al. have provided new insights for characterizing absorption of polymers
onto RBCs.32 In their study tritium labeling was used to quantify the in vitro absorption of
polymers onto the RBCs, while laser scanning confocal microscopy was used to locate the
precise binding sites. Future aggregation studies that allow the implementation of an
absorption interaction with these techniques are therefore warranted.
In the present study, whole blood from T1DM human subjects showed an increased
tendency to aggregate as opposed to whole blood from healthy subjects. It has been
recognized that a disturbed glucose homeostasis in T1DM subjects results in elevated
plasma fibrinogen concentrations and a reduced anionic RBC surface charge.25,33 The
increased AI observed with whole blood from T1DM subjects is thus a result of changes in
both plasma and RBC cellular properties.
Usage of high molecular weight polymers, exclude aggregation differences induced by
variations in plasma protein compositions. In this study, the 200-kDa HES and standard
500-kDa dextran solutions markedly enhanced the AI of RBCs as compared to whole
blood. Nevertheless, both polymer solutions were clearly able to discriminate aggregation
differences between RBCs from healthy and T1DM subjects. Our results therefore
demonstrate that also 200-kDa HES polymers are useful in manifesting RBC aggregation
differences which are of cellular origin. The parameter AI depends both on the kinetics (t1/2)
and extent of aggregation. In this study no significant differences in t1/2 between healthy and
T1DM subjects with either whole blood or polymer induced RBC aggregation could be
observed. This indicated that aggregates are not necessarily formed faster but that the extent
of aggregation is the determining factor when it comes to manifesting differences between
RBCs from healthy and T1DM human subjects.
The ability of RBCs to form aggregates in the presence of plasma proteins may play a
pivotal role in maintaining hemodynamics. RBCs flow from the endothelial wall into the
center of the blood vessel where they form aggregates. While physiological values of RBC
aggregation direct leukocytes and possibly platelets from the axial core towards the
vascular wall,34 intensified RBC aggregation may hinder or obstruct the blood flow in
micro-vessels. More insight in the RBC aggregation process is therefore important from
both a biological and clinical point of view.
29
Chapter 2
In the past, the depletion theory has been particularly favored as the mechanism of nonionic polymer induced RBC aggregation. Although the 200-kDa HES solutions induced
similar aggregation tendencies as the standard 500-kDa dextran solutions, our data supports
the bridging theory as the mechanism underlying HES induced aggregation. Since RBCs
from healthy volunteers show significant variations in terms of their aggregation tendency,4
maybe in vivo both aggregation mechanisms can exist depending on the protein
composition of plasma. Furthermore, our results demonstrate that like the standard 500-kDa
dextran solutions, the 200-kDa HES solutions are useful for manifesting cellular induced
aggregation differences between RBCs from healthy and T1DM subjects.
References
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Marossy A, Svorc P, Kron I, Gresova S. Hemorheology and circulation. Clin.Hemorheol.Microcirc. 2009,
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Mchedlishvili G, Varazashvili M, Gobejishvili L. Local RBC aggregation disturbing blood fluidity and
causing stasis in microvessels. Clin.Hemorheol.Microcirc. 2002, vol 26: 99-106.
3.
Yedgar S, Koshkaryev A, Barshtein G. The red blood cell in vascular occlusion. Pathophysiol.
Haemost.Thromb. 2002, vol 32: 263-268.
4.
Rampling MW, Meiselman HJ, Neu B, Baskurt OK. Influence of cell-specific factors on red blood cell
aggregation. Biorheology. 2004, vol 41: 91-112.
5.
Meiselman HJ. Red blood cell aggregation: 45 years being curious. Biorheology. 2009, vol 46: 1-19.
6.
Reggiori G, Occhipinti G, de Gasperi A, Vincent JL, Piagnerelli M. Early alterations of red blood cell
7.
Ben-Ami R, Barshtein G, Mardi T, Deutch V, Elkayam O, Yedgar S, Berliner S. A synergistic effect of
albumin and fibrinogen on immunoglobulin-induced red blood cell aggregation. Am.J.Physiol Heart
8.
Neu B, Wenby R, Meiselman HJ. Effects of dextran molecular weight on red blood cell aggregation.
rheology in critically ill patients. Crit Care Med. 2009, vol 37: 3041-3046.
Circ.Physiol. 2003, vol 285: H2663-H2669.
Biophys.J. 2008, vol 95: 3059-3065.
9.
Baskurt OK, Tugral E, Neu B, Meiselman HJ. Particle electrophoresis as a tool to understand the aggregation
behavior of red blood cells. Electrophoresis 2002, 23: 2103-2109.
10. Pribush A, Zilberman-Kravits D, Meyerstein N. The mechanism of the dextran-induced red blood cell
aggregation. Eur.Biophys.J. 2007, vol 36: 85-94.
11. Neff TA, Fischler L, Mark M, Stocker R, Reinhart WH. The influence of two different hydroxyethyl starch
solutions (6% HES 130/0.4 and 200/0.5) on blood viscosity. Anesth.Analg. 2005, vol 100: 1773-1780.
12. Armstrong JK, Meiselman HJ, Fisher TC. Covalent binding of poly(ethylene glycol) (PEG) to the surface of
red blood cells inhibits aggregation and reduces low shear blood viscosity. Am.J.Hematol. 1997, 56:26-28.
30
Polymer induced RBC aggregation
13. Rad S, Gao J, Meiselman HJ, Baskurt OK, Neu B. Depletion of high molecular weight dextran from the red
cell surface measured by particle electrophoresis. Electrophoresis 2009, vol 30: 450-456.
14. Mosbah IB, Franco-Gou R, Abdennebi HB, Hernandez R, Escolar G, Saidane D, Rosello-Catafau J, Peralta
C. Effects of polyethylene glycol and hydroxyethyl starch in University of Wisconsin preservation solution
on human red blood cell aggregation and viscosity. Transplant.Proc. 2006, vol 38: 1229-1235.
15. Jungheinrich C, Neff TA. Pharmacokinetics of hydroxyethyl starch. Clin.Pharmacokinet. 2005, vol 44: 681699.
16. Treib J, Baron JF, Grauer MT, Strauss RG. An international view of hydroxyethyl starches. Intensive Care
Med. 1999, vol 25: 258-268.
17. Kozek-Langenecker SA, Jungheinrich C, Sauermann W, Van der Linden P. The effects of hydroxyethyl
starch 130/0.4 (6%) on blood loss and use of blood products in major surgery: a pooled analysis of
randomized clinical trials. Anesth.Analg. 2008, vol 107: 382-390.
18. Morariu AM, van der Plaats A, van Oeveren W, 'T Hart NA, Leuvenink HG, Graaff R, Ploeg RJ, Rakhorst
G. Hyperaggregating effect of hydroxyethyl starch components and University of Wisconsin solution on
human red blood cells: a risk of impaired graft perfusion in organ procurement? Transplantation. 2003, vol
76: 37-43.
19. Baumler H, Neu B, Donath E, Kiesewetter H. Basic phenomena of red blood cell rouleaux formation.
Biorheology. 1999, vol 36: 439-442.
20. Neu B, Meiselman HJ. Depletion-mediated red blood cell aggregation in polymer solutions. Biophys.J. 2002,
vol 83: 2482-2490.
21. Sabolovic D, Sestier C, Perrotin P, Guillet R, Tefit M, Boynard M. Covalent binding of polyethylene glycol
to the surface of red blood cells as detected and followed up by cell electrophoresis and rheological methods.
Electrophoresis. 2000, vol 21: 301-306.
22. Neu B, Armstrong JK, Fisher TC, Baumler H, Meiselman HJ. Electrophoretic mobility of human red blood
cells coated with poly(ethylene glycol). Biorheology. 2001, vol 38: 389-403.
23. Baumler H, Donath E, Krabi A, Knippel W, Budde A, Kiesewetter H. Electrophoresis of human red blood
cells and platelets. Evidence for depletion of dextran. Biorheology. 1996, vol 33: 333-351.
24. Baskurt OK, Bor-Kucukatay M, Yalcin O, Meiselman HJ. Aggregation behavior and electrophoretic
mobility of red blood cells in various mammalian species. Biorheology. 2000, vol 37: 417-428.
25. Singh M, Shin S. Changes in erythrocyte aggregation and deformability in diabetes mellitus: a brief review.
Indian J.Exp.Biol. 2009, vol 47: 7-15.
26. Van Merode AE, Duval JF, van der Mei HC, Busscher HJ, Krom BP. Increased adhesion of Enterococcus
faecalis strains with bimodal electrophoretic mobility distributions. Colloids Surf.B Biointerfaces. 2008, vol
64: 302-306.
27. Hardeman MR, Dobbe JG, Ince C. The Laser-assisted Optical Rotational Cell Analyzer (LORCA) as red
blood cell aggregometer. Clin.Hemorheol.Microcirc. 2001, vol 25: 1-11.
28. Mewis J, Wagner NJ. Colloidal suspension rheology. Cambridge: Cambridge university press; 2011.
31
Chapter 2
29. Jan KM, Chien S. Role of surface electric charge in red blood cell interactions. J.Gen.Physiol. 1973, vol 61:
638-654.
30. Neu B, Sowemimo-Coker SO, Meiselman HJ. Cell-cell affinity of senescent human erythrocytes. Biophys.J.
2003, vol 85: 75-84.
31. Van Oss CJ, Arnold K, Coakley WT. Depletion flocculation and depletion stabilization of erythrocytes. Cell
Biophys. 1990, vol 17: 1-10.
32. Liu Z, Janzen J, Brooks DE. Adsorption of amphiphilic hyperbranched polyglycerol derivatives onto human
red blood cells. Biomaterials. 2010, vol 31: 3364-3373.
33. Le Devehat C, Vimeux M, Khodabandehlou T. Blood rheology in patients with diabetes mellitus.
Clin.Hemorheol.Microcirc. 2004, vol 30: 297-300.
34. Nash GB, Watts T, Thornton C, Barigou M. Red cell aggregation as a factor influencing margination and
adhesion of leukocytes and platelets. Clin.Hemorheol.Microcirc. 2008, vol 39: 303-310.
32
Chapter 3
Red blood cell aggregation; an important
phenomenon
in
damage
control
resuscitation?
Sandra Henkelman
Michael Piagnerelli
Gerhard Rakhorst
Chapter 3
Abstract
To improve the survival of severely bleeding trauma patients, a damage control resuscitation
strategy has been recommended. This strategy includes the early infusion of fresh frozen plasma
(FFP), platelets and RBCs in a 1:1:1 unit ratio to control the bleeding and favor survival of
these patients. Although lower FFP to RBC ratios have been linked to higher rates of mortality,
these high ratios have been associated with adverse outcomes as well. The formation of RBC
aggregates in regions with low shear rate could play a key role in these findings. Administration
of FFP and thus fibrinogen is essential for coagulation. Yet, fibrinogen also promotes RBC
aggregation. Although physiological levels of RBC aggregation support the hemostasis,
promotion of aggregation could be disadvantageous in patients in which the RBC rheology is
already compromised, as was observed in certain trauma states. Notably, enhanced RBC
aggregation may hinder tissue perfusion and contribute to the occlusion of micro-vessels. We
consider that RBC aggregation could play an important role in damage control resuscitation of
severely injured trauma patients.
34
RBC aggregation in trauma patients
Uncontrolled bleeding is the leading cause of death in trauma patients. It was recognized
that approximately 25% of severely injured trauma patients are coagulopathic upon
admission and that these patients are three times more likely to die than those without it.1
This acute coagulopathy of trauma has been attributed to multiple factors such as loss,
dilution and consumption of coagulation factors and platelets as well as to fibrinolysis,
hypothermia and metabolic acidosis.2 To improve the survival of severely bleeding trauma
patients, an early damage control resuscitation strategy has been recommended. This
resuscitation approach prima1rily advocates limited crystalloid use, prevention and
treatment of acidosis and hypothermia, as well as an early administration of fresh frozen
plasma (FFP), platelets and RBCs in a 1:1:1 unit ratio.3 Although the optimal blood
component ratio is still a matter of debate, the general consensus is that an early
resuscitation approach with this high FFP to RBC ratio, controls the bleeding and
potentially favors survival of severely bleeding trauma patients.4
Limited attention has been addressed, to the mechanism by which a high FFP to RBC ratio
influences survival in these trauma patients. Recently, the improved survival has been
linked to inhibition of vascular endothelial permeability and subsequently diminished
interstitial edema.5 Yet the ability of RBCs to form aggregates in the presence of plasma
proteins, especially fibrinogen, could also play a pivotal role in damage control
resuscitation.
The formation of RBC aggregates in regions with low shear rate is a physiological
phenomenon that has been studied for decades. Although RBC aggregation is a major
determinant of the whole blood viscosity at low shear rate, the physiological role of this
process is still elusive. Under normal physiological conditions, RBC aggregates are easily
dispersed by the rise in blood flow rate. However, under pathological conditions stronger
and or larger RBC aggregates are formed which are more resistant to dispersion by shear
forces. Enhanced RBC aggregation may impair the blood flow in the microcirculation and
contribute to the occlusion of micro-vessels, which may induce local hypoxia and damage
to endothelial cells.6,7 In this regard, the LORCA is a useful device, which allows RBC
aggregation to be studied ex vivo.
In cases of massive bleeding, fibrinogen is the first coagulation factor that reaches critically
low levels. Administration of blood components in an 1:1:1 unit ratio will replenish
depleted coagulation factors and platelets and minimize dilutional coagulopathy, as is the
case when only RBCs or volume expanders will be administrated.8 Yet, in a trauma setting
35
Chapter 3
the use of FFP in massive bleedings has also been questioned. Adverse outcome such as
increased incidence of nosocomial infections, multiple organ failure, lung injury and death
have been linked to the usage of FFP.9 Studies have shown mixed results, but in general,
high FFP to RBC ratios have been associated with adverse outcomes whereas low FFP to
RBC ratios have been linked to increased rates of mortality.1,10
For many years, it has been recognized that RBCs can actively participate in clot formation
by enhancing platelet de-granulation and by recruitment of additional platelets into the
forming clot.11 The role of RBC aggregation in hemostasis has been given less attention.
RBCs migrate from the endothelial wall into the center of the blood vessel where they form
aggregates. RBC aggregates exclude leukocytes and possibly platelets from the axial core
and direct them towards the vascular wall.12,13 This process is essential since leukocytes and
platelets need to get into close contact with the damaged endothelium, in order to exert their
function.
RBC aggregation increases proportionally with fibrinogen levels.14 Administration of FFP,
platelets and RBCs in a 1:1:1 unit ratio, a composition that approximates whole blood,
could promote RBC aggregation. On the one hand promotion of aggregation would be
beneficial for supporting hemostasis in severely injured trauma patients. Especially, since it
has been recognized that people with leukocyte adherence deficiency suffer from recurrent
bacterial infections and impaired wound healing and because it has been recognized that
infections remain a concerning complication of combat-related injuries.15,16 On the other
hand, promotion of aggregation could be detrimental to patients in which the RBC rheology
is already compromised, as was observed in certain trauma states.17-19 In this regard,
enhanced RBC aggregation could subsequently hamper tissue perfusion and contribute to
the occlusion of micro-vessels. Enhanced RBC aggregation, which was also evident during
long-term storage of non-leukoreduced RBC units, could furthermore explain the finding
that blood component infusion was less effective than fresh whole blood in supporting
hemostasis of trauma patients.3,20,21 The above mentioned data underline the potential
importance of RBC aggregation in damage control resuscitation.
Most studies regarding resuscitation practices are retrospective. Although these studies are
limited inherently to their retrospective design, they do provide interesting hypotheses.
Early FFP infusion is considered lifesaving in severely bleeding trauma patients. Yet, to
determine the influence of high FFP to RBC ratios on promoting RBC aggregation in these
patients, ex vivo aggregation testing will be necessary. In this regard, the LORCA could be
36
RBC aggregation in trauma patients
useful to demonstrate RBC aggregation tendencies after damage control resuscitation of
severely bleeding trauma patients.
References
1.
Nascimento B, Callum J, Rubenfeld G, Neto JB, Lin Y, Rizoli S. Clinical review: Fresh frozen plasma in
massive bleedings, more questions than answers. Crit Care. 2010, vol.14: 202.
2.
Murthi SB, Stansbury LG, Hess JR. Blood and coagulation support in trauma. Blood Rev. 2009, vol.23: 149155.
3.
Spinella PC, Holcomb JB. Resuscitation and transfusion principles for traumatic hemorrhagic shock. Blood
Rev. 2009, vol.23: 231-240.
4.
Ho AM, Dion PW, Yeung JH, Ng CS, Karmakar MK, Critchley LA, Rainer TH, Cheung CW, Tay BA.
Fresh-frozen plasma transfusion strategy in trauma with massive and ongoing bleeding. Common (sense) and
sensibility. Resuscitation. 2010, vol.81: 1079-81.
5.
Pati S, Matijevic N, Doursout MF, Ko T, Cao Y, Deng X, Kozar RA, Hartwell E, Conyers J, Holcomb JB.
Protective effects of fresh frozen plasma on vascular endothelial permeability, coagulation, and resuscitation
after hemorrhagic shock are time dependent and diminish between days 0 and 5 after thaw. J.Trauma. 2010,
6.
Yedgar S, Koshkaryev A, Barshtein G. The red blood cell in vascular occlusion. Pathophysiol.
vol.69: S55-S63.
Haemost.Thromb. 2002, vol.32:263-268.
7.
Mchedlishvili G. Disturbed blood flow structuring as critical factor of hemorheological disorders in
microcirculation. Clin.Hemorheol.Microcirc. 1998, vol.19: 315-325.
8.
Fries D, Martini WZ. Role of fibrinogen in trauma-induced coagulopathy. Br.J.Anaesth. 2010,105: 116-121.
9.
Spahn DR, Ganter MT. Towards early individual goal-directed coagulation management in trauma patients.
Br.J.Anaesth. 2010, vol.105: 103-105.
10. Wafaisade A, Maegele M, Lefering R, Braun M, Peiniger S, Neugebauer E, Bouillon B. High plasma to red
blood cell ratios are associated with lower mortality rates in patients receiving multiple transfusion (4≤ red
blood cell units <10) during acute trauma resuscitation. J.Trauma. 2011, vol.70: 81-88.
11. Andrews DA, Low PS. Role of red blood cells in thrombosis. Curr.Opin.Hematol. 1999, vol.6: 76-82.
12. Nash GB, Watts T, Thornton C, Barigou M. Red cell aggregation as a factor influencing margination and
adhesion of leukocytes and platelets. Clin.Hemorheol.Microcirc. 2008, vol.39: 303-310.
13. Pearson MJ, Lipowsky HH. Effect of fibrinogen on leukocyte margination and adhesion in postcapillary
venules. Microcirculation. 2004, vol.11: 295-306.
14. Falco C, Vaya A, Simo M, Contreras T, Santaolaria M, Aznar J. Influence of fibrinogen levels on
erythrocyte aggregation determined with the Myrenne aggregometer and the Sefam erythro-aggregometer.
Clin.Hemorheol.Microcirc. 2005, vol.33: 145-151.
37
Chapter 3
15. Van Vliet DN, Brandsma AE, Hartwig NG. Leukocyte-adhesion deficiency: a rare disorder of inflammation.
Ned.Tijdschr.Geneeskd. 2004, vol.148: 2496-2500.
16. Murray CK, Wilkins K, Molter NC, Li F, Yu L, Spott MA, Eastridge B, Blackbourne LH, Hospenthal DR.
Infections complicating the care of combat casualties during operations Iraqi Freedom and Enduring
Freedom. J.Trauma 2011, vol.71:S62-S73.
17. Machiedo GW, Zaets SB, Berezina TL, Xu DZ, Feketova E, Spolarics Z, Deitch EA. Trauma-hemorrhagic
shock-induced red blood cell damage leads to decreased microcirculatory blood flow. Crit Care Med. 2009,
vol.37: 1000-1010.
18. Tatarishvili J, Sordia T, Mchedlishvili G. Comparison of blood rheological changes in the microcirculation
during experimental hemorrhagic and traumatic shock. Clin.Hemorheol.Microcirc. 2006, vol.35: 217-221.
19. Reggiori G, Occhipinti G, de Gasperi A, Vincent JL, Piagnerelli M. Early alterations of red blood cell
rheology in critically ill patients. Crit Care Med. 2009, vol.37: 3041-3046.
20. Hovav T, Yedgar S, Manny N, Barshtein G. Alteration of red cell aggregability and shape during blood
storage. Transfusion. 1999, vol.39: 277-281.
21. Phelan HA, Eastman AL, Aldy K, Carroll EA, Nakonezny PA, Jan T, Howard JL, Chen Y, Friese RS, Minei
JP. Prestorage leukoreduction abrogates the detrimental effect of aging on packed red cells transfused after
trauma: a prospective cohort study. Am.J.Surg. 2012, vol.203:198-204.
38
Chapter 4
Is red blood cell rheology preserved
during routine blood bank storage?
Sandra Henkelman
Margriet J. Dijkstra-Tiekstra
Janny de Wildt-Eggen
Reindert Graaff
Gerhard Rakhorst
Willem van Oeveren
Transfusion 50: 941-948, 2010
(Reproduced with permission of John Wiley and Sons)
Chapter 4
Abstract
RBCs refrigerated stored for more than 2 weeks at 4°C are currently considered of impaired quality.
This opinion has among others been based on altered RBC rheologic properties (i.e. enhanced
aggregability, reduced deformability and elevated EC interaction) observed during storage of nonleukoreduced RBC units. Nevertheless, with the implementation of leukoreduction the storageinduced lesions have considerably diminished. In this study, the aggregability and deformability of
leukoreduced RBCs during routine blood bank storage were investigated. At the blood bank, ten
leukoreduced RBC units were refrigerated stored in SAGM preservation solution for up to 7 weeks.
RBCs were weekly tested for aggregability, deformability and other hematologic variables. The
RBC aggregability was significantly reduced after the first week of storage but recovered during the
following weeks. After 7 weeks of storage the aggregability was slightly reduced (from 46.9 to 44.3
AI; p < 0.05). During storage the osmotic fragility was not significantly enhanced and the
deformability at a shear stress of 3.9 Pa was not significantly reduced. The deformability at a shear
stress of 50 Pa was reduced (from 0.58 to 0.54 EI; p < 0.05) but remained within physiological
values (0.53 ± 0.04). During 5 weeks of storage the ATP content was reduced by 54% whereas the
MCV, pH and MCHC were minimally affected. We conclude that the rheologic properties of
leukoreduced RBC units were well preserved during routine blood bank storage.
40
Rheologic properties of refrigerated stored RBCs
4.1. Introduction
During refrigerated storage at 4°C, RBCs undergo physical and biochemical alterations
collectively referred to as the storage lesion.1 Recent publications suggest that transfusion
of long-term refrigerated stored RBCs are associated with adverse clinical outcome in
critical ill, cardiac surgery, and trauma patients.2
The RBC rheology, that is, the ability to aggregate, deform and adhere to ECs, are
important determinants of the blood flow and hence the oxygen delivery to the tissues.3,4
Aggregation takes place in the venous system where RBCs form linear stacks of cells or
multi-cellular aggregates at low shear rates.5 Normally the increasing blood flow is
sufficient to disperse these aggregates. However, under pathologic conditions stronger and
larger aggregates may form, which are more resistant to dispersion by the blood flow. The
RBC ability to deform due to applied forces makes these cells capable of passing the
capillaries. High RBC deformability and a rapid recovery to the normal shape are therefore
essential factors for maintaining tissue perfusion.6 The RBC deformability is also a major
determinant of the posttransfusion survival, since less deformable cells will be sequestered
and destroyed in the spleen.7 Elevated adherence of RBCs to ECs can reduce the blood flow
in the microcirculation and activate ECs, contributing to the occlusion of micro-vessels.3, 8
Alterations in RBC rheology have been observed in a variety of diseases such as
cardiovascular disease, hypertension, diabetes mellitus, renal failure, malaria, thalassemia
and sickle cell disease.3,9,10 Transfusion of rheologic impaired RBCs may hinder or obstruct
the blood flow in micro-vessels leading to impaired tissue perfusion, ischemia or
infarction.3,4 Therefore, rheologic impaired RBCs may form a hemodynamic risk
particularly in recipients with circulatory and /or cardiovascular disorders. Long-term
refrigerated storage may alter the RBC rheologic properties and adversely influence
transfusion outcome. In vitro studies with non-leukoreduced RBCs demonstrated enhanced
aggregability, reduced RBC deformability and elevated adherence to ECs already after the
second week of storage.11-14
Since 2002, leukoreduction of RBC units is a standard procedure in many European
countries.15 Pre-storage leukofiltration reduces RBC damage caused by cytokines and
enzymes derived from activated leukocytes.16,17 As a result leukoreduced RBC units
demonstrate a lower degree of hemolysis, potassium leakage, osmotic fragility, and free
radical production during storage.18,19 Interestingly, it was also demonstrated that activated
41
Chapter 4
leukocytes induce cellular changes that adversely affect the rheologic properties of the
RBCs.20,21 The in vitro aggregability and deformability of leukoreduced RBCs during
routine blood bank storage remains to be determined and may contribute to the ongoing
discussion of safety of blood transfusions. In this study the aggregability, deformability and
other hematologic variables of leukoreduced refrigerated stored RBC were investigated
during a 7-week period.
4.2. Materials and Methods
Preparation and sampling of RBC units
Blood (500ml ± 10%) was collected from ten volunteers donors at the Sanquin blood bank
in a quadruple top and -bottom bag system (Composelect, Fresenius Hemocare, the
Netherlands) containing 70 ml of CPD anticoagulant. After cooling under butandiol plates
for at least 4 hours, whole blood was separated into plasma, buffycoat and RBCs using an
automated blood processing device (Compomat G4, Fresenius HemoCare, the
Netherlands). SAGM solution (110 ml) was transferred to the RBCs and in-line filtration
was carried out to remove residual leukocytes. The resulting RBC suspension had a Hct of
45-60% and contained less than 106 leukocytes per unit. RBCs were refrigerated stored at 4
± 2 °C for 7 weeks. RBCs were released for use on day 3 after donation, hereafter referred
to as Time 0, which were the freshest RBCs routinely available for transfusion. Weekly,
samples were aseptically withdrawn from the RBC units for analysis after gentle mixing by
inversion.
Rheologic features
RBC aggregability and deformability were monitored in vitro by the LORCA (R&R
Mechatronics, Zwaag the Netherlands).22,23 Aggregation was induced by the addition of
10% HES (MW 200-kDa). Briefly, RBCs suspensions were centrifuged for 1 minute at
3500 x g and the supernatant was discarded. RBCs were resuspended in 10% HES 200-kDa
solution (Fresenius, Bad Homburg, Germany). The Hct in all the samples was corrected to a
constant value of 45%. Aggregability was tested with 1 ml of the RBC suspension.
Aggregation of RBCs was monitored after disaggregating under increased shear stress.
Both the aggregation measuring procedure and the subsequent analyses were computer
42
Rheologic properties of refrigerated stored RBCs
controlled. Aggregability of RBC was expressed by the AI, where a larger AI reflects an
increased ability to aggregate.
The deformability of stored RBCs was determined with RBC suspension diluted 1:100 in
PBS, (pH 6.5), containing 5% polyvinylpyrrolidone (PVP; MW 360 kDa, Sigma-Aldrich,
Germany) and with a viscosity of 30 mPa.sec. One ml of the latter RBC suspension was
inserted into the LORCA and the RBC diffraction pattern was recorded at various shear
stresses at 36.8 ± 0.2°C. The deformability of the RBCs, which is expressed by the EI, was
determined by the LORCA from the size of the vertical (L) and horizontal (W) axes of the
diffraction pattern according to the formula: EI = (L-W) / (L+W). An increased EI at a
given shear stress indicates greater RBC deformability. A deformability curve was obtained
by plotting the calculated values for EI versus the corresponding shear stress. The
deformability at two shear stress values were examined more closely; the deformability at a
shear stress of 3.9 Pa, which reflects the rigidity of the cell membrane, and the maximal
deformability at shear stress of 50 Pa. Since the freshest available RBCs for transfusion
were already 3 days old, the deformability was also performed with RBCs that were
obtained within 2 hours after donation from healthy donors.
Osmotic fragility
The osmotic fragility of RBCs, which reflects the membrane’s ability to maintain structural
integrity, was determined by diluting RBCs in PBS solutions ranging from 0.90% to 0.35%.
RBCs with a Hct level of 30 to 35% were diluted 1:100 in each PBS solution, mixed and
incubated for 30 minutes at 4°C, followed by centrifugation for 12 minutes at 1100 x g. The
free Hb in the supernatant was measured by a spectrophotometer (PowerWave 200
spectrophotometer, Bio-Tek Instruments, USA) The concentration of PBS necessary to
induce 50% hemolysis defined the osmotic fragility index of the RBCs.18 With this method,
a larger osmotic fragility index corresponds to more fragile cells.
Hemolysis
Hemolysis as measured by the amount of free Hb present in the RBC suspensions was
determined according to the method of Harboe.24 Briefly, cell supernatant was obtained by
centrifugation of RBC units for 1 minute at 3500 x g. The supernatant was diluted 1:10 in
0.01% sodium carbonate in a flat-bottom 96-well microtiter plate and mixed for 30 minutes.
43
Chapter 4
The Hb concentration in the supernatant was determined with a spectrophotometer by
measuring the optical density (OD) at 415 nm and correcting for the OD at 380 and 450 nm
according to the formula OD = 2* (OD 415 nm) – (OD 380 nm) – (OD 450 nm). The
hemolysis was expressed as a percentage of the total amount of Hb present in the RBC
lysates.
Hematologic variables
To determine the cellular ATP content, RBC samples were incubated for 30 minutes with
8% ice cold trichloroacetic acid in a ratio of 1:3. Samples were centrifuged for 1 minute at
3500 x g and the protein free supernatant was neutralized with 1.5 mol/L sodium carbonate.
Aliquots were stored at –80°C for later batch analyses of ATP. The ATP content was
determined with a commercially available enzyme assay (Roche Diagnostics, Germany).
For detection of ATP, light emission was measured at 560 nm by an illuminometer
(Fluostar Optima, BMG Labtech, Germany).
The supernatant pH and the RBC MCV were determined with a blood gas analyzer
(Rapidlab 860, Siemens, the Netherlands). Total Hb content and Hct were determined with
a hematology analyzer (Sysmex K4500, Goffin Meyvis, the Netherlands). The internal
viscosity of RBC, as reflected by the MCHC, was determined by dividing the Hb content
by the Hct.
Statistical analysis
Statistical analysis was performed using statistical software (SPSS, version 16.0, SPSS Inc.,
Chicago, IL). Data were tested for normality with the Kolmgorov-Smirnov goodness-of-fit
test. For each variable a repeated measure analysis of variance was performed to identify
subject by time profiles. Post-hoc comparisons were performed to quantify differences
between Time 0 and stored RBC values, using paired t-tests. Differences are considered to
be significant with a p value of less than 0.05. Results are presented as means ± SD.
44
Rheologic properties of refrigerated stored RBCs
4.3. Results
Rheologic features
The RBC ability to aggregate, as represented by the AI, was reduced after the first week of
storage (AI from 46.9 ± 2.4 % to 41.9 ± 3.6 %; p < 0.01; Figure 4.1). In the following
weeks the aggregability recovered; after week 2, 3, and 4, the RBC AIs were 45.2 ± 4.9 %,
46.0 ± 3.8 % and respectively 45.8 ± 3.5 %, which was not significantly different from
Time 0 (46.9 ± 2.4 %). After 5 and 7 weeks of storage the aggregability was significantly
reduced (AI 44.4 ± 4.5 % and respectively 44.3 ± 2.2 %) compared to Time 0.
The RBC deformation curve showed a typical s-shape over a shear stress range of 0.6-50 Pa
for all different time points (Figure 4.2). The deformation curve of 5 week old RBCs was
slightly higher at the low-shear-stress regions and somewhat lower at the high-shear-stress
regions compared to Time 0 (Figures 4.2 and 4.3). After 7 weeks of storage (outdated
RBCs) the deformability at the high-shear-stress regions was further diminished (Figures.
4.2 and 4.3B). The rigidity of the cell membrane, which is reflected by the deformability at
a shear stress of 3.9 Pa, was fluctuating during 5 weeks of storage (Figure 4.3A).
Ultimately, after 7 weeks of storage the deformability at this low shear stress was not
significantly different from Time 0 (from 0.35 ± 0.01 to 0.35 ± 0.02 EI). The deformability
at a shear stress of 50 Pa was reduced from week 1 of storage onward (from 0.58 ± 0.01 to
0.54 ± 0.01 EI) as can be seen in Figure 4.3B. This downward trend in deformability (p <
0.01) remained within the physiological range, as determined with fresh RBCs (0.53 ± 0.04
EI).
Osmotic fragility and hemolysis
The osmotic fragility index, represented by the osmolarity at half-maximum hemolysis of
the RBCs, was not significantly altered during 7 weeks of storage (0.47 ± 0.02 % PBS;
Table 4.1) compared to Time 0.
A gradual increase in hemolysis was observed throughout the storage period (Table 4.1).
After 5 weeks of storage, the hemolysis (0.53 ± 0.24%) still remained below allowable
levels (i.e., 0.8% in Europe and 1% in the United States),15, 25 despite the weekly removal of
samples. Closer evaluation of individual RBC units demonstrated that after 5 and 7 weeks
of storage, respectively, one and four out of ten samples contained hemolysis which
45
Chapter 4
exceeded 0.8%. After 7 weeks of storage one RBC unit even showed hemolysis exceeding
2%, accounting for the large SD. No bacterial contamination was found in this RBC unit.
Figure 4.1. Effect of storage on RBCs ability to aggregate. AI in percentage and corrected for Hct. Values are
expressed as mean ± SD of ten RBC units. Significant difference from Time 0 are shown (* p < 0.05 and † p <
0.01).
Figure 4.2. Shear stress EI curves for Time 0 (■) and after 5 (-) and 7 weeks (▲) of storage. The shear stress
value is plotted on the logarithmic axis. Data represent mean ± SD of ten RBC units
46
Rheologic properties of refrigerated stored RBCs
A B Figure 4.3. Deformability for two representative shear stress levels as a function of storage time. (A) EI at shear
stress of 3.9 Pa. With the exception of week 7, all samples were significantly altered (p < 0.05) compared to Time
0. (B) EI at shear stress of 50 Pa. All samples were significantly reduced compared to Time 0 during 7 weeks of
storage (p < 0.04). Data represent mean ± SD of ten RBC units.
47
Chapter 4
Table 4.1. RBC characteristics during 7 weeks of storage
Variable
t=0
t=1wk
t=2wk
t=3wk
t=4wk
t=5wk
t=7wk
Osmotic fragility (%)
0.48 ± 0.02
0.48 ± 0.01
0.47 ± 0.02
0.46 ± 0.02
0.48 ± 0.02
0.47 ± 0.03
0.48 ± 0.02
Hemolysis (%)
0.24 ± 0.07
0.24 ± 0.07
0.30 ± 0.09 *
0.36 ± 0.12 †
0.42 ± 0.17 †
0.53 ± 0.24 †
0.89 ± 0.51 †
ATP (μmol/g Hb)
4.55 ± 1.34
4.14 ± 0.80
4.06 ± 1.02
3.82 ± 1.09
3.80 ± 0.84
2.12 ± 0.37 †
1.52 ± 0.30 †
pH (22°C)
6.99 ± 0.06
6.80 ± 0.04 †
6.69 ± 0.03 †
6.62 ± 0.03 †
6.55 ± 0.04 †
6.49 ± 0.04 †
6.41 ± 0.04 †
MCV (fl)
89.0 ± 2.4
89.0 ± 2.5 *
91.2 ± 2.7 †
91.1 ± 2.6 †
91.6 ± 2.9 †
92.0 ± 2.8 †
94.0 ± 2.9 †
MCHC (mmol/L)
20.7 ± 0.6
20.5 ± 0.4
20.5 ± 0.4
20.5 ± 0.6
20.5 ± 0.5
20.2 ± 0.3
19.7 ± 0.4 *
Values are expressed as mean ± SD of ten units. Significant difference from Time 0 are reported (* p<0.05 and † p<0.01).
48
Rheologic properties of refrigerated stored RBCs
Hematologic variables
The RBC ATP content reduced during 7 weeks of storage (Table 4.1). Notably, after 5
weeks of storage the ATP content had declined with 54% compared to Time 0 (from 4.6 to
2.1 μmol/gHb; p < 0.01). After the first week of storage the PH of the RBC suspension and
the MCV were significantly altered (Table 4.1). Ultimately, after 5 weeks of storage the pH
was reduced with 7% while the MCV was enhanced by 3%. The MCHC was only
significantly reduced after 7 weeks of storage compared to Time 0 (from 20.7 to 19.7
mmol/L).
4.4. Discussion
This study was undertaken to explore the rheologic properties and quality of leukoreduced
refrigerated stored RBC units. The RBC aggregability was significantly reduced at the first
week of storage but recovered during the following weeks. RBC deformability at shear
stress of 50 Pa was significantly reduced during 7 weeks of storage. However, values were
still within physiological reference ranges. The osmotic fragility did not change
significantly over time. Throughout 5 weeks of storage the ATP content had declined with
54% from initial levels, whereas MCV, PH and MCHC were affected to a lesser degree.
During storage at 4°C RBCs undergo different biochemical and structural alterations that
may adversely affect clinical outcome. Currently, RBCs are considered of impaired quality
after the second week of storage.11,13 As a result, a more restrictive transfusion strategy is
currently favored.26 Nonetheless, pre storage leukofiltration has significantly diminished
storage-induced lesions.18,19 Additionally, it has been shown, that activated leukocytes can
induce damage which adversely affects the rheologic properties of RBCs.20,21 Increased
aggregability and diminished cell deformability during RBC storage have been previously
observed. However, in those studies the RBC units had not been leukoreduced prior to
storage.11-14, 27
In this study, RBC aggregability significantly decreased after 1 week of storage but
recovered throughout the following weeks. At the end of storage, aggregability was slightly
but significantly reduced. RBC aggregation is primarily dependent on the RBC surface
characteristics and the composition of the suspension medium.3 Since the latter was
standardized for all the samples, our results suggest that the observed differences are caused
49
Chapter 4
by alterations in cellular properties. In addition, our results show that RBCs are, to some
extent, able to adapt to environmental changes during storage.
RBC morphology can affect RBC aggregation. During storage at 4°C, RBC morphology
shifts from discocyte towards echinocyte shape, leading to decreased aggregability.28,29 Our
results show that the aggregability in SAGM solution was minimally reduced at the end of
storage. Usage of other additive solutions, which also enhances the MCV during storage,
are therefore expected to give a similar aggregation pattern. The clinical relevance of
altered RBC aggregability, however, remains an ongoing debate.30
The RBC ability to deform depends mainly on the visco-elastic properties of the
cytoskeleton, the intracellular viscosity of the RBC and the overall cell shape.31 In this
study storage induced minor changes in the deformation curve at low shear stress of 3.9 Pa.
The decreasing trend in deformability at a shear stress of 50 Pa has been interpreted as
structural changes in the RBC that cannot be corrected by increasing shear force.32
However, we suggest that the gradual increase in MCV during storage may be responsible
for the observed reduction in deformability, particularly since at high shear stress the cell
volume becomes a limiting factor for the ability of RBCs to deform. Our data suggests that
this effect plays a role for shear stresses above 5 Pa, as can been seen from the deformation
curve. The increased RBC deformation at high shear stress after 5 weeks of storage
compared to the preceding week has been observed by others and can only partly be
explained by hemolysis of less deformable RBCs.11 The occurring deformability changes in
this study were not likely biologically relevant because the reduction in deformation
observed only at high shear stress was minimal and within physiological reference ranges.
Furthermore, shear stresses of 3.9 Pa, which are predominantly found in the
microcirculation,33 are clinically more relevant than those of 50 Pa.
We showed that the osmotic fragility, which is determined by applying deforming stress
from inside the RBC, was not significantly enhanced during 7 weeks of storage. In
addition, the RBC intracellular viscosity, which is predominantly determined by the
MCHC, did not yield significant changes until 7 weeks of storage. Taken together, these
findings further substantiate that the observed reduction in deformability was caused
primarily by morphology changes of the RBC.
It has previously been shown, that RBC deformability was unaffected at pH values ranging
between 6.4 and 7.7.34 In our study, the pH was still within these limits after 5 weeks of
50
Rheologic properties of refrigerated stored RBCs
storage, suggesting that the reduced RBC deformability was not caused by pH alterations in
the storage solution.
ATP as an energy source is important for the overall functioning of the cell. Loss of ATP is
associated with more rigid cell membranes, loss of vasodilatation properties, exposure of
PS on the outer leaflet of the RBC membrane, microvesiculation and decreased RBC
viability.35-39 In our study the ATP content gradually reduced during storage. Although loss
of ATP stiffens the RBC membrane due to calcium accumulation,40 the observed reduction
in deformability at high shear stress was within physiological reference values, indicating
that ATP loss during storage only marginally affected the RBC ability to deform. As
proposed earlier, the RBC ATP content must be at least 2.7 µmol per gram Hb to have a 90
percent chance of acceptable in vivo survival (24-hr in vivo recovery of 75% or higher).41,42
The present study showed that after 5 weeks of storage the RBC ATP content was below
this limit. Similar findings were observed for leukoreduced RBC that were stored for 6
weeks in SAGM solution.43 The in vivo viability of RBCs may be reduced by the low ATP
content. However, it is not likely that this results in the proposed adverse clinical outcome,
in particular, because the observed alterations in rheologic features were minimal and the
hemolysis in the RBC units was still below the allowable limits (i.e. 0.8% in Europe and
1% in the United States).15, 25
Leukocytes can affect the RBC deformability even when they are significantly reduced, e.g.
during the storage of buffycoat-depleted RBC units.44-46 Apparently, removing solely the
buffycoat from the RBCs without performing leukofiltration, still resulted in 108 to 109
leukocytes per RBC units,15, 47 whereas nowadays less than 106 leukocytes per RBC unit are
permitted. Our results demonstrate that cell deformability was minimally affected when the
RBCs were leukofiltrated prior to storage.
Recently, reduced deformability at shear stress of 3 and 30 Pa during the storage of
leukoreduced RBCs has been observed,48 whereas others showed no reduction in RBC
deformability at these shear stresses.49 This reduction in deformability has been linked to
loss of Hb-bound nitric oxide, a variable that participates in controlling the RBC
deformation.50 However, we also suggest that differences in storage procedures could
explain this discrepancy in results. Particularly because the reduction in deformability at
low and high shear stress was observed in the preservative solution AS-3, while in the
SAGM preservation solution28 less changes in RBC deformability were observed.
51
Chapter 4
RBC adherence to ECs is mediated by PS exposure on the cell membrane. Additionally, PS
expression triggers recognition by macrophages and subsequent clearance of RBCs from
the circulation.51,52 PS expression was not determined in this study, since recent work
demonstrated that leukoreduction significantly lowered the PS expression on the RBCs,
resulting in negligible PS exposure after 5 weeks of storage48,53 and because leukoreduction
reduces the progressive adherence of RBCs to ECs with storage time.54, 55
In the Netherlands approximately 37% of all the transfused RBC units are older than three
weeks.56 The primary goal of blood transfusion is to deliver oxygen to the microcirculation
with high-quality stored RBCs. During storage at 4°C, RBCs undergo different alterations
that might influence the patient condition. However, leukoreduction has significantly
reduced storage-induced lesions. Based on our findings, we postulate that the observed
changes in RBC variables during refrigerated storage minimally affected the RBC ability to
aggregate and deform, even after long-term storage. The rheologic properties of
leukoreduced RBC units were well preserved during routine blood bank storage and not
likely to contribute to adverse clinical outcome after transfusion.
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47. Jensen LS, Kissmeyer-Nielsen P, Wolff B, Qvist N. Randomised comparison of leucocyte-depleted versus
buffy-coat-poor blood transfusion and complications after colorectal surgery. Lancet 1996, vol.348: 841-845.
48. Bennett-Guerrero E, Veldman TH, Doctor A, Telen MJ, Ortel TL, Reid TS, Mulherin MA, Zhu H, Buck RD,
Califf RM, McMahon, TJ. Evolution of adverse changes in stored RBCs. Proc Natl Acad Sci. 2007, vol.104:
17063-17068.
49. Raat NJ, Verhoeven AJ, Mik EG, Gouwerok CW, Verhaar R, Goedhart PT, de Korte D, Ince C. The effect of
storage time of human red cells on intestinal microcirculatory oxygenation in a rat isovolemic exchange
model. Crit Care Med. 2005, vol.33: 39-45.
50. Carvalho FA, Maria AV, Braz Nogueira JM, Guerra J, Martins-Silva J, Saldanha C. The relation between the
erythrocyte nitric oxide and hemorheological parameters. Clin Hemorheol Microcirc. 2006, vol.35: 341-347.
51. Manodori AB, Barabino GA, Lubin BH, Kuypers FA. Adherence of phosphatidylserine-exposing
erythrocytes to endothelial matrix thrombospondin. Blood 2000, vol.95: 1293-1300.
52. Schroit AJ, Madsen JW, Tanaka Y. In vivo recognition and clearance of red blood cells containing
phosphatidylserine in their plasma membranes. J Biol Chem. 1985, vol.260: 5131-5138.
53. Cardo LJ, Hmel P, Wilder D. Stored packed red blood cells contain a procoagulant phospholipid reducible
by leukodepletion filters and washing. Transfus Apher Sci. 2008, vol.38: 141-147.
54. Anniss AM, Sparrow RL. Storage duration and white blood cell content of red blood cell (RBC) products
increases adhesion of stored RBCs to endothelium under flow conditions. Transfusion 2006, vol.46: 15611567.
55. Luk CS, Gray-Statchuk LA, Cepinkas G, Chin-Yee IH. WBC reduction reduces storage-associated RBC
adhesion to human vascular endothelial cells under conditions of continuous flow in vitro. Transfusion 2003,
vol.43: 151-156.
56. Raat NJ, Berends F, Verhoeven AJ, de Korte D, Ince C. The age of stored red blood cell concentrates at the
time of transfusion. Transfus Med. 2005, vol.15: 419-423.
55
Chapter 5
Utilization of cryopreserved red blood
cells in transfusion medicine
Sandra Henkelman
Gerhard Rakhorst
Reindert Graaff
Johan W.M. Lagerberg
Submitted
Chapter 5
5.1. Introduction
The ability to store RBCs outside the body has been regarded as a life-saving practice for
many years.1 More recently, the usage of refrigerated stored RBCs in transfusion medicine
has been under extensive evaluation. During refrigerated storage RBCs progressively
deteriorates2 and infusion of prolonged stored RBCs has been linked to adverse clinical
outcome in terms of postoperative infections, length of hospital stay and mortality.3-7
Although the majority of these studies are prone to selection bias due to a retrospective
study design,8 concerns regarding the infusion of aged RBCs still remains and a restrictive
transfusion strategy is currently being favored.9,10 The latter concerns have revived the
interest in cryopreservation. Storage of RBCs at ultra-low temperatures halts the cellular
metabolism and subsequently prevents the progressive cellular deterioration that has been
linked to adverse clinical outcome.
Initially, cryopreservation appeared a promising approach for maintaining RBCs viable for
prolonged periods of time. Yet, the clinical applicability of cryopreserved RBCs
(commonly known as frozen RBCs) was hampered by the expensive, time-consuming and
less efficient nature of this preservation method.11,12 The subsequent unfamiliarity with
regard to the quality of cryopreserved RBCs has also limited clinical usage. However,
ongoing scientific and technological advancement has made cryopreserved RBCs more
utilizable for clinical practice. This could be advantageous especially in situations where
refrigerated stored RBCs are less desired. In this review the utilization of cryopreserved
RBCs in modern transfusion practice will be discussed.
5.2. Cryopreservation methods
Storage of RBCs at ultra-low temperatures ceases the biological activity of RBCs which
enables them to be preserved for prolonged periods of time.13 In order to minimize freezing
damage, cryoprotective additives are pivotal. In general, either high concentrations of
cryoprotective additives or rapid freezing rates are necessary to prevent cell damage.14
Along the years different non-permeating and permeating additives for the cryopreservation
of RBCs have been investigated. Non-permeating additives such as hydroxyethyl starch and
polyvinylpyrrolidone, as well as a variety of glycols and sugars appeared promising
because it was proposed that removal from thawed RBCs prior to transfusion was not
58
Usage of frozen RBCs
required.15-20 To date, the lack of data that support safe usage of non-permeating additives
have prevented these cryoprotectants from being licensed.11,12
Conversely, the permeating additive glycerol is well known for its ability to protect RBCs
at ultra-low temperatures. The concentration of glycerol that is necessary to protect the
RBCs is dependent on the cooling rate and the storage temperature.14 At slow cooling rates,
ice formation will occur extra-cellular. As ice forms, the solute content of the unfrozen
fraction becomes more concentrated. The resulting osmotic imbalance causes fluid to move
out of the RBC and intracellular dehydration occurs. However, at rapid cooling rates the
RBC cytoplasm becomes super-cooled and intracellular ice formation occurs, which
subsequently can lead to mechanical damage (Figure 5.1). Glycerol protects the RBCs by
slowing the rate and extent of ice formation while minimizing cellular dehydration and
solute effects during freezing.11
Figure 5.1. RBC changes in response to cooling rate. The snowflakes indicate the presence of ice crystals in the
extra- and /or intracellular environment. Printed with permission of Scott et al.11
59
Chapter 5
To date, there are two freezing methods approved for the storage of RBCs.21,22 On the one
hand, RBCs can be frozen rapidly (i.e. > 100°C/ min) by the low-glycerol method (LGM).
With this method, RBCs are frozen with a final concentration of approximately 20%
glycerol (wt/vol) at temperatures below -150°C. On the other hand, RBCs can be frozen
slowly (i.e. ~1-3°C/ min) by the high-glycerol method (HGM). With this method, RBCs are
frozen with a final concentration of approximately 40% (wt/vol) glycerol at temperatures
between -65°C and -80°C. Overall, RBC preservation can be extended to at least ten years
if the correct storage temperature is guaranteed.
RBC units are preferentially thawed in a shaking water bath of about 36 to 42°C. The
general consensus is that thawing should be done rapidly in order to prevent ice crystal
growth (so called re-crystallization) upon warming.23 Once thawed, a deglycerolization
washing procedure is performed to reduce the glycerol content in the RBC prior to infusion.
This is necessary, since incomplete deglycerolized RBCs will swell and lyse upon infusion,
resulting in hemolytic transfusion reactions and renal failure.24,25
The deglycerolization washing process causes to some extent osmotic stress to the RBC
which results in cellular losses. Yet, the deglycerolization washing process is also
advantageous in that it considerably reduces the amount of detrimental substances such as
bioactive lipids, microparticles, cytokines, potassium and free Hb as well as leukocytes
from the RBC unit.26-32 Hence, in the absence of leukofiltration, the deglycerolization
process reduces the leukocyte count to a mean of 1 x 107 leukocytes per RBC storage
unit.32,33 In addition, buffycoat depletion before cryopreservation further reduces the
leukocytes count to a mean of 1-3 x 106 leukocytes per unit,32 which in some countries may
even eliminate the need to leukofiltrate RBC units. It became also apparent that after postthaw washing the immunogenicity of the residual leukocytes was reduced.34 So although
frozen storage results in a RBC unit with a lower yield, the remaining RBCs contain less
detrimental substances as compared to refrigerated stored RBCs.12 Altogether, transfusions
of cryopreserved RBCs are associated with less febrile transfusion reactions, human
leukocyte antigen alloimmunization as well as occurrences of TRALI and SIRS.12,35
If the glycerolization and deglycerolization of RBCs is performed in open systems, the
post-thaw storage time is limited to 24-hours due to the potential risk of bacterial
contamination. Yet, with the implementation of the ACP-215 device (Haemonetics,
Braintree, MA), glycerol could be added and removed via an automated closed system,
which minimized the risk of bacterial contamination.36 As a result, the post-thaw storage
60
Usage of frozen RBCs
time of RBCs could be extended to 7 days when stored in SAGM solution and to 14 days
when stored in AS-3.31,37,38
Cryopreserved RBCs are less efficient due to the cellular losses that occur during the
processing procedure. This cell loss was more pronounced in the HGM cryopreserved
group (~ 10-20%) since these RBCs required more extensive washing. However, despite
the higher yield of RBCs with the LGM method, it was recognized that HGM
cryopreserved RBCs could tolerate wide fluctuations in temperature during freezing and
were more stable during post-thaw storage.13,39 In addition, HGM cryopreserved RBCs did
not require liquid nitrogen which eased storage and transportation conditions.
Consequently, the HGM is currently the most applicable RBC freezing method in Europe
and the United States.
5.3. Quality of cryopreserved RBCs
Cryopreservation prolongs the longevity of RBCs. However, once thawed the shelf life of
RBCs is limited. Cryopreserved RBCs have to meet certain guidelines (Table 5.1)21,22. Yet,
these guidelines do not specifically reflect the ability of the RBCs to function after infusion.
Cryopreservation subjects RBC to a range of chemical, thermal and mechanical forces,
which might affect their oxygen delivering capacity after infusion.
The quality of HGM cryopreserved RBCs is primarily dependent on the pre-freeze and
post-thaw storage time, as well as on the anticoagulant and additive solution used.40 The
duration of frozen storage per se minimally attributes to cellular damage.41,42 In order to
limit storage induced lesions, refrigerated stored RBCs need to be frozen as soon as
possible. According to the AABB, RBCs collected in CPDA-1 need to be frozen within 6
days, whereas in Europe the RBCs are preferably frozen within 7 days after collection.21,22
It is possible to freeze prolonged or outdated refrigerated stored RBCs, provided that the
RBCs have been rejuvenated prior to freezing in order to restore the metabolic status of the
cell (i.e. ATP, 2,3-DPG and Hb p50 levels).43-46 After deglycerolization even the outdated
rejuvenated RBCs showed acceptable quality up to seven days of post-thaw storage in
AS-3.36
In recent years more knowledge about the quality of HGM cryopreserved RBCs has
become available. During frozen storage, the ATP and 2,3-DPG content is preserved. Yet,
the length of pre-freezing storage time at 4°C was an important predictor of the ATP and
61
Chapter 5
2,3-DPG content after deglycerolization.36,42 The RBC ATP content is important for the
overall functioning of the cell. Loss of ATP has been associated with rigid cell membranes,
echinocyte shape change, enhanced cation permeability, exposure of phosphatidylserine on
the RBC surface, microvesiculation, loss of vasodilatation control and decreased RBC
viability.47-55 After deglycerolization the RBCs have a high ATP content when stored in
AS-3 or SAGM additive solution. Nevertheless, during post-thaw storage the ATP content
gradually declines. This decline was more prominent in AS-3 due to the diminished
glycolytic activity induced by the lower pH of this storage solution.38
In the RBC binding of 2,3-DPG to the Hb induces a conformation state which will release
oxygen from the Hb. In regions with low oxygen tension, oxygen is released from the Hb
due to the high binding affinity of 2,3-DPG for deoxygenated Hb. Loss of 2,3-DPG will
increase the affinity of oxygen to the Hb which may hamper the oxygen delivery to the
tissues. Due to the low pH of the storage media a considerable loss of 2,3-DPG content was
observed already after one week of refrigerated RBC storage.2 By limiting this pre-freezing
storage time, higher 2,3-DPG values could be obtained post-thaw.56 Nevertheless,
transfusing RBCs with low 2,3-DPG content appeared not to be detrimental to tissue
oxygenation.57-59 Presumably, because the RBC 2,3-DPG content can be replenished hours
following infusion,60-62 or because in hypoxic regions oxygen can still be released from the
RBCs despite the low 2,3-DPG content.
The ability of RBCs to adhere to the vascular endothelium is an important determinant of
the flow behavior of blood and subsequently the oxygenation of the micro-vascular
environment.63,64 Under physiological conditions, the adherence of RBCs to the vascular
endothelium is negligible. Yet, structural changes in the RBC membrane may promote
adherence to ECs and impair the microcirculatory blood flow. PS expression on the RBC
surface mediates adherence of RBCs to ECs and might trigger RBC clearance from the
circulation.65-67 In contrast, surface expression of CD-47, a marker of self, prevents RBCs
from being engulfed by phagocytes.68 In general PS exposure and loss of CD-47 expression
on the RBC surface as well as membrane microvesiculation are all apoptotic signals and
therefore determining factors for the lifespan of the RBCs.69,70 HGM cryopreserved RBCs
that were post-thaw stored in SAGM additive solution showed no significant changes in PS
exposure, CD-47 expression and membrane microvesiculation when the pre-freezing
storage time was limited to three days. However, surface expression of PS and
microvesiculation was observed when longer pre-freezing storage times were used.56
62
Usage of frozen RBCs
Previously, it has been shown that the freeze-thaw process makes the RBC membrane
permeable to cations, which result in RBC swelling and subsequently hemolysis.38,71,72
However, post-thaw storage of RBCs in AS-3 media, which contains the impermeable
solute citrate, prevented cell swelling and limited the level of hemolysis during post-thaw
storage.38 Altogether, it can be concluded that good post-thaw quality of HGM
cryopreserved RBCs can be obtained when the appropriate storage conditions pre-freeze,
frozen and post-thaw were used.
Table 5.1. Requirements of cryopreserved RBCs
Variable
European Guidelines *
AABB guidelines **
Hemolyse
< 0.8 %
< 1.0 %
Volume
> 185 ml
-
Hb content
> 36 g/unit
-
HCT
0.65-0.75 %
-
Post-thaw recovery
-
≥ 80 %
24-hour posttransfusion survival
-
≥ 75 %
Leukocytes
< 0.1 x109 cells/unit
-
Osmolarity
< 340 mOsm/l
-
* European guidelines22
** AABB guidelines21
5.4. Utilization of cryopreserved RBCs
Currently, cryopreserved RBCs are primarily used for controlling an inventory in situations
where the RBC availability is limited or unpredictable. Yet, cryopreserved RBCs are useful
in a variety of clinical settings. The utilization of cryopreserved RBCs in transfusion
medicine will be described in more detail below.
63
Chapter 5
Storage of rare RBCs
Cryopreservation is currently used for preserving RBCs with rare blood phenotypes. In
general, a blood group is regarded as rare if the RBC phenotype has a frequency of
approximately 1 in 100-1000 or less in the general population.73 Refrigerated stored RBCs
need to be as soon as possible, ideally within 6 to 7 days. However, cryopreservation of
RBCs even beyond the regulated expiration date is still possible for exceedingly rare RBC
phenotypes.11
Cryopreservation of rare RBCs is advantageous to patients for whom compatible blood is
not instantly available. Over the years, numerous countries in Europe, America and Asia
have set up frozen rare RBC banks.73-77 In Europe, most notably are the blood banks in
Amsterdam, Birmingham and Paris, which house a large collection of cryopreserved rare
RBC units. Most cryopreserved rare RBC units are for national use. This is because the
RBC unit usually needs to be thawed in the donor blood center, imposing a time limit in
which the RBCs must be transfused. Nevertheless, when no compatible blood can be found
via the national blood banks, it is general practice to appeal to countries abroad. Although
usage of cryopreserved rare RBCs is extremely costly and the international shipment is
usually cumbersome, it has been lifesaving for a variety of patients.
Military blood bank
In military combat massive blood loss is a major cause of death.78,79 Having RBCs available
in the military theater at all times is therefore of vital importance. Previously, an inventory
of RBCs was difficult to maintain in combat areas due to the unpredictable demand and the
limited shelf life of refrigerated stored RBCs. Yet, cryopreserved RBCs are a valuable
blood resource due to the prolonged storage time. Cryopreserved RBCs have been used in
the military theater since the Vietnam war.80,81 Although back then the processing of
cryopreserved RBCs was still in its early stages, it was already concluded that usage of
cryopreserved RBCs in combat casualty care was technically feasible and clinically
acceptable. Ongoing scientific and technological advancement have made cryopreserved
RBCs become a more utilized blood product in modern military operations.13,82-84
Especially the Dutch army has been using cryopreserved RBCs in theater operations with
great success.83,85,86 This was also demonstrated at the conflicts in Afghanistan, where
already 1360 cryopreserved RBC units have been transfused by the Dutch military blood
64
Usage of frozen RBCs
bank without any shortages or transfusion reactions reported.87 Hence, the Dutch military
blood bank only uses type O leukodepleted RBCs in the theater in order to improve the
effectiveness and decrease the chance of clerical errors. In this regard, leukodepleted type O
whole blood is processed into RBCs and frozen within 24 hours after donation.
Additionally, the short pre-storage time reduces storage induced lesions to a minimum.
Cryopreserved RBC units are transported to combat areas on dry ice. These RBCs have
been frozen in polyvinyl chloride plastic (PVC) bags inside polyester plastic bags which
were placed in rigid cardboard boxes. In the past, PVC bags that were stored at -80°C and
subjected to transportation had a breakage incidence of approximately 6.7%.88 Due to the
use of an additional vacuum sealed over-wrap bag, the breakage incidence of cryopreserved
RBC units subjected to transportation is currently negligible.83 Subsequently, all in theater
storage, thawing and washing procedures are performed in a temperature controlled blood
bank container, designed by the Dutch army. Thawed and washed RBCs are ultimately
preserved in AS-3 and can be refrigerated stored at 2-6 °C for a maximum of 14 days.
Usage of cryopreserved RBCs in combat casualty care offers a better inventory control.
This is because cryopreserved RBC units can be prepared either on demand or in advance,
thereby providing a continuous RBC supply even when standard refrigerated stored RBCs
cannot be replenished on time. Furthermore, cryopreserved RBCs may be advantageous to
prolonged refrigerated stored RBCs. In combat hospitals, it is not uncommon to receive
refrigerated stored RBC units with a mean storage age of 27 days on arrival.89,90 Compared
to prolonged refrigerated stored RBCs, cryopreserved RBCs have less detrimental
substances that can cause transfusion reactions. Nevertheless, randomized-controlled trials
comparing transfusion of cryopreserved RBCs with refrigerated stored RBCs have still to
be performed. Cryopreserved RBCs are undeniable more costly. Hence, a unit of
cryopreserved RBCs cost approximately twice the amount of a refrigerated stored RBC
unit. In addition, thawing and washing of cryopreserved RBC units requires skilled
personnel and takes up about 70-120 minutes.12,82,83 Yet despite these disadvantages,
stockpiling a frozen RBC inventory proved to be an efficient and safe blood resource in
combat casualty care.
65
Chapter 5
Blood shortages
Blood shortages due to natural or civil disasters as well as due to seasonal shortages can
pose a major health challenge. However, in emergency blood management planning
cryopreserved RBCs are rarely implemented. This is primarily due to the longer processing
time of cryopreserved RBCs, making it difficult to prepare large quantities of RBC units
within a given time frame. Cryopreserved RBCs are also less implemented during times of
blood shortages, due to the improved emergency procedures of blood centers with regard to
refrigerated stored RBCs.91-94
A main strategy of the emergency procedures is to mobilize stocks of refrigerated stored
RBC through coordination with nearby blood centers. Hence, blood centers will usually
have a 2-3 day supply of refrigerated stored RBC units on hand. This strategy could
compensate for RBC shortages as long as the local stocks of the nearby blood centers are
repleted appropriately. After a disaster the influx of blood donations is often increased
because of the altruistic response of the public. Although this influx of blood donations
could be used to replenish local stocks, this is true for only part of the donated blood.
Notably, part of the donated blood is non-transfusible due to the higher reactive screening
tests and sometimes inadequately processing procedures.95-99 In this regard, blood centers
that do send refrigerated stored RBC units could face the risk of becoming under-supplied
themselves.
Recently, it was demonstrated that managing a frozen RBC reserve could be useful in
emergencies scenarios.100 In this regard, cryopreserved RBCs would serve as a bridge-over
supply during short term RBC deficits until support by the blood centers could be reestablished. Despite the complexity and costs of implementing and maintaining a frozen
RBC reserve, it was concluded that the benefits of self-sufficiency outweighs the
disadvantages.
Autologous transfusion
Cryopreserved RBCs have occasionally been used for preoperative autologous storage.101
In general, preoperative autologous RBC transfusion offers advantage above allogeneic
RBC transfusions in that it prevents immunosuppression and infectious disease
transmission, while it reduces postoperative infections and subsequently length of hospital
stay.102,103 During the last couple of years preoperative autologous RBCs usage has been
66
Usage of frozen RBCs
questioned and its demand has declined. This is predominantly due to the improved safety
of allogeneic RBCs as well as due to the organizational and logistic hurdles, the higher
disposal rate and the more costly nature associated with autologous RBCs transfusions.103107
Additionally, the beneficial effect of preoperative autologous RBC donation has been
compromised by the short time period between the last donation and the planned surgical
procedure. As a result, patients often develop anemia before the surgery and are more likely
to receive transfusion.105,106,108
In order to avoid anemia, RBCs need to be harvested months in advance of the expected use
so that the Hb level of the patient can be restored. Yet, prolongation of the time period
between the last donation and the surgical procedure is hampered by the short storage time
of refrigerated stored RBCs. In contrast, cryopreservation enables storage of RBCs for
years, which allow RBCs to be donated far in advance of the surgical procedure109 without
affecting its quality. Usage of cryopreserved RBCs has an additional advantage in that less
detrimental substances, that have accumulate during storage and which may affect
transfusion outcome, are transfused.
Although the transmission of infectious diseases is currently very low and the benefit of
preoperative autologous RBC transfusions is questioned, new pathogens keep emerging110113
and with the implementation of cryopreservation, preoperative autologous RBC
transfusions could become more attractive again.
Usage when refrigerated stored RBCs are less desired
Cryopreserved RBCs could be advantageous in situations where refrigerated stored RBCs
are less desired. This could be the case for patients who receive transfusions frequently,
such as patients with thalassemia, sickle-cell disease or with certain types of glucose-6phosphate dehydrogenase deficiency. This is because frequent transfusions with
refrigerated stored RBCs are a risk of human leukocyte antigen alloimmunization. Hence,
the development of antibodies against infused RBC surface antigens is of considerable
medical importance because it will result in hemolytic transfusion reactions.114,115 In
contrast, usage of cryopreserved RBCs reduces the risk of alloimmunization due to a
reduction in the number of leukocytes by the deglycerolization washing procedure.12,13
Cryopreserved RBCs may also be helpful as part of the routine RBC inventory because of
its advantages over prolonged refrigerated stored RBCs. As mentioned earlier, transfusion
67
Chapter 5
of prolonged refrigerated stored RBCs is associated with occurrences of TRALI and
SIRS.12,35 Although the etiology of TRALI and SIRS remains incompletely understood, it is
recognized that substances that accumulate in the supernatant of refrigerated stored RBCs
are involved in the pathogenesis of these syndromes.116-120 Ultimately, these syndromes will
lead to an increased hospitalization and subsequently higher burden on the healthcare costs.
Cryopreserved RBCs on the other hand have less than 5% cytokines and biologically active
substances in the supernatant, which make them the ideal blood product to prevent TRALI
and SIRS.35,121
In line with this, cryopreserved RBCs are also useful for patients with immunoglobulin A
(IgA) deficiency. These patients usually have undetectable IgA and high titer of class
specific anti-IgA. Transfusion of only a small amount of blood can cause severe
anaphylactic reactions due to the presence of IgA in plasma.40 Since cryopreserved RBCs
are extensively washed, these blood products are especially recommended for patients with
IgA deficiency.13,40,122
5.5. Conclusion
In transfusion medicine the balance between the RBC availability and demand is variable.
However due to the perishable nature of refrigerated stored RBCs, hospitals often maintain
only a minimal RBC reserve to maximize the efficiency while minimizing the cost of
wastage. This policy also means that hospitals will be more vulnerable to RBC shortages
due to fluctuations in the RBC availability and or demand.
Cryopreserved RBCs may be helpful as part of the routine refrigerated stored RBC
inventory. Hence, having cryopreserved RBCs available in civilian blood banks and or
hospitals could result in better blood management and patient care need. Today,
cryopreserved RBCs are still infrequently implemented in transfusion medicine. This is
mainly because of the expensive nature of this preservation method. Although, the higher
costs of cryopreserved RBCs are of major concern, the cost difference with regard to
refrigerated stored RBCs is often overrated. This is because the costs of treating and
managing adverse events are not taken into account, indicating that the total cost of a
refrigerated stored RBC unit would be substantial higher than currently is represented.123
Cryopreserved RBCs are also less efficient and more time-consuming, however ongoing
scientific and technological advancement has made cryopreserved RBCs more utilizable for
68
Usage of frozen RBCs
clinical practice. Notably, more knowledge about the quality of cryopreserved RBCs could
further expand its use in clinical practice.
In the foreseeable future the overall use of cryopreserved RBCs could expand as a result of
a change in RBC supply and demand, due to a shift of increasingly older patient
population.124,125 For now, we showed that cryopreservation of RBCs is already useful in a
variety of clinical settings. Especially, since cryopreserved RBCs are available, save, in
compliance with European and US guidelines and can be used effectively.
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78. Kelly JF, Ritenour AE, McLaughlin DF, Bagg KA, Apodaca AN, Mallak CT, Pearse L, Lawnick MM,
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75
Chapter 5
114. Gupta R, Singh DK, Singh B, Rusia U. Alloimmunization to red cells in thalassemics: emerging problem and
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76
Chapter 6
The effects of cryopreservation on red
blood cell rheologic properties
Sandra Henkelman
Johan W.M. Lagerberg
Reindert Graaff
Gerhard Rakhorst
Willem van Oeveren
Transfusion 50: 2393-2401, 2010
(Reproduced with permission of John Wiley and Sons)
Chapter 6
Abstract
In transfusion medicine, cryopreserved RBCs are an alternative for refrigerated stored RBCs.
Less is known about the rheologic properties of cryopreserved RBCs. In this study the
aggregability and deformability of HGM cryopreserved RBCs that were post-thaw stored in
SAGM solution were compared to that of refrigerated stored and fresh RBCs. Fresh RBCs were
obtained from healthy volunteers while leukoreduced refrigerated stored and cryopreserved
RBC units were obtained from the Sanquin Blood Bank. RBCs were tested for aggregability,
deformability and various hematologic variables. The AI of cryopreserved RBCs was
considerably reduced, compared to fresh and refrigerated stored RBCs. The EI of stored RBCs
was significantly enhanced over a shear stress range of 2.0 to 50 Pa compared to fresh RBCs.
No significant differences in EI between cryopreserved and 21- or 35-day refrigerated stored
RBCs were observed. The osmotic fragility, hemolysis, MCV and MCHC of cryopreserved
RBCs were markedly altered, compared to fresh and refrigerated stored RBCs (p < 0.05). The
ATP content of cryopreserved RBCs was similar to fresh and 3-or 21-day refrigerated stored
RBCs. These findings suggest that although cryopreserved RBCs are more fragile than fresh
and refrigerated stored RBCs, the freeze-thaw-wash process did not did not adversely affect the
aggregability, deformability or the ATP content of cryopreserved RBCs. Based on these
rheologic properties, we conclude that cryopreserved RBCs are a valuable alternative to
refrigerated stored RBCs.
78
Rheologic properties of frozen RBCs
6.1. Introduction
In transfusion medicine RBCs are refrigerated stored for a maximum of 5 to 6 weeks before
being discarded. Alternatively, cryopreservation enables storage of RBCs for years.1,2
Cryopreservation is currently a valuable approach for long-term storage of RBCs from
donors with rare blood groups and for military deployment.3-5 However, stockpiling
cryopreserved RBCs can also be beneficial in emergency or clinical situations, where the
demand exceeds the supply of RBCs. At the moment, the shelf life of HGM cryopreserved
RBCs has been approved for up to ten years.3,4
Usage of glycerol as a cryoprotectant requires a deglycerolization washing procedure postthaw to prevent hemolytic transfusion reactions and renal failure after infusion.6-8 Usage of
the ACP 215 device (Haemonetics, Braintree, MA) to glycerolize and deglycerolize RBC
units has minimized the risk of bacterial contamination. As a result, the post-thaw storage
time of RBCs has extended to 2 days in SAGM solution and to 14 days in AS-3
respectively.9 Current regulations require that cryopreserved RBCs have a post-thaw
recovery of at least 80% and that the hemolysis in the RBC unit remains below allowable
levels (i.e., 0.8% in Europe and 1% in the United States). Additionally, at least 75% of the
cryopreserved RBCs should remain within the circulation 24 hour after infusion.3,4
Current regulations however, do not specifically address the quality of stored RBCs.10 The
quality of HGM cryopreserved RBCs is primarily dependent on the pre-freeze and postthaw storage time as well as on the anticoagulant and additive solution used. The duration
of frozen storage per se, however, minimally attributes to cellular damage.1,2,11 Previously it
was shown that cryopreserved RBCs have no significant loss of cellular ATP content or
CD47 antigen expression on the external RBC membrane during 24 hours of post-thaw
storage in AS-3. Moreover, when the pre-freezing storage time was limited to 3 days, no
2,3-DPG loss, microvesiculation or PS externalization was observed after 24 hours of postthaw storage.1,12 Cryopreserved RBC units usually contain low potassium and free Hb
content due to the removal of these extra cellular substances during washing. 9
The aggregability and deformability of cryopreserved RBCs remain to be elucidated. These
rheologic properties are important determinants of the blood flow and hence the
oxygenation of the tissues.13 In the venous system RBCs are able to form linear aggregates
(so called Rouleaux) or more complex multi-cellular aggregates at low shear rates.14
Normally, the blood flow is sufficient to disperse these aggregates. However, under
79
Chapter 6
pathologic conditions stronger and larger aggregates may form that are more resistant to
dispersion by the blood flow.15 In the microcirculation, the RBC ability to deform due to
applied forces, make these cells capable of passing narrow capillaries. A high deformability
of RBCs and a rapid recovery of the normal shape are essential factors for maintaining
tissue perfusion and cell survival.16,17 Transfusion of rheologic impaired RBCs may hinder
or obstruct the blood flow in the microcirculation, leading to reduced tissue perfusion,
ischemia or infarction.13,18-20
In the Netherlands, leukoreduced RBCs are refrigerated stored in SAGM preservation
solution for a maximum of 5 weeks. In chapter 4 we demonstrated that refrigerated storage
minimally affected the RBC ability to aggregate and deform, even after 5 weeks of routine
blood bank storage. Cryopreservation offers the advantage of storing RBCs for prolonged
periods of time. However, once thawed the shelf life of RBCs is limited. In the current
study, the aggregability, deformability and other hematologic variables of HGM
cryopreserved RBCs that were post-thaw stored in SAGM solution for 2 days were assessed
and compared to those of refrigerated stored and fresh RBCs.
6.2. Materials and Methods
RBC preparation and processing
Human blood (50 ml ± 10 %) was collected after informed consent from ten healthy
volunteers and anticoagulated with 7 ml of CPD. Functional measurements were performed
with whole blood, whereas the rheologic features were determined with washed RBCs.
Briefly, whole blood was washed by centrifugation at 1100 x g for 12 minutes and decanted
three times with PBS pH 7.4. The final Hct of the RBC solution was set between 45% and
60 % and all measurements were performed within 3 hours after donation.
Ten leukoreduced RBC units stored in SAGM solution were obtained from the Sanquin
Blood Bank and refrigerated stored according to standard blood bank procedures as
described in chapter 4. These RBC units, which were released for use on day 3 after
donation, had a Hct of 45% to 60 % and contained fewer than 106 leukocytes per unit. In
general, the average storage time of RBC units that are used in transfusion medicine is
approximately 21 days.21,22 For analysis samples were aseptically withdrawn from the RBC
units at 3, 21, and 35 days of refrigerated storage, reflecting short-, average- and long term
stored RBCs, respectively.
80
Rheologic properties of frozen RBCs
Ten cryopreserved RBC units were obtained from the Sanquin blood bank and stored in
polypropylene tubes at 2 to 6° for 48 hours. These RBC have been cryopreserved according
to the HGM freezing method and post-thaw resuspended in SAGM solution as described
previously.9 Briefly, leukoreduced refrigerated stored RBC units were centrifuged at 3200 x
g for 5 minutes to remove the SAGM solution. Subsequently, the RBCs were glycerolized
to a final concentration of 40% glycerol via the Haemonetics ACP-215 device.23 All
glycerolized RBC units were frozen and stored at –80 ± 10°C in a mechanical freezer for
34.1 ± 20.5 months. Cryopreserved RBC units were thawed in a temperature-controlled
water-bath of 40°C, until the units reached a temperature between 25°C and 30°C.
Subsequently, thawed RBCs were deglycerolized via the Haemonetics ACP-215 device and
resuspended in SAGM solution. The supernatant osmolarity of all cryopreserved units was
below 400 mOsm/ kg H2O, indicating an efficient removal of glycerol.
Rheologic features
RBC aggregability and deformability were monitored in vitro by the LORCA (R&R
Mechatronics, Zwaag the Netherlands).24,25 Aggregation was induced by the addition of
10% HES (MW 200-kDa). Briefly, RBCs suspensions were centrifuged for 1 minute at
3500 x g and the supernatant was discarded. RBCs were resuspended in 10% HES 200-kDa
solution (Fresenius, Bad Homburg, Germany) to obtain a Hct of 32 ± 6%. Aggregability
was tested with 1 ml of the RBC suspension. RBC aggregation was monitored after
complete disaggregation under increased shear stress. Both the aggregation measuring
procedure and the subsequent analyses were computer controlled. Aggregability of RBCs
was expressed by the AI, where a larger AI reflects an increased ability to aggregate. The
AI was determined after correcting the Hct in all the samples to a constant value of 45%.
The kinetics of aggregation (T½) was expressed by the time necessary to induce 50%
aggregation.
The deformability of RBCs was determined with RBC suspension that were diluted 1:100
in PBS (pH 6.5), containing 5% PVP (MW 360 kDa, Sigma-Aldrich, Germany) and with a
viscosity of 30 mPa.sec. One ml of the latter RBC suspension was inserted into the LORCA
and the RBC diffraction pattern was recorded at various shear stresses at 36.8 ± 0.2 °C. The
deformability of the RBCs, which is expressed by the EI, was determined by the LORCA
from the size of the vertical (L) and horizontal (W) axes of the diffraction pattern according
81
Chapter 6
to the formula: EI = (L-W) / (L+W). An increased EI at a given shear stress indicates
greater RBC deformability. A deformability curve was obtained by plotting the calculated
values for EI versus the corresponding shear stress. The deformability at two shear stress
values were examined more closely; the deformability at a shear stress of 3.9 Pa, which
reflects the rigidity of the cell membrane, and the maximal deformability at shear stress of
50 Pa.
Osmotic fragility
The osmotic fragility of RBCs, which reflects the membrane’s ability to maintain structural
integrity, was determined by diluting RBCs in PBS solutions ranging from 0.90% to 0.35%.
RBCs with a Hct level of 30 to 35% were diluted 1:100 in each PBS solution, mixed and
incubated for 30 minutes at 4°C, followed by centrifugation for 12 minutes at 1100 x g. The
free Hb in the supernatant was measured by a spectrophotometer (PowerWave 200
spectrophotometer, Bio-Tek Instruments, USA). The concentration of PBS necessary to
induce 50% hemolysis defined the osmotic fragility index of the RBCs.26 With this method,
a larger osmotic fragility index corresponds to more fragile cells.
Hematologic variables
The RBC MCV, the Hb concentration and the Hct were determined with a hematologic
analyzer (Medonic CA 530-Oden, Sweden). The cytoplasmic viscosity of RBCs, which is
determined by the MCHC,16,27 was calculated by dividing the Hb concentration by the Hct.
The amount of free Hb in the RBC suspension was determined according to the Harboe
method.28 In short, cell supernatant was obtained by centrifugation of RBC suspensions for
1 minute at 3500 x g. The supernatant was diluted 1:10 in 0.01% sodium carbonate in a flatbottom 96-well microtiter plate and mixed for 30 minutes. The free Hb concentration in the
supernatant was determined by measuring the OD at 415 nm and correcting for the OD at
380 and 450 nm (PowerWave 200 spectrophotometer, Bio-Tek Instruments, USA),
according to the formula OD = 2* (OD 415 nm) – (OD 380 nm) – (OD 450 nm). The
hemolysis was expressed as a percentage of the total amount of Hb present in the RBC
lysates.
To determine the RBC ATP content, RBC samples were incubated with 8% ice-cold
trichloroacetic acid, in a ratio of 1:3, during a 30-minute period. The samples were
82
Rheologic properties of frozen RBCs
centrifuged for 1 minute at 3500 x g and the protein-free supernatant was neutralized with
1.5 mol/L sodium carbonate. Aliquots were stored at –80°C for later batch analyses of
ATP. The ATP content was determined with a commercially available enzyme assay
(Roche Diagnostics, Germany). For detection of ATP, light emission was measured at 560
nm by an illuminometer (Wallac 1420 Multilabel Counter, Perkin Elmer Life Sciences,
Finland). Due to the detrimental influence of pre-freezing storage time on the RBC energy
content,1,11 ATP was determined in RBC units with a pre-freezing storage time that did not
exceeded 8 days.
Statistical analysis
Statistical analysis was performed using statistical software (SPSS, version 16.0, SPSS Inc.,
Chicago, IL). Data were tested for normality with the Kolmgorov-Smirnov goodness-of-fit
test. In the case of normally distributed data, differences between storage groups were
demonstrated by using unpaired t-tests. Within storage groups, paired t-tests were
performed to show differences over time. For none normally distributed data, the MannWhitney test was used to quantify differences between groups whereas the Wilcoxon
signed ranks test was used to quantify differences within storage groups. Differences are
considered to be significant with a two-tailed p value of less than 0.05. Results are
presented as means ± SD.
6.3. Results
Rheologic features
The RBC ability to aggregate, as represented by the AI, was significantly reduced in
cryopreserved RBCs when compared to both fresh and refrigerated stored RBCs (Table
6.1). During storage, the AI of refrigerated stored RBCs was significantly reduced, whereas
the AI of cryopreserved RBCs remained stable. The T½ of cryopreserved RBCs was
significantly higher on Day 0, compared to refrigerated stored RBCs (Table 6.1). The latter
observation indicated that aggregation formation immediately after deglycerolization was
slightly slower. During storage, the T½ of refrigerated stored RBCs was significantly
reduced in accordance with the AI, whereas the T½ of cryopreserved RBCs did not
significantly change. It should be noted that the SD of both AI and T½ obtained with
83
Chapter 6
cryopreserved RBCs, were much larger than those of fresh and refrigerated stored RBCs.
Probably the variability in (pre) freezing time may provide an explanation.
The RBC deformation curve showed a typical sigmoid shape over a shear stress range of
0.6- 50 Pa for all the tested RBC groups (Figure 6.1). Yet, the deformation curves of
cryopreserved and refrigerated stored RBCs were significantly elevated over the shear
stress range of 2.0 Pa to 50 Pa compared to fresh RBCs. The deformability of RBCs at a
shear stress of 3.9 Pa and 50 Pa was not significantly different between cryopreserved and
21- or 35-day refrigerated stored RBCs (Figure 6.2). During post-thaw storage, the RBC
deformability at a shear stress of 3.9 Pa and 50 Pa did not yield significant changes.
However, during refrigerated storage the RBC deformability at a shear stress of 3.9 Pa
slightly increased (from 0.35 ± 0.01 to 0.38 ± 0.01 EI; p < 0.01), whereas the deformability
at a shear stress of 50 Pa slightly decreased (from 0.58 ± 0.01 to 0.56 ± to 0.02; p < 0.01).
Table 6.1. Aggregation behavior of fresh, refrigerated stored and cryopreserved RBCs.
Measure
Fresh
Refrigerated storage
Day 3
AI (%)
T ½ (sec)
45.3 ± 2.6
1.3 ± 0.2
Post-thaw storage
Day 21
Day 35
Day 0
Day 2
46.9 ± 2.4
46.0 ± 3.8
44.4 ± 4.5 †
36.0 ± 8.6 *† ‡§
36.9 ± 8.0 *† ‡§
1.03 ± 0.3 *
0.86 ± 0.21 *†
0.91 ± 0.12 *†
2.4 ± 1.7 †‡§
2.3 ± 2.4
Aggregation behavior was expressed as mean ± SD of ten units. Significant differences with a P- value < 0.05
were reported
* significantly different from fresh RBCs
† significantly different from day 3 refrigerated stored RBCs
‡ significantly different from day 21 refrigerated stored RBCs
§ significantly different from day 35 refrigerated stored RBCs
84
Rheologic properties of frozen RBCs
Table 6.2. Hematologic variables of fresh, refrigerated stored and cryopreserved RBCs
Measure
Fresh
Refrigerated storage
Day 0
Day 3
Day 21
Day 35
Day 0
Day 2
MCV (fl)
89.1 ± 2.4
89.0 ± 2.4
91.1 ± 2.6 †
92.0 ± 2.8 *†‡
104.2 ± 10.2 *†‡§
106.4 ± 8.3 *†‡§
MCHC (mmol/L)
20.4 ± 0.4
20.7 ± 0.6
20.5 ± 0.6
20.2 ± 0.3
16.1 ± 0.6 *†‡§
16.0 ± 0.8 *†‡§
Osmotic fragility (%)
0.45 ± 0.03
0.48 ± 0.02
0.46 ± 0.02
0.47 ± 0.02
0.66 ± 0.05 *†‡§
0.67 ± 0.08 *†‡§
Hemolysis (%)
0.26 ± 0.04
0.24 ± 0.07
0.36 ± 0.12 *†
0.53 ± 0.24 *†‡
0.57 ± 0.26 *†‡
0.99 ± 0.28 *†‡§
6.1 ± 3.1
4.6 ± 1.3
ATP (μmol/gHb)
3.8 ± 1.1
Post-thaw storage
2.1 ± 0.4 *†‡
4.5 ± 2.2 §
4.0 ± 1.9 §
Values were expressed as mean ± SD of ten units. ATP values were determined from seven RBC units. Significant differences with a P-value less than 0.05 were reported
* significantly different from fresh RBCs
† significantly different from day 3 refrigerated stored RBCs
‡ significantly different from day 21 refrigerated stored RBCs
§ significantly different from day 35 refrigerated stored RBCs
85
Chapter 6
Figure 6.1. Shear stress EI curves for fresh, refrigerated stored and thawed deglycerolized RBCs. The shear stress
value is plotted on logarithmic axis and data represent the mean ± SD of ten RBC units. The EI over a shear stress
range of 2.0 Pa to 50 Pa of refrigerated stored and thawed deglycerolized RBCs was significantly elevated for all
time points, compared to fresh RBCs (* p < 0.05). Fresh (▲); Day 3 liquid storage (■); Day 21 liquid storage (*);
Day 35 liquid storage (--); Day 0 postthaw storage (Δ); Day 2 postthaw storage (♦).
Osmotic fragility
The osmotic fragility index of cryopreserved RBCs, as represented by the osmolarity at
half-maximum hemolysis, was markedly enhanced (p < 0.01) in comparison to both fresh
and refrigerated stored RBCs (Table 6.2). These results indicate that cryopreserved RBCs
are more fragile than fresh and refrigerated stored RBCs. During storage, the osmotic
fragility index of cryopreserved and refrigerated stored RBCs did not change significantly.
86
Rheologic properties of frozen RBCs
A B Figure 6.2. Deformability for two representative shear stress levels as a function of storage time. (A) EI at a shear
stress of 3.9 Pa. (B) EI at a shear stress of 50 Pa. Values are expressed as the mean ± SD of ten RBC units.
Significant changes are illustrated in the figure (p < 0.05); * significantly different from fresh RBCs, †
significantly different from day 3 refrigerated stored RBCs, ‡ significantly different from day 21 refrigerated
stored RBCs
87
Chapter 6
Hematological variables
Cryopreserved RBCs had a considerably higher MCV (p < 0.05) and a lower MCHC (p <
0.01) compared to fresh and refrigerated stored RBCs (Table 6.2). During storage, the
MCV of refrigerated stored RBCs was significantly enhanced. At the end of storage, the
MCV and MCHC of cryopreserved RBCs differed from refrigerated stored RBCs by 15.7
% and respectively 20.8 %.
The hemolysis was generally higher in cryopreserved RBC units than in refrigerated stored
RBC units (Table 6.2). Moreover, during storage the hemolysis increased with 1.7-fold in
cryopreserved RBC units (p < 0.05) and with 2.2-fold in refrigerated stored RBC units (p <
0.01). As a result, after 2 days of post-thaw storage the hemolysis in the RBC unit slightly
exceeded the allowable limits, whereas after 35 days of refrigerated storage the hemolysis
in the RBC unit clearly remained below these limits (i.e., 0.8% in Europe and 1% in the
United States).3,4
The ATP content of cryopreserved RBCs was similar to that of fresh and 3- or 21-day
refrigerated stored RBCs (Table 6.2). During storage, the ATP content of cryopreserved
and refrigerated stored RBCs reduced with 8.6 and 53.4% respectively. As a result, the
ATP content at the end of refrigerated RBC storage was significantly different from that of
fresh and cryopreserved RBCs. None of the aerobic and anaerobic blood cultures of
cryopreserved RBC units showed evidence of bacterial contamination.
6.4. Discussion
The RBC aggregability and deformability are important hemodynamic determinants.
Alterations in RBC rheology, that is, enhanced RBC aggregability and reduced
deformability, have been observed in a variety of pathological states such as sepsis,
myocardial ischemia, renal failure, inflammation, diabetes mellitus, obesity, hypertension,
sickle cell disease and malaria.15,19,20,29,30 Throughout the years, the rheologic properties of
refrigerated stored RBCs have been extensively investigated.31-40 So far, the rheologic
properties of cryopreserved RBCs have scarcely been investigated.41-43 The current study
was undertaken to explore the rheologic features and hematologic variables of HGM
cryopreserved RBCs and to compare this to conventional refrigerated stored and fresh
RBCs.
88
Rheologic properties of frozen RBCs
In this study the aggregability of cryopreserved RBCs was markedly reduced, in
comparison to that of refrigerated stored and fresh RBCs. The time necessary to induce
aggregation was also slightly prolonged after deglycerolization, compared to refrigerated
stored RBCs. In circulating blood, this may result in less effective transport of RBCs in the
microcirculation. The ability of RBCs to aggregate is dependent on cellular properties and
the composition of the suspension medium.20 In our study the suspension medium was
standardized for all samples, indicating that the observed differences were caused by
alterations in cellular properties only. Cell morphology is a major determinant of RBC
aggregability. Cell swelling suppresses cell contact and subsequently Rouleaux
formation.44,45 Previously, it has been shown that the freeze-thaw (wash) process made the
RBC membrane permeable to cations and hence induced cell swelling.9,46 Our study
supports these results, as the MCV of cryopreserved RBCs markedly exceeded the MCV of
fresh and refrigerated stored RBCs. This gain in RBC volume, as obtained by the freezethaw-wash process, could be responsible for the observed reduction in aggregability.
Although the clinical relevance of altered RBC aggregability is an ongoing debate,47 RBC
swelling during storage is probably irrelevant, as long as it is reversible upon transfusion. In
vitro data obtained from refrigerated stored RBCs support the hypothesis that RBC swelling
can be reversed in plasma.40
The RBC ability to deform depends on the cytoplasmic viscosity of the cell, which is
reflected by the MCHC, as well as on the overall cell shape and the viscoelastic properties
of the cytoskeleton.16 Our data showed no significant difference in the deformability
between cryopreserved and long-term refrigerated stored RBCs. The deformation curves of
both cryopreserved and refrigerated stored RBCs were, however, significantly elevated as
compared to fresh RBCs. At a shear stress of 3.9 Pa an increasing deformability of
refrigerated stored RBC and an increased deformability of cryopreserved RBC, with respect
to fresh RBCs, was observed during storage, despite an enhanced MCV. At a shear stress of
50 Pa the deformability during refrigerated storage reduced, but was still elevated, whereas
the deformability of cryopreserved RBCs was elevated and comparable to long-term
refrigerated stored RBCs. These observations indicated that the RBC deformability in the
low shear stress regions was less affected by changes in MCV. The deformability at a shear
stress of 3.9 Pa, a shear stress which is predominantly found in the microcirculation,48 is
thus a direct reflection of the rigidity of the cell membrane. Altogether, the increased MCV
and the resultant lowered MCHC value of cryopreserved RBCs did not seem to adversely
89
Chapter 6
influence the RBC flexibility, because the deformability of cryopreserved RBCs was still
higher than the deformability of fresh RBCs. The improved flexibility of 21- and 35-day
refrigerated stored as well as that of cryopreserved RBCs, could be explained by di(2ethylhexyl)phthalate (DEHP) leaking from the PVC storage bag. This plasticizer, which is
added to the PVC to impart flexibility, is known to improve RBC storage by suppressing
hemolysis, microvesiculation and morphology changes.49-53 It was also shown that the
presence of DEHP improved the flexibility of RBCs during long-term refrigerated
storage.54 We hypothesize that DEHP enhances the viscoelastic properties of the
cytoskeleton, explaining the improved deformability of long-term refrigerated stored and
cryopreserved RBCs, despite the gain in cell volume. The observation that the
deformability at a shear stress of 3.9 Pa of both fresh and 3-day refrigerated stored RBCs
was significantly lower than long-term refrigerated stored and cryopreserved RBCs further
substantiates this hypothesis.
In the past, the RBC flexibility has been investigated after subjection of RBCs to subzero
temperatures.41-43 Nevertheless, in those studies the RBC storage conditions were not
representative for current clinical standards, explaining the discrepancy between our results.
The osmotic fragility was significantly increased in cryopreserved RBCs compared to fresh
and refrigerated stored RBCs. This indicates that the freeze-thaw-wash process made the
RBCs more fragile. Moreover, the hemolysis was also significantly higher during post-thaw
storage. At the end of storage, the hemolysis in cryopreserved RBC units exceeded the
allowable limits. Previous studies demonstrated, however, that hemolysis during 48 hours
of post-thaw storage remained clearly within the allowable limits.5,9,23 In our study,
polypropylene containers were used for storage of thawed RBCs. It cannot be ruled out that
the lower gas permeability or the absence of DEHP plasticizer of these containers, was
responsible for the increased hemolysis observed in our experiments.55
ATP as an energy source is important for the overall functioning of the RBC. Loss of ATP
is associated with more rigid cell membranes, echinocyte shape change, enhanced cation
permeability, loss of vasodilatation control, exposure of phosphatidylserine on the external
RBC membrane, microvesiculation, and decreased RBC viability.56-64 In the past it was
shown that the RBC ATP content must be at least 2.7 µmol per gram Hb to have a 90
percent chance of acceptable in vivo survival (24-hr in vivo recovery of 75% or higher).
65,66
In our study, the ATP content of 35-day refrigerated stored RBCs was slightly below
this limit, whereas the ATP content of cryopreserved RBCs remained close to those of fresh
90
Rheologic properties of frozen RBCs
RBCs. These results are in line with current findings, which showed a minimal loss of ATP
in cryopreserved RBCs.9,12,23 More recently, it was indicated that the RBC ATP content
plays a prominent role in restoring oxygen deficits in the microcirculation.67,68 Transfusion
of cryopreserved RBCs with physiological ATP content would therefore favor the RBC
viability and the oxygen delivery to the tissues.
In transfusion medicine, cryopreserved RBCs are a valuable blood resource for controlling
an inventory in situations where the RBC availability is limited or unpredictable. This is the
case for storage of RBC with rare blood types or for usage in military settings and
occasionally during civil disasters.5,69,70 Routine usage of cryopreserved RBCs in
transfusion medicine is limited due to the more expensive, time consuming and less
efficient nature of this preservation method. Consequently, the unfamiliarity with regard to
the quality of cryopreserved RBCs has further limited clinical usage over the years. Yet,
cryopreserved RBCs showed satisfactory in vitro quality and posttransfusion in vivo
survival,1,9,12,71 Improving the RBC freezing technology with preservation of the current
quality, could ultimately make cryopreserved RBCs more utilizable for modern transfusion
medicine.
Our data demonstrate that although cryopreserved RBCs are more fragile than refrigerated
stored and fresh RBCs, the freeze-thaw-wash process did not adversely affect the ATP
content or the aggregability and deformability of cryopreserved RBCs. From a rheologic
point of view, we concluded that cryopreserved RBCs are a valuable alternative to
refrigerated stored RBCs for usage in transfusion medicine.
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obtained by leukocyte depletion before storage. Anasthesiol.Intensivmed.Notfallmed.Schmerzther. 2001,
vol.36: S11-S19.
40. Zehnder L, Schulzki T, Goede JS, Hayes J, Reinhart WH. Erythrocyte storage in hypertonic (SAGM) or
isotonic (PAGGSM) conservation medium: influence on cell properties. Vox Sang. 2008, vol.95: 280-287.
41. Wegner G, Kucera W. Changes in erythrocyte deformability in blood preservation. Z.Gesamte Inn.Med.
1987, vol.42: 575-581.
42. Chien S, Usami S, Rowe AW. Rheologic properties of erythrocytes preserved in liquid nitrogen. J.Lab
Clin.Med. 1971, vol.78: 175-180.
43. Mantzavinos D, Bailey AI, Rampling MW. Flash freezing of erythrocyte suspensions. Biorheology 1997,
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44. Reinhart WH, Singh-Marchetti M, Straub PW. The influence of erythrocyte shape on suspension viscosities.
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45. Cicha I, Suzuki Y, Tateishi N, Maeda N. Changes of RBC aggregation in oxygenation-deoxygenation: pH
dependency and cell morphology. Am.J.Physiol Heart Circ.Physiol 2003, vol.284: H2335-H2342.
46. Kofanova OA, Zemlyanskikh NG, Ivanova L, Bernhardt I. Changes in the intracellular Ca2+ content in
human red blood cells in the presence of glycerol. Bioelectrochemistry. 2008, vol.73: 151-154.
47. Baskurt OK, Meiselman HJ. Hemodynamic effects of red blood cell aggregation. Indian J.Exp.Biol. 2007,
vol.45: 25-31.
48. Pries AR, Secomb TW. Microcirculatory network structures and models. Ann.Biomed.Eng 2000, vol.28:
916-921.
49. Deepa Devi KV, Manoj Kumar V, Arun P, Santhosh A, Padmakumaran Nair KG, Lakshmi LR, Kurup PA.
Increased lipid peroxidation of erythrocytes in blood stored in polyvinyl chloride blood storage bags
plasticized with di-(2-ethyl hexyl) phthalate and effect of antioxidants. Vox Sang. 1998, vol.75: 198-204.
50. Draper CJ, Greenwalt TJ, Dumaswala UJ. Biochemical and structural changes in RBCs stored with different
plasticizers: the role of hexanol. Transfusion 2002, vol.42: 830-835.
51. Estep TN, Pedersen RA, Miller TJ, Stupar KR. Characterization of erythrocyte quality during the refrigerated
storage of whole blood containing di-(2-ethylhexyl) phthalate. Blood 1984, vol.64: 1270-1276.
52. Hill HR, Oliver CK, Lippert LE, Greenwalt TJ, Hess JR. The effects of polyvinyl chloride and polyolefin
blood bags on red blood cells stored in a new additive solution. Vox Sang. 2001, vol.81: 161-166.
53. Rock G, Tocchi M, Ganz PR, Tackaberry ES. Incorporation of plasticizer into red cells during storage.
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54. Labow RS, Card RT, Rock G. The effect of the plasticizer di(2-ethylhexyl)phthalate on red cell
deformability. Blood 1987, vol.70: 319-323.
55. Sowemimo-Coker SO. Red blood cell hemolysis during processing. Transfus.Med.Rev. 2002, vol.16: 46-60.
56. Betz T, Lenz M, Joanny JF, Sykes C. ATP-dependent mechanics of red blood cells. Proc.Natl.Acad.Sci.
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57. Bucki R, Bachelot-Loza C, Zachowski A, Giraud F, Sulpice JC. Calcium induces phospholipid redistribution
and microvesicle release in human erythrocyte membranes by independent pathways. Biochemistry 1998,
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58. Gov NS, Safran SA. Red blood cell membrane fluctuations and shape controlled by ATP-induced
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59. Gov NS. Active elastic network: cytoskeleton of the red blood cell. Phys.Rev.E.Stat.Nonlin.Soft.Matter Phys.
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60. Heaton WA. Evaluation of posttransfusion recovery and survival of transfused red cells. Transfus.Med.Rev.
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61. Johnson RM. Membrane stress increases cation permeability in red cells. Biophys.J. 1994, vol.67:1876-1881.
62. Park Y, Best CA, Auth T, Gov NS, Safran SA, Popescu G, Suresh S, Feld MS. Metabolic remodeling of the
human red blood cell membrane. Proc.Natl.Acad.Sci. 2010, vol.107: 1289-1294.
63. Verhoeven AJ, Hilarius PM, Dekkers DW, Lagerberg JW, de Korte D. Prolonged storage of red blood cells
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64. Wan J, Ristenpart WD, Stone HA. Dynamics of shear-induced ATP release from red blood cells.
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65. Stichting Sanquin bloedvoorziening. Richtlijn Bloedproducten. PT009.RL.SQ_002. Amsterdam: 2005.
66. van der Meer PF, Pietersz RN. The effect of plastic overwraps on storage measures of red cell concentrates.
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67. Raat NJ, Verhoeven AJ, Mik EG, Gouwerok CW, Verhaar R, Goedhart PT, de Korte D, Ince C. The effect of
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69. Hess JR. Red cell freezing and its impact on the supply chain. Transfus.Med. 2004, vol.14: 1-8.
70. Zimrin AB, Hess JR. Planning for pandemic influenza: effect of a pandemic on the supply and demand for
blood products in the United States. Transfusion 2007, vol.47: 1071-1079.
71. Valeri CR, Ragno G, Pivacek LE, Srey R, Hess JR, Lippert LE, Mettille F, Fahie R, O'Neill EM, Szymanski
IO. A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol)
glycerol and stored after deglycerolization for 15 days at 4°C in AS-3: assessment of RBC processing in the
ACP 215. Transfusion 2001, vol.41: 933-939.
95
Chapter 7
A new freezing technique allows storage
of red blood cells with 20% glycerol at
-80°C
Sandra Henkelman
Willem van Oeveren
John Blanton
Reindert Graaff
Gerhard Rakhorst
Submitted
Chapter 7
Abstract
Freezing RBCs with 40% glycerol at -80°C allow long-term storage of RBCs. However, due to
the high glycerol content, up to 20% of the RBCs are lost during the addition and removal of
glycerol. A liquid Bio-freezer with a rapid heat exchange could be suitable to preserve RBCs with
lower glycerol concentrations. In this study, the quality and in vivo survival of RBCs
cryopreserved in a liquid Bio-freezer were investigated. Human and rat RBCs were cryopreserved
with 20 or 40% glycerol (Bio-20 or Bio-40) in the Bio-freezer at -25°C or with 20% glycerol,
followed by storage at -80°C in a mechanical freezer (Combi-20). Cryopreserved human RBCs
were tested in vitro for deformability, hemolysis and various other hematologic variables. The
posttransfusion survival was determined in rats with chromium-51 labeled RBCs. After
deglycerolization, Bio-20 cryopreserved RBCs demonstrated 0.80 ± 0.3 % hemolysis and reduced
EI, compared to fresh RBCs (p < 0.05). Bio-40 cryopreserved RBCs maintained the EI but the 48hour posttransfusion survival values were lower than those of fresh RBCs (p < 0.05). Combi-20
cryopreserved RBCs showed 0.50 ± 0.1% hemolysis and the MCV, EI and the 48-hour
posttransfusion survival values were not significant different from fresh RBCs. The temperature
of the Bio-freezer was not cold enough to maintain the RBC quality during storage, neither with
20% nor with 40% glycerol. However, combining the Bio-freezer with -80°C storage enabled
cryopreservation of RBCs with 20% glycerol, while maintaining the RBC integrity, deformability
and high 48-hour posttransfusion survival values. This new freezing method could contribute to
an increased use of cryopreservation in transfusion medicine.
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Quality and viability of RBCs frozen in a Bio-freezer
7.1. Introduction
In transfusion medicine, cryopreservation has been particularly useful for long-term storage
of RBCs with rare phenotypes and to stock RBCs for military deployment.1-4 In addition,
stockpiling frozen RBCs could be beneficial in emergency or clinical situations, where the
demand exceeds the supply of RBCs.5-7 Currently, the HGM is the most applied RBC
freezing method in Europe and the United States. This method allows preservation of RBCs
with a final concentration of approximately 40% (wt/vol) glycerol at temperatures between 65 and -80°C for up to ten years.1,2
Usage of glycerol as a cryoprotectant requires an intensive deglycerolization washing
procedure post-thaw in order to reduce the glycerol concentration within the cell.
International guidelines require that cryopreserved RBCs have a post-thaw recovery of at
least 80% and that the hemolysis in the RBC unit remains below allowable levels (i.e., 0.8%
in Europe and 1% in the United States). Additionally, at least 75% of the cryopreserved
RBCs should remain within the circulation 24 hour after infusion.1,2 The RBC viability, as
defined by the posttransfusion survival of RBCs, is often determined with Chromium-51
(51CR) labeled RBCs. In the Netherlands this method is not allowed in humans because of the
radiation exposure. However, murine transfusion models have been proven useful for
assessing the in-vivo survival of short term preserved RBCs.8-11
In transfusion medicine, routine usage of cryopreserved RBCs is still limited, due to the more
expensive and labor intensive nature of this preservation method. Also HGM frozen storage
is less efficient due to the freeze-thaw-wash related cell loss of approximately 10 to 20 %.12-14
A freezing method which would allow RBCs to be frozen with lower glycerol concentrations
could reduce the cell loss and subsequently make cryopreserved RBCs more applicable for
routine clinical usage.
Currently, a new liquid Bio-freezer (Supachill, Lubbock, USA) has become available which
enables preservation of sperm cells at temperatures of -25°C to-32°C with a high post-thaw
quality.15,16 This new Bio-freezer consists of a reservoir of fluid with a high heat absorption
capacity. Sperm samples are immersed in the fluid and heat is rapidly dissipated from the
sample, which limits the freezing damage. The rate of heat release during freezing is also an
important contributor to the post-thaw quality of RBCs.17 Traditional freezing of RBCs at
-25°C in a mechanical freezer, normally requires high glycerol concentrations to minimize
ice formation.18 However, high concentrations of cryoprotectants can exert detrimental
99
Chapter 7
effects on the cell.19 The rapid heat exchange of the liquid Bio-freezer, might allow RBCs to
be frozen with lower glycerol concentrations at higher temperatures. The latter could reduce
the osmotic stress and subsequently the freeze-thaw-wash related cell loss. This characteristic
could make the liquid Bio-freezer interesting for the cryopreservation of RBCs.
The deformability of the cell membrane is a major determinant of the RBC posttransfusion
survival, because less deformable cells are removed from the circulation via the spleen.20-22
In addition, a high RBC deformability and a rapid recovery to the normal shape are essential
for capillary passage and subsequently to maintain adequate tissue perfusion.23 In chapter 6
we demonstrated that the freeze-thaw-wash process did not adversely affect the deformability
of HGM cryopreserved RBCs. Previously, it was also demonstrated that cryopreserved RBCs
showed satisfactory 24-hour posttransfusion survival, even after 7 days of post-thaw storage
in SAGM solution.24
The objective of this study was to assess whether the liquid Bio-freezer could be suitable
for the preservation of RBCs, especially with low glycerol concentrations. The RBC
deformability and other in vitro quality measures were determined with human RBCs
whereas a rat transfusions model was used to test the in vivo survival of preserved rat
RBCs.
7.2. Materials and methods
RBC collection and processing
Human whole blood was obtained from eight healthy volunteers, after informed consent
and in accordance with University Medical Center Groningen protocols. Briefly, whole
blood (54 ± 2 ml) was anticoagulated with 14% CPD and centrifuged at 1100 x g for 12
minutes, to remove the buffycoat and supernatant. The concentrated RBCs were washed
twice with PBS (pH 7.4), after which they were resuspended in PBS to a final Hct value of
50 ± 10 %. This volume allowed the preparation of identical samples in the glycerolization
procedure.
The animal experiments to study in vivo RBC survival were approved by the ethical
committee for animal research of the University Medical Center Groningen. Animal care
and handling was performed in accordance with Dutch guidelines for the care of laboratory
animals. Rats were anaesthetized with 2.5% isoflurane and 0.8 L oxygen per minute. A
total of twenty six male inbred Lewis rats (LEW/Hantm Hsd: Harlan, the Netherlands), with
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Quality and viability of RBCs frozen in a Bio-freezer
a body weight of 270 ± 23g, were included in this study. Twenty rats were used for
transfusion experiments, whereas six rats were used to collect the required amount of blood.
Briefly, whole blood (10 ± 1 ml) was obtained by cardiac puncture of anaesthetized rats.
Rat blood was collected into 14% CPD and processed similar as described above for human
blood. After blood withdrawal, the rats were euthanized.
Glycerolization
Human and rat RBCs were glycerolized at room temperature to obtain a final concentration
of 20% or 40% glycerol. Briefly, an equal amount of standard 57% (wt/vol) glycerol
mixture (S.p.A. Laboratorio Farmacologico, Italy) was added to the RBCs,25 to achieve a
final concentration of approximately 40% (wt/vol) glycerol. A 25% (wt/vol) glycerol
mixture, containing 50mM sodium phosphate and 46mM sodium gluconate, was added in a
ratio of 5:1 to the RBCs to achieve a final concentration of approximately 20% (wt/vol)
glycerol. To estimate the glycerol concentration within the RBCs,26 the supernatant
osmolarity of the RBC suspension was measured with a cryoscopic osmometer (Osmomat
030-D, Gonotec, Germany).
RBC preservation
The liquid Bio-freezer consists of a reservoir of circulating fluid which is set at a
temperature of -25°C. RBC suspensions are immersed in the fluid allowing heat to be
rapidly dissipated from the sample. A mechanical freezer was used to store RBCs at
temperatures of -80°C.
Human and rat RBCs were frozen for 6 weeks and respectively 48 hours according to
different protocols (Table 7.1). In short, human and rat RBCs with a Hct of 50 ± 10% were
incubated with the appropriate glycerol mixture for 15 minutes. RBC suspensions were
frozen in 20 ml syringes (Omnifix, Braun, Germany) within 2 hours after donation. In
protocol Combi-20, human RBCs were frozen for 24-hours in the liquid Bio-freezer prior to
storage at –80°C, while rat RBCs were frozen for 2-hours in the liquid Bio-freezer prior to
storage at –80°C.
To mimic routine refrigerated blood bank storage, washed rat RBCs were diluted 2:1 in
SAGM solution (Fresenius Hemocare, Germany) and stored at 4 ± 2 °C. RBC samples
were withdrawn from the bag one week after storage for determination of the
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Chapter 7
posttransfusion survival. The RBCs were washed and resuspended in PBS to a Hct of 3035% and radioactive labeled, as will described further on.
Table 7.1. Freezing protocols for human and rat RBCs
Freezing protocol
Final glycerol % (wt/vol)
Bio-20
Combi-20
Bio-40
20% Glycerol
20% Glycerol
40% Glycerol
Freezing
-25°C liquid Bio-freezer
-25°C liquid Bio-freezer
-25°C liquid Bio-freezer
Storage
-25°C liquid Bio-freezer
-80°C mechanical freezer
-25°C liquid Bio-freezer
Deglycerolization
Human and rat RBC suspensions were thawed at room temperature for 40 minutes with
gentle inversion of the syringes every 10 minutes. Thawed RBC suspensions were manually
deglycerolized by washing with NaCl solutions of decreasing osmolarity. Briefly, 2 ml of
thawed Bio-20 or Combi-20 RBC suspension were diluted with 0.5 ml of 8% NaCl,
followed by dilutions with 2.5 ml, 3 ml and 16 ml of 0.9% NaCl, with a 3 minute
incubation period between each step. After centrifugation for 12 minutes at 1100 x g the
supernatant was discarded. Human RBCs were resuspended in PBS to a final Hct of 4045%, while rat RBCs were resuspended to a final Hct of 30-35%. Thawed Bio-40 RBC
suspensions were treated similar as above, with the exception of using 12% instead of 8%
NaCl in the first washing step. In all thawed deglycerolized samples, the supernatant
osmolarity was measured to ensure an efficient removal of glycerol. Functionality testing
with human and rat RBCs were performed within 2- and respectively 4 hours after
deglycerolization.
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Quality and viability of RBCs frozen in a Bio-freezer
Hematologic variables
The RBC MCV and the total Hb concentration were determined with a hematologic
analyzer (Medonic CA 530-Oden, Sweden). The Hct was manually determined using a
micro-centrifuge (Mikro-20, Hettich, Germany). The MCHC, which is a primary
determinant of cytoplasmic viscosity,27,28 was calculated by dividing the Hb concentration
by the Hct.
Hemolysis as measured by the amount of free Hb present in human RBC suspensions,29
was determined after glycerolization, thawing and deglycerolization respectively. Briefly,
cell supernatant was obtained by centrifugation of RBC suspensions for 1 minute at 3500 x
g. The supernatant was diluted 1:10 in 0.01% sodium carbonate in a flat-bottom 96-well
microtiter plate and mixed for 30 minutes. The free Hb concentration in the supernatant was
determined by measuring the OD at 415 nm and correcting for the OD at 380 and 450 nm
(PowerWave 200 spectrophotometer, Bio-Tek Instruments, USA), according to the formula
OD = 2* (OD 415 nm) – (OD 380 nm) – (OD 450 nm). Hemolysis was expressed as a
percentage of the total amount of Hb present in the RBC lysates.
RBC deformability
RBC deformability was monitored in vitro by the LORCA (R&R Mechatronics, Zwaag the
Netherlands).30 Deformability was determined with fresh and thawed deglycerolized human
RBC suspensions diluted 1:100 in PBS, pH 6.5, containing 5% polyvinylpyrrolidone (MW
360 kDa, Sigma-Aldrich, Germany) and with a viscosity of 30 mPa.sec. About 1 ml of the
PVP suspension was transferred into the LORCA device. The diffraction pattern of the
RBCs was recorded at increasing shear stresses at a temperature range of 36.7°C – 37°C.
The deformability of the RBCs, which is expressed by the elongation index (EI), was
determined by the LORCA from the size of the vertical (L) and horizontal (W) axes of the
diffraction pattern according to the formula: EI = (L-W) / (L+W). An increased EI at a
given shear stress indicates greater RBC deformability. The deformability at two shear
stress values were examined more closely; the deformability at a shear stress of 3.9 Pa,
reflecting the rigidity of the cell membrane, and the maximal deformability at shear stress
of 50 Pa.
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Chapter 7
Assessment of RBC survival by chromium-51 labeling
Fresh, refrigerated stored and thawed deglycerolized rat RBC suspensions with a Hct of 30
-35 % were labeled with 51CR according to standard protocols.31 The in vitro label decay
was determined by measuring the radioactivity of 51CR labeled fresh RBCs stored for up to
one week at 4°C. The posttransfusion survival per storage group was determined by
infusing five rats intravenously with each 0.5 ml of 3.0 µCi/ml 51CR labeled RBC solution.
The 51CR radioactivity was measured in blood samples obtained from the rat tail vein after
15 and 20 minutes, 24- and 48 hours and 1 week after infusion with a gamma counter
(Compugamma 1282 CS; LKB Wallac, Finland). The survival of RBCs after infusion, was
determined by the 51CR single-label method after correction for label decay.32
Statistics
Statistical analysis was performed using statistical software (SPSS, version 16.0, SPSS Inc.,
Chicago, IL). Data were tested for normality with the Kolmogorov-Smirnov goodness-of-fit
test. For variables used in the in vitro tests, a repeated measure analysis of variance was
performed to identify subject by storage group profiles. Post-hoc multiple comparisons
were performed to quantify differences between the storage groups using paired t-tests. For
variables used in the in vivo test, a one way analysis of variance was performed to compare
groups. Post-hoc multiple comparisons were performed to quantify differences between the
groups using a Turkey HSD test. For all post-hoc tests a Bonferroni correction was applied
to correct for multiple comparisons. Differences are considered to be significant with a twotailed p value of less than 0.05. Results are presented as means ± SD.
7.3. Results
In vitro measurement of human RBCs
After glycerolization the hemolysis for Bio-20, Combi-20 and Bio-40 cryopreserved RBCs
were respectively 0.28 ± 0.1 %; 0.29 ± 0.1% and 0.41 ± 0.1%. The hemolysis was more
pronounced when a higher glycerol concentration was used (p < 0.01). After thaw the
hemolysis for Bio-20, Combi-20 and Bio-40 cryopreserved RBCs were respectively 0.96 ±
0.5 %; 0.77 ± 0.2 % and 1.05 ± 0.1 % and did not significantly differ from each other. After
deglycerolization (Table 7.2) the lowest hemolysis was observed with protocol Combi-20
104
Quality and viability of RBCs frozen in a Bio-freezer
(0.50 ± 0.1%), where RBCs were first frozen in the liquid Bio-freezer followed by storage
in a -80°C mechanical freezer. Hence, this was also the only group that met the
international guidelines. After deglycerolization, the MCV and MCHC of Bio-20 and Bio40 cryopreserved RBCs did not show significant differences compared to fresh RBCs. In all
thawed deglycerolized samples, the supernatant osmolarity was below 400 mOsm/ kg H2O,
indicating an efficient removal of glycerol.26
The deformability of Bio-40 and Combi-20 cryopreserved RBCs was not adversely affected
at shear stress of 3.9 and 50 Pa (Figures 7.1A and 7.1B). Hence, Bio-40 cryopreserved
RBCs showed slightly higher deformability values at low shear stress compared to fresh
RBCs (p < 0.05). Conversely, Bio-20 cryopreserved RBCs showed considerably lower
deformability values compared to fresh RBCs at both shear stresses (p < 0.05).
Table 7.2. Variables of fresh and thawed deglycerolized human RBCs
Fresh RBCs
Thawed deglycerolized RBCs
Bio-20
Combi-20
Bio-40
Haemolysis (%)
0.26 ± 0.1
0.80 ± 0.3 *
0.50 ± 0.1 *
1.23 ± 0.5 *
MCV (fl)
88.9 ± 2.2
84.2 ± 4.1
89.9 ± 2.6
96.9 ± 5.3
MCHC (mmol/L)
20.3 ± 0.4
21.1 ± 0.9
21.0 ± 1.9
19.3 ± 2.3
302 ± 12
312 ± 14
304 ± 5.0
Osmolarity (mOsm/kg H2O)
-
Values are expressed as mean ± SD of eight units. After deglycerolization significant differences from fresh RBCs
were reported (* P-value < 0.05)
Posttransfusion in vivo survival of rat RBCs
Preserved rat RBCs were tested for their ability to survive after infusion (Figure 7.2). Due
to the detrimental effects of cryopreservation on the deformability of Bio-20 cryopreserved
RBCs, as was mentioned above, this group was omitted from in vivo viability testing. The
posttransfusion survival values of RBCs that have been refrigerated stored for one week in
SAGM solution, were comparable to fresh RBCs even 48 hours after infusion. Combi-20
cryopreserved RBCs showed a similar pattern, whereas the posttransfusion survival values
105
Chapter 7
of Bio-40 cryopreserved RBCs were lower than for fresh RBCs as soon as 48-hours after
infusion (p < 0.05). One week after infusion, the posttransfusion survival values of all
storage groups significantly differed from fresh RBCs.
A B Figure 7.1. Deformability for two representative shear stress levels as a function of storage method. (A) EI at a
shear stress of 3.9 Pa. (B) EI at a shear stress of 50 Pa. Values are expressed as the mean ± SD of eight human
RBC samples. Significant differences from fresh RBCs are illustrated in the figure (* p < 0.05).
106
Quality and viability of RBCs frozen in a Bio-freezer
Figure 7.2. The posttransfusion survival (%) of preserved rat RBCs using the 51CR single-label procedure.
Survival percentages are expressed as the mean ± SD in five inbred rats. Significant changes are illustrated in the
figure (p < 0.05); * significantly different from fresh RBCs 48 hours after infusion, † significantly different from
fresh RBCs 168 hours after infusion.
7.4. Discussion
During freezing and thawing, RBCs undergo alterations which may hamper their ability to
survive and function after transfusion. Cellular damage of HGM cryopreserved RBCs has
been attributed to cellular dehydration and extra-cellular ice formation, as well as to
osmotic stress obtained during the glycerolization and deglycerolization process.13,33-35 New
freezing techniques that improve the efficiency of the freeze-thaw-wash process could
make cryopreserved RBCs more applicable for routine clinical usage. In this study, the
quality and functionality of RBCs cryopreserved in a new liquid Bio-freezer were
investigated.
Addition and removal of glycerol can be detrimental to the RBCs. In our study,
glycerolization with 20% glycerol maintained the integrity of the RBCs, whereas usage of
107
Chapter 7
40% glycerol slightly increased the hemolysis. The freezing process per se, did not result in
much cellular losses, since after thaw the hemolysis in the freezing protocols was only
slightly increased. After deglycerolization, low hemolysis was observed with RBCs which
were cryopreserved with 20% glycerol in the liquid Bio-freezer. Yet, only the RBCs that
were frozen in the liquid Bio-freezer and stored at -80°C (Combi-20), demonstrated
hemolysis values that met international guidelines (e.g. 0.8% in Europe and 1% in the
United States).1,2 Even though we observed low hemolysis values after thaw, our
preservation methods could have adversely affected the hemolysis. Due to the small sample
size, RBCs mixtures were frozen in syringes instead of PVC bags. It has been demonstrated
that the material of the freezing container can influence the hemolysis post-thaw.36
Changing to PVC bags for human RBCs could possibly further reduce hemolysis due to the
more rapid heat exchange and or the reduction in mechanical force experienced during
freezing and thawing.
The ability of RBCs to deform is an important determinant of the flow behavior of blood.
RBC deformability depends on the viscosity of the cytoplasm, which is reflected by the
MCHC, as well as on the overall cell shape and the viscoelastic properties of the
cytoskeleton.28 Long-term preservation of RBCs with 20% glycerol in the liquid Biofreezer considerably lowered the deformability compared to fresh and the other frozen
storage groups. Since the MCV and MCHC of these RBCs were not significantly different,
alterations in the RBC cytoskeleton were presumably responsible for the observed changes.
It is therefore likely that the lower deformability of these RBCs will not be reversed upon
infusion and subsequently results in removal from the circulation. Altogether, our results
thus indicated that a concentration of 20% glycerol was not sufficient to protect the
flexibility of RBCs cryopreserved at -25°C, despite the rapid heat exchange of the liquid
Bio-freezer. Either lowering the storage temperature (Combi-20) or raising the glycerol
concentration (Bio-40), maintained the deformability of RBCs cryopreserved in the liquid
Bio-freezer. Hence, it appeared that at -25°C, the detrimental effects become more evident
at lower glycerol concentrations. The slightly higher deformability of RBCs cryopreserved
with 40% glycerol in the liquid Bio-freezer versus fresh RBCs, was only present at a shear
stress of 3.9 Pa. It is likely that the enhanced MCV of some RBC samples may have
counteracted this difference at a shear stress of 50 Pa. Particularly, since the RBC
deformability in the high shear stress regions are more influenced by changes in cell
volume.
108
Quality and viability of RBCs frozen in a Bio-freezer
The survival of RBCs after infusion, depends on the morphological and metabolic status of
the RBC, as well as on the protein and lipid composition of the RBC membrane.37-43 In the
present study rat RBCs, which have been refrigerated stored for one week in SAGM
solution, demonstrated high 24-hour posttransfusion survival which was consistent with
earlier findings.8,9 In addition, the posttransfusion survival of the latter group was even
comparable to fresh RBCs 48-hours after infusion. However, one week after infusion, the
posttransfusion survival of refrigerated stored rat RBCs was lower than that of fresh RBCs.
Although one week of refrigerated storage is often not considered to be detrimental to
human RBCs, the latter results indicate that the more accelerated aging process of rat
RBCs, adversely affect the in vivo RBC viability on the long-term.8,9 Frozen storage of rat
RBCs with 40% glycerol (Bio-40) in the liquid Bio-freezer, demonstrated posttransfusion
survival values which were below the international limit of 75%. Yet, the deformability of
human RBCs was not adversely affected with the Bio-40 protocol. Since changes in
morphological and metabolic state of the RBCs would have affected the RBC
deformability,44-46 we hypothesize that the low posttransfusion survival values could be the
result of alterations in the protein and lipid composition of the RBC membrane.
Combi-20 cryopreserved RBCs demonstrated posttransfusion survival values which were
comparable to fresh and refrigerated stored RBCs even 48-hours after infusion. This
indicates that the rapid heat exchange of the liquid Bio-freezer may be favorable for
freezing the RBCs. However, in order to maintain a high degree of viability, storage of
RBCs at lower temperatures was required. The use of only 20% glycerol, could be
beneficial in reducing the osmotic stress associated with the addition and removal of
glycerol which result in cellular losses.12,13,35 One week after infusion, the survival of all
preserved RBCs was lower than that of fresh RBCs. Although current guidelines focus on
the 24-hour posttransfusion RBC survival, maintaining a high in vivo survival for longer
time periods may be beneficial to the patient outcome. This is a first study, which evaluates
the feasibility of the liquid Bio-freezer for human RBC preservation. Nevertheless, further
studies are needed to establish if Combi-20 cryopreserved RBCs, further satisfy
international guidelines and demonstrate acceptable quality during prolonged post-thaw
storage.
In transfusion medicine, cryopreservation enables RBCs to be stockpiled for years.47-49
Nevertheless, current freezing techniques still limit the routine clinical usage of
cryopreserved RBCs. Our data demonstrated that the liquid Bio-freezer alone was not
109
Chapter 7
sufficient to preserve the RBC deformability or maintain high in vivo RBC survival.
However, usage of the liquid Bio-freezer in combination with the -80°C mechanical
freezer, enabled preservation of RBCs with low glycerol concentrations, while maintaining
the RBC fragility, deformability and high 48-hour posttransfusion survival values. The
liquid Bio-freezer could become a valuable tool for the cryopreservation of RBCs.
Acknowledgements
We gratefully thank Supachill and Haemoscan for their financial support. Furthermore, we
thank Michel de Vries from the department of Nuclear Medicine and Molecular Imaging at
the University Medical Center Groningen for his assistance with the 51CR-labeling
procedures.
110
Quality and viability of RBCs frozen in a Bio-freezer
References
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Kakaiya R, Aronson CA, Julleis J. Whole blood collection and component processing, AABB Technical
Manual 16th ed. Bethesda, MD.: AABB Press; 2008.
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Lelkens CC, Koning JG, de Korte D, Floot IB, Noorman F. Experiences with frozen blood products in the
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Peyrard T, Pham BN, Le Pennec PY, Rouger P. Transfusion of rare cryopreserved red blood cell units stored
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Zimrin AB, Hess JR. Planning for pandemic influenza: effect of a pandemic on the supply and demand for
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Schmidt PJ. Blood and disaster-supply and demand. N Engl J Med. 2002, vol.346: 617-620.
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d'Almeida MS, Jagger J, Duggan M, White M, Ellis C, Chin-Yee IH. A comparison of biochemical and
functional alterations of rat and human erythrocytes stored in CPDA-1 for 29 days: implications for animal
models of transfusion. Transfus Med. 2000, vol.10: 291-303.
9.
Gilson CR, Kraus TS, Hod EA, Hendrickson JE, Spitalnik SL, Hillyer CD, Shaz BH, Zimring JC. A novel
mouse model of red blood cell storage and posttransfusion in vivo survival. Transfusion. 2009, vol.49: 15461553.
10. Hall CE, Nash JB, Hall O. Erythrocyte survival and blood volume in the rat as determined by labeling the red
cells with Cr 51. Am J Physiol. 1957, vol.190: 327-329.
11. Hod EA, Arinsburg SA, Francis RO, Hendrickson JE, Zimring JC, Spitalnik SL. Use of mouse models to
study the mechanisms and consequences of RBC clearance. Vox Sang. 2010, vol.99: 99-111.
12. Valeri CR, Ragno G, Pivacek LE, Srey R, Hess JR, Lippert LE, Mettille F, Fahie R, O'Neill EM, Szymanski
IO. A multicenter study of in vitro and in vivo values in human RBCs frozen with 40-percent (wt/vol)
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13. Lelkens CC, Noorman F, Koning JG, Truijens de Lange R, Stekkinger PS, Bakker JC, Lagerberg JW, Brand
A, Verhoeven AJ. Stability after thawing of RBCs frozen with the high- and low-glycerol method.
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14. Hess JR. Red cell freezing and its impact on the supply chain. Transfus Med. 2004, vol.14: 1-8.
15. Goolsby HA, Blanton JR, Cotter PZ, Prien SD. Preliminary trial: motility comparisons of a unique freezing
technology (UFT) to liquid nitrogen mist methodology for cryopreservation of porcine spermatozoa. Reprod
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17. Rinfret AP. Factors affecting the erythrocyte during rapid freezing and thawing. Ann NY Acad Sci. 1960,
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113
Chapter 8
General discussion
Chapter 8
8.1. General Discussion
The ability to store RBCs outside the body has resulted in a worldwide network of blood
banks that collect blood and distributed it when needed. RBCs are frequently life-saving in
many circumstances. Yet, over the last few years concerns regarding the quality of longterm refrigerated stored RBCs have arisen. The RBC rheologic properties for example,
have been repeatedly documented to be impaired during prolonged refrigerated storage.
Nevertheless, RBC storage conditions have continuously improved and the majority of
these rheologic studies were either outdated or not adequately corrected for known
confounding factors. New studies that demonstrated the RBC rheologic properties during
contemporary refrigerated storage were therefore desirable.
A well-recognized problem of refrigerated storage is that due to the limited shelf life, RBCs
continuously need to be replenished to balance the inventory. In an effort to circumvent
storage induced lesions and thus to prolong the shelf life of RBCs, cryopreservation seems
to be a promising method to preserve RBC. Particularly, since in recent years research has
focused on optimizing the use of cryopreserved RBCs for clinical practice. This thesis
aimed to gain a better understanding of the RBC quality from a rheologic perspective in
transfusion medicine, as well as to explore the utilization of cryopreservation for long-term
storage of RBC.
8.2. RBC rheology
The ability of RBCs to deform and aggregate are key determinants of the flow behavior of
blood. The deformability enables RBCs to adapt their size to minimize flow resistance as
well as to pass narrow capillaries. In contrast, RBC aggregation increases the flow
resistance by enhancing whole blood viscosity at low shear rate, albeit that the
physiological role of RBC aggregation is still elusive.1
A better understanding of RBC aggregation my help to guide hemorheologic studies and
therapeutic approaches in pathophysiology. In general, considerable variations in RBC
aggregation tendencies exists between healthy individuals.2 Interestingly, RBCs also exhibit
different aggregation tendencies among mammalian species.3 Yet, these interspecies
differences are not solely ascribed to variations in plasma composition because differences
continue to exist even when RBC are washed and re-suspended in a standard medium of
116
General discussion
high molecular weight polymers. Replacement of plasma proteins by standard polymers to
induce RBC aggregation may help to unravel the fundamentals of the aggregation process.
At present, a depletion and a bridging theory have been proposed to explain the RBC
aggregation mechanism induced by high molecular weight polymers. Neu et al.4-7support
the depletion theory underlying 500-kDa dextran induced RBC aggregation. In contrast, our
data support the bridging theory as the mechanism underlying 200-kDa HES induced RBC
aggregation, which is in line with the formation of stronger and larger RBC aggregates with
HES polymers of increasing molecular weight.8 Despite these differences in aggregation
mechanisms, the 200-kDa HES and the 500-Kda dextran polymer solutions are useful to
manifest cellular induced aggregation differences between healthy individuals and T1DM
patients.
Disturbed rheologic properties can be detrimental to the flow dynamics and subsequently
the oxygen delivery in the microcirculation. Reduced deformation and enhanced
aggregation of RBCs have been observed in a variety of chronic diseases. However, in
some acute situations, such as during damage control of severely bleeding trauma patients,
enhanced RBC aggregation could also play a pivotal role. Presumably, adoption of a liberal
FFP to RBC ratio could promote RBC aggregation. This could be disadvantageous in
trauma patients in which the RBC rheology is already compromised. In this regard, the
LORCA (R&R Mechatronics, Zwaag, the Netherlands) could be useful to demonstrate
RBC aggregation tendencies of trauma patients who undergo damage control resuscitation.
In addition, analysis of the RBC cellular factors that affect the RBC aggregability3 could
further improve our understanding of altered aggregation tendencies in disease.
8.3. Refrigerated storage of RBCs
The rheologic properties of prolonged refrigerated stored RBCs have been repeatedly
documented to be impaired (i.e. enhanced aggregation, reduced deformation and elevated
endothelial cell interaction).9-15 Yet, these studies analyzed the rheologic properties of nonleukofiltrated refrigerated stored RBCs, which is not representative for European blood
banking practice. More recently, it was demonstrated that leukoreduction minimizes storage
associated adhesiveness of RBCs to endothelial cells.16-18 Our data showed that although
the aggregability of leukoreduced refrigerated stored RBCs was slightly altered over time,
an enhanced RBC aggregability was clearly not observed. In addition, we found that the
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Chapter 8
reduction in RBC deformability, which was only observed at high shear stress, was still
within physiological ranges. Previously, it was demonstrated that the phosphorylation of
cytoskeleton protein 4.1, which was powered by ATP, played an important role in
regulating the RBC deformability.19,20 Although we demonstrated that the RBC ATP
content became progressively depleted during refrigerated storage, we did not observe a
reduction in the RBC deformability at low shear stress. This could suggest that DEHP
leaching from the PVC storage bag has counteracted the storage induced deterioration in
flexibility21,22 or that the total adenine phosphate pool, which consist of ATP, ADP and
AMP, has not depleted yet.23,24 We also consider that the sphericity of the RBCs, induced
by the progressive ATP depletion,25,26 could play a fundamental in role in our observations.
Particularly, because at high shear stress the cell volume becomes a limiting factor for the
RBCs to deform and because RBC swelling suppresses cell contact and subsequently
aggregation formation.27,28 Due to the high osmotic content of plasma, RBC swelling is
most likely to be reversed after infusion.29 Based on these and recent findings we concluded
that the rheologic properties of leukoreduced refrigerated stored RBC are not likely to
contribute to adverse clinical outcomes after transfusion.
ATP does not only play a role in the deformability of the RBCs, it is fundamental for the
overall functioning of the cell. We observed considerable low ATP values after 5 weeks of
refrigerated RBC storage. Although a low ATP content can be replenished within 48 hours
following infusion of RBCs,30 in some patient groups, such as critical ill, an immediate
restoration of the oxygen delivery may be desired. Recently, Spinella et al proposed that
transfusion of 1 to 2 RBC units of increased storage age was sufficient to compromise
clinical outcome in trauma patients.31 Nonetheless, this retrospective study was further
limited by the lack of discrimination between leukoreduced and non-leukoreduced RBCs
within each storage group. Despite these limitations, it is possible that there is a storage
limit beyond which RBC transfusion may be less beneficial or can even compromise the
clinical outcome of critical ill patients.32-34 Especially, if a low ATP content hinders the
RBCs to properly function. Yet the clinical consequences of prolonged stored RBCs are
poorly understood and well-designed randomized controlled trials are currently lacking.35-42
Implementation of procedures that improve the RBC quality rather than extending the
maximal storage duration of refrigerated RBC storage are therefore desirable.43 In this
regard, a new storage solution (i.e PAGGGM) was developed that maintains high ATP and
2,3-DPG content of RBCs and prevents microvesicle formation during 5 weeks of
118
General discussion
refrigerated storage.44,45 Other quality determinants, including the RBC rheologic properties
will still need to be determined.
Alternatively, storage of RBCs at ultra-low subzero temperatures halts the biochemical
processes in the cell which may circumvent storage induced lesions. From this perspective,
cryopreservation could be a promising approach to prolong the lifespan of RBCs.
8.4. Cryopreservation of RBCs
Currently, the HGM is the most widely applied freezing method for long-term storage of
RBCs. Cryopreserved RBCs have to meet certain guidelines,46,47 yet as in the case of
refrigerated stored RBCs, these guidelines do not reflect the RBCs ability to function after
infusion. Over the years more knowledge about the quality of HGM cryopreserved RBCs
has become available. Data about the rheologic properties of cryopreserved RBCs were
however scarce. In this regard, we investigated the deformability and aggregability of HGM
cryopreserved RBCs that were post-thaw stored in SAGM additive solution.
In our study the deformability of cryopreserved RBCs was higher than that of fresh RBCs
but similar to prolonged refrigerated stored RBCs. Fresh RBCs were not leukofiltrated as
opposed to the stored RBCs. Although the presence of leukocytes could have been
detrimental to the flexibility of RBCs, it is unlikely that this caused the lower deformability
of fresh RBCs. Especially, because their contact time with fresh RBCs was only minimal.
We hypothesized that DEHP leaching from the PVC storage bag has enhanced the
flexibility of the RBCs, explaining the improved deformability of both refrigerated stored
and cryopreserved RBCs over that of fresh RBCs. Unfortunately, we did not have the
infrastructure to study DEPH leaching per se. Surprisingly, the deformability of
cryopreserved and long-term refrigerated stored RBCs was similar despite their differences
in MCV. Clearly, additional studies are warranted to elucidate the precise role of RBC
volume in limiting the flexibility of the RBCs.
We also observed a higher osmotic fragility of cryopreserved RBCs that were post-thaw
stored in SAGM additive solution, compared to fresh and refrigerated stored RBCs. The
increasing cell volume could have made the RBCs more prone to hemolysis upon
concomitant swelling. Especially, since no alterations in osmotic fragility have been
observed with cryopreserved RBCs that were post-thaw stored in AS-3 or CPDA-1
119
Chapter 8
solution.48 Presumably, the absence of citrate, an impermeable anion, in the SAGM additive
solution allows for more substantial cell swelling.49,50
We demonstrated that cryopreserved RBCs were less able to aggregate then fresh and
refrigerated stored RBCs. This was not surprisingly in the light of the enhanced RBC
volume after deglycerolization. Although enhanced RBC aggregation has been commonly
observed in various diseases, less is known about the effect of diminished RBC
aggregation. Previously, it has been hypothesized that low RBC aggregation is a pathogenic
co-factor in endothelial activation during hemodilution,51. However, it was recognized that
hemodilution per se induced a hypercoagulable state.52 One could argue that infusion of
RBCs with low aggregation tendencies may be less beneficial in directing leukocytes and
possibly platelets to a damaged vessel wall.53 On the other hand, infusion of RBCs with
lower aggregation tendencies could also be beneficial to patients with microcirculatory
disorders.54,55 Nevertheless, the high 24-hour in vivo survival of cryopreserved RBCs
indirectly suggests that RBC swelling and subsequently the lower aggregability tendencies
of cryopreserved RBCs are of less physiologic importance.56
Infusion of cryopreserved RBCs has potential advantages over that of refrigerated stored
RBCs. We demonstrated that the ATP content of cryopreserved RBCs was similar to that of
fresh RBCs. Indicating that cryopreserved RBCs could have superior oxygen delivering
capacity compared to that of prolonged refrigerated stored RBCs.57 Also, due to the
deglycerolization washing procedure less biological active substances will be present in the
RBC unit that may cause transfusion related reactions in the recipient.58 In this regard,
cryopreserved RBCs could be useful in situations where refrigerated stored RBCs are less
desired. This could be the case for chronically transfused patients. In addition,
cryopreserved RBCs could also be useful in order to minimize occurrences of TRALI and
SIRS. Nevertheless, cryopreserved RBCs are primarily used for controlling an inventory of
RBCs with rare blood phenotypes and for military conflicts. Occasionally, cryopreserved
RBCs are used during temporarily blood shortages.
Cryopreserved RBCs are more expensive, time-consuming and less efficient than
refrigerated stored RBCs. However, among the years cryopreserved RBCs have become
more utilizable. Implementation of the automated ACP-215 device (Haemonetics,
Braintree, MA) for example has simplified the washing process and prolonged the postthaw
storage time of RBCs in AS-3 to 14 days. This development has eased the clinical
120
General discussion
applicability of cryopreserved RBCs particularly, because RBCs could now be thawed and
washed in advance.
The developments of new techniques that could make cryopreserved RBCs more utilizable
for transfusion medicine are still ongoing. Implementation of a liquid Bio-freezer with a
rapid heat exchange for example could make cryopreserved RBCs more interesting for
clinical usage. Although our results documenting the quality of RBCs cryopreserved in the
liquid Bio-freezer demonstrated that the temperature of the Bio-freezer was not sufficient to
preserve the RBC deformability or maintain high in vivo RBC survival, combining the biofreezer with -80°C storage enabled preservation of RBCs with only 20% glycerol while
maintaining good in vitro and in vivo viability. In this context, the lower glycerol content
could be favorable since it potentially reduces the osmotic stress during the glycerolization
and deglycerolization process that causes cellular losses. Additional studies will be needed
to determine the RBC quality during prolonged post-thaw storage.
8.5. Conclusion
Refrigerated storage induces a variety of cellular changes that could hamper the RBCs to
properly function after infusion. In this regard, the rheologic properties are important
quality determinants, because alterations in the rheologic properties can be detrimental to
the flow dynamics and subsequently the oxygen delivery in the microcirculation. This
thesis demonstrates that the rheologic properties of leukoreduced RBCs were well
preserved during refrigerated storage and not likely to contribute to adverse clinical
outcome after infusion.
Cryopreservation of RBCs has been practiced to counteract storage induced lesions and
subsequently to extend the shelf life of preserved RBCs. This thesis furthermore
demonstrates that the freeze-thaw-wash process was not detrimental to the aggregability
and deformability of HGM cryopreserved RBCs. RBC cryopreservation is still infrequently
used. However, ongoing scientific and technological advancement has eased the clinical
applicability of cryopreserved RBCs which could extend their utilization in clinical
practice. As we progress towards a more aging society the demand for RBC transfusions
will inevitably increase.59,60 and cryopreserved RBCs could become a more routinely used
blood resource. Especially, since cryopreserved RBCs are available, safe, in compliance
with international regulations and guidelines and can be used effectively.61-63
121
Chapter 8
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125
Summary
Summary
RBC transfusion practice has been widely applied in clinical settings to compensate for
excessive blood loss as well as to improve the oxygen carrying capacity. Although the
infusion of refrigerated stored RBCs has been considered a life-saving practice for years,
concerns about the efficacy and safety of prolonged stored RBCs are currently emerging.
This is because RBCs biochemical processes are not completely suppressed during
refrigerated storage. Consequently, the RBCs undergo various cellular and biochemical
changes that could hamper the RBC to adequately function after infusion. Yet, evidence of
a significant detrimental clinical effect associated with the infusion of prolonged stored
RBCs is still inconclusive. The rheologic properties are important determinants of the
quality of RBCs. Particularly because impaired RBC rheologic properties, which are
enhanced aggregability, reduced deformability and elevated adherence to endothelial cells,
proposes a circulatory risk by hindering adequate tissue perfusion and contributing to
ischemia or even infarction in the micro-vascular environment.
Chapter 1 reviews ex vivo RBC preservation and emphasizes the importance of the RBC
rheologic properties and in particular those that can be measured by the LORCA.
Alternatively, the utilization of cryopreservation for long-term storage of RBCs is
discussed. The general objectives of this thesis were to gain a better understanding of the
RBC quality from a rheologic perspective in transfusion medicine, as well as to explore the
utilization of cryopreserved RBCs for routine clinical practice.
The first part of this thesis mainly focuses on the RBC aggregation process and the
rheologic properties of refrigerated stored RBCs. RBC aggregation has been studied for
decades in healthy and diseased subjects. Despite these studies, the underlying mechanism
and the physiological role are still elusive. Replacement of plasma proteins by standard
polymers to induce RBC aggregation helps to unravel the fundamentals of the aggregation
process. In chapter 2 the usefulness of 200-kDa HES polymers to induce RBC aggregation
was investigated. The EPM of RBCs in 200-kDa HES solution was measured to get more
insight into the RBC aggregation mechanism. The measured EPM values of RBCs in HES
solutions were less negative than could be predicted by the suspension viscosity, which
supports the bridging theory as the mechanism underlying 200-kDa HES induced RBC
aggregation. These findings are in line with previous observations in which stronger and
larger RBC aggregates are formed with HES solutions of increasing molecular weight.
Furthermore, the LORCA was used to demonstrate aggregation differences between RBCs
from healthy individuals and patients with a disease. We demonstrate that like the standard
128
Summary
500-kDa dextran polymers, the 200-kDa HES polymers are able to discriminate cellular
induced aggregation differences between RBCs from healthy and T1DM subjects.
Altogether, our results demonstrate that 200-kDa HES polymers are useful as a proaggregant in RBC rheologic studies.
Enhanced RBC aggregation has been observed in a variety of chronic diseases, among
which T1DM disease. Yet, RBC aggregation could also play a pivotal role in certain acute
situations, such as during damage control resuscitation of severely bleeding trauma patients.
Chapter 3 discusses the potential importance of RBC aggregation in supporting hemostasis
after blood component infusion in severely bleeding trauma patients. In general, RBCs
aggregates exclude leukocytes and possibly platelets from the axial core and direct them
towards the vascular wall. This process is essential, since leukocytes and platelets need to
get into close contact with the damaged endothelium, in order to exert their function.
Adoption of a liberal policy with regard to FFP to RBC infusion could promote RBC
aggregation. Although physiological levels of RBC aggregation would be beneficial for
supporting hemostasis, promotion of aggregation could be detrimental to patients in which
the RBC rheology is already compromised, as was observed in certain trauma states. Future
studies with the LORCA will be helpful to elucidate the role of RBC aggregation in damage
control resuscitation of severely injured trauma patients.
A better insight of the RBC rheologic properties during ex vivo preservation may improve
transfusion outcome. Earlier studies have shown that the RBC rheologic properties become
impaired as soon as the second week of refrigerated storage. Yet, most of these studies were
not representative for modern RBC storage due to the absence of leukofiltration prior to
storage. In chapter 4 the RBC rheologic features and other hematologic variables of
leukoreduced RBCs were studied during seven weeks of refrigerated blood bank storage.
Our data shows, that although the aggregability of leukoreduced refrigerated stored RBCs
was slightly altered over time, an enhanced aggregability was clearly not observed. In
addition, we found that the reduction in RBC deformability, which was only observed at
high shear stress, was still within physiological ranges. We did observe that the ATP
content became progressively depleted during refrigerated storage, whereas the MCV, pH
and MCHC were affected to a lesser degree. We postulate, that the observed changes in
RBC variables during refrigerated storage minimally affected the RBC ability to aggregate
and deform, even after prolonged refrigerated storage. Based on these and recent findings,
129
Summary
we conclude that the rheologic properties of leukoreduced RBC units were well preserved
during routine blood bank storage.
In order to circumvent storage induced lesion and thus to extend the shelf-life of RBCs,
cryopreservation of RBCs has become a feasible alternative. In Chapter 5 cryopreservation
of RBC and in particular its utilization in modern transfusion practice is discussed. In the
past, the clinical applicability of cryopreserved RBCs was hampered by the expensive, less
efficient and more time consuming nature of this preservation method. Yet, the subsequent
unfamiliarity with regard to the quality of cryopreserved RBCs has further limited clinical
usage. In this regard, the rheologic features and various hematologic variables of HGM
cryopreserved RBCs were investigated in chapter 6. We showed that the aggregability of
cryopreserved RBCs was reduced, compared to fresh and refrigerated stored RBCs. The
deformability of stored RBCs was enhanced compared to fresh RBCs, but no significant
differences in deformability between cryopreserved and 21- or 35-day refrigerated stored
RBCs was observed. We also show that the osmotic fragility, hemolysis, MCV and MCHC
of cryopreserved RBCs were altered, compared to fresh and refrigerated stored RBCs, but
that the ATP content of cryopreserved RBCs remained close to those of fresh RBCs. We
demonstrated that although cryopreserved RBCs were more fragile than refrigerated stored
and fresh RBCs, the HGM procedure did not adversely affect the ATP content or the
aggregability and deformability of cryopreserved RBCs. Therefore, cryopreserved RBCs
could become a more utilized blood resource in clinical settings.
To date however, utilization of cryopreserved RBCs is primarily restricted to controlling an
inventory in situations where the RBC availability is limited or unpredictable. Such is the
case for storage of RBC with rare blood groups or for usage in military conflicts. In this
regard, the feasibility of a new Bio-freezer was investigated in chapter 7, as a way to
improve the clinical applicability of cryopreserved RBCs. Human and rat RBCs were
preserved with different glycerol concentrations in the Bio-freezer at -25°C or in
combination with storage at -80°C in a mechanical freezer. Cryopreserved human RBCs
were tested in vitro for deformability and various hematologic variables. The
posttransfusion survival was determined in rats with chromium-51 labeled preserved rat
RBCs. Our data demonstrated that the liquid Bio-freezer alone was not sufficient to
preserve the RBC deformability or to maintain high in vivo RBC survival. However, usage
of the liquid Bio-freezer in combination with the -80°C mechanical freezer, enabled
preservation of RBCs with 20% glycerol, while maintaining the RBC integrity,
130
Summary
deformability and high 48-hour posttransfusion survival values. The use of only 20%
glycerol could be beneficial in reducing the osmotic stress that is associated with the
glycerolization and deglycerolization procedure and which causes cellular losses. We
therefore consider that the liquid Bio-freezer could become a valuable tool for the
cryopreservation of RBCs.
Ultimately important findings in this thesis were discussed in a broader perspective in
chapter 8.
131
Samenvatting
Samenvatting
In Nederland ontvangen jaarlijks vele mensen een bloedtransfusie. De rode bloedcellen
(RBCs) worden toegediend om bloedarmoede, veroorzaakt door ernstig bloedverlies of een
probleem in de bloedaanmaak, te corrigeren. De belangrijkste functie van de RBCs is het
vervoeren van zuurstof vanuit de longen naar de lichaamsweefsels. De RBCs hebben in de
circulatie een levensduur van 120 dagen. RBCs die in de koelkast bewaard worden hebben
echter een aanzienlijke kortere levensduur. Afhankelijk van de bewaarvloeistof worden
RBCs in de bloedbank momenteel 5 á 6 weken bij 2-6°C bewaard. Bij deze temperaturen
worden de biochemische processen in de RBC verlaagd maar niet volledig onderdrukt.
Hierdoor treden er tijdens het bewaren veranderingen op in de eigenschappen van de RBCs,
die de functionaliteit na infusie zouden kunnen beïnvloeden. Er zijn diverse studies
gepubliceerd die veronderstellen dat de infusie van oudere RBCs de gezondheidstoestand
van de patiënt negatief kunnen beïnvloeden. Echter, de meeste van deze studies zijn
retrospectief van aard en kunnen dus dan ook geen causaal verband leggen tussen de
bewaarduur van RBCs en de uitkomst van de patiënt op het toegediende bloed. Het is dus
van essentieel belang dat er meer inzicht in de kwaliteit van de RBCs gedurende het
bewaren bij 2-6°C verkregen wordt. De reologie van de RBCs speelt hierbij een belangrijke
rol, met name omdat veranderingen in de reologische eigenschappen van de RBCs
(verhoogde aggregatie, lagere deformabiliteit en een toegenomen binding aan
endotheelcellen) de bloedtoevoer en dus de oxygenatie in de microcirculatie kunnen
belemmeren of zelfs kunnen blokkeren.
In hoofdstuk 1 wordt een literatuuroverzicht gegeven over de preservatie van RBCs en de
cellulaire veranderingen die hierbij optreden. De reologie van de RBC staat hierin centraal,
en dan in het bijzonder de aggregatie en deformabiliteit van RBCs, aangezien deze
eigenschappen met behulp van de laser-assisted optical red blood cell analyzer (LORCA)
gemeten kunnen worden. Het doel van dit proefschrift was om RBCs te onderzoeken vanuit
een reologisch perspectief om zo meer inzicht te verkrijgen in de kwaliteit van
gepreserveerde RBCs. Daarnaast werd het gebruik van ingevroren RBCs voor transfusie
doeleinden onderzocht. Het eerste gedeelte van dit proefschrift legt zowel de nadruk op de
aggregatie van RBCs onder fysiologische en pathologische omstandigheden als wel op de
reologie van RBCs die bij 2-6°C bewaard worden.
Aggregatie van RBC (ook wel Rouleaux vorming genoemd) vindt plaats in gebieden waar
de stroomsnelheid van het bloed laag of gestagneerd is. Naarmate de stroomsnelheid van
het bloed weer toeneemt zullen, onder fysiologische omstandigheden, de RBC aggregaten
134
Samenvatting
uiteenvallen. Echter, onder sommige pathologische omstandigheden kunnen er grotere en
sterkere aggregaten gevormd worden die niet zo gemakkelijk uiteenvallen en zo de zuurstof
afgifte in de microcirculatie zouden kunnen bemoeilijken. Tot op heden is er nog veel
onduidelijkheid over de fysiologische functie en het mechanisme dat ten grondslag ligt aan
RBC aggregatie. Het vervangen van plasma-eiwitten door standaard polymeer oplossingen
om de aggregatie van RBCs te induceren zal meer inzicht verschaffen in dit fenomeen. In
hoofdstuk 2 wordt het gebruik van 200-kDa HES polymeren in RBC aggregatie studies
onderzocht. Hierbij werd de electroforetische mobiliteit van RBCs gemeten om meer
inzicht te verschaffen in het onderliggende aggregatie proces. De electroforetische
mobiliteit van RBCs in een 200-kDa HES oplossing bleek minder negatief te zijn dan aan
de hand van de toegenomen viscositeit verwacht zou worden. Dit impliceert dat 200-kDa
HES polymeren de aggregatie van RBCs induceren via een absorptie mechanisme. Deze
uitkomst sluit aan bij bevindingen van anderen die aantoonden dat de mate van RBC
aggregatie afhankelijk is van het molecuul gewicht van HES polymeren. Ook werd de
LORCA ingezet om aggregatie verschillen tussen RBCs van gezonde en zieke mensen op te
sporen. We toonden aan dat naast de standaard 500-kDa dextran polymeren ook de 200kDa HES polymeren in staat waren om aggregatie verschillen tussen RBCs van gezonde en
type-1 diabetes mellitus patiënten (T1DM) te onderscheiden. Kort samengevat kan gesteld
worden dat 200-kDa HES polymeren nuttig zijn om als pro-aggreganten in reologische
studies gebruikt te worden.
Een verhoogde aggregatie van RBCs wordt aangetroffen in diverse chronische
aandoeningen, waaronder T1DM. Echter, RBC aggregatie zou ook een belangrijke rol
kunnen spelen in acute situaties, zoals bij trauma patiënten met ernstig bloedverlies.
Hoofdstuk 3 beschrijft de potentiële rol van RBC aggregatie na infusie van bloed
componenten in hevig bloedende trauma patiënten. Onder fysiologische omstandigheden
vind de aggregatie van RBCs plaats in de aanwezigheid van plasma-eiwitten. Deze RBC
aggregaten worden voornamelijk gevormd in het midden van het bloedvat waardoor
leukocyten en mogelijk bloed plaatjes naar de wand van het bloedvat gedreven worden. Dit
proces is essentieel aangezien leukocyten en bloedplaatjes in nauw contact dienen te komen
met het endotheel om effectief te kunnen zijn bij het bestrijden van een infectie en het
bevorderen van de bloedstolling. Momenteel wordt voor trauma patiënten met ernstig
bloedverlies de infusie van RBC, bloedplaatjes en ingevroren plasma in een 1:1:1
verhouding aanbevolen. Infusie van ingevroren plasma (en dus van fibrinogeen) zou de
135
Samenvatting
aggregatie van RBCs kunnen promoten. Zoals gezegd kan RBC aggregatie een belangrijke
rol spelen in het ondersteunen van de hemostase omdat leukocyten en mogelijk
bloedplaatjes zo makkelijker in de nabijheid van het beschadigde endotheel terecht komen.
Echter, een verhoogde aggregatie kan ook nadelig zijn in bepaalde trauma patiënten
waarvan de RBC reologie al verstoord is. De LORCA zou uitkomst kunnen bieden om te
bepalen of in deze trauma patiënten de infusie van bloedcomponenten in een 1:1:1
verhouding invloed heeft op het aggregatie proces van RBCs.
De reologische eigenschappen van RBCs zijn belangrijke kwaliteitsvariabelen in de
transfusiegeneeskunde. Meer inzicht in de reologische veranderingen tijdens RBC
preservatie zou de transfusie uitkomst kunnen beïnvloeden. Er zijn diverse studies die
aantonen dat de reologische eigenschappen van RBCs die bij 2-6°C bewaard worden, al na
enkele weken verslechterd zijn. Echter, de meeste van deze studies zijn verouderd omdat er
RBCs getest werden die niet gefilterd waren van hun leukocyten. In hoofdstuk 4 worden
daarom de reologische eigenschappen en diverse hematologische variabelen van
leukogefiltreerde RBCs onderzocht die gedurende 7 weken bij 2-6°C bewaard worden. We
toonden aan dat de bewaarde RBCs geen verhoogde aggregatie vertoonden. Wel namen we
een afname waar in de RBC deformabiliteit gedurende het bewaren. Deze afname in RBC
deformabiliteit viel echter binnen de fysiologische waarden en werd alleen onder hoge
shear stress condities waargenomen. Het ATP gehalte van de RBC nam wel aanzienlijk af
tijdens het bewaren. De MCV, pH en MCHC daarentegen veranderde minder gedurende 7
weken. Kort samengevat kan gesteld worden dat er tijdens het bewaren bij 2-6°C
veranderingen optreden in de eigenschappen van de RBCs. Deze veranderingen hebben
echter geen nadelige uitwerkingen op het vermogen van de RBC om te kunnen aggregeren
en vervormen. Aan de hand van deze en recente bevindingen van anderen kunnen we
concluderen dat de reologische eigenschappen van RBCs goed behouden blijven bij 2-6°C.
Een manier om de veranderingen die tijdens het koud bewaren optreden tegen te gaan is om
de bewaartemperatuur zodanig te verlagen dat de biochemische processen in de RBC
volledig onderdrukt worden. Cryopreservatie is een methode om RBCs langdurig te kunnen
preserveren. In het tweede gedeelte van dit proefschrift wordt daarom de nadruk gelegd op
ingevroren RBCs. Hierbij wordt in hoofdstuk 5 de cryopreservatie van RBCs en het
gebruik ervan in de transfusiegeneeskunde besproken. Ondanks het feit dat ingevroren
RBCs decennia lang bewaard kunnen worden en zo de beschikbaarheid van RBCs
aanzienlijk kunnen vergroten, is het gebruik van dit bloedproduct in de transfusie136
Samenvatting
geneeskunde beperkt. Dit komt voornamelijk door de hogere kostprijs, de langere
verwerkingstijd en de lagere opbrengst die aan het invriezen van RBCs verbonden zijn.
Ook de beperkte kennis omtrent de kwaliteit van ingevroren RBC heeft het gebruik van dit
bloedproduct in de kliniek beperkt. Momenteel worden RBCs voornamelijk ingevroren
voor militaire doeleinden en om zeldzame bloedgroepen langdurig te bewaren. De laatste
jaren is er echter steeds meer onderzoek verricht naar de kwaliteit van ingevroren RBCs. De
reologische eigenschappen van ingevroren RBCs waren echter nog nauwelijks onderzocht.
In hoofdstuk 6 worden daarom de reologische eigenschappen en hematologische
variabelen van ingevroren RBCs onderzocht. We toonden aan dat de aggregatie van
ingevroren RBCs lager was dan die van verse en in de koeling (2-6°C) bewaarde RBCs. De
deformabiliteit van ingevroren RBCs daarentegen was hoger dan die van verse RBCs maar
vergelijkbaar met de deformabiliteit van RBCs die 21 of 35 dagen in de koeling bewaard
werden. De osmotische flexibiliteit, hemolyse, MCV en MCHC van ingevroren RBCs was
veranderd vergeleken met verse en in de koeling bewaarde RBCs. Het ATP gehalte van
ingevroren en verse RBCs was echter vergelijkbaar. We concludeerden dat ondanks dat
ingevroren RBCs fragieler waren dan verse en in de koeling bewaarde RBCs, dit geen
nadelige uitwerking had op de aggregatie, deformabiliteit en het ATP gehalte van
ingevroren RBCs.
Meer kennis omtrent de kwaliteit van ingevroren RBC zou het gebruik in de kliniek kunnen
bevorderen. Ook het gebruik van nieuwe vriestechnieken zou hierbij een rol kunnen spelen.
In dit opzicht werd in hoofdstuk 7 het gebruik van een nieuwe Bio-vriezer (Supachill)
getest om RBCs in te vriezen. Humane en ratten RBCs werden met verschillende glycerol
concentraties in de Bio-vriezer bij -25°C ingevroren of in combinatie met een mechanische
vriezer bij -80°C opgeslagen. Van de humane RBCs werden de deformabiliteit en diverse
hematologische variabelen getest. De ratten RBCs werden gebruikt om de overlevingsduur
van ingevroren RBCs na infusie te bepalen. We toonden aan dat RBCs die in de Bio-vriezer
ingevroren en bewaard waren, ofwel een lagere deformabiliteit, dan wel een verminderde
overleving 48 uur na infusie vertoonden. Het gebruik van de Bio-vriezer in combinatie met
een standaard -80°C vriezer, maakt het mogelijk om RBCs met slechts 20% glycerol in te
vriezen terwijl de RBC integriteit, deformabiliteit en de overlevingsduur tot en met 48 uur
na infusie behouden werd. Het gebruik van een glycerol concentratie van slechts 20% kan
voordelig zijn bij het invriezen van RBCs omdat dit de osmotische stress en dus de
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Samenvatting
hemolyse van de vriesprocedure zou kunnen verminderen. We veronderstellen dat de Biovriezer een belangrijke rol zou kunnen gaan spelen in de preservatie van RBCs.
De belangrijkste bevinding van dit proefschrift worden in een breder perspectief besproken
in hoofdstuk 8.
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Acknowledgements
Eindelijk is het zo ver, “het boekje” is af.
Gedurende de afgelopen jaren is er veel gebeurd en er zijn diverse mensen die ik toch zeer
dankbaar ben. Allereerst wil ik Supachill en Haemoscan bedanken voor hun financiële
bijdrage waardoor dit proefschrift mede mogelijk gemaakt werd. Ook wil ik ook de
leescommissie bedanken voor hun tijd en inzet.
Daarnaast Gerhard. Beste Gerhard, het is niet altijd even makkelijk geweest voor ons
beiden maar ik ben je zeer dankbaar voor je advies en begeleiding. Vooral op het eind bleek
het of we het steeds vaker eens waren. Het was een nuttig leerproces voor ons beiden.
Reindert, gedurende mijn gehele AIO periode stond je deur altijd open voor raad en daad,
maar zeker ook voor gezelligheid. Dat heb ik super gewaardeerd. Ook heb ik veel geleerd
van je kritische blik met betrekking tot het schrijven van artikelen. Bedankt hiervoor en
blijf zoals je bent!
Beste Johan. Bedankt voor al je wijsheid, tijd en gezelligheid. Ik heb veel van je geleerd.
Het was altijd een waar genoegen om richting Amsterdam te reizen om nieuwe sampels op
te halen of om nog het een en ander door te nemen. Thankxxx
Margriet en Janny. Ook jullie bedankt voor jullie raad en tijd. Mede dankzij jullie kritische
blik is het eerste artikel goed tot uiting gekomen.
Hans, soms moet je wel eens gedacht hebben: daar komt ze weer. Dat krijg je als je
statistiek zo goed kunt uitleggen. Je maakte altijd tijd ondanks dat je agenda zelf super vol
zat. En na een half uur bij jouw geweest te zijn werd statistiek ineens hèèel simpel. Bedankt
dat je wat rust in de chaos verzorgde.
Michel, ook jij bedankt voor je medewerking en inzet bij het labelen. Je vond altijd wel
even tijd voor me ondanks je eigen werkzaamheden.
Lieve mensen van het CDL: waar zouden we zijn zonder jullie. Bedankt voor het
meedenken, assisteren en de gezelligheid. Jullie zijn een fijne club om mee te werken.
Ook op de afdeling zijn er diverse mensen die ik wil bedanken. Allereerst wil ik de vele
bloeddonoren bedanken voor hun bijdrage. Zonder jullie geen onderzoek: super bedankt!!
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Henny en Betsy, bedankt voor jullie tijd, inzet en meedenken met mijn proeven. Jullie
waren erg geduldig en altijd bereid om me te helpen. Dit heeft geresulteerd in een mooi
artikel, mede dankzij jullie.
Bastiaan en Prashant. Ook jullie deur stond altijd open. Ik heb daar af en toe gebruik van
gemaakt. Bedankt hiervoor en voor alle gezelligheid. Het is motiverend om wetenschappers
zoals jullie tegen te komen.
Marten, Marieke, Arina, Stefan, Adam, Anieska en Deepak. Bedankt voor jullie
gezelligheid, conversaties en de lekkere borrels. Vooral de spontane vrijdag middag borrels
waren super!
Also many thanks to my Indonesian friends: Dear Punto, when you came over it was
always very nice (especially the Indonesian food). The congress in Amsterdam was special
en we had lots of fun! It was a pleasure of having you here in the department.
Dear Shanti, You rock girl!! It was great having you here in our department. I hope your
new job will be great as well, you deserve it!. I want to especially thank for your beautiful
drawings in this thesis.
Beste Ingrid. Ook jij bedankt voor al je tijd en energie en dan met name met betrekking tot
de lay-out: het is een super mooi geheel geworden.
Lieve Babs, thanks voor al je gezelligheid en advies. Zoals jij is er maar een. Laat je niet
gek maken!!
Lieve Ed, waar zou ik zijn zonder jou. Niet alleen voor alle blond gerelateerde computer
vraagjes maar zeker ook voor de snoepjes. Thanks voor making my life sweeter!!
Lieve Paranimfen: jullie zijn allen speciaal, daarom heb ik er ook vier.
Ward, je bent een bijzonder persoon. Laat je niets anders wijs maken. Soms iets meer in
jezelf geloven en dan zie je dat je alles kunt bereiken wat je wilt! Bedankt voor je positieve
mentaliteit en je gezelligheid.
Gerwin: je handigheid met computers werd vaak door mij benut. Daar komt bij dat we
dezelfde mentaliteit en humor hebben. Bedankt dat je de laatste jaren mijn roommate was.
Het was in ieder geval een stuk gezelliger met jouw erbij.
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Oana, Ook voor jou was het niet altijd even gemakkelijk, een nieuwe cultuur, het werken
op het lab. Je was echter altijd bereid te helpen, en we hebben veel aan elkaar gehad.
Bedankt voor je gezelligheid.
Lieve Anita, door je gezellige babbel waren we niet altijd even productief. Echter het was
erg gezellig en gelukkig ben jij ook zo’n snoepert. Jouw tijd komt nog wel. Gewoon nog
even volhouden.
Alle dierbare Calameriaantjes: in het bijzonder Ghis en Peer, Henk en Manon, Marjolein,
Wietse, Rob en Ciske. Bedankt voor jullie gezelligheid. Vooral de borrels in de Singelier, de
gezellige etentjes, de duikkampjes en de Egypte vakanties hebben me weer nieuwe energie
gegeven om er tegen aan te kunnen. Wanneer gaan we weer??
Alle lieve vriendjes en vriendinnetjes die mij de afgelopen jaren iets minder hebben kunnen
zien: El, Syl, Simo en Ramona. Thanks dat jullie er waren en vanaf nu wordt er weer tijd
gemaakt voor leuke dingen.
Ritsert, bedankt dat je de tijd neemt om me op het juiste pad te brengen. Een uur met jouw
geeft enorm veel motivatie. Thanks
Ed, Michell, Jackie en Robert ook jullie bedankt voor alle steun, raad en gezelligheid. Ook
de borrels (ja ja ik hou van borrels), etentjes en ski vakanties werden zeer gewaardeerd.
Mam en Pap, bedankt dat jullie er altijd voor me waren. Mede dankzij jullie opvoeding is
dit alles mogelijk geweest. Ik ben trots op jullie.
Last but not least: lieve Stephen, ze zeggen dat je niet in je eentje promoveert. Daar heb jij
het nodige van ondervonden. Een vriendin die vele uren maakt en slapeloze nachten als
gevolg. Het was zeker ook niet gemakkelijk voor jou: bedankt voor je oneindige geduld en
dat je er altijd voor me was: Tá tú an solas na gréine de mo shaol agus is breá liom tú.
Dikke kus
San
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