Anaesthesia 2015, 70 (Suppl. 1), 2937 doi:10.1111/anae.

12891

Review Article
The pathophysiology and consequences of red blood cell storage
D. Orlov1 and K. Karkouti2

1 Clinical Fellow, 2 Associate Professor, Department of Anesthesia and Pain Management, Toronto General Hospital,
University Health Network, University of Toronto, Toronto, Ontario, Canada

Summary
Red cell transfusion therapy is a common treatment modality in contemporary medical practice. Although blood col-
lection and administration is safer and more efcient than ever before, red cells undergo multiple metabolic and
structural changes during storage that may compromise their functionality and viability following transfusion. The
clinical relevance of these changes is a hotly debated topic that continues to be a matter of intense investigation. In
the current review, we begin with an in-depth overview of the pathophysiological mechanisms underlying red cell
storage, with a focus on altered metabolism, oxidative stress and red cell membrane damage. We proceed to review
the current state of evidence on the clinical relevance and consequences of the red cell storage lesion, while discuss-
ing the strengths and limitations of clinical studies.
.................................................................................................................................................................
Correspondence to: K. Karkouti; Email: keyvan.karkouti@uhn.ca; Accepted: 8 August 2014

Introduction
Modern blood banking represents a major achievement
in the eld of medicine. In less than 500 years, we
have progressed from vein-to-vein xeno-human trans-
fusions that were nearly uniformly fatal (to both par-
ties), to a highly procient system that supplies a wide
array of remarkably safe blood components for an
expanding number of indications. Several pivotal
discoveries have contributed to this advance, one of
which was the development of storage mediums in the
1940s that allowed red blood cells to be stored for pro-
longed periods while maintaining much (but not all)
of their functionality and viability.
Modern storage mediums for the most part consist
of various combinations of saline, adenine, glucose/
dextrose, phosphate and mannitol [1, 2]. The primary
regulatory criteria for setting the maximum amount of
time that red cells can be stored (refrigerated at
4  2 C) using these mediums are that the amount
of haemolysis during storage be < 1% of the total hae-
moglobin in the unit, and that at least 75% of the red
cells survive for more than 24 h post-transfusion [3,
4]. These criteria, which have not been substantially
altered since the 1940s [5], are somewhat arbitrary,
and do not consider whether transfusing better func-
tioning and more viable fresh red cells have any clini-
cal advantages over transfusing older red cells.
Nevertheless, based on these criteria, the allowable
storage duration has increased from 21 days in the
1940s to 42 days or higher with the currently available
storage mediums [1]. Over the past decade, the detri-
mental effects of storage on red cell functionality and
viability have come under greater scrutiny [6], raising
questions about the clinical relevance of the storage
lesion and the appropriateness of currently approved
storage durations. In this narrative review, we will
explore this issue by outlining the pathophysiological
changes that red cells undergo during storage, and the
mechanisms by which these changes may lead to
adverse clinical outcomes. We will also review the

2014 The Association of Anaesthetists of Great Britain and Ireland 29

Anaesthesia 2015, 70 (Suppl. 1), 2937 Orlov and Karkouti | Storage of red cells

current state of evidence on the clinical relevance of Altered RBC metabolism

the red cell storage lesion.
Pathophysiological changes during red
cell storage
The function of red cell storage is to maintain the
functionality and viability of red cells throughout the
approved storage period [7]. The difculty that is
shared by modern storage mediums, however, is that
red cell functionality and viability are progressively
impaired during storage by three interrelated mecha-
nisms: altered metabolism; increased oxidative stress;
and membrane damage (Fig. 1).
Most cells meet their metabolic needs by oxidative
phosphorylation of glucose, but red cells lack mito-
chondria and therefore have to rely on anaerobic gly-
colysis to generate metabolites, primarily adenosine
triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG),
and reduced nicotinamide adenine dinucleotide
(NADH) [8]. Adenosine triphosphate is the energy
source for red cells numerous biochemical reactions,
2,3-DPG regulates the afnity of haemoglobin to oxy-
gen, and NADH is an important co-factor that reverses
the spontaneous oxidation of oxyhaemoglobin to meth-
aemoglobin within the red cell (see below) [8, 9].

Figure 1 Red cell changes associated with storage. Under normal conditions (left panel), the process of glycolysis
converts glucose to lactate, with adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), reduced nicotin-
amide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) formed as by-
products. Auto-oxidation of oxyhaemoglobin to methaemoglobin (dotted-arrow), with resultant production of hae-
min, iron and reactive oxygen species, occurs very slowly and is of limited signicance. The structure and orientation
of membrane protein anion exchanger-1 (AE1), ankyrin and spectrin, as well as phosphatidylserine (PS), maintain
the integrity of the red cell membrane. With prolonged storage (right panel), the accumulation of lactate inhibits
glycolysis. Reduced ATP further inhibits glycolysis, whereas depletion of 2,3-DPG attenuates the ability for oxygen
delivery. Reduced ATP and cold-storage impair the exchange of sodium (Na+) and potassium (K+) across the mem-
brane, resulting in increased intracellular Na+, which affects cell volume and shape. Reduced NADH content pro-
motes auto-oxidation and formation of methaemoglobin, whereas reduction of NADPH decreases production of
reduced glutathione (GSH), which is a potent cytosolic antioxidant. Clustering of AE1 compromises red cell mem-
brane integrity, whereas externalisation of PS acts as a senescence signal. Increased formation of microvesicles in the
supernatant decreases endothelial-derived nitric oxide (NO) gas, which is a potent vasodilator and antioxidant.
Finally, elevated levels of free haemoglobin (Hb) and iron in the supernatant released from haemolysed cells contrib-
ute to oxidative stress. Together, these changes reduce red cell viability (pre- and post-transfusion) and functionality
(post-transfusion).

30 2014 The Association of Anaesthetists of Great Britain and Ireland

Orlov and Karkouti | Storage of red cells
Cold-storage of red cells at 4  2 C helps main-
tain red cell functionality and viability by reducing
the red cell metabolic rate. For each one degree drop
in storage temperature, there is approximately a 10%
decrease in red cell metabolic rate [10], and at 4 C,
the metabolic rate is ten times lower than at 25 C
[11]. As metabolic activity does not completely cease
when red cells are stored, glucose or dextrose are
added to storage mediums to allow red cells to con-
tinue glycolysis and thus produce sufcient ATP, 2,3-
DPG and NADH to maintain adequate functionality
and viability during storage. Continued glycolysis,
however, results in the accumulation of its primary
by-product, lactic acid, in the supernatant [8]. The
resulting acidosis inhibits glycolysis via a negative
feedback loop [8, 12, 13], which results in a progres-
sive reduction in ATP, 2,3-DPG and NADH levels
[1317]. By the sixth week of storage, lactate levels in
the supernatant are increased by several fold, the pH
is below 6.5, the ATP and NADH contents are sub-
stantially reduced, and 2,3-DPG content is depleted
[1521]. Reduced ATP levels impair all red cell meta-
bolic activities including glycolysis itself (as ATP is
needed for the initial steps of glycolysis, creating a
vicious cycle), formation of cytosolic antioxidants
(thus reducing antioxidant defences), and mainte-
nance of membrane integrity (thereby reducing red
cell deformability and promoting alterations in their
discoid shape) [8, 22]. Although there exists a direct
relationship between reduced ATP levels and red cell
viability, the overall role of ATP depletion in the
storage lesion seems to be limited [22, 23]. Deple-
tion of 2,3-DPG reduces oxygen delivery, but levels
are rapidly replenished after transfusion [8]. The
most important contribution to the storage lesion is
likely to be the depletion of reduced NADH, which
impairs the conversion of methaemoglobin back to
oxyhaemoglobin within the red cell, thereby aggravat-
ing oxidative stress (see next section) [9, 24].
Another effect of cold-storage is that it impairs the
exchange of sodium and potassium across the red cell
membrane by the sodium/potassium adenosine tri-
phosphatase (Na+/K+ ATPase) pump [13, 16]. Under
normal circumstances, the concentration of potassium
and sodium inside the red cell are maintained at
approximately 90 mM and 5 mM, respectively,
2014 The Association of Anaesthetists of Great Britain and Ireland
Anaesthesia 2015, 70 (Suppl. 1), 2937
whereas outside, they are approximately 5 and
140 mM, respectively [13]. By the sixth week of stor-
age, red cell potassium content is decreased by about
40% and sodium content is increased by about 300%
[16]. At the same time, the potassium content in the
supernatant of the stored units is markedly increased,
which may lead to hyperkalaemia after transfusion [13,
17, 21]. Increased intracellular sodium content affects
cell volume and shape, such that the mean corpuscular
volume of stored red cells is increased after 3 weeks of
storage [21]. This effect is more pronounced in older
red cells, impairing their deformability and viability
[16, 25].
Increased red cell oxidative stress
For haemoglobin to be able to reversibly bind oxygen
(oxyhaemoglobin deoxyhaemoglobin) within the
red cell, its component haeme-irons must be main-
tained in their reduced, or ferrous (Fe2+), form. Under
normal circumstances, a small amount of oxyhaemo-
globin undergoes spontaneous oxidation, generating
methaemoglobin (which has oxidised or ferric (Fe3+)
iron and cannot bind oxygen) and reactive oxygen
species [24, 2628]. Methaemoglobin is inherently
unstable and is denatured into globin and, more
importantly, haemin (also known as ferric or oxidised
haeme) [8], some of which may in turn be catabolised
by erythroid haeme-oxygenase-1 to release its iron
[29]. Free haemin and iron, in conjunction with reac-
tive oxygen species, can generate highly hazardous
hydroxyl radicals that can cause oxidative injury to
membrane lipids and proteins (see below) [9, 27, 30].
Under normal circumstances, red cells are protected
against this oxidative injury because the rate of sponta-
neous oxidation of haemoglobin is slow [24, 31], the
NADH-dependent cytochrome-b5 reductase (CYTb5)
reduces methaemoglobin back into oxyhaemoglobin,
and cytosolic antioxidants (primarily reduced glutathi-
one or GSH) and membrane anti-oxidants (primarily
ascorbic acid or vitamin C) neutralise the generated
reactive oxygen species [9, 24, 26, 27, 32].
All of these protective mechanisms are impaired
during storage. Spontaneous oxidation of haemoglobin
to methaemoglobin increases under conditions of
increased oxygen partial pressure and acidosis [31],
both of which are present during storage [9]. As a
31
Anaesthesia 2015, 70 (Suppl. 1), 2937
result, the rate of this reaction is substantially
increased during storage [24, 26, 27, 33]. Moreover,
due to the metabolic changes outlined earlier, levels of
NADH, GSH and ascorbic acid are markedly reduced
during this period [18, 34, 35]. Increased spontaneous
oxidation of haemoglobin in the setting of reduced
antioxidant defences exposes red cells to increased oxi-
dative stress, which is the predominant cause of red
cell membrane damage (see below) [9, 18, 3638].
Red cell membrane damage
Red cell functionality and viability is critically depen-
dent on the integrity of the red cell membrane, to
maintain normal cell shape, deformability and
mechanical stability [37]. Storage-related alterations in
metabolism and oxidative stress have considerable del-
eterious effects on red cell membrane integrity.
The red cell membrane consists of a lipid bilayer
that is interspersed with proteins. The lipid bilayer
includes phospholipids, cholesterol and fatty acids that
are asymmetrically distributed between the inner and
outer layers [8]. Phosphatidylserine is an important
component of the lipid layer that under normal cir-
cumstances is present entirely on the inner layer, but
in senescent red cells, it is expressed on the outer layer
of the membrane [8]. When phosphatidylserine is
expressed on the outer layer, it is highly thrombogenic
and also acts as an important senescence signal that
leads to the removal of red cells by reticuloendothelial
macrophages [38, 39]. Cholesterol is another important
component of the lipid layer as increased cholesterol
to phospholipid ratios impair red cell viscosity and de-
formability, and promote alterations in red cell shape
[8]. Important membrane proteins include the trans-
membrane protein anion exchanger 1 (AE1, also
known as band 3), and the primary structural cytoskel-
etal proteins spectrin and ankyrin. The AE1 is a trans-
port protein that regulates the exchange of chloride
and bicarbonate across the membrane and also links
the lipid bilayer to the cytoskeleton by binding to
ankyrin, which in turn binds to spectrin [8]. Normal
protein organisation is crucial for maintaining red cell
stability, deformability and shape [8]. AE1 is also
involved in senescence signalling, as its breakdown (or
clustering) generates a neo-antigen that results in the
32
Orlov and Karkouti | Storage of red cells
rapid clearance of the red cell from the circulation
[38].
Metabolic alterations due to cold-storage and
reduced glycolysis as well as increased oxidative stress
have profoundly deleterious effects on the red cell
membrane. Increased auto-oxidation of haemoglobin
within the red cell leads to precipitation of structurally
distorted forms of methaemoglobin (known as haemi-
chromes) near the cell membrane, and causes disrup-
tion of AE1 and cytoskeletal membrane proteins [8, 9].
Denaturation of methaemoglobin results in the forma-
tion of haemin and iron, which freely partition into
the membrane lipid bilayer to cause peroxidation of
membrane lipids, proteins, and carbohydrates [9, 20].
Membrane disruption and peroxidation causes mem-
brane cation leak, increased cholesterol to plasma
ratios, phosphatidylserine externalisation, clustering of
AE1, and increases production of microvesicles. These
changes reduce red cell functionality and viability by
causing membrane instability and loss, reduced de-
formability, alterations in red cell discoid shape and
increased senescence signalling [9, 10, 1820, 36, 37,
4042]. Microvesicle formation and accumulation in
the supernatant increases exponentially during storage
[19, 21, 4346]. The accumulated microvesicles, which
are haemoglobin-laden and externalise phosphatidyl-
serine, can also cause post-transfusion physiological
perturbations by contributing to the oversaturation of
the bodys clearance systems for haemolysed red cells
(discussed below), by increasing thrombogenicity due
to externalisation of phosphatidylserine, and by scav-
enging of endothelial-derived nitric oxide (NO) gas
which is a potent vasodilator, antioxidant and inhibitor
of platelet aggregation by its component haemoglo-
bin [24, 41, 4648]. When haemoglobin comes in con-
tact with NO, it near instantaneously consumes the
NO to form methaemoglobin and nitrate [24, 48].
Under normal circumstances, encapsulation of haemo-
globin within the red cell prevents this reaction
because NO does not diffuse well across the red cell
membrane [24, 48]. It does readily permeate across the
microvesicular membrane, such that the rate of reac-
tion between haemoglobin and NO is increased by
1000-fold [49]. Thus, the accumulated microvesicles in
the supernatant can reduce endothelial-derived NO
2014 The Association of Anaesthetists of Great Britain and Ireland
Orlov and Karkouti | Storage of red cells
bioavailability in the recipient, which can lead to tissue
ischaemia and end-organ injury [24, 4852].
Reduced red cell viability
The nal common pathway of injury due to the stor-
age-related alterations in red cell metabolism, increased
oxidative stress and membrane damage may be reduced
red cell viability resulting in pre- and post-transfusion
haemolysis.[53] Pre-transfusion haemolysis leads to the
release of red cell contents in the supernatant, which
leads to an approximately eightfold increase in the free
haemoglobin levels in the supernatant after 6 weeks of
storage [20, 32, 49, 51, 54, 55]. As a result of similar
processes that occur within the red cell (outlined
above), some of the free haemoglobin in the superna-
tant undergoes spontaneous oxidation, resulting in the
accumulation of methaemoglobin, haemin, iron and
reactive oxygen species in the supernatant, thus aggra-
vating the oxidative stress that red cells are exposed to
during storage [49]. Methaemoglobin levels, which nor-
mally constitute < 1% of total haemoglobin in the
supernatant [8], are increased by twofold after 3 weeks
of storage [56]. Free iron, which is essentially undetect-
able during the rst few days of storage, is ubiquitous
in the supernatant after 3 weeks of storage [35, 57].
Similar to the post-transfusion effects of microvesicles
(outlined above), the contents of the supernatant may
also reduce endothelial-derived NO bioavailability and
contribute to the oversaturation of the clearance
systems for haemolysed red cells in the recipient. How-
ever, given that the amount of free haemoglobin in the
supernatant constitutes < 1% of the total haemoglobin
in a unit of blood [1921], this contribution is likely to
be minor.
A much more onerous burden on the recipient is
likely to be the progressively increasing number of red
cells that become senescent but do not undergo haem-
olysis during storage. These comprise nearly 25% of
the total cells in a unit of blood by the fourth week of
storage [5, 17, 58], and are haemolysed and removed
from the recipients circulation within 1 h of transfu-
sion [17]. Their rapid removal may overwhelm the
normal systems of haemolysis that include several
highly efcient but saturable pathways for clearing the
contents of red cells from the circulation, thereby cur-
tailing their toxic effects.
2014 The Association of Anaesthetists of Great Britain and Ireland
Anaesthesia 2015, 70 (Suppl. 1), 2937
Under normal circumstances, approximately 1% of
the bodys 2.5 9 1013 circulating red cells
(5 9 106.ll 1 of blood) become senescent and are
cleared from the circulation each day (which works
out to approximately 1 x 1010 cells per hour), with the
majority (8090%) undergoing extravascular haemoly-
sis in the macrophages of the reticuloendothelial
system and the remaining 1020% undergoing intra-
vascular haemolysis [8] (Fig. 2). When red cells are
destroyed in the intravascular space, the released
haemoglobin is rapidly bound to haptoglobin and
removed from the circulation by macrophages via
receptor-mediated endocystosis [8, 5962]. The scav-
enged haemoglobin is then safely degraded within the
macrophages as described below. Once the haptoglobin
scavenging capacity has been exceeded, the remaining
free haemoglobin scavenges endothelial-derived NO to
form methaemoglobin [24, 48, 52]. As noted above,
reduced NO bioavailability can cause endothelial dys-
function, platelet aggregation and oxidative injury,
which can lead to tissue ischaemia and end-organ
injury [48, 49, 52]. The formed methaemoglobin is in
turn converted to free haemin, which is then rapidly
cleared from the circulation and transferred to the
liver by haemopexin and albumin, thereby protecting
against its potent pro-oxidant and pro-inammatory
effects [8, 6164]. Thus, intravascular clearance of
haemolysed red cells protects against the toxic effects
of free haemoglobin and haemins at the cost of
reduced NO bioavailability.
Extravascular haemolysis involves the reticuloendo-
thelial macrophages that recognise and phagocytose not
only senescent red cells, but also the haptoglobin-hae-
moglobin and haemopexin-haemin complexes that are
formed during intravascular haemolysis as well as any
haemoglobin-laden microvesicles [8, 41]. Once inside
the macrophages, the scavenged haemoglobin and hae-
mins are catalysed by haeme-oxygenase-1 to carbon
monoxide, biliverdin (a precursor of bilirubin), and
most importantly, iron [8, 65]. The majority of the
recovered iron is buffered and stored within the macro-
phage in ferritin, and the remainder is released into the
circulation where it is bound by the iron-carrier protein
transferrin [8, 66]. Excessive extravascular haemolysis
can lead to the presence of highly toxic free iron inside
the macrophages as well as in the circulation.
33
Anaesthesia 2015, 70 (Suppl. 1), 2937 Orlov and Karkouti | Storage of red cells

Senescent RBCs
Normally 80-90% Normally 10-20%

Extravascular Haemolysis Intravascular Haemolysis

Free Hb
Free Hb

Haeme NO

2NO3-
Iron
+ Met-Hb (Fe3+)
CO
+
Biliverdin Haemin

Macrophage
Microvesicles

Figure 2 Haemolytic pathways of senescent red cells following transfusion. Following red cell transfusion, intravas-
cular haemolysis results in the formation of free haemoglobin (Hb) which binds to haptoglobin and is delivered to
macrophages for safe degradation. If this system is saturated, free Hb may form methaemoglobin (Met-Hb) and hae-
min at the expense of endothelial nitric oxide (NO) consumption. Haemin a known pro-oxidant and pro-inam-
matory mediator is further scavenged by haemopexin and albumin and delivered to macrophages for processing.
Extravascular haemolysis occurs within reticuloendothelial macrophages and results in the production of iron, carbon
monoxide and biliverdin from the breakdown of free Hb. Outside of the macrophage, free iron binds to transferrin
before transport. Macrophages are further responsible for phagocytosis and degradation of haemoglobin-laden micro-
vesicles. The combination of macrophage over-activation and overwhelmed safety mechanisms (pink shading) may
contribute to end-organ dysfunction secondary to oxidative injury (from accumulation of free iron, free Hb, and
haemin) and tissue ischaemia (from vasoconstriction due to NO-consumption).

As noted above, under normal circumstances, decreased endothelial-derived NO bioavailability and

the systems of intravascular and extravascular haem-
olysis clear approximately 1 9 1010 red cells per
hour. A single unit of blood that has been stored
for more than 4 weeks, however, contains approxi-
mately 1.5 9 1012 red cells, of which about 25%, or
approximately 4 9 1011, will have become senescent
during storage and are cleared from the circulation
within 1 h of transfusion [17]. Moreover, each unit
also contains a large number of haemoglobin-laden
microvesicles, free haemoglobin, haemin and iron in
the supernatant that also need to be rapidly cleared
from the circulation. Since the number of senescent
red cells alone are one order of magnitude greater
than the number that are cleared per hour under
normal circumstances, it is quite possible that trans-
fusing a single unit of old blood may oversaturate
the protective systems of extravascular and intravas-
cular haemolysis in some patients, leading to
the presence of free haemoglobin, haemin and iron
in the recipients circulation as well as within the
reticuloendothelial macrophages [48, 49, 55, 6772].
The potential clinical consequences of these effects
are many, and include: increased risk of infection
due to macrophage activation or dysfunction and
cytokine release; tissue ischaemia due to endothelial
dysfunction; and end-organ dysfunction due to oxi-
dative injury [28, 41, 49, 54, 55, 61, 67, 69, 71, 73
79]. Whether or not these effects are clinically rele-
vant remains unproven and a matter of intense
investigation.
Current state of clinical evidence on
the clinical relevance of the red cell
storage lesion
There has been a plethora of studies investigating the
effects of prolonged blood storage on clinical out-

34 2014 The Association of Anaesthetists of Great Britain and Ireland

Orlov and Karkouti | Storage of red cells
comes. These have included healthy volunteers [67,
8082], numerous retrospective and prospective
observational studies [77, 83114], and a handful of
older [115123], more recent [124], and ongoing
randomised controlled trials. Overall, study results
have been conicting and affected by multiple meth-
odological issues including confounding, systematic
bias, limited external validity and multiple statistical
shortcomings.
Volunteer studies
Human volunteer studies have examined the effect of
blood storage on post-transfusion physiological vari-
ables, including anaemia-induced cognitive dysfunc-
tion [81], extravascular haemolysis [67], serum iron
elevation [67], propensity for bacteraemia (evaluated
in vitro) [67], attenuation of NO-mediated hypera-
emia [80] and gas exchange [82]. All of these studies
involved cross-over designs where autologous blood
was used for transfusion following both a short (any-
where from 34 h to 37 days) and prolonged (any-
where from 23 to 42 days) storage duration. In all
cases, each subject participated in both study arms,
thereby serving as their own control. Of all these
parameters, only transient increases in extravascular
haemolysis and serum iron elevations were observed
in relation to storage latency [67], supporting the piv-
otal role of reduced red cell viability in the storage
lesion.
Volunteer studies have the advantage of investigat-
ing outcomes in humans while achieving great tempo-
ral separation between the study arms to allow for
maximal investigation of any treatment effect. Never-
theless, they suffer from a myriad of drawbacks which
warrant careful interpretation. Specically, volunteers
are frequently devoid of comorbidities, receive slow,
low-volume infusions of autologous blood (thereby
minimising the potential adverse effects of volume
overload and immunomodulation, respectively), and
are unsuitable for studying certain outcomes such as
mortality. Moreover, inherent in a crossover study is
the potential for order and carry-over effects if tech-
niques such as counterbalancing and an adequate
wash-out period are overlooked. Most importantly,
volunteer studies have limited external validity for
many of the study populations of interest.
2014 The Association of Anaesthetists of Great Britain and Ireland
Anaesthesia 2015, 70 (Suppl. 1), 2937
Human observational studies
No study subtype is more ubiquitous in the realm of
red cell storage research than human observational
studies. Indeed, we have come a long way from the
seminal investigation by Purdy et al. [99, 125], with
upwards of thirty observational studies published in
the literature [77, 83114], and multiple recent system-
atic reviews [1, 126131] attempting to consolidate this
data. All but one of these reviews [130] have not sup-
ported the hypothesis that transfusion of older red
cells is worse than that of fresh red cells. The review
by Wang et al. [130] was the only one that found a
signicant association between transfusion of older red
cells and mortality, but this review included several
primary studies that are evidently confounded [129].
In all, no rm conclusions can be drawn from existing
studies owing to important limitations in their design
or analyses.
Observational studies are fraught with numerous
potential sources of bias that may hinder their ability
to answer the research question. For instance, in the
absence of randomisation, sampling bias might occur,
such that older red cells are predominantly transfused
to sicker patients or those with a more precarious
peri-operative course, based on a combination of
increased demand and institutional pressures on blood
banks for efciency and subsequent utilisation of the
rst in-rst out principle for cost-containment. If a
causal association between storage latency and adverse
outcomes does indeed exist, the presence of this bias
would overestimate this effect. Similarly, although a
multicentre, international, observational study would
improve the power of an analysis, its results should
not be analysed in aggregate, independent of the coun-
try of origin. Given that the maximum duration of red
cell storage is a function of the preservative solution
which varies by country, unadjusted pooling of
national outcomes would render the results both non-
generalisable and meaningless.
Multiple confounders should also be considered
when evaluating observational data. For example, in a
study of prolonged duration, the year of transfusion
should be accounted for, as it may affect both storage
latency (through advances in preservative solution) and
outcomes (secondary to rapid advances in technology
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Anaesthesia 2015, 70 (Suppl. 1), 2937
and standards of care). The nature of ABO-matching
between donor and recipient should also be carefully
noted. Specically, Group O red cells are considered the
universal donor, thereby decreasing their turnover time
and inating the number of Group O transfusions in
the fresh blood subgroup of patients. It follows that the
old transfusion subgroup usually contains a greater pro-
portion of non-O blood recipients, as demonstrated by
the largest and perhaps most inuential observational
study on the topic [90]. Due to the known association
of non-O blood subtypes with an increased rate of
venous thrombo-embolic events and possible increased
mortality [132134], the nature of ABO-matching may
overestimate the causal relationships of interest in the
absence of appropriate statistical adjustment. Finally,
the number of red cell transfusions is a parameter
requiring special attention. For one, each additional
unit of transfused red cells simultaneously brings the
average age of transfused red cells closer to the blood
bank mean, while selecting for a more critical patient
population with increased likelihood of experiencing
adverse outcomes. Moreover, this confounded direc-
tionality between storage latency and adverse outcomes
can be further inuenced by the precise denition of
what constitutes fresh and old blood [89, 95, 114].
Randomised controlled trials (RCT)
Although close to a dozen RCTs have been published
on the relationship between storage duration and
adverse outcomes [115124], the majority have been
either small or not adequately powered to evaluate
mortality. Moreover, none of them have found any sig-
nicant differences in outcomes between fresh and old
subgroups of transfusion recipients across a variety of
clinical settings. A handful of large, ongoing RCTs,
including one that has been completed and published
[124], aim to contribute valuable information to this
area of interest.
One recently published double-blinded RCT that
included 377 premature infants found no difference in
the rates of morbidity (necrotising enterocolitis, reti-
nopathy, bronchopulmonary dysplasia or intraventric-
ular haemorrhage), or mortality among those who
received fresh ( 7 days old as stated by protocol;
mean (SD) 5.1 (2.0) days in the trial) vs older (stan-
dard practice as stated by protocol; mean (SD) 14.6
36
Orlov and Karkouti | Storage of red cells
(8.3) days in the trial) red cell transfusions [124].
Although robust in its design, this trial is limited in its
external generalisability for two reasons. First, the pop-
ulation was limited to premature infants a very
unique subgroup of patients, mainly cared for in spec-
ialised institutions. Second, the standard-of-care arm
still received relatively fresh blood only 12 infants
received red cells that were exclusively more than
14 days old. Given that the average blood storage in
the US is 18 days [1], and certain centres routinely
store red cells for longer than 21 days [117], these
results may not be applicable to the broader popula-
tion.
Five other large RCTs have either been completed,
or are ongoing. The age of blood evaluation study
(ABLE; ISRCTN44878718) has completed recruiting
and randomising 2510 ICU patients across Canada to
receive either fresh ( 7 days old) or older (standard
blood bank procedure of using the oldest blood units
rst, irrespective of age) red cells with the primary
outcome being all-cause 90-day mortality. These
results are yet to be published. The red cell storage
duration and outcomes in cardiac surgery study
(NCT00458783) has been enrolling patients for 7 years
with a goal to recruit and randomise a total of 2800
primary or re-operative adult cardiac surgical patients
to receive either fresh (< 14 days old) or older
(> 20 days old) red cells at a single centre, with the
outcome being all-cause postoperative 30-day mortal-
ity. The red cell storage duration study (RECESS;
NCT00991341) has completed randomising 1696
patients undergoing complex cardiac surgery across 33
centres in the USA to receive either fresh ( 10 days
old) or older ( 21 days old) red cells. The primary
outcome is a change from baseline in the multi-organ
dysfunction score at 7 days postoperatively. The
informing fresh vs old red cell management study
(INFORM; ISRCTN08118744) is continuing to rando-
mise a projected 24 400 acute care inpatients across
Canada, Australia and the US to receive either fresh
(youngest available) or old (oldest product compatible
in stock) red cells, with the primary outcome being in-
hospital mortality. Finally, the standard issue transfu-
sion vs fresher red blood cell use in intensive care
study (TRANSFUSE; NCT01638416) is an interna-
tional investigation set to randomise 5000 ICU patients
2014 The Association of Anaesthetists of Great Britain and Ireland
Orlov and Karkouti | Storage of red cells
to oldest vs freshest available red cells, with 90-day
mortality being the primary outcome.
These large-scale RCTs are likely to enhance the
state of knowledge and contribute to standard-of-care
transfusion practice, whereas they are still subject to
numerous limitations. First, given the ethical con-
straints, it is unlikely that any randomised trial will
allow for a substantial inter-group contrast that reects
the spectrum of the red cell storage shelf-life. This
drawback substantially limits the power of an RCT
due to both a small intervention effect, and also the
unknown temporal trajectory of red cell degradation.
A recent simulation study examined the power of mul-
tiple pre-existing RCT designs for ve hypothetical red
cell degradation patterns [135]. Only a single pattern
of red cell degradation occurring between 17 and
25 days allowed for adequate power of the RCTs in
question. Second, it is becoming increasingly clear that
certain patient subpopulations might be more suscepti-
ble to the effects of the red cell storage lesions com-
pared to others. These subpopulations including
patients with an infection, renal failure, or those
requiring a massive transfusion need to be dened a
priori, but could only be identied using alternative
study designs. Finally, as with any longitudinal out-
come measure (e.g. 90-day mortality), a lengthy follow
up may be resource-intensive, costly and may result in
multiple patient dropouts. In this case, adequate trial
funding, the indirect use of large administrative data-
bases or statistical imputation may be warranted to
maintain data integrity.
Conclusion
Over the past half-millennium, we have witnessed
incredible progress in the efciency and safety of blood
transfusion therapy. Nevertheless, although we continue
to acquire insight into the intricacies of metabolic
2014 The Association of Anaesthetists of Great Britain and Ireland
Anaesthesia 2015, 70 (Suppl. 1), 2937
mechanisms underlying red cell storage, the answers to
many fundamental questions continue to evade us.
Although we have succeeded in prolonging the maxi-
mum storage shelf-life of blood with advances in pre-
servative solution, the criteria on which this is based
are almost completely arbitrary and have remained
stagnant since the 1940s. We are also no closer to
delineating the temporal pattern of changes with red
cell storage, nor, more importantly, the point at which
these changes begin to manifest in clinically-signicant
sequelae following transfusion. We have likely only hit
the tip of the iceberg in beginning to identify predis-
posing comorbidities and contexts that may worsen
prognosis following red cell transfusions of prolonged
storage duration. All of these questions warrant system-
atic investigation with a simultaneous understanding of
research design and its many limitations.
The multiple ongoing, large-scale RCTs are a tes-
tament to the importance of the storage lesion and its
potential effects on patient outcomes. Over the next
decade, ndings from these RCTs are likely to have
signicant implications for international transfusion
practice, policy makers, and regulatory bodies. Never-
theless, it must be appreciated that all of the aforemen-
tioned study designs are complementary and
invaluable for the holistic exploration of the broader
research question. Only through a large, concerted
effort, will we be closer to understanding the patho-
physiology and consequences of the red cell storage
lesion in clinical practice.
Acknowledgements
KK is supported in part by a merit award from the
Department of Anesthesia, University of Toronto.
Competing interests
No competing interests declared.
37
Anaesthesia 2015, 70 (Suppl. 1), e9e12
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