Anaesthesia 2015, 70 (Suppl. 1), 29–37

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 collection and administration is safer and more efficient 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 discussing the strengths and limitations of clinical studies. .................................................................................................................................................................

Correspondence to: K. Karkouti; Email: [email protected]; Accepted: 8 August 2014

Introduction Modern blood banking represents a major achievement in the field of medicine. In less than 500 years, we have progressed from vein-to-vein xeno-human transfusions that were nearly uniformly fatal (to both parties), to a highly proficient 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 prolonged 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© 2014 The Association of Anaesthetists of Great Britain and Ireland

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 functioning and more viable fresh red cells have any clinical 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 detrimental 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 29

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current state of evidence on the clinical relevance of 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 difficulty that is shared by modern storage mediums, however, is that red cell functionality and viability are progressively impaired during storage by three interrelated mechanisms: altered metabolism; increased oxidative stress; and membrane damage (Fig. 1).

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Altered RBC metabolism Most cells meet their metabolic needs by oxidative phosphorylation of glucose, but red cells lack mitochondria and therefore have to rely on anaerobic glycolysis 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 affinity of haemoglobin to oxygen, and NADH is an important co-factor that reverses the spontaneous oxidation of oxyhaemoglobin to methaemoglobin 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 nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) formed as byproducts. Auto-oxidation of oxyhaemoglobin to methaemoglobin (dotted-arrow), with resultant production of haemin, iron and reactive oxygen species, occurs very slowly and is of limited significance. 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 membrane, resulting in increased intracellular Na+, which affects cell volume and shape. Reduced NADH content promotes 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 membrane 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 contribute to oxidative stress. Together, these changes reduce red cell viability (pre- and post-transfusion) and functionality (post-transfusion). 30

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Cold-storage of red cells at 4  2 °C helps maintain 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 continue glycolysis and thus produce sufficient ATP, 2,3DPG 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 progressive reduction in ATP, 2,3-DPG and NADH levels [13–17]. 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 substantially reduced, and 2,3-DPG content is depleted [15–21]. Reduced ATP levels impair all red cell metabolic 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 maintenance 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]. Depletion 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 aggravating 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 triphosphatase (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

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whereas outside, they are approximately 5 and 140 mM, respectively [13]. By the sixth week of storage, 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 maintained in their reduced, or ferrous (Fe2+), form. Under normal circumstances, a small amount of oxyhaemoglobin undergoes spontaneous oxidation, generating methaemoglobin (which has oxidised or ferric (Fe3+) iron and cannot bind oxygen) and reactive oxygen species [24, 26–28]. 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 reactive 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 spontaneous oxidation of haemoglobin is slow [24, 31], the NADH-dependent cytochrome-b5 reductase (CYTb5) reduces methaemoglobin back into oxyhaemoglobin, and cytosolic antioxidants (primarily reduced glutathione 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

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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 oxidative stress, which is the predominant cause of red cell membrane damage (see below) [9, 18, 36–38].

Red cell membrane damage Red cell functionality and viability is critically dependent 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 deleterious 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 circumstances 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 deformability, and promote alterations in red cell shape [8]. Important membrane proteins include the transmembrane protein anion exchanger 1 (AE1, also known as band 3), and the primary structural cytoskeletal proteins spectrin and ankyrin. The AE1 is a transport 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

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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 haemichromes) near the cell membrane, and causes disruption of AE1 and cytoskeletal membrane proteins [8, 9]. Denaturation of methaemoglobin results in the formation 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 membrane 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 deformability, alterations in red cell discoid shape and increased senescence signalling [9, 10, 18–20, 36, 37, 40–42]. Microvesicle formation and accumulation in the supernatant increases exponentially during storage [19, 21, 43–46]. The accumulated microvesicles, which are haemoglobin-laden and externalise phosphatidylserine, can also cause post-transfusion physiological perturbations by contributing to the oversaturation of the body’s clearance systems for haemolysed red cells (discussed below), by increasing thrombogenicity due to externalisation of phosphatidylserine, and by scavenging of endothelial-derived nitric oxide (NO) gas – which is a potent vasodilator, antioxidant and inhibitor of platelet aggregation – by its component haemoglobin [24, 41, 46–48]. When haemoglobin comes in contact with NO, it near instantaneously consumes the NO to form methaemoglobin and nitrate [24, 48]. Under normal circumstances, encapsulation of haemoglobin 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 reaction between haemoglobin and NO is increased by 1000-fold [49]. Thus, the accumulated microvesicles in the supernatant can reduce endothelial-derived NO

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bioavailability in the recipient, which can lead to tissue ischaemia and end-organ injury [24, 48–52].

Reduced red cell viability The final common pathway of injury due to the storage-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 supernatant undergoes spontaneous oxidation, resulting in the accumulation of methaemoglobin, haemin, iron and reactive oxygen species in the supernatant, thus aggravating the oxidative stress that red cells are exposed to during storage [49]. Methaemoglobin levels, which normally constitute < 1% of total haemoglobin in the supernatant [8], are increased by twofold after 3 weeks of storage [56]. Free iron, which is essentially undetectable during the first 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. However, given that the amount of free haemoglobin in the supernatant constitutes < 1% of the total haemoglobin in a unit of blood [19–21], 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 haemolysis 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 recipient’s circulation within 1 h of transfusion [17]. Their rapid removal may overwhelm the normal systems of haemolysis that include several highly efficient but saturable pathways for clearing the contents of red cells from the circulation, thereby curtailing their toxic effects. © 2014 The Association of Anaesthetists of Great Britain and Ireland

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Under normal circumstances, approximately 1% of the body’s 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 (80–90%) undergoing extravascular haemolysis in the macrophages of the reticuloendothelial system and the remaining 10–20% undergoing intravascular 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, 59–62]. The scavenged 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 dysfunction, 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-inflammatory effects [8, 61–64]. 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 reticuloendothelial macrophages that recognise and phagocytose not only senescent red cells, but also the haptoglobin-haemoglobin 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 haemins 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 macrophage 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

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Orlov and Karkouti | Storage of red cells Senescent RBCs Normally 80-90%

Extravascular Haemolysis

Normally 10-20%

Intravascular Haemolysis

Free Hb Free Hb NO

Haeme

2NO3Iron + CO + Biliverdin

Met-Hb (Fe3+)

Haemin

Macrophage Microvesicles

Figure 2 Haemolytic pathways of senescent red cells following transfusion. Following red cell transfusion, intravascular 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 haemin at the expense of endothelial nitric oxide (NO) consumption. Haemin – a known pro-oxidant and pro-inflammatory 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 microvesicles. 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, the systems of intravascular and extravascular haemolysis 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 approximately 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 transfusing a single unit of old blood may oversaturate the protective systems of extravascular and intravascular haemolysis in some patients, leading to 34

decreased endothelial-derived NO bioavailability and the presence of free haemoglobin, haemin and iron in the recipient’s circulation as well as within the reticuloendothelial macrophages [48, 49, 55, 67–72]. 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 oxidative injury [28, 41, 49, 54, 55, 61, 67, 69, 71, 73– 79]. Whether or not these effects are clinically relevant 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© 2014 The Association of Anaesthetists of Great Britain and Ireland

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comes. These have included healthy volunteers [67, 80–82], numerous retrospective and prospective observational studies [77, 83–114], and a handful of older [115–123], more recent [124], and ongoing randomised controlled trials. Overall, study results have been conflicting and affected by multiple methodological 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 variables, including anaemia-induced cognitive dysfunction [81], extravascular haemolysis [67], serum iron elevation [67], propensity for bacteraemia (evaluated in vitro) [67], attenuation of NO-mediated hyperaemia [80] and gas exchange [82]. All of these studies involved cross-over designs where autologous blood was used for transfusion following both a short (anywhere from 3–4 h to 3–7 days) and prolonged (anywhere 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 pivotal role of reduced red cell viability in the storage lesion. Volunteer studies have the advantage of investigating outcomes in humans while achieving great temporal separation between the study arms to allow for maximal investigation of any treatment effect. Nevertheless, they suffer from a myriad of drawbacks which warrant careful interpretation. Specifically, 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 techniques 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

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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, 83–114], and multiple recent systematic reviews [1, 126–131] attempting to consolidate this data. All but one of these reviews [130] have not supported 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 significant association between transfusion of older red cells and mortality, but this review included several primary studies that are ‘evidently confounded’ [129]. In all, no firm 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 efficiency and subsequent utilisation of the first in-first 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 country 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 nongeneralisable 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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and standards of care). The nature of ABO-matching between donor and recipient should also be carefully noted. Specifically, Group O red cells are considered the universal donor, thereby decreasing their turnover time and inflating the number of Group O transfusions in the fresh blood subgroup of patients. It follows that the old transfusion subgroup usually contains a greater proportion of non-O blood recipients, as demonstrated by the largest and perhaps most influential 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 [132–134], 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 directionality between storage latency and adverse outcomes can be further influenced by the precise definition 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 [115–124], the majority have been either small or not adequately powered to evaluate mortality. Moreover, none of them have found any significant 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, retinopathy, bronchopulmonary dysplasia or intraventricular 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 (standard practice as stated by protocol; mean (SD) 14.6 36

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(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 population was limited to premature infants – a very unique subgroup of patients, mainly cared for in specialised 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 population. 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 first, 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 mortality. 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 randomise 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 inhospital mortality. Finally, the standard issue transfusion vs fresher red blood cell use in intensive care study (TRANSFUSE; NCT01638416) is an international investigation set to randomise 5000 ICU patients © 2014 The Association of Anaesthetists of Great Britain and Ireland

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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 constraints, it is unlikely that any randomised trial will allow for a substantial inter-group contrast that reflects 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 multiple pre-existing RCT designs for five 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 susceptible to the effects of the red cell storage lesions compared to others. These subpopulations – including patients with an infection, renal failure, or those requiring a massive transfusion – need to be defined a priori, but could only be identified using alternative study designs. Finally, as with any longitudinal outcome 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 databases or statistical imputation may be warranted to maintain data integrity.

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mechanisms underlying red cell storage, the answers to many fundamental questions continue to evade us. Although we have succeeded in prolonging the maximum storage shelf-life of blood with advances in preservative 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-significant sequelae following transfusion. We have likely only hit the tip of the iceberg in beginning to identify predisposing comorbidities and contexts that may worsen prognosis following red cell transfusions of prolonged storage duration. All of these questions warrant systematic investigation with a simultaneous understanding of research design and its many limitations. The multiple ongoing, large-scale RCTs are a testament to the importance of the storage lesion and its potential effects on patient outcomes. Over the next decade, findings from these RCTs are likely to have significant implications for international transfusion practice, policy makers, and regulatory bodies. Nevertheless, it must be appreciated that all of the aforementioned 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 pathophysiology and consequences of the red cell storage lesion in clinical practice.

Acknowledgements Conclusion Over the past half-millennium, we have witnessed incredible progress in the efficiency and safety of blood transfusion therapy. Nevertheless, although we continue to acquire insight into the intricacies of metabolic

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KK is supported in part by a merit award from the Department of Anesthesia, University of Toronto.

Competing interests No competing interests declared.

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