RBC bio-preservation represents an artificial means of storing and preserving the in vitro red blood cell quality for subsequent transfusion. The rationale for red blood cell biopreservation is subjecting biological cells to low or sub-zero temperatures will suppress most intracellular biochemical and biomechanical reactions. As a result, the natural ageing process will be slowed thereby prolonging its 'shelf life'. Blood banks have adopted this approach worldwide when formulating and implementing RBC storage parameters in a bid to maintain the overall in vitro RBC quality and stability in order to promote maximum in vivo RBC survival and performance.
The use of typical blood banking processing and storage procedures in Canada involves the buffy coat removal method (1) whereby whole blood is collected into a blood bag containing an anticoagulant such as CPD (citrate-phosphate-dextrose) (2,3). Once collected, it is converted into a variety of components including packed red blood cells (2,3). Centrifugation or filtration to remove white blood cells is performed on whole blood cell unit which is then extracted to another bag with an additive solution. Packed red blood cells can be used immediately or stored at 1-6oC in additive solutions for up to 42-49 days (1-4) or cryopreserved using Meryman's high glycerol (40 % w/v) method or low glycerol (20 % w/\v) method. Alternatively they can be either freeze dried (lyophilized) or vitrified for extended storage.
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The safety and efficacy of RBC transfusion depends upon the use of suitable biopreservation techniques that maintain in vitro RBC quality in order to improve in vivo RBC recovery and viability. RBC viability is based upon a minimal 0.8 % hemolysis and retention of at least 75 % red blood cells in the circulation 24 hours after transfusion criteria (5). Essentially RBC transfusions save lives as it represents an effective therapeutic strategy to increase RBC mass in patients suffering from low oxygen-carrying capacity conditions due to increased RBC loss (traumatic/surgical hemorrhage), decreased bone marrow production (aplastic anemias), abnormal or defective hemoglobin (hemaglobinopathies and thalassemias) and decreased RBC survival (hemolytic anemias) (1,6,6,6,7)
Each year approximately 16 million RBC units are transfused worldwide (8) because of application of biopreservation techniques to transfusion medicine. Possibly the main biophysical effect of storage is the loss of membrane and hemoglobin through the progressively increased vesiculation and the subsequent changes in RBCs mechanical and rheological properties. The purpose of this review is to highlight the biophysical and biochemical consequences of hypothermic storage and cryopreservation and their impact on in vitro RBC survival and performance.
1.1.1Background on red blood cells - Synthesis, Identification and Energetics
Red cells are a product of a differentiation process that starts in the bone marrow where hematopoietic stem cells differentiate to nucleate RBCs. After extrusion of nuclei and degradation of endoplasmic reticulum, reticulocytes emerge in the circulation; here they rapidly develop into mature RBCs 8 Âµm biconcave disk with a 120 days life span (9,10). Morphologic identification of blood cells depends on a well stained peripheral blood film or bone marrow smear. In hematology a modified Romanowsky stain such as Wright or Wright Giemsa are commonly used (10). The cell's main function of oxygen delivery throughout the body requires a membrane that is flexible and deformable. Under instances of oxygen deprivation, primary oxygen sensing system of the body (the peritubular interstitial cells of the kidney) secretes EPO to stimulate RBC production (10). Under hemorrhage increased RBC destruction or other factors that diminish the oxygen carrying capacity of the blood the production of EPO can be increased which allows for the early release of reticulocytes from the bone marrow that aids in preventing apoptotic cell death. In addition EPO can reduce the time needed for cells to mature in the bone marrow. In essence EPO adds more RBCs into the circulation at a faster rate than occurs without stimulation. A second way of increasing circulating RBCs is by increasing the number of cells that will be able to mature into circulating erythrocytes.
Despite being anucleated, RBCs posses a ``central core`` of chaperone proteins, heat shock proteins and proteins, RBCs which enables them to maintain and protect the RBC throughout its 120 day lifespan(11) Without organelles, RBCs take advantage of diversions or shunts from the glycolytic pathway to generate metabolites that maximize oxygen delivery from several vantage points. The three diversions are the hexose monophosphate pathway (HMP) that produces NADPH to prevent oxidative injury, the Rappaport-Luebering Pathway that promotes 2, 3 DPG synthesis and the Methemoglobin pathway that prevents Hb denaturation as well as preventing oxidative injury (12).
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Reactive oxidative stress is known to induce diverse damaging processes in tissues and cells, including oxidation of intracellular and surface components of blood cells. Specifically during hypothermic storage, the different ROS species might induce the oxidation of membrane lipids and alteration of their in/out distribution (exposure of phosphatidylserine at the cell surface), oxidation and cleavage of proteins, degradation of surface proteoglycans, oxidation of haemoglobin and ts adherence to the cell membrane (13-15). These changes especially those of the membrane have been associated with altered red blood cell rheological properties and a deeper understanding and appreciation of the composition and physical properties of the cell membrane is necessitated (16).
1.1.2 Physical Structure of the RBC
An appreciation of the RBC physical structure must be gained In order to understand the overall changes that are occurring due to RBC biopreservation (hypothermic storage and cryopreservation), The RBC membrane content is similar with most animal membranes and it is composed of: 19.5 % (w/w) of water, 39.5 % of proteins, 35.1 % lipids and 5.8 % of carbohydrates (17). Of the 35 % lipid portion, 60 % are phospholipids, essentially phosphatidylcholine (PC), phosphatidyethanolamine (PE), sphingomyelin (SM) and phosphatidylserine (PS). It has also some phospholipidic minor components such as phosphatidylinositol (PI), PI-monophosphate (PIP), PI-4, 5 bisphosphate (PIP2), phosphatidic acid (PA) lysophophatidylcholine (Lyso-PC) and lysophophatidylethanolamine (lyso-PE). Non sterified cholesterol represents about 30 % of the lipidic RBC composition and the last 10 % are glycolipids (17).
Cholesterol, esterified and largely hydrophobic resides parallel to the acyl tails of the phospholipids equally distributed between the outer and inner layers and evenly dispersed within each layer, approximately one molecule per phospholipid molecule. Cholesterol's beta hydroxyl group the only hydrophilic portion of the molecule anchors within the polar head groups, while the rest of the molecule intercalates among and parallel to the acyl tails. Cholesterol confers tensile strength to the lipid bilayer(18) At the physiologic pH, the majority of phospholipid content is electrically neutral, although PS, PA and PI are negatively charged. With the exception of SM and lyso-PC, the bulk of phospholipids have two fatty acid chains attached to a glycerol backbone. The most common fatty acids in human lipid bilayer membranes are usually the following: (16:0, 18:0, 18:1, 18:2 and 20:4). The phospholipids are asymmetrically distributed across the bilayer plan which is known as Trans asymmetry. The asymmetric distribution has a very important structural and functional role. In human RBCs, PS and PE are located almost entirely in the inner monolayer while PC and SM are more common in the outer leaflet. PS exposure leads to several mechanisms to achieve cell death and apoptosis (17).
Distribution of the phospholipids is energy dependent, relying on a number of membrane-associated enzymes, called flippases, floppases, scramblases for their position (19). When phospholipid distribution is disrupted as in sickle cell, anemia and thalassemia or in RBCs that have reached the end of their 120-day life span, PS, the only negatively charged phospholipid, redistributes (flips) to the outer layer. Splenic macrophages possess receptors that bind PS and destroy senescent RBCs. Two important enzymes are responsible for the translocation of phospholipids: flippase is responsible for PS and PE translocation from the outer to the inner leaflet, floppase catalyzes the translocation of the other lipids from the inner to the outer leaflet of the bilayer. The flop mechanism that controls PS externalization usually implicated in RBC adherence is a Mg2+ /ATP dependent reaction that results from aminophospholipid translocase impairment proposed to occur but very slowly during hypothermic storage. Flippase is a member of the Mg2+ dependent enzyme and P-glycoprotein ATPases family whereas floppase is a multidrug resistance protein 1 (MRP1) family member (20). Conservation of the aminophospholipids on the inner surface of the RBC membrane is thought to become disrupted during routine blood banking conditions and might induce the thrombogenic cascade and lead to enhanced red cell-endothelium interaction and their subsequent clearance from the circulation by the reticuloendothelial system(21,22).
Glycolipids (sugar bearing lipids) comprise 5 % of the external half of the membrane (19) and they are comprised of mainly sphingosines, such as glycosphingolipids. It is known that numerous sugar residues present in glycolipids are responsible for numerous functions such as adhesiveness to the extracellular endothelium. They associate in clumps or rafts and support carbohydrate side chains that extend into the aqueous plasma to help form the glycocalyx. The glycocaylx is a layer of carbohydrates whose net negative charge prevents microbial attack and protects the RBC from mechanical damage caused by adhesion to neighbouring RBCs or to the endothelium. Transmembranous (integral) and skeletal (cytoskeletal, peripheral) proteins make up 52 % of the membrane structure by mass. Nowadays Pasini et al. had already made the identification of at least 340 different red cell membrane proteins (9) It is evident that many of the problems arising out of the storage issue lies within the membrane itself and the only way to address this is through a deeper understanding of what happens from a biophysical standpoint with the RBC membrane during routine blood banking. The current in vitro predictors of membrane integrity are crude and not sensitive enough to determine proper RBC quality. Hence more definitive measures of RBC membrane integrity to assess RBC quality prior to transfusion from cryopreservation and hypothermic storage are required most notably for determining adherence and deformability values.
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RBC cryopreservation is a red blood cell biopreservation technique that has been around for at least 150 years (23) and is the process of preserving the biologic structure and function of living systems by freezing and storage at ultralow temperatures (6). According to the literature, red blood cells stored at 4oC in the additive solution (CPD) for longer than 2 weeks have significantly impaired function. These red cells require a period of time in the recipient's circulation to repair the functional defect to release oxygen at high tissue pO2 tensions (24,25). For optimum survival and function, liquid preserved red blood cells should not be stored in any of the current preservative solutions at 4oC for more than 2 weeks (26). Determining in vitro RBC quality with more contemporary determinants of membrane integrity will be integral to having a better and safer product for transfusion. The onset of cellular injury is thought to occur at this time period and hence the idea to couple hypothermic storage to RBC cryopreservation was borne to maintain in vitro red blood cell quality and reduce further damage associated with the hypothermic storage lesion and low temperature storage.
The concept of cryopreservation revolves around the idea that subjecting living tissues and cells to sub-zero temperatures will halt all intracellular biochemical and biophysical reactions and prolong red blood cell storage life. Minimal damage to RBC physiology, including haemoglobin structure, membrane and cellular energetics usually occur as a result of cryopreservation allowing this technique to be a practical approach for long term storage and preservation of red blood cells. Although it is an invaluable tool for extended RBC storage, cryopreservation is an expensive and labor intensive biopreservation procedure due to the technically demanding nature of processing and low temperature storage and the limited 24 hour shelf life of deglycerolized units (27)
1.2.1 RBC Cryoinjury (Structural and functional changes)
Red blood cells or erythrocytes due to their highly permeable membranes have very high optimal cooling rates (28). During freezing the formation of extracellular ice causes an increase in extracellular osmolality. As soon as ice forms outside of a cell in solution, the cell begins to dehydrate. If the cell membrane is highly permeable to water, then the intracellular solution will maintain osmotic equilibrium with the extracellular solution as the temperature continues to drop and progressively more ice is formed. The intracellular and extracellular solution concentrations will continue to increase as temperature drops until a eutectic point is reached and the remaining solution solidifies, or more commonly in cryobiological systems a glass transition occurs and the remaining solution is vitrified. At this point the system can be cooled to liquid nitrogen temperatures without significant changes to the chemical structure inside or outside the cell. During very slow cooling the process of gradually dehydrating the cell as ice is slowly formed inside the cell leads to extensive cellular damage.
High concentrations of electrolytes are very lethal to the cell. The damage observed during slow freezing has been well correlated with the damage seen when cells are exposed to equivalent electrolyte concentrations without freezing (29,30). Slow freezing damage however is not based solely on increased electrolyte concentration. Survival of slowly frozen red blood cells depends on electrolyte concentration and the unfrozen fraction of water (30). The unfrozen fraction is the fraction of water in the system that does not form ice but instead is incorporated into a eutectic solid or into a glass. By adding glycerol to the cellular solution electrolyte concentration can be controlled independently of the unfrozen fraction and survival decreases dramatically as the unfrozen fraction decreases. Damage is caused by aggregation of the cells into small channels as they are excluded from the ice and are forced into some type of adverse cell-cell interaction. Additionally, total volume changes experienced by the cells as they undergo shrinkage during slow freezing may cause membrane damage and still others have proposed that there may be a critical minimum volume for cells and if cells are shrunken to sizes smaller than that critical level during slow freezing they suffer irreparable damage (29). If cells are frozen slowly without cryoprotectant, they will shrink as ice forms, and they will almost always die. Excessive cellular shrinkage can be reduced by the addition of penetrating cryoprotectants.
Since the discovery of glycerol as a cryoprotectant in the 1950s by Dr. Audrey Smith, freezing has been used as an effective approach to supplement hypothermic storage system for the cryopreservation of rare RBC phenotypes and military storage for more than 40 years and their storage for as long as 37 years before successful re-infusion (31). RBC cryopreservation also allows cells used to immunize donors of Rh immune globulin to be quarantined, enables storage of large volumes of autologous blood and is a potential way of limiting the exposure of a given RBC recipient to a single donor (32). To meet the demands of modern medicine and surgery, RBC must be available, safe, effective and cheap. In clinical practice, units of RBC are frozen using either low or high glycerol methods (33). RBC cryopreservation utilizes the use of a cryoprotectant that may be membrane permeable (glycerol) or impermeable (HES, PVP) (32) to limit the rate and extent of ice crystal formation during freezing (34)
Cryoprotectants are chemicals that are added to cellular solution to try and protect the cell during freezing. The most commonly used are dimethyl sulfoxide (DMSO), glycerol, sucrose and trehalose and of there are two fundamental types namely, penetrating cryoprotectants and non-penetrating cryoprotectants. Penetrating cryoprotectants pass through the cellular membrane unaided and non-penetrating ones do not. When considering slow freezing damage which seems to be caused by cellular shrinkage and increased electrolyte concentration, the two types of cryoprotectants act very differently. The addition of non-penetrating cryoprotectants increases the osmolality of the extracellular solution and causes the cells to shrink before freezing.
Freezing may be harder to initiate due to the presence of the cryoprotectant (at the very least a higher osmolality will lead to a lower equilibrium freezing temperature) but once freezing is initiated solution rejection will commence and the cell will begin to shrink as the osmolality of the unfrozen solution continues to increase. The extracellular solutions may form a glass rather than a water and salt eutectic depending on the type of cryoprotectant used. The composition of the intracellular solution however will not be altered by non-penetrating cryoprotectants so the electrolyte concentration of the intracellular solution and the cell size will both be functions of the extracellular osmolality exactly as if the cryoprotectant were not added. If water is removed from the cell until the intracellular solution forms a glass or eutectic solid, then the end state inside the cell will be the same whether non-penetrating cryoprotectant is added or not. In most cases, unless the cryoprotectant dramatically alters the liquidus curve of the extracellular solution, the addition of non-penetrating cryoprotectants will not prevent injury to slowly frozen cells.
Penetrating cryoprotectants on the other hand, pass through the cellular membrane. Adding these cryoprotectants of which DMSO and glycerol are the most common causes the concentration electrolytes to decrease for a given temperature during freezing, since for any given osmolality a certain fraction of the solutes inside and outside the cell are cryoprotectants. They also increase the final size of the cell when dehydration due to freezing is complete, because in addition to the usual intracellular components there is an additional non-water volume component to the intracellular volume. The unfrozen fraction of the solution is also increased as is the case for non-penetrating cryoprotectants. The addition of DMSO substantially decreases the amount of ice that is formed and the electrolyte concentration as the sample is cooled. Since these are the leading causes of cellular damage during slow cooling it is clear that addition of DMSO can mitigate this damage; but DMSO is toxic to cells (35). The amount of DMSO that cells can tolerate varies by cell type. Often the amount of DMSO or other penetrating cryoprotectants required to prevent electrolyte damage during slow cooling is lethal to cells. The end result of a slow freezing process is one in which cells are shrunk and trapped in the unfrozen fraction of the extracellular media. With the addition of penetrating cryoprotectants, the interior of cells are usually in a glass-phase solid or in partially crystallized glassy matrix. Upon warming the cells are rehydrated as ice melts and it is possible for the cells to undergo membrane damage or phospholipid reorientation upon cellular resizing.
To avoid solution effects and excessive cellular shrinkage one can freeze the cells more quickly. Since the cellular membrane resists water transport, water can be retained inside cells during cooling if one cools the cells at a high rate. If the cooling is slow then the cells dehydrate and ice is formed only outside the cells but if cooling is fast intracellular water will be retained and freezing will occur both inside and outside the cells. Many studies have shown that the presence of ice outside the cell promotes ice formation inside the cell. .Non penetrating cryoprotectants can be used to partially dehydrate the cell prior to freezing, thereby reducing the chances that ice will nucleate in the cellular interior. Cryoprotectants can raise the glass-phase transition temperature so that the cells have a greater chance to reach glass phase without nucleating intracellular ice. Cryoprotectants also change fluid properties and can thereby directly reduce the chances for ice nucleation. Although it has been proven that intracellular ice formation during rapid freezing leads to cell death it is not at all clear that the formation of intracellular ice causes cell death. During warming many cells with intracellular ice suddenly lyse. If the damage occurs during warming and not during freezing, then the most likely mechanism involves recrystallization. When ice is formed intracellularly during rapid freezing, many nucleation sites are formed as there is not much time during freezing for crystal growth. The frozen cell is filled with many tiny crystals. During warming the small crystals reorganize into larger crystals, which may damage organelles or other cellular structures. How quickly this happens is not known but if lethal damage to cells with intracellular ice occurs during warming it is probably the formation of large crystals inside the cell that leads to the damage. The crystal formation during the active process of freezing and warming during clinical cryopreservation may lead to irreparable membrane damage that can potentially hamper the RBC rheological properties.
1.2.3 Clinical cryopreservation
The overarching concept behind cryopreservation in clinical cryopreservation revolves around the fact that cooling to extremely low temperatures should stop the degradative processes and preserve the cells and tissues for long periods of time (28). Clinical cryopreservation involves the use of either high or low glycerol methods (33) as well as Hydroxyethyl Starch (HES) as cryoprotectants in efforts to limit the rate and extent of ice crystal formation during freezing. Glycerol slows the rate of ice crystal formation and allows the RBC suspension to freeze as a glass (32). Glycerol colligatively reduces cellular injury by lowering the vapor pressure of the aqueous solution, reducing the amount of ice formed so that solute concentrations and the resulting reduction in cell volume are insufficient to exceed the tolerance of the cell.
Extracellular cryoprotectant hydroxyethyl starch (HES) allows for freezing in a 14 % solution, in liquid nitrogen at -197oC and frozen storage of the red blood cell at -150oC. This method does not require post thaw washing. Low, 15-20 % concentrations of glycerol are sufficient to limit rates of crystal growth during rapid cooling. High 40-50 % glycerol is necessary to slow crystal growth even more when rates of cooling are slower. The low glycerol method is compatible for storage in thin-walled, siliconized aluminium cans, flat steel canisters or Teflon bags that can withstand rapid freezing and proper cooling which minimizes RBC loss due to fluid transfers (32). The high glycerol method is compatible with storage in plastic bags protected by aluminium cassettes or cardboard boxes to withstand freezing and RBC loss in mechanical -80 oC freezers and cooling rates of about -1oC min-1 (32). The glycerol method requires more extensive washing to reduce the glycerol concentration to below 1 % and is the more favored approach in North America where frozen units are shipped on dry ice and processed near the facility of use whereas the low glycerol method has been favoured in Europe where units are processed at the storage facility and shipped in liquid form for immediate use. However both systems are inefficient and expensive and may result in losses of up to 10 % during processing and deglycerolization (36). Another approach uses 20 % weight/volume (wt/vol) glycerol with freezing in liquid nitrogen at -197oC and storage at -150o C and requires post thaw washing to reduce the glycerol concentration to less than 1 % wt/vol (37) During slow cooling in clinical cryopreservation, RBCs undergo excessive cellular shrinkage and toxicity due to the increasing concentration of solutes which has been described the "solution effects injury"(6,38-42). Additionally, cellular damage might occur due to the physical forces exerted by interactions with the ice crystals and/or tight packing of RBCs in unfrozen channels (43-46). Whether clinical cryopreservation results in changes to RBC rheological properties still remains to be elucidated.
RBC hypothermic storage
RBC hypothermic storage involves storage and preservation of red blood cells at below physiological temperatures (37oC) but above freezing temperatures, usually at 1-6 o C in an effort to maintain in vitro RBC quality for transfusion purposes. The rationale for this type of storage as previously described for all biopreservation techniques relies on low temperature storage of RBCs to suppress all intracellular biochemical reactions. During hypothermic storage however, biochemical processes are continuously running albeit at a lessened rate. To reduce nutrient consumption and cellular waste accumulation that adversely affect in vitro quality, red blood cells must be supplemented in specially designed storage solutions that reduce metabolic consumption (glucose) and limit waste accumulation (lactate) in efforts to extend the in vitro storage time or "shelf life" of red blood cells. Specially designed storage solutions involve licensed anticoagulant and additive storage solutions with varying differences in salts, sugars, guanosines and adenines to maintain the in vitro reactions that are still occurring between its constituents (47). Preserving ATP and 2, 3 DPG levels are necessary for red cell viability and function.
1.3.1 Background on RBC Preservative Systems
The discovery by Arthus & Pages in 1890 (48) that calcium ions in the plasma of whole blood were responsible for clotting began a search for the ability to extract and retain red blood cell viability for patient transfusion. In 1914 and later on in 1916, Hustin (48) and Rouse and Turner (48) introduced two substances, citrate and glucose, which laid the foundation for modern day blood banking. Citrate and glucose as anticoagulant and preservative respectively, made it possible to separate donor and recipient in space and time for transfusion. Later on in 1918, Oswald Robertson successfully transfused stored citrated human red blood cells into soldiers on the battlefield during the First World War (49,50). Since that time period, manipulations to the components of anticoagulant and additive solutions have led to advances which have made it possible to prolong the shelf life of stored RBCs. RBC storage time is important as it reduces the need for frequent blood donations; it preserves RBCs for longer and increases RBCs therapeutic opportunities in remote locations if the duration of RBC hypothermic storage can be lengthened (51) .
The ability to lengthen RBC storage life has been achieved using additive solutions (AS-1, AS-2, AS-3, AS-5, SAGM, PAGGSM (4) which are coupled to anticoagulants such as CPD or ACD to enhance preservation of RBCs under hypothermic storage (52). Additive solutions provide additional volume and nutrients for longer storage and better flow of packed RBCs (4) as well as reverse some of the hypothermic storage lesion associated with long term storage (4), (53) which promotes the longevity of RBCs under hypothermic storage from 42-49 days (36), (54). The use of specially designed plastic storage containers made of leachable plasticizers, DEHP (55-57) has been used as effective strategies as well for longer RBC storage. Storage in plastic containers made of PVC (polyvinyl chloride) which contains leachable plasticisers such as DEHP (di-2-ethylhexylphthalate) that binds to the RBC membrane preventing hemolysis have been in use (58).
The belief that maintaining cellular ATP concentrations by manipulations of pH and tonicity of additive solutions that may prevent calcium induced membrane loss by microvesiculation (4,51,58), has led to the development of new nutrient storage media (51,54,58) as well as improvements of currently used additive solutions. Storage in anoxic conditions (53,59) has also been used as effective strategies for counteracting the lesions associated with hypothermic storage. Specifically, blood stored directly in an atmosphere of inert gas at a pO2Â less than 4 % using a patented method (WO/1996/039026) has been shown to slow the decrease of 2,3 DPG and ATP values (59-61). They also demonstrated that the storage period for red blood cells could be extended to 120 days when rejuvenating solution is added at day 63 which restored levels of 2,3 DPG and ATP.
Red cell concentrates are prepared by the removal of plasma and, in some cases, also leukoreduction. The product stored at 4 + 2oC is usually in a slightly hypertonic additive solution, generally SAGM (sodium, adenine, glucose, mannitol, 376 mOsm/L)
There is still an underlying concern about the real need to store blood components for as long as possible in order to obtain a gradual increase in the interval between the donation and the transfusion, and how much this elastic time span can be prolonged without definitively, compromising the quality of the product and in the final analysis, the recipients` health from (62). The current standard requirements for patenting new solutions in the USA and also suggested in the recommendations of the European Council are essentially based on the level of haemolysis (below the threshold of 0.8 % at the end of the storage period and a survival rate of the transfused cells of more than 75% 24 hours post transfusion which is assessed by measuring the half life of chromium labelled RBCs post transfusion(5,63).
1.3.2 STORAGE LESION
1.3.2 RBC HSL (Structural and Functional changes)
The resultant biochemical and biomechanical changes that plague the RBC unit as a direct consequence of hypothermic storage is known collectively as the "hypothermic storage lesion". Briefly, RBC storage lesions include: morphological changes, slowed metabolism with a decrease in the concentration of adenosine triphosphate (ATP), acidosis with a decrease in the concentration of 2,3-diphosphoglycerate (2,3 DPG), loss of function (usually transient) of cation pumps and consequent loss of intracellular potassium and accumulation of sodium within the cytoplasm, oxidative damage with changes to the structure of band 3 (64) and lipid peroxidation, apoptotic changes with the racemisation of membrane phospholipids and loss of parts of the membrane through vesiculation (65). Some of these changes occur within the first few hours of hypothermic storage for example the decrease in pH or the increases in potassium and lactate while others take days or weeks. Together these events risk compromising the safety and efficacy of long-stored red blood cells, reducing their capacity to carry and release oxygen, promoting the release of potentially toxic intermediates (for example, free hemoglobin can act as a source of reactive oxygen species) and negatively influencing physiological rheology (through the increased capacity of the red blood cells to adhere to the endothelium(66,67) or through their enhanced thrombogenic (68) or pro-inflammatory (69) potential).
Fluctuating ATP levels during hypothermic storage also appear to inhibit proper membrane skeleton complex formation such as spectrin-actin 4.1, spectrin-spectrin, spectrin-actin 4.1 linkages which affects RBC shape and by extension function. The lipid bilayer asymmetry is fundamental for maintaining RBC shape which may become disrupted and also effect RBC-endothelial cell interaction. Ca2+ influx as well as pH may also play a role in shape loss as the diffusion of intracellular calcium frustrates the Ca2+/Mg2+ pump resulting in lowered ATP levels. RBC survival appears to be caused by a loss of surface area attributable to cholesterol and lipid microvesiculation which is independent of ATP but due to calcium accumulation or oxidative injury.
The progressive loss of membrane lipids can prove detrimental to the survival of the RBC rendering it osmotically fragile and limiting its lifespan in storage or after transfusion. RBC integrity and survival are also affected by pH and the components of storage media (anticoagulants and additives) as leachable plasticizers used in storage containers (BHTC, TOTM and DEHP) that serve to stabilize the RBC membrane by reducing percent hemolysis can be potentially toxic(58)
The most evident changes affecting red blood cells during the storage period are alterations of the cell phenotype which varies from a smooth discoid shape to a phenotype characterized by various membrane protrusions or specula (echinocyes) and finally to a spheroid-shaped cell (spheroechnocyte)(70). The reversibility of these changes is inversely proportional to the duration of storage. The storage lesion also involves the fluxes of sodium ions (massive entry into the cell) and potassium ions (exit from the cell), since the Na+/K+ pump is inactive at 4 oC (71). Although this is a reversible process (it takes 24 hours to restore the physiological gradients for sodium and up to 4 days for potassium(63), this phenomenon means that blood stored for a prolonged period should not be used for neonates or paediatric patients, unless first washed or the potassium removed from the storage medium(72).
Another biochemical effect is the clear decrease in the levels of 2,3 DPG(which is consumed already within the first week) translating into increased affinity of haemoglobin for oxygen and consequently decreased capacity of the red blood cells to release oxygen according to local metabolic needs. The decrease in 2,3DPG levels is also reversible event and completely normal levels can be restored within 3 days after transfusion (73) . Alongside these reversible changes, various irreversible events occur during the storage process, including fragmentation and aggregation of proteins and lipids, activated by radical species generated by prolonged, continuous oxidative stress (74-76). In this way oxygen constantly leaves one molecule of hemoglobin to bind to another. It is known that, occasionally oxygen leaving the hemoglobin molecules carries with it an electron, forming a superoxide ion (O3-ÂÂ) and ferric methaemoglobin.
Finally it is known that the cell activates a process of vesiculation, in order to eliminate proteins and lipids that have been altered by oxidative stress, as to protect the cell from a further chain reaction of stress and consequent removal from the circulation (77), constituting membrane signals to"remove" the cell, through IgG, or complement-mediated phagocytosis by the recipients' Kuppfer cells. These membrane neoantigens, by stimulating the immune system, seem to be related to the onset of proinflammatory events, which are often harmful if not fatal in critically ill patients undergoing transfusion therapy (78,79). Alongside these signals, which are particular to red cell aging, a series of other markers appear, these markers are common in other physiological phenomena associated with programmed cell death or apoptosis, such as exposure of phosphatidylserine on the external leaflet of the lipid bilayer of the cell membrane thought to play a prominent role in RBC-EC interactions and can have detrimental effects upon the patient on transfusion by altering its rheological properties. Rheological properties are the flow properties of red blood cells and in this thesis they are RBC deformability and adherence.
Clinical significance of RBC HSL and Cryoinjury
Cryoinjury results from changes within the membrane due to intracellular and extracellular ice formation. Currently the criteria for safe and effective RBC transfusions are evaluated by the number of RBCs in the unit, the survival and function of the RBCs, the residual hemolysis and sterility, the residual number and function of WBCs and the presence of residual biologically active plasma and non-plasma substances. Hence the quality of the donor RBC influences the recipient's clinical outcome. According to the literature, a liberal transfusion policy (80) directly related to the age of hypothermically stored RBCs negatively affects patient's clinical outcomes (58,81-84). Authors suggest a direct correlation between the age of transfused RBCs and the residual white blood cell content. It has been proposed that the recurring shortages experienced by most banks could be alleviated by maintaining a supply of frozen red blood cells with normal or improved oxygen transport function and acceptable 24 hour post transfusion survival and lifespan value (26).
However the 24 hour post-wash storage period limits the use of frozen allogeneic universal donor red blood cells as a primary source to balance the fluctuations in supply and demand and hampers efforts to provide rare, selected and autologous red blood cells (26). There is also an association that stable critically ill children receiving prestorage leukoreduced RBC units with increased storage time may be at greater risk of developing new or progressive Multiple Organ Dysfunction Syndrome (MODS). However conclusions could not be adequately drawn from that experiment as it described an independent association and not a cause-effect relationship and a randomized clinical trial had to be drawn to evaluate the true effect (85). In general, more severely ill patients, as measured by either APACHE II (Acute Physiology and Chronic Health Evaluation II) or sepsis-related organ failure assessment (SOFA) score, received more RBC transfusions (82). Additionally, an epidemiological study provided evidence of an association between blood transfusion and diminished organ function and increased mortality in critically ill patients. A significant association between the number of RBC transfusion and risk of subsequent infection has been reported in patients following trauma, burns and a variety of surgical procedures both elective and emergency (86-89). Similarly in the critically ill, Taylor et al have demonstrated an association between RBC transfusion and nosocomial infection and mortality (90). The potential risks associated with transfusion include hemolytic transfusion reaction reactions, transfusion-related graft-versus host disease, non-hemolytic febrile transfusion reactions, and transmission of disease, immune suppression, and post-transfusion infection (91-99). A recent study evaluating clinical outcomes following the institution of a universal prestorage leukoreduction program in Canada noted a reduction in hospital mortality following introduction of this program (100). This was a retrospective before and after cohort study conducted from August 1998 to August 2000 in 23 academic and community hospitals throughout Canada, enrolling 14,786 patients who received RBC transfusions following cardiac surgery or repair of hip fracture, or who required intensive care following a surgical intervention or multiple traumas. As such careful consideration and precautions must be taken to ensure that patients receiving RBC units are not prone to the deleterious effects of impaired adherent and deformability values which may negatively affect patient health subsequent to transfusion.
Transfusion of stored red blood cells exhibiting signs of the storage lesion have been implicated in adversely affecting patients in intensive care(82,84,101-104) those undergoing cardiac interventions (81,81,105-109,109-111) those submitted to colorectal surgery(112-114) or those with multiple trauma(115-120). Additionally, side effects varied in multiply transfused patients, ranging from decreases in gastric pH(82) to increases in mortality rates(84) from multiple organ failure(115) to an increased incidence of pneumonia in patients transfused following aorto-coronary artery bypass(81,106,108) from an increased susceptibility to infections(116) to major complications following heart surgery(102,109,110) and from an increase in the duration of hospital admissions(117,118) to the development of complications such as transfusion related acute lung injury (TRALI)(121,122). Many of the problems arising out of the storage lesion lies within the membrane itself and the only way to address this is through a deeper understanding of what RBC ageing coupled with storage does to the membrane in terms of RBC deformability and adhesion.
RBC deformability and adhesion
RBC deformability refers to the ability of red blood cells to adapt their shape to the dynamically changing flow conditions in order to minimize their resistance to flow. This is particularly important for their passage through capillaries, which have a diameter smaller than that of the red cell. Structural changes in RBC and subsequent impaired deformability contribute to hindrance of blood flow, particularly in low flow states (13,123). RBC deformability has been proposed to be a major determinant of RBC survival, since less deformable cells sequester in the spleen leading to the destruction of RBCs (13). Red blood cells with reduced deformability can thus impair perfusion and thus oxygen delivery to peripheral tissues and rigid, undeformable RBCs can directly block capillaries.
RBC deformability depends not only on RBC geometry but also on relative cytoplasmic (hemoglobin viscosity). The normal mean cell hemoglobin concentration (MCHC) ranges from 32-36 % and as hemoglobin concentration rises above 36 % deformability is compromised and RBC shelf life is shortened as the more viscous cells cannot accommodate to narrow capillaries or splenic pores. As RBCs age they lose membrane surface area while retaining hemoglobin. The hemoglobin becomes more and more concentrated and eventually the RBC unable to pass through the splenic pores becomes destroyed by splenic macrophages.
Many studies have linked reduced RBC deformability to circulatory disorders and anemia observed in diverse pathologies, for example, thalassemia, sickle cell anemia, cerebral malaria, sepsis, diabetes, and disturbed cerebral flow in patients with stroke (124) Membrane elasticity, fragility, increased permeability to cations, a loss of cell water and a stiffened cytoskeleton are also intrinsic factors that contribute to cellular deformability. Extrinsic factors (i.e. related to the surface area, volume and environment) include: oxygen tension, osmolality, pH, temperature and concentration of plasma proteins. These combined with its unique cell geometry (size, shape, excess surface-area to volume ratio), provide the RBC with special mechanical properties and confer remarkable flexibility (28).
Generally, RBC-EC interactions are insignificant in normal conditions however they have been known to become greatly enhanced in pathophysiological and oxidative stress states. Similar behavior have also been noticed in stored RBCs and these might lead to increased microvasculature blockage, resulting in reduced oxygenation, tissue infarct and necrosis. Additionally, RBCs are considered non adhesive cells however several studies have reported the expression of a large number of adhesion molecules in RBC such as CD44, CD47, CD58, LW/ICAM-4, RAGE and Lu ((9,125-141) . Other adhesion molecules include LW/ICAM-4, BCAM/LU, CD47, and sialyl moieties (20). Many of these adhesion proteins belong to the Immunoglobulin superfamily (IgSF) of proteins (125) and they play a crucial role in cell-cell and cell-tissue interactions which has been considered to be a prominent catalyst of microvessel occlusion. These adhesion molecules are differentially expressed at distinct stages of the life of RBCs (133). Additionally they have been implicated in a large range of biological functions such as erythropoiesis (differentiation, maturation, enucleation and release of RBCs) self-recognition mechanism, red cell turnover and cell aging (133,133,138) . However, identification and characterization of these adhesion molecules lies outside the scope of this review and thesis.
RBC-EC interactions have also been known to be enhanced in pathophysiology related to RBC abnormalities such as in sickle cell disease, cerebral malaria, diabetes, and thalassemia. Alterations in flow properties of human RBC, measured in vitro have been observed and implicated in the pathophysiology of numerous diseases including acute myocardial infarct, unstable angina, sepsis, schizophrenia, obesity, thalassemia and pregnancy induced hypertension (142-146).
Currently the criteria for in vitro RBC quality is based upon a minimal 1 % hemolysis and a survival rate that is greater than 75 % for post transfused blood (5,63). However current conventional assays are not insightful to detect the subtle membrane changes associated with cryopreservation and hypothermic storage that can compromise clinical patient outcomes. Better in vitro determinants of cellular membrane changes that results from these biopreservation techniques are necessary hence this thesis seeks to delineate the more subtle membrane processes resulting from cryoinjury and the hypothermic storage lesion via two novel assessments of biophysical injury, specifically ektacytometry and adhesion assay.
Hypothesis and thesis objectives
This thesis will test the following hypothesis:
RBC cryoinjury and HSL result in significant in vitro changes in RBC adhesion. Additionally it will show that Eadie Hofstee ektacytometric analysis can be coupled to adhesion assay to provide adequate insights into the subtle biophysical changes that are occurring within the RBC membrane during cryopreservation which may jeopardize the flow properties of transfused patients that have been correlated to adverse clinical outcomes. This thesis is made up of three experimental studies consisting of three specific research aims (SRAs):
SRA1: To develop and establish the adhesion assay protocol which will be used for further assessment of RBC adhesion properties prior and post hypothermic and cryopreservation storage(Chapter 2)
SRA2: To determine RBC adhesion in hypothermically stored RBCs (Chapter 3)
SRA3: To determine RBC deformation and adhesion changes in cryopreserved RBCs (Chapter 4)