Impact Of Storage Lesions On Biochemical And Hematological Parameters Of Packed Red Blood Cells

Figure 1. Structure of a normal red blood cell showing its biconcave shape, hemoglobin-rich cytoplasm, and major physiological functions.

Table 1. Structural and Functional Characteristics of Red Blood Cells and Their Importance During Blood Storage

3. Packed Red Blood Cells and Storage Conditions

3.1 Preparation of Packed Red Blood Cells (PRBCs)

Among the several blood components that are manufactured from healthy donors, packed red blood cells are among the most frequently transfused. Following donation, centrifugation separates the blood into its component parts: red blood cells, plasma, and buffy coat. The majority of the plasma is removed and a concentrated suspension of erythrocytes is formed, called PRBCs. Some blood banks leukoreduce prior to storage while others do it after storage, either way it is done in the blood bank, it helps to reduce the white blood cell count, hence the chances of febrile non-hemolytic transfusion reaction, alloimmunization and the accumulation of inflammatory mediators in storage. The prepared PRBC then are combined with preservative or additive solutions and preserved under controlled conditions until they are given as transfusion [20].

3.2 Storage Solutions

The viability, metabolism and functional integrity the effectiveness of the preservation solution used to keep red blood cells is crucial. These solutions give nutrients, help sustain osmotic equilibrium and limit metabolic degradation during storage. Citrate phosphate dextrose adenine is a popular anticoagulant-preservative solutio. Citrate binds up calcium ions to prevent coagulation, phosphate buffers, dextrose provides an energy source for glycolysis and adenine helps to produce ATP. The recommended storage temperature for PRBCs preserved in CPDA-1 is 20°C, and the storage period is usually up to 35 days. After the plasma has been removed, Saline Adenine Glucose Mannitol (SAGM) is often used to further improve red blood cell preservation. Glucose and adenine keep the erythrocyte membrane stabilized and help to minimize hemolysis during storage, so that PRBCs can be stored for up to 42 days. Other additive solutions such as AS-1, AS-3 and AS-5 have also been created to support the survival of red blood cells by supplying them with further nutrients and reducing biochemical deterioration during extended storage [21].

3.3 Storage Conditions

The maintenance of the quality and safety of PRBCs depends on proper storage conditions. The international guidelines for blood banking suggest to store PRBC at temperature of 2–6°C in a blood bank refrigerator with temperature control. This temperature range is effective in reducing cellular metabolism, bacterial growth, and maintaining red blood cell viability. Depending on the preservative solution, the maximum storage time is one month. The storage period for PRBCs in CPDA-1 is usually 35 days and for PRBCs preserved with additive solutions like SAGM is 42 days. Blood banks are keeping a constant watch on the refrigerator temperature, inventory control and quality control parameters during storage, to ensure that blood products comply with the transfusion standards and to keep the products safe [22].

3.4 Factors Affecting Storage Quality

PRBCs have several factors that affect their quality in storage. The most important factor is storage duration and over time biochemical, metabolic and structural changes can occur, which are grouped together as storage lesions. While temperatures are lower, cellular metabolism is affected, but inappropriate temperature fluctuations can cause hemolysis and affect red blood cell integrity. Leukoreduction can reduce the levels of leukocytes derived cytokines, enzymes and reactive oxygen species which can cause inflammatory responses and oxidative damage, thereby improving the storage quality. On the other hand, certain patients may receive irradiation so that graft-versus-host disease, which can occur after blood transfusions, might be lessened, which can result in higher fragility, potassium loss, and hemolysis, especially in long storage. Thus, it is important to follow standard processing and storage procedures to maintain the quality of PRBCs and maximize the benefits of blood transfusion [23].

Table 2. Common preservative and additive solutions used for the storage of packed red blood cells (PRBCs), their principal components, primary functions, and recommended maximum storage duration [24].

Fig 2: Flow diagram of PRBC preparation and storage process.

4. Mechanisms of Storage Lesions

4.1 Definition of Storage Lesions

Prolonged storage causes a group of alterations in packed red blood cells  called "storage lesions." Any number of biochemical, metabolic, structural, or functional changes could be at work here. The erythrocytes' shelf life is prolonged by the low temperature, but their gradual degeneration is still there. These changes start soon after the blood is drawn and during the entire storage period will impact on red blood cell viability, integrity of the cell membrane, deformability and oxygen delivery capacity. Storage lesions are typically more severe with longer storage time and could affect the quality or clinical utility individuals receiving blood transfusions, particularly those in critical care and those undergoing extensive transfusions [25].

4.2 Cellular Mechanisms

Progressive metabolic abnormalities of erythrocytes is the main cause of development of storage lesions. There are no mitochondria or nuclei in mature red blood cells, which means that they produce all of their energy via anaerobic glycolysis. As the available amount of glucose diminishes and enzyme activity decreases during refrigerated storage, the glycolytic activity progressively decreases. As a result, the metabolic rate of stored erythrocytes becomes progressively diminished. Loss of metabolism is one of the most important effects resulting in a loss of adenosine triphosphate . ATP is crucial for maintaining the asymmetry of membrane phospholipids, for regulating the sodium–potassium ATPase pumps, for maintaining membrane flexibility, and for preventing premature destruction of cells. When ATP is reduced, the integrity of the cell membrane is compromised, the ion balance in the cell is disrupted, and erythrocytes gradually become non-biconcave. ATP depletion is also responsible for increased rigidity of the membrane, increased hemolysis, and decreased post-transfusion survival. Another major contributor to storage lesions is oxidative stress. Reactive oxygen species (ROS) are always formed during storage through auto-oxidation of hemoglobin, and other oxidative reactions. The antioxidant defence system of erythrocytes decreases during storage, leading to the progressive accumulation of ROS in erythrocytes. This oxidative imbalance leads to the damage of membrane lipids, cytoskeletal proteins and hemoglobin, which leads to impaired cellular function and increased sensitivity to hemolysis. Both ATP depletion and oxidative stress contribute to the advancement of storage deterioration [26].

4.3 Membrane Alterations

The membrane of the erythrocytes is very sensitive to oxidative damage especially in the prolonged storage. Lipid peroxidation is one of the earliest changes in membrane that is caused by the attack of membrane phospholipids by being around when ROS are present leads to reduced stability and increased permeability of the membrane. Lipid peroxidation causes loss of structural integrity and leakage of intracellular potassium and other intracellular constituents into the storage medium. Protein oxidation also causes damage to membrane and cytoskeletal proteins. Oxidative modifications of structural proteins, such as spectrin, ankyrin, band 3 protein, and actin are responsible for changes in membrane architecture. These modifications affect interactions between the membrane and the cytoskeleton, thus decreasing the mechanical stability and fragility of the membrane. As the damage to the membrane increases, the erythrocytes get progressively less biconcave and become less flexible. The decrease in cell deformability decreases their ability to pass through the narrow capillaries and splenic sinusoids, thus reducing their viability after transfusion. Morphological changes of discocytes to echinocytes and finally to spherocytes is a characteristic feature of the advanced storage lesion [27].

4.4 Microvesicle Formation

A key aspect of storage lesions is the generation of microvesicles from the red blood cell (RBC) population. When erythrocytes sustain oxidative damage to their membranes, it triggers the release of these extracellular vesicles, destruction of the cytoskeletal elements and loss of membrane phospholipids. The vesiculation of microvesicles is a process of protection which eliminates damaged membrane components; however, when there is too much vesiculation, it will progressively decrease the areas of the membranes and add to the fragility of the erythrocytes. Microvesicles can be stored and contain phospholipids, membrane proteins, hemoglobin and bioactive molecules that could induce inflammatory responses, endothelial activation and coagulation after transfusion. There is growing evidence to indicate that there is a relationship between higher concentrations of micro vesicles with prolonged storage and other adverse transfusion-related outcomes [28].

4.5 Progressive Changes During Storage

Lesions that form over time while stored in a refrigerator are known as storage lesions. Decreases in metabolic activity and 2,3-Diphosphoglycerate (2,3-DPG) levels are observed during the initial days of storage. Continued storage results in further deterioration of erythrocyte metabolism due to glucose consumption, lactate production and progressive acidification of the storage medium. At the same time, there is a potassium leakage from the cells, oxidative stress is enhanced, structural modifications of membrane proteins, and an acceleration of the production of micro vesicles. At the end of storage, red blood cells are extremely rigid, fragile, morphologically altered and hemolyse quickly. These biochemical and structural changes decrease functional properties of stored PRBCs and could affect the efficacy of PRBC transfusion. Therefore, knowledge of the mechanisms of storage lesion is important to improve storage protocols and to optimize transfusion outcomes [29].

Fig 3: Mechanisms Involved in the Development of Storage Lesions in Packed Red Blood Cells During Refrigerated Storage.

Table 3. Principal cellular and membrane mechanisms involved in the development of storage lesions in packed red blood cells (PRBCs) and their effects on erythrocyte quality and transfusion outcomes.

5. Biochemical Changes During Storage

Packed red blood cells experience a series of progressive biochemical changes during the refrigerated storage that are collectively called storage lesions. Mature erythrocytes do not have mitochondria or a nucleus, and thus rely solely on anaerobic glycolysis to produce energy and maintain cellular homeostasis. Over time, metabolic activity slows with a depletion of important metabolites, build-up of metabolic waste, imbalance of ions and more oxidative damage. These biochemical changes have a negative impact on the viability of the erythrocytes, integrity of the erythrocyte membrane, capacity to deliver O2 and post-transfusion recovery.

5.1 ATP Depletion

The main energy source needed for the structural and functional integrity of RBCs is adenosine triphosphate (ATP). It regulates the activity of membrane ion pumps, helps to keep membranes phospholipid asymmetric, maintains intracellular ion balance, and keeps membranes flexible. In refrigerated storage the glycolytic enzyme activity gradually decreases, which results in a gradual decrease in ATP production, but cellular ATP levels remain utilized for key metabolic processes. The amount of ATP in the cell decreases significantly as the storage period is prolonged. Normally the reduction in ATP will reduce the activity of the sodium–potassium ATPase, causing ionic imbalance, membrane rigidity, and decreased erythrocyte deformability. Moreover, when ATP levels are low, the rapidity of membrane vesiculation, osmotic fragility, and hemolysis are increased. As such, ATP-depleted erythrocytes used for transfusion have a shorter survival time in the blood of the recipient and lower therapeutic effectiveness [37].

5.2 Decrease in 2,3-Diphosphoglycerate (2,3-DPG)

2,3-Diphosphoglycerate (2,3-DPG) is a crucial glycolytic intermediate that controls the oxygen-binding ability of hemoglobin. At normal 2,3-DPG levels, the oxygen dissociation curve of haemoglobin moves to the right, releasing oxygen to the tissues surrounding the heart. After 14 days of refrigeration, the levels of 2,3-DPG in blood are usually extremely low, to the point where they are almost impossible to detect. Low temperatures primarily cause a drop because they suppress metabolism and reduce glycolytic activity. When 2,3-DPG levels fall, hemoglobin's oxygen affinity rises, limiting the quantity of oxygen that may be delivered to tissues immediately following a transfusion. Anyone requiring a large blood transfusion or severely ill patients during periods of low 2,3-DPG should be aware of the potential drawbacks of the short-term fall in 2,3-DPG [38].

5.3 Glucose Consumption

Mature erythrocytes use glucose as their sole energy substrate, and continually metabolize it via anaerobic glycolysis. Glucose slowly breaks down during storage to produce ATP for cell maintenance and normal function of the cell membrane. Preservative solutions have extra glucose but as the glucose is used up by the metabolic activity, it continues to be depleted over time. Depleted glucose supplies result in decreased ATP production and exacerbate further metabolic decline. As glycolysis slows down, the integrity of the erythrocyte membrane and the ability to sustain its ionic regulation is gradually impaired, exacerbating the storage lesion.

5.4 Lactate Accumulation

In red blood cells, the major product of anaerobic glycolysis is lactate. During refrigerated storage, glucose continues to be metabolised, causing the concentration of lactate in the storage solution to increase over time. There is a continuous rise in lactate concentration, which is related to the continued anaerobic metabolism in spite of diminished enzyme activity. High levels of lactate can directly lead to acidification of the storage medium, and also interfere with glycolytic enzymes further decreasing ATP production. High lactate concentrations are thus a useful biochemical parameter to assess storage-induced metabolic deterioration [39].

5.5 pH Reduction

Lactate builds up during storage, causing a progressive decrease in the pH of the storage solution. Preservative solutions buffer for a while, but over time these buffers are overwhelmed. A decrease in pH will have a negative impact on several intracellular enzymes essential for glycolysis and energy production. Acidification further increases oxidative injury, decreases the function of membrane proteins and increases ATP depletion. Low pH thus leads to a marked decrease in the viability of the erythrocytes, and hemolysis is enhanced during long storage.

5.6 Potassium Leakage

The intracellular potassium concentration is maintained by the ATP-dependent sodium–potassium pumps present in the erythrocyte membrane. During storage, the activity of these ion pumps gradually decreases as the ATP content decreases. Loss of membrane integrity and defects in ion transport leads to leakage of potassium out of the erythrocytes into the surrounding storage solution. The potassium concentration in the outside of the cell therefore rises gradually during the storage time. If you are transfusing older PRBC units, such as in a massive transfusion or a transfusion to a baby, an elevation of potassium can lead to hyperkalemia and cardiac problems. Thus, one of the clinically significant biochemical changes that occurs with the prolonged storage of blood is the build-up of potassium [40].

5.7 Sodium Alterations

Unlike potassium leakage, sodium slowly flows into erythrocytes due to a decrease in sodium–potassium ATPase activity. This ionic homeostasis disrupts leads to osmotic imbalance and changes in the intracellular water content. Sodium changes are usually not as clinically relevant as potassium changes, but indicate progressive membrane dysfunction and decreased cell metabolism. Changes in both sodium and potassium levels are indications of decreasing membrane transport potential during storage, when combined.

5.8 Oxidative Stress and Reactive Oxygen Species

A storage lesion develops due in large part to oxidative stress. The auto-oxidation of hemoglobin and other oxidative reactions cause reactive oxygen species (ROS) to be constantly created during cold storage. Glutathione, catalase, superoxide dismutase, and glutathione peroxidase are antioxidant defense systems found in normal red blood cells that help to neutralize excess reactive oxygen species (ROS). But with prolonged storage these anti-oxidant mechanisms start to reduce, leading to oxidative imbalances. This led to damage to the ROS of phospholipids, cytoskeletal proteins, membrane transport proteins, and hemoglobin molecules. Oxidative injury leads to increased lipid peroxidation, protein oxidation, vesiculation of the membranes and decreased erythrocyte deformability. Oxidative stress also is a primary cause of hemolysis during storage and will play a significant role in the progressive decline in PRBC quality during storage [41].

5.9 Free Hemoglobin Release

Free hemoglobin will be released from the erythrocytes during prolonged storage, when some erythrocytes gain integrity in the storage material. The level of extracellular free hemoglobin rises with the duration of storage and is considered to be a reliable marker of the impairment of erythrocytes. This free Hb has biological implications as it freely binds nitric oxide (NO) leading to decreased NO availability and endothelial dysfunction and vasoconstriction. Overwhelming free hemoglobin can also be responsible for excess oxidative injury and inflammatory responses after transfusion. The regulatory agencies set allowable limits for hemolysis of blood stored in units, but it is also important to minimize the release of free hemoglobin in stored units to ensure the safe and effective use of PRBC transfusion. As a whole, these metabolic changes are quite interdependent. Red blood cells in storage suffer from deterioration in quality and functional performance due to oxidative stress, glucose consumption, “loss of 2,3-DPG, pH drop, accumulation of lactate, and depletion of ATP and 2,3-DPG”. Better storage solutions and preservation procedures can be developed to lower the occurrence of storage lesions and improve transfusion results, therefore understanding these biochemical changes is helpful [42].

Table 4. Major Biochemical Changes in Packed Red Blood Cells During Storage [43]

6. Hematological Changes During Storage

Packed red blood cells  undergo several biochemical and hematological changes during storage that impact the structural integrity, morphology and functional characteristics of the erythrocytes. These changes occur gradually in course of refrigerated storage and are closely related with storage lesions. Changes in blood cell morphology, stability, deformability, hematological parameters and EVs production can have a profound impact on the survival of donor RBCs and their function. It is important to recognize these hematological changes to assess the quality and clinical effectiveness of stored PRBCs.

6.1 Morphological Changes

The progressive change on the morphology of RBCs is one of the earliest and most prominent signs of storage lesion. Normally, fresh erythrocytes are disc-shaped (discocytes), able to traverse small capillaries and possess a wide surface area that promotes gas transfer. Over the course of refrigerated storage these changes gradually occur, as ATP is depleted, oxidative injuries take place, and the composition of lipids in the membrane changes. Consequently, discocytes start to change into echinocytes, which have several small membrane projections evenly spaced. The formation of the echinocytes may be reversible to a certain extent, but if stored for a long period of time, irreversible damage is done to the membrane. The erythrocytes continue to change, some of them becoming even more spheroidal, smaller, with less surface area and greater rigidity, as they continue to be stored. Spherocytes have reduced deformability and are more maybe taken up by the spleen and undergo splenolysis following transfusion. The progressive morphological changes will be important indicators of the reduction of erythrocyte quality during storage [44].

6.2 Membrane Fragility

Erythrocyte membrane slowly deteriorates during the refrigerated storage period. Oxidative stress causes membrane phospholipid and cytoskeletal protein damage, which in turn causes loss of membrane integrity, and loss of ATP leads to the loss of phospholipid asymmetry and ion homeostasis. The more unstable the membrane the more delicate the erythrocyte and the more susceptible to mechanical and osmotic stress it is. The fragility of the membrane makes stored RBCs more susceptible to early rupture during processing, transport and transfusion. The fragility of the membrane tends to correlate with the length of time stored and is a measure of the build up of metabolic and oxidative damage to the erythrocyte membranes.

6.3 Hemolysis

Hemolysis is a crucial hematological alteration that happens when blood is stored. This happens when the membrane deteriorates with time, causing the erythrocytes to burst and releasing intracellular chemicals into the storage solution. Hemolysis levels rise steadily with storage time due to a number of factors, including oxidative stress, lipid peroxidation, protein oxidation in membranes, and diminished membrane flexibility. There are generally limits set by regulatory authorities for the amount of hemolysis that can be found in stored PRBC units, but the more the unit is hemolyzed, the fewer viable erythrocytes will be available for the transfusion and the higher the extracellular levels of free hemoglobin, potassium and other intracellular constituents. Hemolysis is therefore a significant quality-control parameter in blood banking, and excessive hemolysis could affect the therapeutic efficacy of blood transfusion and transfusion safety [45].

6.4 Reduced Deformability

Red blood cell deformability refers to the fact that RBCs can alter their shape in order to fit through capillaries that are narrower than their resting diameter. The maintenance of the normal biconcave shape depends mainly on the elasticity of the membrane, the integrity of the cytoskeleton, the viscosity of the intracellular fluids, and the maintenance of the normal biconcave shape. As the ATP contents deplete due to storage conditions, and as the storage time increases, membrane flexibility is also lost, and the organization of the cytoskeletal components is also affected. Thus erythrocytes stored in blood banks are increasingly rigid and less deformable to mechanical stresses. The decreased deformability reduces the ability of microvascular passage, shortens post transfusion survival, and decreases efficiency of oxygen delivery to peripheral tissues. Loss of deformability is one of the most significant functional changes of storage lesions, directly affecting the clinical function of transfused PRBCs.

6.5 Changes in Red Blood Cell Indices

Hematological indices which change measurably during prolonged storage can give helpful clues to erythrocyte quality. A slight rise in Mean Corpuscular Volume (MCV) may occur during storage since this may lead to sodium accumulation in cells and hence to water influx.A slight rise in Mean Corpuscular Volume (MCV) may happen in the course of storage as disturbances in ion transport promote the accumulation of sodium in cells, which in turn leads to water influx and a mild swelling of the cells. The Mean Corpuscular Hemoglobin  is fairly stable as the hemoglobin content of individual erythrocytes does not vary greatly during storage. But there is a small variation that may occur due to hemolysis. Marginal decrease in Mean Corpuscular Hemoglobin Concentration (MCHC) may result due to alterations in cell volume and membrane integrity. This is a progressive structural change in stored erythrocytes, although such changes are typically small. During storage, RDW rises, as the cells lose membrane, swell and shrink, and morphologically change to various extents, making the cell population more heterogeneous. A rise in RDW thus indicates greater variation in erythocyte size and morphology which is correlated with storage lesions [46].

6.6 Microparticle Formation

One of the hallmarks of long-term storage of PRBCs is the liberation of red blood cell-derived microparticles (also known as extracellular vesicles). The membrane-bound vesicles are produced on membrane budding and vesiculation due to oxidative stress, ATP depletion, calcium influx, and cytoskeletal disruption. Vesiculation is one way of eliminating damaged parts of the membrane, but excessive vesiculation causes progressive loss of membrane surface area, increased formation of spherocytes and increased susceptibility (fragility) of erythrocytes. Moreover, these microparticles are enriched with biologically active molecules, membrane proteins, hemoglobin, and phospholipids which can trigger inflammation, endothelial activation, coagulation and immune modulation after transfusion.The number of microparticles rises steadily during refrigerated storage and it is now being used as a useful marker of the severity of the storage lesion. Therefore, tracking the generation of microparticles during storage of PRBCs could be useful for understanding the quality and integrity of the stored PRBCs and contribute to the development of better blood processing methods.

Table 5. Major Hematological Changes in Packed Red Blood Cells During Storage [47]

Fig 4. Schematic representation of the progressive morphological transformation of red blood cells during refrigerated storage, illustrating the transition from normal discocytes to echinocytes, sphero-echinocytes, and spherocytes, ultimately leading to hemolysis and microparticle formation.