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![Biomechanical effects
Membrane changes occur in parallel with metabolic changes
RBC shape maintenance -> dependent on [ATP]
Shape changes due to ATP depletion
Echinocytes/burr cells
Greek – sea urchin
Disk shaped cells with projections on the membrane
reversible
Sphero-echinocytes
With further depletion of total ATP and ADP pool
spherical red cells with thorny projections
decreased surface to volume ratio and deformability -> reduce RBC
post-transfusion survival
Irreversible change
Microvesiculation occurs from the tips of echinocytic spines](https://image.slidesharecdn.com/storagelesions-170117170618/85/Red-cell-and-platelet-storage-lesions-and-their-effect-in-transfusion-practise-13-320.jpg)
Uploaded byArjuna Samaranayaka
Red cell and platelet storage lesions and their effect in transfusion practise
The document discusses storage lesions in red blood cells and platelets, outlining the biochemical and biomechanical changes that occur during ex vivo preservation, which ultimately reduce their functionality and survival. Key factors include oxidative stress, metabolic alterations, and membrane integrity issues that arise from storage conditions, leading to adverse effects post-transfusion. Strategies to mitigate these storage lesions include optimizing storage solutions, conditions, and potential rejuvenation techniques.
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Red cell and platelet storage lesions and their effect in transfusion practise
- 1. Storage lesions G.D.A. SAMARANAYAKA DIPLOMATRAINEE IN TRANSFUSION MEDICINE
- 2. What are storagelesions? Series of biochemical and biomechanical changes in red cells or platelets during ex vivo preservation that reduce their survival and function.
- 3. Storage lesions Redcells Platelets
- 4. Red cell storagelesions - Introduction Erythrocytes are prone to modifications due to High oxygen environment (prone to oxidative stress and hemoglobin auto- oxidation) Absence of nucleus and other organelles – no repair mechanism Ex vivo storage of blood Non-biological containers at non- biological temperatures Changes in cellular biochemistry Change in normal ageing processes that cells undergo in the body
- 5. The storage effects Metabolic effects Biomechanical or membrane effects Oxidative effects
- 6. Metabolic effects Lackof mitochondria -> Energy production only by glycolysis One molecule of glucose produce two molecules of lactate two molecules of adenosine 50- triphosphate (ATP) two protons -> increase the acidity of the storage solution over time
- 7. Metabolic effects Acidosisleads to inhibition of phosphofructokinase and hexose kinase slower glycolysis reduced ATP production Decreased glutathione reductase levels -> reduce the ability of the RBC membrane to avoid oxidative damage
- 8. Metabolic effects Thedecrease in pH -> breakdown of 2,3- diphosphoglycerate low level of 2,3-DPG leads to a left shifted oxygen disassociation curve increase haemoglobin O2 saturation and affinity 2,3-DPG restored in vivo after transfusion one hour - 30% 24 hrs - 50% 3 days - full restoration
- 9. Metabolic effects Storageof 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 -> approximately a 10% decrease in red cell metabolic rate At4°C, the metabolic rate is ten times lower than at 25°C Metabolic activity does not completely cease when red cells are stored at cold temperature glucose or dextrose are added to storage mediums to allow red cells to continue glycolysis.
- 10. Metabolic effects Themajor membrane Na+/K+ ATPase is inhibited at low temperature continuous leakage of intracellular potassium Na+ entry in to cells K+ accumulation in storage solution Increase the risk of hyperkalemia-induced arrhythmia Specially in vulnerable patients Ex:- neonates, renal failure patients, massive transfusions After transfusion Na+ content normalizes in 24 hours but K+ recovery takes at least 4 days Washing or simply removal of the supernatant is an alternative to reduce K+ toxicity for high risk patients
- 11. Biomechanical effects Normalshape of the RBC is a biconcave disc. Maximum surface area -> efficient gas exchange and flexibility to travel through the capillaries
- 12. Biomechanical effects Thered cell membrane consists of lipid bilayer that is interspersed with proteins The lipid bilayer includes phospholipids, cholesterol and fatty acids -> asymmetrically distributed between the inner and outer layers Phosphatidylserine is an important component 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 When expressed on the outer layer -> highly thrombogenic & leads to the removal of red cells by reticuloendothelial macrophage. Auto-oxidation of haemoglobin within the red cell leads to precipitation of structurally distorted forms of methaemoglobin a.k.a - haemichromes near the cell membrane causes disruption of AE1 and cytoskeletal membrane proteins
- 13. Biomechanical effects Membranechanges occur in parallel with metabolic changes RBC shape maintenance -> dependent on [ATP] Shape changes due to ATP depletion Echinocytes/burr cells Greek – sea urchin Disk shaped cells with projections on the membrane reversible Sphero-echinocytes With further depletion of total ATP and ADP pool spherical red cells with thorny projections decreased surface to volume ratio and deformability -> reduce RBC post-transfusion survival Irreversible change Microvesiculation occurs from the tips of echinocytic spines
- 15. Biomechanical effects Microvesicle(MV) release is a controlled process stimulated by pro-apoptotic signals, shear stress or oxidative damage mechanism to remove damaged cellular components. RBC microvesiculation ->decreased surface to volume ratio and increased cell rigidity reduction in RBC deformability occlusion in capillary beds or intravascular rupture of RBCs. Sialic acid content decreases with storage -> reduction of the electrostatic repulsive forces that protect RBCs from aggregation blood viscosity and potentially block flow in small vessels impairment of tissue perfusion.
- 16. Biomechanical effects Inceasednumber of Microvesicles causes oversaturation of the body’s clearance systems for haemolysed red cells Haemoglobin - scavenging of endothelial-derived nitric oxide externalised phosphatidylserine - Thrombogenic
- 17. Oxidative effects Forhaemoglobin 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 (Fe 2+), 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 Methaemoglobin Inherently unstable haemin (also known as ferric or oxidized haeme) 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. Under normal circumstances, red cells are protected against this oxidative injury the rate of spontaneous oxidation of haemoglobin is slow NADH-dependent cytochrome-b5 reductase (CYTb5) reduces methaemoglobin back into oxyhaemoglobin cytosolic antioxidants (primarily reduced glutathione or GSH) and membrane anti-oxidants (primarily ascorbic acid or vitamin C) neutralise the generated reactive oxygen species.
- 18. Oxidative effects Underaerobic storage conditions -> RBCs are constantly exposed to a pro-oxidative environment. Superoxide dismutase and methaemoglobin reductase repair any oxidative damage via the hexose-monophosphate shunt. Glutathione stores decline during storage likelihood of hydroxyl radical formation via the Fenton reaction increases Fenton’s reaction – ferrous and ferric ions react with peroxide to form hydroxyl radicles Fe2+ + H2O2 ----> Fe3+ + .OH + OH- Fe3+ + H2O2 ----> Fe2+ + .OOH + H+
- 19. Oxidative effects Oxidativeinsult to proteins and lipids protein oxidation and lipid peroxidation formation of lysophospholipid, may contribute to transfusion- related acute lung injury RBC function and viability deteriorate as the oxidative injury persists over storage.
- 21. The impact ofstorage- induced changes Reduction in RBC quality over time poor in vivo efficacy of stored RCC Lead to adverse post-transfusion events Accumulation of bioactive substances in stored blood actively modify the immune function may lead to TRIM Metabolic modulation, shape changes, altered rheological properties and oxidative injury Profressive RBC lysis release of cytosolic content and large amounts of haemoglobin ultimate manifestation of the storage lesion -> RBC is no longer viable and can provide no therapeutic effect
- 22. The impact ofstorage- induced changes • S-Nitrosothiol-Hb (SNO-Hb) release NO -> vasodilatation • SNO-Hb decays almost instantaneous following blood withdrawal • Old red cells -> insufficient NO bioavailability (INOBA ) • Impaired NO production and increased NO scavenging by stored RBCs • Reduced NO levels below a critical threshold in vascular beds • vasoconstriction occurs, leading to reduced blood flow and insufficient O2 delivery to end organs.
- 23. The impact ofstorage- induced changes Free haemoglobin potent inducer of oxidative stress augment the storage lesion development Increase rate of nitric oxide (NO) scavenging by endothelial cells leads to vasoconstriction, platelet activation and inflammation -> clinically significant outcomes in critically ill transfusion recipients
- 24. The impact ofstorage- induced changes Non-transferrin-bound iron/free iron Generates reactive oxygen species, which may in turn cause the production of cytokines and altered immunity. promote the growth of bacteria.
- 25. However Even ifa significant storage lesion is observed during prolonged storage, the reversible character of some of the changes and the remaining quality of RBC after prolonged storage may still be adequate for the transfused RBC to perform all of the clinically required functions Requirements of stored RBC • 24 hours after transfusion >75% of transfused RBC should be present in the circulation and • At expiration the plasma Hb may not exceed 0.8% - 1%
- 26. Platelets Platelets arecellular fragments derived from the cytoplasm of megakaryocytes Do not contain a nucleus Have mitochondria and various cytoplasmic granules. Do not possess either a golgi body or rough endoplasmic reticulum Platelets are released and circulate approximately 9 to 12 days as small, disk-shaped cells
- 27. Biochemical changes Inthe resting state 15% ATP - by glycolysis 85% by TCA cycle – with O2 consumption In the activated state 50% ATP by glycolysis - increase lactate production. Decreased pO2 in the plastic platelet container Increasing the rate of glycolysis to compensate for the decrease in ATP regeneration from the oxidative (TCA) metabolism This increases glucose consumption and causes an increase in lactic acid This results in a fall in pH - <6.4 after 5-7 days of storage at 22°C. Lactic acid is buffered by bicarbonate - When the bicarbonate buffers are depleted during PC storage pH rapidly falls to less than 6.2
- 28. Activation Platelets getactivated following exposure to foreign surfaces – plastic bag low pH – metabolic alteration shear stress - during component separation Upon activation, the platelets lose their discoid morphology and become more spherical with multiple pseudopods. Conformational changes in GPIIb/IIIa complex exposes binding sites for adhesive proteins (fibrinogen, vWF) resulting in platelet aggregates.
- 29. Activation Platelet activation causes 1.Release of granular contents Function -> recruitment of leucocytes and platelets promote, immunity against infection contribute to wound healing presence of these contents in storage medium -> various transfusion reactions 2. Expression of sequestered membrane proteins (CD62, CD63) & phospholipids Negatively charged phospholipids providing a surface for the prothrombinase complex (X-Va) thereby contributing to procoagulant activity
- 30. Activation Agitation whilestorage cause platelet lysis and calpain (protease) activation Platelet lysis discharge cytosolic lactate dehydrogenase (LDH) and granular contents accumulation in the storage solution Activation of calpain degradation of cytoskeletal proteins like actin generate platelet microvesicles. Microvesicle formation leads to decrease in mean platelet volume (MPV) and also contributes to procoagulant activity
- 31. Assessment of platelet storagelesions Biochemical tests - assess platelet viability pH, pO2, LDH accumulation, glucose consumption, and ATP depletion Assess alterations in the discoid morphology Swirling phenomenon decrease in MPV Platelet activation markers – various assays can be used release of specific granular contents (β thromboglobulin, platelet factor 4) changes in GP expression on platelet surface (GPIb, GPIIb, and GPIIIa)
- 33. RCC and Platelets Stored PRBC and platelet transfusions seem to upregulate pro- inflammatory gene expression in the leukocytes of the transfusion recipient. Cytokines and chemokines that have been shown to increase during storage of RBCs and platelets are Interleukin (IL)-1β, IL-6, IL-8, Tumor necrosis factor-α Myeloperoxidase (MPO) neutrophil-activating peptide-2 (NAP-2) Monocyte Chemoattractant Protein-1 (MCP-1) RANTES (regulated on activation, normal T cell expressed and secreted; CCL5) - associated with allergic reactions IL-6, IL-8, and MCP-1 - may be associated with TRALI Fas ligand and TGF-β - may contribute to transfusion-related immune modulation.
- 34. How to extendthe storage duration
- 35. Additive solutions Greaterplasma recovery from whole blood donations for transfusion or fractionation Minimization of the adverse effects mediated by plasma allergic and FNHTRs transfusion-related acute lung injury Use of photochemical pathogen reduction technologies, because the presence of plasma may interfere with the technology system Potential improvements in platelet & RBC storage through manipulation of the storage medium.
- 36. Red cell additivesolutions First generation Most commonly used – SAG-M, AS-1 and AS-5 First widely used additive solution – SAG Idea was to replace the volume and sugar lost with plasma removal adenine -synthesizes ATP, increases level of ATP Mannitol reduced hemolysis Other first-generation additive solution CP2D/AS-3 - in the United States CPD/MAP (mannitol, adenine, and phosphate) - in Japan.
- 37. Red cell additivesolutions Second generation Addition of phosphate and bicarbonate Buffers protons and reduce acidity Guanosine guanosine triphosphate was detected in red cells and known to decrease during storage. However, guanosine nucleotides - minimal role in critical events in RBC storage, inhibiting the primitive coagulation enzyme transglutaminase.
- 38. Platelet additive solutions Acetate efficiently substitute in the citric acid cycle decreasing both the glycolytic rate and lactic acid generation. Acetate must also be transformed to acetic acid to enter the cycle, removing hydrogen ions produced by the anaerobic metabolism of glucose This bicarbonate-sparing buffering effect also helps preserve pH Most commercially available PASs do not contain additional glucose glucose carmelizes upon heat sterilization at the neutral or slightly basic PAS pHs Citrate important in maintaining anticoagulation However upregulates glycolysis and make platelets more susceptible to activating stimuli. use of the lowest possible concentrations of citrate in the medium Phosphate serves as a buffer Magnesium and potassium - decrease platelet activation and may downregulate glycolysis
- 39. Storage containers Playsa major role in storage lesions Allows gas exchange – depends on the thickness of the bag & gas transport capacity of the material. Polyvinyl chloride (PVC) bags plasticized with Di(2-ethylhexyl) phthalate (DEHP) - standard RBC and platelet storage containers. The presence of DEHP reduces hemolysis by fourfold during storage by intercalating into the red cell membrane. Used in most of the countries. PVC bags plasticized with butyryl-n-trihexyl citrate more expensive Sweden, Spain, and Norway have an unusual smell when initially unwrapped. Newer plastic bags have higher gas permeability polyolefin with no plasticizer (Baxter's PL 732) thin walled PVC with tri-(2-ethylhexyl)trimellate plasticizer (TOTM)
- 40. Storage under anaerobic conditions- RCC Researchers have demonstrated that anaerobic storage can slower the decreases in 2,3-DPG and ATP levels decrease production of free radicles and membrane damage Vox Sang. 2007 Jan;92(1):22-31. Extended storage of red blood cells under anaerobic conditions. Yoshida T1, AuBuchon JP, Tryzelaar L, Foster KY, Bitensky MW. Transfusion. 2009 Mar;49(3):458-64. doi: 10.1111/j.1537-2995.2008.02038.x. Epub 2009 Jan 2. Anaerobic storage of red blood cells in a novel additive solution improves in vivo recovery. Dumont LJ1, Yoshida T, AuBuchon JP.
- 41. Rejuvenation Red cellsat the end of storage time - low pH, ATP and 2,3-DPG concentrations. Rejuvenation - metabolic recharging of red cells at the end of their storage period. By incubation in a high-pH solution of phosphate, inosine, pyruvate, and adenine for 2 hours. Increases red cell ATP and 2,3-DPG concentrations and increases their in vivo recovery Restored normal distribution of phospholipids - prevents red cells from participating in plasma coagulation reactions. Rejuvenation does not reverse the oxidative damage to cell membrane Ex - Rejuvesol - Cytosol Labs
- 42. Pre transfusion washing Reduce inflammation markers. Association with fewer transfused blood units, and decreased mortality. Remove accumulated storage-related compounds (potassium and lactate). Older erythrocytes are lysed during the washing process. induce higher hemolysis and MV release reduced transfusion-induced impaired vascular function
- 43. Frozen storage ofred cells and platelets Freezing significantly reduces the metabolic rate Cryopreservative agents helps to reduce intracellular dehydration and mechanical damage due to water crystal forming during freezing process platelets at -80°C with DMSO - shelf life up to 2 years. Red cells at -80°C with glycerol – up to 10 years Cryopreservation and Freeze-Drying Protocols - Volume 368 of the series Methods in Molecular Biology™ pp 283-301
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Editor's Notes
- #9 Theoretically, negligible 2,3-DPG levels in RCCs should not be a major concern in transfusion, as this metabolic alteration is reversible once the RBCs are back in vivo post-transfusion
- #16 Phosphatidylserine (PS) translocation to the RBC surface increases during storage, adding to the progressive adherence of RBCs to endothelial cells. Elevated interaction between RBC and endothelial cells could potentially cause local turbulent flow, subsequently activating endothelial cells and facilitating unwanted inflammation.
- #19 Fenton's reaction Fe2+ + H2O2 ----> Fe3+ + .OH + OH- Fe3+ + H2O2 ----> Fe2+ + .OOH + H+
- #34 Chemokine (C-C motif) ligand 5-CCL5 is an 8kDa protein classified as a chemotactic cytokine or chemokine. CCL5 is chemotactic for T cells, eosinophils, and basophils, and plays an active role in recruiting leukocytes into inflammatory sites. With the help of particular cytokines (i.e., IL-2 and IFN-γ) that are released by T cells, CCL5 also induces the proliferation and activation of certain natural-killer (NK) cells to form CHAK (CC-Chemokine-activated killer) cells.[4] It is also an HIV-suppressive factor released from CD8+ T cells. This chemokine has been localized to chromosome 17 in humans.[3]