Bio mechanical and metabolic changes that occur in red cell concentrates and platelets during ex-vivo storage, their effect in transfusion practise and strategies to minimize them.

1. Storage lesions
G.D.A. SAMARANAYAKA
DIPLOMA TRAINEE IN TRANSFUSION MEDICINE

2. What are storage lesions?
 Series of biochemical and biomechanical changes in
red cells or platelets during ex vivo preservation that
reduce their survival and function.

3. Storage lesions
 Red cells
 Platelets

4. Red cell storage lesions -
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
 Lack of 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
 Acidosis leads 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
 The decrease 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
 Storage of red cells at 4 ± 2°C
 helps maintain red cell functionality and viability by reducing
the red cell metabolic rate.
 For each one degree drop in storage temperature ->
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
 The major 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
 Normal shape of the RBC is a biconcave disc.
 Maximum surface area -> efficient gas exchange and
flexibility to travel through the capillaries

12. Biomechanical effects
 The red 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
 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

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
 Inceased number 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
 For haemoglobin to be able to reversibly bind oxygen (oxyhaemoglobin ↔
deoxyhaemoglobin) within the red cell, its component haeme-irons must be maintained in
their reduced, or ferrous (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
 Under aerobic 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
 Oxidative insult 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 of storage-
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 of storage-
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 of storage-
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 of storage-
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 if a 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 are cellular 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
 In the 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 get activated 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 while storage 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
storage lesions
 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 extend the storage
duration

35. Additive solutions
 Greater plasma 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 additive solutions
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 additive solutions
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
 Plays a 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 cells at 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 of red 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

44. Referances

45. Thank you