Red blood cell and platelet preservation

RED BLOOD CELL AND PLATELET PRESERVATIONTraditionally, the amount of whole blood in a unit has been 450 mL +/10% of blood. More recently, 500 mL +/10% of blood are being collected with the volume of anticoagulant-preservative solution being increased from 63 mL to 70 mL.

For a 110-pound donor, a maximum volume of 525 mL can be collected, including samples drawn for processing. The total blood volume of most adults is 4-6 L, and donors can replenish the fluid lost from the 450-500 mL donation in 24 hours.

The donors red cells are replaced within 1 to 2 months after donation. A volunteer donor can donate whole blood every 8 weeks.

Units of the whole blood collected can be separated into three components: packed RBCs, platelets, and plasma. In recent years, less whole blood has been used to prepare platelets with the increased utilization of apheresis platelets. Apheresis: is a method in blood collection in which whole blood is withdrawn, a desired component is separated and retained, and the remainder of the blood returned to the donor

Hence, many units are converted only into RBCs and plasma. The plasma can be converted by cryoprecipitation to a clotting factor concentrate that is rich in antihemophilic factor (AHF, factor VIII).

A unit of whole bloodprepared RBCs may be stored for 21 to 42 days, depending on the anticoagulant-preservative solution used when the whole blood unit was collected, and whether a preserving solution is added to the separated RBCs. 1RED BLOOD CELL BIOLOGY AND PRESESERVATIONThree areas of RBC biology are crucial for normal erythrocyte survival and function:Normal chemical composition and structure of the RBC membraneHemoglobin structure and functionRBC metabolism

RBC MEMBRANEThe RBC membrane represents a semipermeable lipid bilayer supported by a meshlike protein cytoskeleton structurePhospholipids, the main lipid components of the membrane, are arranged in a bilayer structure comprising the framework in which globular proteins traverse and move.

Proteins that extend from the outer surface and span the entire membrane to the inner cytoplasmic side of the RBC are termed integral membrane proteins.Beneath the lipid bilayer, a second class of membrane proteins, called peripheral proteins, is located and limited to the cytoplasmic surface of the membrane forming the RBC cytoskeletonDEFORMABILITYThe loss of adenosine triphosphate (ATP) (energy) levels leads to a decrease in the phosphorylation of spectrin and, in turn, a loss of membrane deformability.Accumulation or increase in deposition of membrane calcium also results, causing an increase in membrane rigidity and loss of pliability.

PERMEABILITYThe RBC membrane is freely permeable to water and anions (Cl- and HCO3-) but is relatively impermeable to cations such as sodium (Na+) and potassium (K+).

DEFORMABILITYThese cells are at a marked disadvantage when they pass through the small (3 to 5 m in diameter) sinusoidal orifices of the spleen, an organ that functions in extravascular sequestration and removal of aged, damaged, or less deformable RBCs or fragments of their membrane.

PERMEABILITYThe permeability properties of the RBC membrane and the active RBC cation transport prevent colloid hemolysis and control the volume of the RBC.

Na-K ATPase is responsible for maintaining the balance between Na and K within the RBC. It pumps out Na and pumps in K.

The RBC intracellular-to-extracellular ratios for Na+ is 1:12 and K+ is 25:1. PISO5Metabolic pathwaysThe RBCs metabolic pathways that produce ATP are mainly anaerobic, because function of the RBC is to deliver oxygen not to consume it.

RBC metabolism may be divided into the Anaerobic Glycolytic Pathway and three ancillary pathways that serve to maintain the structure and function of hemoglobin: the Pentose Phosphate Pathway, the Methemoglobin Reductase Pathway, and the Luebering-Rapoport shunt.RBCs are devoid of nucleus and mitochondria for oxidative metabolism and their energy must be generated almost exclusively through the breakdown of glucose.6EMBDEN-MEYERHOF PATHWAYMajor pathway generating energy for RBCs90% glycolysis; anaerobic pathway2 ATP is produced for every glucose broken down to lactic acid

PENTOSE PHOSPHATE PATHWAY10% glycolysis; aerobic pathwayProvides reduced glutathione to prevent denaturation of Hgb

LUEBERING-RAPOPORT PATHWAYGenerates 2,3-DPG (2,3-diphosphoglyserate) that regulates the Hgb affinity for oxygen 2,3-DPG - Hgb affinity for Oxygen 2,3-DPG - Hgb affinity for Oxygen

METHEMOGLOBIN REDUCTASE PATHWAYMaintains Hgb iron in ferrous state to be functional

ATP: controls the flow of Na and K; protects membrane lipids

2,3-DPG occupies the space vacated by oxygen when delivered to the tissue

The unloading of oxygen by hemoglobin is accompanied by widening of a space between chains and the binding of 2,3-DPG on a mole-for-mole basis, with the formation of anionic salt bridges between the chains.7HEMOGLOBIN OXYGEN DISSOCIATION CURVESHIFT TO THE LEFT Hb affinity for Oxygen delivery of Oxygen to tissuesFactors: pH 2,3-DPG CO2 TemperatureSHIFT TO THE RIGHT Hb affinity for Oxygen Delivery of Oxygen to tissuesFactors: pH 2,3-DPG CO2 TemperatureRBC PRESERVATIONThe goal of blood preservation is to provide viable and functional blood components for patients requiring blood transfusion.

RBC viability is a measure of in vivo RBC survival following transfusion.

To maintain optimum viability, blood is stored in the liquid state between 1C and 6C for a specific number of days, as determined by the preservative solution(s) used.

The loss of RBC viability has been correlated with the lesion of storage, which is associated with various biochemical changesTo determine post-transfusion RBC survival, RBCs are taken from healthy subjects, stored, and then labeled with radioisotopes, reinfused to the original donor, and measured 24 hours after transfusion.9RBC STORAGE LESIONCHARACTERISTICCHANGE OBSERVED% viable cellsDecreasedGlucoseDecreasedATPDecreasedLactic AcidIncreasedpHDecreased2,3-DPGDecreasedOxygen Dissociation CurveShift to the LeftPlasma K+IncreasedPlasma HemoglobinIncreasedANTICOAGULANT PRESERVATIVE SOLUTIONSCHEMICALS IN ANTICOAGULANT SOLUTIONSCHEMICALFUNCTIONPRESENT INACD-ACPDCP2DCPDA-1CitrateChelates Ca++; prevents clottingXXXXMonobasic Sodium PhosphateMaintains pH during storage; necessary for maintenance of adequate levels of 2,3-DPGXXXXDextroseSubstrate for ATP production (cellular) energyXXXXAdenineProduction of ATP; for improved survival of RBCs (extends shelf life from 21 to 35 days)XAPPROVED ANTICOAGULANT PRESERVATIVE SOLUTIONSNAMEABBREVIATIONSTORAGE TIME (DAYS)Acid Citrate Dextrose (formula A)ACD-A21 daysCitrate-Phosphate DextroseCPD21 daysCitrate-Phosphate Double DextroseCP2D21 daysCitrate-Phosphate-Dextrose-AdenineCPDA-135 daysCitrate-Phosphate-Dextrose-AdenineCPDA-242 daysADDITIVE SOLUTIONSAdditive solutions (AS) are preserving solutions that are added to the RBCs after removal of the plasma with or without platelets.One of the reasons for their development is that removal of the plasma component during the preparation of RBC concentrates removed much of the nutrients needed to maintain RBCs during storage.These solutions reduce hematocrits from around 70% to 85% to around 50% to 60%.Additive solutions consists of: Saline, Adenine and Glucose. AS-1 and AS-5 also contain mannitol, which protects against storage-related hemolysis, while AS-3 contains citrate and phosphate for the same purpose.Additive solutions are approved for 42 days of storage.FREEZING AND REJUVENATIONRBC freezing is primarily used for autologous units and the storage of rare blood types. Autologous transfusion (auto meaning self) allows individuals to donate blood for their own use in meeting their needs for blood transfusion.Cryoprotective agents such as glycerol are used to prevent rupture of RBCs during freezing.Two concentrations of glycerol have been used to freeze RBCs: a high-concentration glycerol (40% weight in volume [w/v]) and a low-concentration glycerol (20% w/v) in the final concentration of the cryopreservative.Frozen RBCs can be stored for 10 years from the date of freezing; that is, frozen RBCs may be stored up to 10 years before thawing and transfusion.Advantages of High-Concentration Glycerol Technique Used by Most Blood Banks Over Low-Concentration Glycerol TechniqueADVANTAGEHIGH GLYCEROLLOW GLYCEROLInitial freezing temperature80C 196CNeed to control freezing rateNoYesType of freezerMechanicalLiquid NitrogenMaximum storage temperature65C120CShipping requirementsDry iceLiquid NitrogenEffect of changes in storage temperatureCan be thawed and frozenCriticalAdvantages and Disadvantages of RBC FreezingADVANTAGESDISADVANTAGESLong-term storage (10 years)A time-consuming processMaintenance of RBC viability and functionHigher cost of equipment and materialsLow residual leukocytes and plateletsStorage requirements (65C)Removal of significant amount of plasma proteinsHigher cost of productRBC REJUVENATIONRejuvenation of RBCs is the process by which ATP and 2,3-DPG levels are restored or enhanced by metabolic alterations.Currently, Rejuvesol (enCyte Systems) is the only FDA approved rejuvenation solution sold in the United States.It contains phosphate, inosine, pyruvate, and adenine.Rejuvesol is currently approved for use with CPD, CPDA-1, and CPD/AS-1 RBCs.RBCs stored in the liquid state can be rejuvenated at outdate or up to 3 days after outdate, depending on RBC preservative solutions used.Current Trends in RBC Preservation ResearchResearch and development in RBC preparation and preservation is being pursued in five directions:Development of improved additive solutionsDevelopment of procedures to reduce and inactivate the level of pathogens that may be in RBC unitsDevelopment of procedures to convert A-, B-, and Abtype RBCs to O-type RBCsDevelopment of methods to produce RBCs through bioengineering (blood pharming)Development of RBC substitutesPLATELET PRESERVATIONPLATELETSDisk-shaped cells derived from the megakaryocytes found within bone marrow.2-4 m in diameterLife span: 9-12 daysApproximately 1/3 of the platelets released from the bone marrow are sequestered in the spleen and the remaining 2/3 are found in the circulation.Platelets are intimately involved in primary hemostasis, which is the interaction of platelets and the vascular endothelium in halting and preventing bleeding following vascular injury.The role of platelets in hemostasis includes:initial arrest of bleeding by platelet plug formation andstabilization of the hemostatic plug by contributing to the process of fibrin formation maintenance of vascular integrity.

PLATELETSGlycogen granules provide energy substrate.Alpha () granules contain contact-promoting factors, including:Platelet fibrinogenPlatelet-derived growth factor (PDGF)von Willebrands factor (factor VIII:R)-Thromboglobulin (BTG)Platelet factor 4 (heparin-neutralizing)FibronectinDense granules contain nonprotein factors including:Adenosine diphosphate (ADP)Adenosine triphosphate (ATP)5-Hydroxytryptamine (5-HT; or serotonin)Calcium

PLATELET STORAGE LESIONCHARACTERISTICCHANGE OBSERVEDLactateIncreasedpHDecreasedATPDecreasedMorphology scores change from discoid to spherical (loss of swirling effect)DecreasedDegranulationIncreasedPlatelet Activation MarkersIncreasedPlatelet AggregationDrop in responses to some agonistspH below 6.2: associated with a loss of platelet viability, the platelets swell and there is a disk-to-sphere transformation in morphology that is associated with a loss of membrane integrity. Platelets then become irreversibly swollen, aggregate together, or lyse, and when infused, will not circulate or function.

22CLINICAL USE OF PLATELETSPlatelet components are effectively used to treat bleeding associated with thrombocytopenia, a marked decrease in platelet number.

Platelets are also transfused prophylactically to increase the circulating platelet count in hematology-oncology thrombocytopenic patients to prevent bleeding secondary to drug and radiation therapy.

Platelets are also utilized in some instances to treat other disorders in which platelets are qualitatively or quantitatively defective because of genetic reasons.STORAGE CONDITIONSWhole blood derived platelet apheresis are stored at 20-24degC with continuous agitation for up to 5 days.

Platelet concentrates can also be stored at 1C to 6C for 48 hours without agitation

agitation has been shown to facilitate oxygen transfer into the platelet bag and oxygen consumption by the platelets. The positive role for oxygen has been associated with the maintenance of platelet component pH. Maintaining pH was determined to be a key parameter for retaining platelet viability in vivo when platelets were stored at 20C to 24C.24PLATELET STORAGE AND BACTERIAL CONTAMINATIONSources of bacterial contamination:Contamination at the phlebotomy siteDonor has bacterial infectionEnvironmental contamination during storage and processing3 commercial systems approved for screening platelets for bacterial contamination:BacT/ALERT (bioMrieux) - culture based systemeBDS (Pall Corp.) - culture based systemScansystem (Hemosystem) - is a laser-based, scanning cytometry methodDisadvantages of Culture MethodsProduct loss due to samplingDelay in product release, further reducing already short shelf-lifeFalse-negative resultsCostLogistical problems of culturing WBD platelets

Pan Genera Detection (PGD) test (Verax Biomedical)first rapid test approved to detect bacteria in WBD plateletsAn immunoassay that detects lipoteichoic acids on grampositive bacteria and lipopolysaccharides on gram-negative bacteria.Following pretreatment, the sample is loaded into a disposable plastic cartridge with built-in controls that turn from yellow to blue-violet when the test is ready to be read, in approximately 20 minutes. A pink-colored bar in either the grampositive or gram-negative test window indicates a positive result.CURRENT TRENDS IN PLATELET PRESERVATION RESEARCHResearch and development in platelet preservation is being pursued in many directions, including the following:Development of methods that would allow platelets to be stored for 7 daysDevelopment of additive solutions, also termed synthetic mediaDevelopment of procedures to reduce and inactivate the level of pathogens that may be in platelet unitsDevelopment of platelet substitutesNew approaches for storage of platelets at 1C to 6CThe development of processes to cryopreserve platelets