Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends

Red Blood Cell and Platelet Preservation: Historical and Current Trends

• Chapter focuses on past, present and future of red blood cell and platelet preservation.
• Progress has been made toward perfecting a safe, efficient, and uncomplicated transfusion technique.

Introduction

• People have always been fascinated by blood and modern medicine transfuses it.

Historical Overview

• First recorded blood transfusion was in 1492, for Pope Innocent VII, who died.
• In 1869 Braxton Hicks recommended sodium phosphate as a nontoxic anticoagulant.
• Karl Landsteiner discovered the ABO blood groups in 1901 and won a Nobel Prize for his find.
• Edward E. Lindemann used multiple syringes and a special cannula for vein-to-vein transfusions.
• Unger designed a syringe-valve apparatus making transfusions practical.
• In 1914, sodium citrate was reported by Hustin as an anticoagulant solution for transfusions.
• In 1915, Lewisohn determined the minimum amount of citrate needed for anticoagulation.
• In 1916, Rous and Turner introduced a citrate-dextrose solution for blood preservation.
• The function of glucose in RBC metabolism was not understood until the 1930s, delaying the use of glucose in preservative solutions.
• Charles Drew developed blood transfusion and preservation methods during WWII leading to blood banks.
• In February 1941, he was appointed director of the first American Red Cross blood bank.
• In 1943, Loutit and Mollison introduced the acid-citrate-dextrose (ACD) preservative formula.
• The July 1947 issue of the Journal of Clinical Investigation had a dozen papers about blood preservation.
• In 1957, Gibson introduced citrate-phosphate-dextrose (CPD) as a less acidic preservative that replaced ACD.
• Circulatory overload brought on by frequent transfusions was resolved via component therapy.

Current Status

• In 2008, AABB, formerly the American Association of Blood Banks, estimates there were 19 million volunteer donors.
• Approximately 17 million units of whole blood and RBCs were donated in 2008 in the United States.
• Approximately 24 million blood components were transfused in 2008.
• The New York Blood Center estimates 1 in 3 people will need blood in their lifetime.
• Fewer than 10% of healthy Americans who are eligible donate each year.
• Volunteer donors are unpaid, and provide nearly all the blood used for transfusion.
• The traditional whole blood unit contains 450 mL +/-10% of blood, now 500 mL +/-10% are being collected.
• Modified plastic collection systems are used when collecting 500 mL of blood, volume of anticoagulant-preservative solution being increased from 63 mL to 70 mL.
• For a 110 lb donor, a maximum volume of 525 mL can be collected.
• The donor's red cells are replaced within 1-2 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.
• Plasma can be converted by cryoprecipitation to an antihemophilic factor (AHF, factor VIII) concentrate.
• Whole blood–prepared RBCs may be stored for 21 to 42 days depending on the anticoagulant-preservative solution.
• The donation process includes educational reading materials, a health history questionnaire, and an abbreviated physical examination.

RBC Biology and Preservation

• Three areas of RBC biology are crucial for normal erythrocyte survival and function: membrane structure, Hemoglobin structure, and RBC metabolism.
• Defects in any of these areas will result in RBCs surviving less than the normal 120 days in circulation.
• Currently, 10 screening tests for infectious disease are performed on each unit of donated blood.
• The current risk of transfusion-transmitted hepatitis C virus (HCV) is 1 in 1,390,000, hepatitis B virus (HBV), it is between 1 in 200,000 and 1 in 500,000.
• The use of nucleic acid amplification testing (NAT), licensed by the Food and Drug Administration (FDA) since 2002, has increased the blood supply's safety.

RBC Membrane - Basic Concepts

• The RBC membrane is a semipermeable lipid bilayer supported by a mesh-like protein cytoskeleton structure.
• Integral membrane proteins extend from the outer surface and span the entire RBC membrane to the inner cytoplasmic side.
• Peripheral proteins are located and limited to the cytoplasmic surface of the membrane forming the RBC cytoskeleton.

RBC Membrane - Advanced Concepts

• Both proteins and lipids are organized asymmetrically within the RBC membrane.
• Lipids are not equally distributed in the two layers of the membrane.
• The membrane is composed of approximately 52% protein, 40% lipid, and 8% carbohydrate.
• They maintain deformability and permeability.

Deformability

• Normal RBCs must remain flexible, deformable, and permeable to remain viable.
• Membrane calcium in excess causes an increase in membrane rigidity and loss of pliability.
• Loss of RBC membrane results in spherocytes and bite cells.

Permeability

• RBC membrane permeability and active RBC cation transport prevent colloid hemolysis and control the volume of RBC.
• The RBC membrane is freely permeable to water and anions.
• Chloride (Cl-) and bicarbonate (HCO3-) can traverse the membrane in less than one second.
• The RBC membrane is relatively impermeable to cations such as sodium (Na+) and potassium (K+).

Metabolic Pathways - Basic Concepts

• The RBC's metabolic pathways that produce ATP are mainly anaerobic.
• Mature erythrocytes have no nucleus and there is no mitochondrial apparatus for oxidative metabolism, so, energy must be generated almost exclusively through the breakdown of glucose.

Metabolic Pathways - Advanced Concepts

• RBC metabolism may be divided into the anaerobic glycolytic pathway and three ancillary pathways.
• The ancillary pentose, methemoglobin reductase, and Luebering-Rapoport pathways serve to maintain hemoglobin structure and function.
• Glycolysis generates about 90% of the ATP needed by the RBC.
• Approximately 10% is provided by the pentose phosphate pathway.
• The methemoglobin reductase pathway is another important pathway of RBC metabolism.
• The Luebering-Rapoport shunt permits accumulation of 2,3-diphosphoglycerate (2,3-DPG).

Hemoglobin Oxygen Dissociation Curve

• Hemoglobin’s primary function is gas transport: oxygen delivery to the tissues and carbon dioxide (CO2) excretion.
• One of the most important controls of hemoglobin affinity for oxygen is the RBC organic phosphate 2, 3-DPG.
• Normal hemoglobin function depends on adequate 2, 3-DPG levels in the RBC.
• A shift to the left of the hemoglobin-oxygen dissociation curve occurs due to the multiple transfusions of 2,3-DPG-depleted stored blood.

RBC Preservation - Basic Concepts

• The goal of blood preservation is to provide viable and functional components for blood transfusion.
• RBC viability is a measure of in vivo RBC survival following transfusion.
• The FDA requires an average 24-hour post-transfusion RBC survival of more than 75%.
• FDA mandates red cell integrity as less than 1% of total hemoglobin.
• Blood is stored in the liquid state between 1°C and 6°C for a specific number of days, determined by the preservative solution.

RBC Preservation - Advanced Concepts

• Low 2,3-DPG levels diminish the oxygen dissociation curve of hemoglobin, DPG-depleted RBCs may have an impaired capacity to deliver oxygen to the tissues.

Anticoagulant Preservative Solutions - Basic Concepts

• ACD-A, CPD and CP2D preservative time is 21 days, CPDA-1 is 35 days of storage at temperatures between 1°C and 6°C.
• Incorporated various chemicals into anticoagulant-preservative CPD to stimulate glycolysis so that ATP levels were maintained.

Antigoagulent Preservative Solutions - Advanced Concepts

• After two weeks blood stored in all CPD preservatives also becomes depleted of 2,3-DPG.
• Transfusion of low 2,3-DPG levels causes increase in cardiac output and a decrease in mixed venous tension.

Additive Solutions - Basic Concepts

• Additive solutions (AS) extend shelf-life, and removes nutrients needed for RBC storage.
• Lack of nutrients and RBCs causes increased viscosity, making it difficult to infuse.
• The licensed additive solutions are Adsol (AS-1; Baxter Healthcare), Nutricel (AS-3; Pall Corporation), and Optisol (AS-5; Terumo Corporation).
• Surface area of storage bags needs to allow for the number of platelets that will be stored.

RBC Freezing - Basic Concepts

• RBC freezing is primarily used for autologous units and the storage of rare blood types.
• Involves a 6-day old cryoprotective agent (mostly Glycerol) that permeates the cells.
• Storage temperature are below −65°C and depends on the glyceral used.
• Most banks use high-concentration glycerol technique.

Additive Solutions - Advanced Concepts

• Red blood cells stored in additive solutions have been frozen up to 42 days after liquid storage. High and low Glycerol Advantages are initial freezing temperature, control of freezing rate, type of freezer, maximum storage temperature, shipping requirements, effect of storage changes.

RBC Rejuvenation - Basic Concepts

• Rejuvenation restores ATP and 2,3-DPG levels are or enhanced by metabolic alterations.
• Currently, Rejuvesol (enCyte Systems) contains phosphate, inosine, pyruvate, and adenine,
• Rejuvesol is only FDA-approved rejuvenation solution sold in the United States for blood stored in CPD, CPDA-1, and CPD/AS-1 RBCs.
• It can be rejuvenated at outdate temperatures or 3 days after outdate after being depended on RBC preservant solutions.
• Only RBCs prepared from 450-mL collections can be rejuvenated.

RBC Rejuvenation - Advanced Concepts

• RBC unit gets incubated with 50 mL of the rejuvenating solution for 1 hour at 37°C.
• Rejuvenated or frozen RBCs are used within 24 hours of thawing.
• Rejuvenation salvages O-type and rare blood type units for later use as it is expensive,
• Its effectiveness is not often implemented.

Current Trends in RBC Preservation Research - Advanced Concepts

• RBC preparation and preservation research is being pursued in five directions: Improved additive solutions, Reductive and inactivation of pathogens, Conversion of RBC's, Production of RBCs blood pharming', and RBC substitutes.

Improved Additive Solutions

• Research is being conducted to develop improved additive solutions for longer storage to improve logistics.

Procedures to Reduce and Inactivate Pathogens

• Research is being conducted to develop procedures that would lower levels of or inactivate residual viruses, bacteria and parasites in RBC units.
• The goal is inactivation of unrecognised pathogens that may have emerged in recent years.

Platelet Preservation - Basic Concepts

• Platelets arrest bleeding. Platelets are cellular fragments derived from cytoplasm of megakaryocytes in bone marrow.
• Released plates recirculate for approximately 9 to 12 days as disk-shaped cells with a diameter of approximately 2 to 4 millimeters.
• The normal platelet count is 150,000ul to 350,000ul. 30 per cent is sequestered in the microvasculature of the spleen.
• Platelets have roles in the hemostatic process that are dependent on adequate quantity and function.
• Includes:
  • Initial arrest of bleeding by platelet plug formation,
  • Stabilization of the hemostatic plug and contributing to fibrin formation and maintenance of vascular integrity.

Platelet Harvest - Advanced Concepts

• The organelle region of the platelet is the location for its metabolic activities. Platelets have cytoplasmic granules, like mitochondria. They have no nucleus, golgi body, or rough endoplasmic reticulum (RER).
• Contains 3 types of storage Granules: Dense granules, Lysosomes, and A granules.
• The three, the most numerous and most abundant, store beta thromboglobulin (bTG), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), thrombospondin,and factor V.

The Platelet Storage Lesion - Basic Conscept

• Challenges come with storing platelets.
• Platelets undergo variable activation.
• FDA limits to just five days of storing before platelets are discarded due to bacterial contamination at 22°C and loss of platelet.
• Activation causes the release of some intracellular granules, reduce oxygen pO2, increasing glucose consumption and lactic and causing a pH drop.
• Falling Ph is correlated to loss of life (viability) and functional integrity

Platelet Harvest- Advanced Concepts

• Platelets lose qualities within 5 days of storage.
• Loss results in reduced platelets (quality) during storage; membranes lose their normal lipid asymmetry and results in phosphatidylserine expression on platelet membranes, loss of ability to fix bleeding as needed within proper time-frame.
• Visual inspections assess the state of platelet.

Clinical Use of Platelets

• Platelet transfusions alleviate/cure the effects of marked levels of Thrompocitopenia.
• Efficiacy is often measured by measuring the difference between the old and new measured increments. (corrected count increment) or the Transfused Platelets.

Current Conditions for Platelet Preservation - Basic Concepts

• Platelet concentrates, prepared from whole blood, as part of the aphaeresis is conducted at average 20 degrees C.
• And agitation for average 5 days, or otherwise directed, until expired for the day.
• Containers are either flatbeds or as circulator agitators.
• The United States currently makes use of 100% plasma medium but does not use platelet addition.

History of Platelet Storage: Rationale for Current Conditions - (Advanced Concepts)

• Since the 1960's many improvements have evolved to retain platelet quantities and conditions.
• In 1969 Dr Murphy was the first to show that cold storage at 1 to 6 °C, caused a marked reduction in living qualities after just only 18 hours of storage.
• He was also for first time the person to advocate for warmer 20 to 24 degrees C storage to best keep them longer.

Limitations in Storage: Rationale of why

• As the qualities diminish over time, post-transfusion drops accordingly.
• So scientists are often trying to maintain viability.
• A pH level of average 6+ has been shown as a goal for maintaining satisfactory levels.
• That level of preservation continues to improve slightly with changing variables.

Storage in Second-Generation Containers

• Since the 1980s there have been great improvements in platelet concentrations, gas transport, and second generation containers.
• The new material increases transportation of O2 and escape through Carbon Dioxide. These efforts continue to be improved.

Storing Platelets Without Agitation for Limited Times

• Tests now show in vitro that properties are still retained for platelets, even when agitation ceases for 24 Hours.
• Likely due to retained O2.

Measurement of Viability and Functional Properties of Stored Platelets

• Viability indicates that Platelets can circulate post transfusion unharmed if undamaged, and in normal conditions and not prematurely removed.
• Platelets live about 8 to 10 days when healthy but is reduced after their stored period.
• Platelet viability can then, be based on measuring pre and post number counts.
• Or the time of tracing said infused radiolabeled amount, vs normal donors amounts.

Platelet Storage and Bacterial Contamination - Basic Concepts

• The main danger associated with storage of platelets between 20°C to 24°C is the potential for bacterial contamination.
• The potential is in the phlebotomy where bacteria can enter.
• Or donors that aren't tested or recognized to harbor bacteria can spoil the sample.

Platelet Storage and Bacterial Contamination - Advanced Concepts

• There are only and exactly only three systems approved by the FDA to check against the possibilities of such potential contamination of platelets harvested are: BacT/ALERT (bioMerieux), eBDS (Pall Corp) and Scan system from Hemosystems.

Advantages of Culture Methods for Detection of Bacterial Contamination of Platelets

• Culture, delay in product release, cost, and problems with testing WBDs is limited due to time and costs.
• Sensetivity is approximately 50%, resulting in transfusing only Aphaeresis harvest.
• Pan tested for the PGD- test via Verax Biomedics can remedy a lot of the problems, with testing LRs (Leukocyte depleted/reduced) to maintain all is well.

Cuurent Trends

• Current trends focus on research and development of platelet preservation .

Improved Additive Solutions - Additive Solutions

• Platelets can improve the shelf-life by optimizing medium composition and by using inhibitors to better maintain the in vitro properties.

Procedures to Reduce and Inactivate Pathogens

• In 2009 this type of action could be performed because the target is always going to be nucleic acids to provide a safer product against many different pathogens.

Development of Platelet Substitutes in View of Short Shelf Lifes

• Platelet substitutes will be more widely used because platelets more more complex both biochemically and physiologically more benefits and less harm can be generated, and more applications are being used to generate useful products, such as creating a better lyophilization.