1060 Unit 1 Text PDF

2 PART I Fundamental Concepts OBJECTIVES—cont’d 7. Define storage lesion and list the associated biochemical changes. 8. Explain the importance of 2,3-diphosphoglycerate (2,3-DPG) levels in transfused blood, including what happens to levels...

2 PART I Fundamental Concepts OBJECTIVES—cont’d 7. Define storage lesion and list the associated biochemical changes. 8. Explain the importance of 2,3-diphosphoglycerate (2,3-DPG) levels in transfused blood, including what happens to levels post-transfusion and which factors are involved. 9. Name the approved anticoagulant preservative solutions, explain the function of each ingredient, and state the maximum storage time for RBCs collected in each. 10. Name the additive solutions licensed in the United States, list the common ingredients, and describe the function of each ingredient. 11. Explain how additive solutions are used and list their advantages. 12. Explain rejuvenation of RBCs. 13. List the name and composition of the FDA-approved rejuvenation solution and state the storage time following rejuvenation. 14. Define the platelet storage lesion. 15. Describe the indications for platelet transfusion and the importance of the corrected count increment (CCI). 16. Explain the storage requirements for platelets. 17. Explain the swirling phenomenon and its significance. 18. List the two major reasons why routine platelet storage is limited to 5 days in the United States. 19. List the various ways that blood banks in the United States meet the FDA regulation requiring that blood establishments and transfusion services must assure that the risk of bacterial contamination of platelets is adequately controlled using FDA approved or cleared devices. 20. Explain the use and advantages of platelet additive solutions (PASs), and name two that are approved for use in the United States. Introduction first example of blood preservation research. Karl Landsteiner in 1901 discovered the ABO blood groups and explained People have always been fascinated by blood: Ancient the serious reactions that occur in humans as a result of Egyptians bathed in it, aristocrats drank it, authors and incompatible transfusions. His work in the beginning of the playwrights used it as themes, and modern humanity trans- 20th century won a Nobel Prize. fuses it. The road to an efficient, safe, and uncomplicated Next came devices designed for performing the transfu- transfusion has been difficult, but great progress has been sions. Edward E. Lindemann was the first to succeed. He made. This chapter reviews the historical events leading to carried out vein-to-vein transfusion of blood by using the current status of how blood is stored. A review of red multiple syringes and a special cannula for puncturing the blood cell (RBC) biology serves as a building block for the vein through the skin. However, this time-consuming, com- discussion of red blood cell preservation, and a brief plicated procedure required many skilled assistants. It was description of platelet metabolism sets the stage for review- not until Unger designed his syringe-valve apparatus that ing the platelet storage lesion. Current trends in red blood transfusions from donor to patient by an unassisted physi- cell and platelet preservation research are presented for cian became practical. the inquisitive reader. An unprecedented accomplishment in blood transfusion was achieved in 1914 when Hustin reported the use of Historical Overview sodium citrate as an anticoagulant solution for transfusions. Later, in 1915, Lewisohn determined the minimum amount In 1492, blood was taken from three young men and given of citrate needed for anticoagulation and demonstrated its to the stricken Pope Innocent VII in the hope of curing nontoxicity in small amounts. Transfusions became more him; unfortunately, all four died. Although the outcome of practical and safer for the patient. this event was unsatisfactory, it is the first time a blood The development of preservative solutions to enhance the transfusion was recorded in history. The path to successful metabolism of the RBCs followed. Glucose was evaluated as transfusions that is so familiar today is marred by many early as 1916, when Rous and Turner introduced a citrate- reported failures, but our physical, spiritual, and emotional dextrose solution for the preservation of blood. However, the fascination with blood is primordial. Why did success elude function of glucose in RBC metabolism was not understood experimenters for so long? until the 1930s. Therefore, the common practice of using Clotting was the principal obstacle to overcome. Attempts glucose in the preservative solution was delayed. World War II to find a nontoxic anticoagulant began in 1869, when Braxton stimulated blood preservation research because the demand Hicks recommended sodium phosphate. This was perhaps the for blood and plasma increased. During World War II,  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 3 the pioneer work of Dr. Charles Drew on developing tech- blood drives conducted at their place of work, school, and niques in blood transfusion and blood preservation led to church, as well as at community and hospital-based blood the establishment of a widespread system of blood banks.1 centers. Volunteer donors are not paid and provide nearly all In February 1941, Dr. Drew was appointed director of of the blood used for transfusion in the United States. the first American Red Cross blood bank at Presbyterian Traditionally, the amount of whole blood in a unit has been Hospital.1 The pilot program Dr. Drew established became 450 mL ± 10% of blood (1 pint). More recently, 500 mL ± 10% the model for the national volunteer blood donor program of blood is being collected.5 These units are collected from of the American Red Cross.1 donors with a minimum hematocrit of 38%.5 Modified plastic In 1943, Loutit and Mollison of England introduced the for- collection systems are used when collecting 500 mL of blood, mula for the preservative acid-citrate-dextrose (ACD). Efforts with the volume of anticoagulant preservative solution being in several countries resulted in the landmark publication of the increased from 63 to 70 mL. The total blood volume of most July 1947 issue of the Journal of Clinical Investigation, which adults is 10 to 12 pints, and donors can replenish the fluid lost devoted nearly a dozen papers to the topic of blood preserva- from the 1-pint donation in 24 hours. The donor’s red blood tion. Hospitals responded immediately, and in 1947 blood cells are replaced within 1 to 2 months after donation.4 A banks were established in many major cities of the United volunteer donor can donate whole blood every 8 weeks. (Refer States; subsequently, transfusion became commonplace. to Chapter 13 on Donor Selection.) The daily occurrence of transfusions led to the discovery Units of the whole blood collected can be separated into of numerous blood group systems. Antibody identification three components: packed RBCs, platelets, and plasma. In surged to the forefront as sophisticated techniques were recent years, less whole blood has been used to prepare developed. The interested student can review historic events platelets because of the increased utilization of apheresis during World War II in Kendrick’s Blood Program in World platelets. Hence, many units are converted only into RBCs War II, Historical Note.2 In 1957, Gibson introduced an im- and plasma. The plasma can be converted by cryoprecipita- proved preservative solution called citrate-phosphate-dextrose tion to a clotting factor concentrate that is rich in fibrinogen. (CPD), which was less acidic and eventually replaced ACD A unit of whole blood–prepared RBCs may be stored for as the standard preservative used for blood storage. 21 to 42 days, depending on the anticoagulant preservative Frequent transfusions and the massive use of blood soon solution used when the whole blood unit is collected and resulted in new problems, such as circulatory overload. whether a preserving solution is added to the separated Component therapy has helped these problems. In the past, RBCs. Donated blood is free. However, there is a cost asso- a single unit of whole blood could serve only one patient. ciated with collection, testing, processing, storing, and ship- With component therapy, however, one unit may be used for ping of the blood components. The donation process multiple transfusions. Today, health-care providers can select consists of three predonation steps. Donors receive the the specific component for their patient’s particular needs following (Box 1–1): without risking the inherent hazards of whole blood trans- 1. Educational reading materials fusions. Health-care providers can transfuse only the re- 2. A donor health history questionnaire quired fraction in the concentrated form, decreasing the 3. An abbreviated physical examination possibility of overloading the circulatory system. Appropriate blood component therapy now provides more effective treat- ment and more complete use of blood products. Extensive use of blood during this period, coupled with component BOX 1–1 separation, led to increased comprehension of erythrocyte The Donation Process metabolism and a new awareness of the problems associated with RBC storage. Step 1: Educational Materials Educational material (such as the AABB pamphlet “An Important Current Status Message to All Blood Donors”) that contains information on the risks of infectious diseases transmitted by blood transfusion, including the symptoms and signs of AIDS, is given to each prospective donor AABB, formerly the American Association of Blood Banks, to read. estimates that 6.8 million volunteers donate blood each year. Based on the 2015 National Blood Collection and Utilization Step 2: The Donor Health History Questionnaire Survey (NBCUS) approximately 12.6 million units of red A uniform donor history questionnaire, designed to ask questions blood cells (RBCs) were collected, and around 11.4 million that protect the health of both the donor and the recipient, is given to every donor. The health history questionnaire is used to identify were transfused.3 This represents a decline of 11.6% and donors who have been exposed to diseases that can be transmitted 13.9%, respectively since 2013.4 With an aging population in blood (e.g., variant Creutzfeldt-Jakob, West Nile virus, malaria, and advances in medical treatments requiring transfusions, babesiosis, or Chagas disease). the demand for blood and blood components is expected to Step 3: The Abbreviated Physical Examination continue to be high. It is estimated that one in three people The abbreviated physical examination for donors includes blood will need blood at some point in their lifetime.4 These units pressure, pulse, and temperature readings; hemoglobin or hemat- are donated by fewer than 10% of healthy Americans who ocrit level; and the inspection of the arms for skin lesions. are eligible to donate each year.4 Volunteers can donate at 4 PART I Fundamental Concepts The donation process, especially steps 1 and 2, has been RBC Membrane carefully modified over time to allow for the rejection of donors who may transmit transfusion-associated disease to The RBC membrane represents a semipermeable lipid bilayer recipients. For a more detailed description of donor screen- supported by a mesh-like protein cytoskeleton structure ing and processing, refer to Chapter 13. (Fig. 1–1).7 Phospholipids, the main lipid components of the The nation’s blood supply is safer than it has ever been be- membrane, are arranged in a bilayer structure comprising cause of the donation process and extensive laboratory testing the framework in which globular proteins traverse and of blood. Current infectious disease screening tests performed move. Proteins that extend from the outer surface and span on each unit of donated blood are listed in Table 1–1. For a the entire membrane to the inner cytoplasmic side of the more detailed description of transfusion-associated disease, RBC are termed integral membrane proteins. Beneath the refer to Chapter 14. lipid bilayer, a second class of membrane proteins, called peripheral proteins, is located and limited to the cytoplasmic RBC Biology and Preservation surface of the membrane forming the RBC cytoskeleton.7 Three areas of RBC biology are crucial for normal erythrocyte survival and function: Advanced Concepts 1. Normal chemical composition and structure of the RBC Both proteins and lipids are organized asymmetrically membrane within the RBC membrane. Lipids are not equally distrib- 2. Hemoglobin structure and function uted in the two layers of the membrane. The external layer 3. RBC metabolism6 is rich in glycolipids and choline phospholipids.7 The internal cytoplasmic layer of the membrane is rich in amino Defects in any or all of these areas will result in RBCs phospholipids.7 The biochemical composition of the RBC surviving fewer than the normal 120 days in circulation. membrane is approximately 52% protein, 40% lipid, and 8% carbohydrate.6 As mentioned previously, the normal chemical compo- sition and the structural arrangement and molecular inter- Table 1–1 Current Donor Screening Tests actions of the erythrocyte membrane are crucial to the for Infectious Diseases normal length of RBC survival of 120 days in circulation. In addition, they maintain a critical role in two important Test Date Test Required RBC characteristics: deformability and permeability.8 Syphilis 1950s Deformability Hepatitis B surface antigen (HBsAg) 1971 Hepatitis B core antibody (anti-HBc) 1986 To remain viable, normal RBCs must also remain flexible, deformable, and permeable. The loss of adenosine triphos- Hepatitis C virus antibody (anti-HCV) 1990 phate (ATP) (energy levels) leads to a decrease in the phos- Human immunodeficiency virus 19921 phorylation of spectrin and, in turn, a loss of membrane antibodies (anti-HIV-1/2) deformability.6 An accumulation or increase in deposition of membrane calcium also results, causing an increase in Human T-cell lymphotropic virus 19972 membrane rigidity and loss of pliability.8 These cells are at antibody (anti-HTLV-I/II) a marked disadvantage when they pass through the small Human immunodeficiency virus 1999 (3 to 5 µm in diameter) sinusoidal orifices of the spleen, (HIV-1) NAT* an organ that functions in extravascular sequestration, and Hepatitis C virus (HCV) NAT* 1999 removal of aged, damaged, or less deformable RBCs or fragments of their membrane. The loss of RBC membrane West Nile virus NAT 2004 is exemplified by the formation of spherocytes (cells with Trypanosoma cruzi antibody 2007 a reduced surface-to-volume ratio; Fig. 1–2) and bite cells, (anti-T. cruzi) in which the removal of a portion of membrane has left a permanent indentation in the remaining cell membrane Hepatitis B virus (HBV) NAT 2009 (Fig. 1–3). The survival of these forms is also shortened. Babesia microti antibody and NAT 2012 (recommended) Permeability Zika virus NAT 2016 The permeability properties of the RBC membrane and the active RBC cation transport prevent colloid hemolysis and NAT = nucleic acid amplification testing *Initially under IND starting in 1999. control the volume of the RBCs. Any abnormality that 1Anti-HIV-1 testing implemented in 1985. increases permeability or alters cationic transport may 2Anti-HTLV testing implemented in 1988.  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 5 I = integral proteins Spectrin P = peripheral proteins ankyrin-band 3 Phospholipids interaction Fatty acid chains GP-C Membrane F-actin surface I Lipid I I I bilayer GP-B 3 3 GP-A 7 P 2.1 4.2 6 P P Membrane P cytoskeleton Adducin Protein 4.1 Spectrin dimer-dimer Alpha chain Spectrin- Ankyrin interaction Spectrin Beta chain actin-4.1-adducin interaction Figure 1–1. Schematic illustration of red blood cell membrane depicting the composition and arrangement of RBC membrane proteins. GP-A = glycophorin A; GP-B = glycophorin B; GP-C = glycophorin C; G = globin. Numbers refer to pattern of migration of SDS (sodium dodecyl sulfate) polyacrylamide gel pattern stained with Coomassie brilliant blue. Relations of protein to each other and to lipids are purely hypothetical; however, the positions of the proteins relative to the inside or outside of the lipid bilayer are accurate. (Note: Proteins are not drawn to scale and many minor proteins are omitted.) (Reprinted with permission from Harmening, DH: Clinical Hematology and Fundamentals of Hemostasis, 5th ed., FA Davis, Philadelphia, 2009.) decrease RBC survival. The RBC membrane is freely per- meable to water and anions.9 Chloride (Cl–) and bicarbon- ate (HCO3–) can traverse the membrane in less than a second. It is speculated that this massive exchange of ions occurs through a large number of exchange channels located in the RBC membrane. The RBC membrane is rela- tively impermeable to cations such as sodium (Na+) and potassium (K+). RBC volume and water homeostasis are maintained by controlling the intracellular concentrations of sodium and potassium.9 The erythrocyte intracellular-to-extracellular ratios for Na+ and K+ are 1:12 and 25:1, respectively.6 The 300 cationic pumps, which actively transport Na+ out of Figure 1–2. Spherocytes. the cell and K+ into the cell, require energy in the form of ATP. Calcium (Ca2+) is also actively pumped from the inte- rior of the RBC through energy-dependent calcium- ATPase pumps.6 Calmodulin, a cytoplasmic calcium-binding protein, is speculated to control these pumps and to pre- vent excessive intracellular Ca2+ buildup, which changes the shape and makes it more rigid.6 When RBCs are ATP-depleted, Ca2+ and Na+ are allowed to accumulate intracellularly, and K+ and water are lost, resulting in a dehydrated rigid cell that is subsequently sequestered by the spleen, resulting in a decrease in RBC survival.9 Metabolic Pathways The RBCs’ metabolic pathways that produce ATP are mainly anaerobic because the function of the RBC is to deliver oxygen, not to consume it. Because the mature erythrocyte Figure 1–3. “Bite” cells. has no nucleus and there is no mitochondrial apparatus 6 PART I Fundamental Concepts for oxidative metabolism, energy must be generated almost (2,3-DPG). The amount of 2,3-DPG found within RBCs exclusively through the breakdown of glucose. has a significant effect on the affinity of hemoglobin for oxygen and therefore affects how well RBCs function post-transfusion. Advanced Concepts RBC metabolism may be divided into the anaerobic gly- Hemoglobin-Oxygen Dissociation Curve colytic pathway and three ancillary pathways that serve to maintain the structure and function of hemoglobin Hemoglobin’s primary function is gas transport: oxygen (Fig. 1–4): the pentose phosphate pathway, the methemo- delivery to the tissues and carbon dioxide (CO2) excretion. globin reductase pathway, and the Luebering-Rapoport One of the most important controls of hemoglobin affinity shunt. All of these processes are essential if the erythrocyte for oxygen is the RBC organic phosphate 2,3-DPG. The is to transport oxygen and to maintain critical physical unloading of oxygen by hemoglobin is accompanied by characteristics for its survival. Glycolysis generates about widening of a space between β chains and the binding of 90% of the ATP needed by the RBC. Approximately 10% 2,3-DPG on a mole-for-mole basis, with the formation of is provided by the pentose phosphate pathway. The methe- anionic salt bridges between the chains.10 The resulting moglobin reductase pathway is another important pathway conformation of the deoxyhemoglobin molecule is known of RBC metabolism, and a defect can affect RBC post- as the tense (T) form, which has a lower affinity for oxygen.6 transfusion survival and function. Another pathway that When hemoglobin loads oxygen and becomes oxyhemo- is crucial to RBC function is the Luebering-Rapoport globin, the established salt bridges are broken, and β chains shunt. This pathway permits the accumulation of an im- are pulled together, expelling 2,3-DPG. This is the relaxed portant RBC organic phosphate, 2,3-diphosphoglycerate (R) form of the hemoglobin molecule, which has a higher PHOSPHOGLUCONATE PATHWAY (oxidative) H 2O 2 GP EMBDEN-MEYERHOF PATHWAY GSH GSSG (non-oxidative) Glucose GR ATP HK NADP NADPH ADP Glucose 6-P 6-P-Gluconate G-6-PD GPI 6-PGD CO2 Fructose 6-P Pentose-P ATP PFK ADP Fructose 1,6-diP METHEMOGLOBIN A REDUCTASE PATHWAY Glyceraldehyde DHAP LUEBERING-RAPAPORT Hemoglobin NAD PATHWAY R GAPD Methemoglobin NADH HK Hexokinase 1,3-diP-Glycerate DPGM GPI Glucose-6-phosphate isomerase ADP 2,3-diP-Glycerate PFK Phosphofructokinase PGK DPGP ATP A Aldolase 3-P-Glycerate TPI Triose phosphate isomerase GAPD Glyceraldehyde-3-phosphate dehydrogenase PGM PGM Phosphoglycerate mutase E Enolase 2-P-Glycerate PK Pyruvate kinase LDH Lactic dehydrogenase E DPGM Diphosphoglyceromutase P-Enolpyruvate DPGP Diphosphoglycerate phosphatase ADP G-6-PD Glucose-6-phosphate dehydrogenase PK ATP 6-PGD 6-Phosphogluconate dehydrogenase GR Glutathione reductase Pyruvate GP Glutathione peroxidase NADH LDH DHAP Dihydroxyacetone-P NAD PGK Phosphoglycerate kinase Lactate R NADH-methemoglobin reductase Figure 1–4. Red blood cell metabolism. (Reprinted with permission from Hillman, RF, and Finch, CA: Red Cell Manual, 7th ed., FA Davis, Philadelphia, 1996.)  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 7 affinity for oxygen.6 These allosteric changes that occur as are much less efficient because only 12% of the oxygen the hemoglobin loads and unloads oxygen are referred to can be released to the tissues.6 Multiple transfusions of as the respiratory movement. The dissociation and binding 2,3-DPG–depleted stored blood can shift the oxygen dis- of oxygen by hemoglobin are not directly proportional to sociation curve to the left.10 the partial pressure of oxygen (pO2) in its environment but instead exhibit a sigmoid-curve relationship, known as the hemoglobin-oxygen dissociation curve (Fig. 1–5). RBC Preservation The shape of this curve is very important physiologically The goal of blood preservation is to provide viable and func- because it permits a considerable amount of oxygen to be tional blood components for patients requiring blood trans- delivered to the tissues with a small drop in oxygen tension. fusion. RBC viability is a measure of in vivo RBC survival For example, in the environment of the lungs, where the following transfusion. Because blood must be stored from the pO2 tension, measured in millimeters of mercury (mm Hg), time of donation until the time of transfusion, the viability of is nearly 100 mm Hg, the hemoglobin molecule is almost RBCs must be maintained during the storage time as well. 100% saturated with oxygen. As the RBCs travel to the The U.S. Food and Drug Administration (FDA) requires an tissues, where the pO2 drops to an average of 40 mm Hg average 24-hour post-transfusion RBC survival of more than (mean venous oxygen tension), the hemoglobin saturation 75%.11 In addition, the FDA mandates that red blood cell drops to approximately 75% saturation, releasing about integrity be maintained throughout the shelf-life of the stored 25% of the oxygen to the tissues.6 This is the normal RBCs. This is assessed as free hemoglobin less than 1% of situation of oxygen delivery at a basal metabolic rate. The total hemoglobin.11 These two criteria are used to evaluate normal position of the oxygen dissociation curve depends new preservation solutions and storage containers. To deter- on three different ligands normally found within the RBC: mine post-transfusion RBC survival, RBCs are taken from H+ ions, CO2, and organic phosphates. Of these three healthy subjects, stored, and then labeled with radioisotopes, ligands, 2,3-DPG plays the most important physiological reinfused to the original donor, and measured 24 hours after role. Normal hemoglobin function depends on adequate transfusion. Despite FDA requirements, the 24-hour post- 2,3-DPG levels in the RBC. In situations such as hypoxia, transfusion RBC survival at outdate can be less than 75% and a compensatory shift to the right of the hemoglobin- in critically ill patients is often less than 75%.12,13 oxygen dissociation curve alleviates the tissue oxygen To maintain optimum viability, blood is stored in the liquid deficit. This rightward shift of the curve, mediated by in- state between 1°C and 6°C for a specific number of days, as creased levels of 2,3-DPG, decreases hemoglobin’s affinity determined by the preservative solution(s) used. The loss of for the oxygen molecule and increases oxygen delivery to RBC viability has been correlated with the storage lesion, which the tissues. A shift to the left of the hemoglobin-oxygen is associated with various biochemical changes14 (Table 1–2). dissociation curve results, conversely, in an increase in hemoglobin-oxygen affinity and a decrease in oxygen de- livery to the tissues. With such a dissociation curve, RBCs Advanced Concepts Because low 2,3-DPG levels profoundly influence the oxy- gen dissociation curve of hemoglobin,14 DPG-depleted 100 Normal 90 “Left-shifted” “Right-shifted” 80 Table 1–2 RBC Storage Lesion ↑Abn Hb Oxyhemoglobin (% saturation) 70 ↑pH Characteristic Change Observed ↓DPG ↓pH ↓Temp ↑DPG 60 Viable cells (%) Decreased ↓P50 ↑Temp ↑P50 50 Glucose Decreased P50 40 ATP Decreased 30 Lactic acid Increased 20 pH Decreased 10 2,3-DPG Decreased Normal P50 = 28 mm Hg 0 Oxygen dissociation curve Shift to the left (increase in 0 10 20 30 40 50 60 70 80 90 100 hemoglobin and oxygen affinity; less oxygen delivered to tissues) PO2 (mm Hg) Plasma K+ Increased Figure 1–5. Hemoglobin-oxygen dissociation curve. (Reprinted with permission from Harmening, DH: Clinical Hematology and Fundamentals of Hemostasis, Plasma hemoglobin Increased 5th ed., FA Davis, Philadelphia, 2009.) 8 PART I Fundamental Concepts RBCs may have an impaired capacity to deliver oxygen to Advanced Concepts the tissues. As RBCs are stored, 2,3-DPG levels decrease, with a shift to the left of the hemoglobin-oxygen dissocia- It is interesting to note that blood stored in all CPD preser- tion curve, and less oxygen is delivered to the tissues. It is vatives also becomes depleted of 2,3-DPG by the second well accepted, however, that 2,3-DPG is re-formed in stored week of storage. The reported pathophysiological effects of RBCs after in vivo circulation, resulting in restored oxygen the transfusion of RBCs with low 2,3-DPG levels and delivery. The rate of restoration of 2,3-DPG is influenced by increased affinity for oxygen include an increase in cardiac the acid-base status of the recipient, the phosphorus meta- output, a decrease in mixed venous (pO2) tension, or a bolism, the degree of anemia, and the overall severity of the combination of these.18 The physiological importance of disorder.6 It has been reported that within the first hour after these effects is not easily demonstrated. This is a complex transfusion, most RBC clearance occurs.13 Approximately mechanism with numerous variables involved that are 220 to 250 mg of iron are contained in one RBC unit.15 beyond the scope of this text. Therefore, rapid RBC clearance of even 25% of a single unit Stored RBCs regain the ability to synthesize 2,3-DPG after of blood delivers a massive load of hemoglobin iron to the transfusion, but levels necessary for optimal hemoglobin- monocyte and macrophage system, potentially producing oxygen delivery are not reached immediately. Approxi- harmful effects.12 mately 24 hours are required to restore normal levels of Despite the biochemical, structural, and functional 2,3-DPG after transfusion.18 The 2,3-DPG concentrations changes that occur to RBCs during storage, there is no after transfusion have been reported to reach normal levels significant difference in rates of death between patients as early as 6 hours post-transfusion.18 Most of these who were transfused with only fresh blood versus studies have been performed on normal, healthy individu- those patients who were transfused with the oldest blood als. However, evidence suggests that, in the transfused available.16 subject whose capacity is limited by an underlying physio- logical disturbance, even a brief period of altered oxygen hemoglobin affinity is of great significance.12 It is quite Anticoagulant Preservative Solutions clear now that 2,3-DPG levels in transfused blood are important in certain clinical conditions. Some studies Table 1–3 lists the approved anticoagulant preservative demonstrate that myocardial function improves following solutions for whole blood and RBC storage at 1°C to 6°C. transfusion of blood with high 2,3-DPG levels during The addition of various chemicals, along with the approved cardiovascular surgery.18 Several investigators suggest that anticoagulant preservative CPD, was incorporated in an the patient in shock who is given 2,3-DPG–depleted eryth- attempt to stimulate glycolysis so that ATP levels were better rocytes in transfusion may have already strained the com- maintained.17 One of the chemicals, adenine, incorporated pensatory mechanisms to their limits.18,19–21 Perhaps for into the CPD solution (CPDA-1) increases ADP levels, this type of patient, the poor oxygen delivery capacity of thereby driving glycolysis toward the synthesis of ATP. 2,3-DPG–depleted cells makes a significant difference in CPDA-1 contains 0.25 mM of adenine plus 25% more recovery and survival. glucose than CPD. Adenine-supplemented blood can be It is apparent that many factors may limit the viability stored at 1°C to 6°C for 35 days; the other anticoagulants are of transfused RBCs. One of these factors is the plastic approved for 21 days. Table 1–4 lists the various chemicals material used for the storage container. The plastic must used in anticoagulant solutions and their functions during be sufficiently permeable to CO2 in order to maintain the storage of red blood cells. higher pH levels during storage. Glass storage containers are no longer used in the United States. Currently, the majority of blood is stored in polyvinyl chloride (PVC) Table 1–3 Approved Anticoagulant plastic bags. One issue associated with PVC bags relates Preservative Solutions to the plasticizer di(ethylhexyl)-phthalate (DEHP), which is used in the manufacture of the bags. It has been found Storage to leach from the plastic into the lipids of the plasma Name Abbreviation Time (Days) medium and RBC membranes of the blood during storage. Acid citrate-dextrose ACD-A 21 However, its use or that of alternative plasticizers that (formula A)* leach are important because they have been shown to sta- bilize the RBC membrane and therefore reduce the extent Citrate-phosphate CPD 21 dextrose of hemolysis during storage. Another issue with PVC is its tendency to break at low temperatures; therefore, com- Citrate-phosphate- CP2D 21 ponents frozen in PVC bags must be handled with care. double-dextrose In addition to PVC, polyolefin containers, which do not Citrate-phosphate- CPDA-1 35 contain DEHP, are available for some components, and dextrose-adenine latex-free plastic containers are available for recipients with latex allergies.5 *ACD-A is used for apheresis components.  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 9 Table 1–4 Chemicals in Anticoagulant Solutions Chemical Function Present In ACD-A CPD CP2D CPDA-1 Citrate (sodium citrate/citric acid) Chelates calcium; prevents clotting. X X X X Monobasic sodium phosphate Maintains pH during storage; necessary for maintenance X X X X of adequate levels of 2,3-DPG. Dextrose Substrate for ATP production (cellular energy). X X X X Adenine Production of ATP (extends shelf-life from 21 to 35 days). X ACD-A = acid citrate-dextrose (formula A); CPD = citrate-phosphate dextrose; CP2D = citrate phosphate double dextrose; CPDA-1 = citrate-phosphate-dextrose-adenine; 2,3-DPG = 2,3-diphosphoglycerate; ATP = adenosine triphosphate Additive Solutions The additive solution is contained in a satellite bag and is added to the RBCs after most of the plasma has been ex- Additive solutions (AS) are preserving solutions that are added pressed. All three additives contain saline, adenine, and glu- to the RBCs after removal of the plasma with or without cose. AS-1, AS-5, and AS-7 also contain mannitol, which platelets. Additive solutions are now widely used. One of the protects against storage-related hemolysis, whereas AS-3 reasons for their development is that removal of the plasma contains citrate and phosphate for the same purpose.22 All component during the preparation of packed RBCs removed of the additive solutions are approved for 42 days of storage much of the nutrients needed to maintain RBCs during storage. for packed RBCs.22 Table 1–5 lists the currently approved This was dramatically observed when high-hematocrit RBCs additive solutions. were prepared. The influence of removing substantial amounts of adenine and glucose present originally in, for example, the CPDA-1 anticoagulant preservative solution, led to a decrease Advanced Concepts in viability, particularly in the last 2 weeks of storage.16 Table 1–6 shows the biochemical characteristics of RBCs Packed RBCs prepared from whole blood units collected stored in the additive solutions after 42 days of stor- in primary anticoagulant preservative solutions can be rela- age.22,23,24 Additive system RBCs are used in the same way tively void of plasma with high hematocrits, which causes as traditional RBC transfusions. Blood stored in additive the units to be more viscous and difficult to infuse, especially solutions is now routinely given to newborn infants and in emergency situations. Additive solutions (100 mL to the pediatric patients, although some clinicians still prefer packed RBCs prepared from a 450-mL blood collection) also CPDA-1 RBCs because of their concerns about one or more overcome this problem. Additive solutions reduce hemato- of the constituents in the additive solutions.25 crits from around 65% to 80% to around 55% to 65% with a None of the additive solutions maintain 2,3-DPG volume of approximately 300 to 400 mL.22 The ability to throughout the storage time. As with RBCs stored only with pack RBCs to fairly high hematocrits before adding additive primary anticoagulant preservatives, 2,3-DPG is depleted solution also provides a means to harvest greater amounts by the second week of storage.23 of plasma with or without platelets. Box 1–2 summarizes the benefits of RBC additive solutions. Currently, four additive solutions are licensed in the United States: 1. Adsol (AS-1; Fenwal Inc.) Table 1–5 Additive Solutions in Use 2. Nutricel (AS-3; Haemonetics Corporation) in North America 3. Optisol (AS-5; Terumo Corporation) 4. SOLX (AS-7; Haemonetics Corporation) Storage Name Abbreviation Time (Days) Adsol (Fenwal AS-1 42 Inc.) BOX 1–2 Nutricel (Haemonetics AS-3 42 Benefits of RBC Additive Solutions Corporation) Extends the shelf-life of RBCs to 42 days by adding nutrients Optisol (Terumo AS-5 42 Allows for the harvesting of more plasma and platelets from Corporation) the unit Produces a packed RBC of lower viscosity that is easier to infuse SOLX (Haemonetics) AS-7 42 10 PART I Fundamental Concepts Table 1–6 Red Blood Cell Additives: Biochemical Characteristics AS-1 AS-3 AS-5 AS-7 Storage period (days) 42 42 42 42 pH (measured at 37°C) 6.6 6.5 6.5 6.6 24-hour survival*(%) 83 85.1 80 80 ATP (% initial) 68 67 68.5 91% 2,3-DPG (% initial) 6 6 5 **1.5 µmol/L/g Hb Hemolysis (%) 0.5 0.7 0.6 0.3 *Survival studies reported are from selected investigators and do not include an average of all reported survivals. **Reported by researchers using different units Freezing and Rejuvenation Currently, the FDA licenses frozen RBCs for a period of 10 years from the date of freezing; that is, frozen RBCs may RBC Freezing be stored up to 10 years before thawing and transfusion.26 RBC freezing is primarily used for autologous units and the Once thawed, these RBCs demonstrate function and viability storage of rare blood types. Autologous transfusion (auto near those of fresh blood. Experience has shown that 10-year meaning “self”) allows individuals to donate blood for storage periods do not adversely affect viability and their own use to meet their needs for blood transfusion function.27 Table 1–8 lists the advantages and disadvantages (see Chapter 16, “Transfusion Therapy”). of RBC freezing. The procedure for freezing a unit of packed RBCs is not complicated. It involves the addition of a cryoprotective agent to RBCs that are less than 6 days old. Glycerol is used most Advanced Concepts commonly and is added to the RBCs slowly with vigorous Transfusion of frozen cells must be preceded by a deglyc- shaking, thereby enabling the glycerol to permeate the RBCs. erolization process; otherwise, the thawed cells would be The cells are then rapidly frozen and stored in a freezer. The accompanied by hypertonic glycerol when infused, and usual storage temperature is below –65°C, although storage RBC lysis would result. Removal of glycerol is achieved by (and freezing) temperature depends on the concentration systematically replacing the cryoprotectant with decreasing of glycerol used.23 Two concentrations of glycerol have concentrations of saline. The usual protocol involves wash- been used to freeze RBCs: a high-concentration glycerol ing with 12% saline, followed by 1.6% saline, with a final (40% weight in volume [wt/vol]) and a low-concentration wash of 0.2% dextrose in normal saline.5 A commercially glycerol (20% wt/vol) in the final concentration of the cryo- available cell-washing system, such as those manufactured preservative.23 Most blood banks that freeze RBCs use the by several companies, has traditionally been used in the high-concentration glycerol technique. deglycerolizing process. Excessive hemolysis is monitored Table 1–7 lists the advantages of the high-concentration by noting the hemoglobin concentration of the wash su- glycerol technique in comparison with the low-concentration pernatant. Osmolality of the unit should also be monitored glycerol technique. See Chapter 15 for a detailed to ensure adequate deglycerolization. Traditionally, because description of the RBC freezing procedure. Table 1–7 Advantages of High-Concentration Glycerol Technique Over Low-Concentration Glycerol Technique Advantage High Glycerol Low Glycerol 1. Initial freezing temperature –80°C –196°C 2. Need to control freezing rate No Yes 3. Type of freezer Mechanical Liquid nitrogen 4. Maximum storage temperature –65°C –120°C 5. Shipping requirements Dry ice Liquid nitrogen 6. Effect of changes in storage temperature Can be thawed and refrozen Critical  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 11 Table 1–8 Advantages and Disadvantages 3. Development of procedures to convert A, B, and AB type of RBC Freezing RBCs to O type RBCs 4. Development of methods to produce RBCs through Advantages Disadvantages bioengineering (blood pharming) Long-term storage (10 years) A time-consuming process 5. Development of RBC substitutes Maintenance of RBC viability Higher cost of equipment and and function materials Improved Additive Solutions Low residual leukocytes and Storage requirements (–65°C) Research is being conducted to develop improved additive platelets solutions for RBC preservation. One reason for this is Removal of significant amounts Higher cost of product because longer storage periods could improve the logistics of plasma proteins of providing RBCs for clinical use. Procedures to Reduce and Inactivate Pathogens Research is being conducted to develop procedures that a unit of blood is processed in an open system (one in would reduce the level of or inactivate residual viruses, bac- which sterility is broken) to add the glycerol (before freezing) teria, and parasites in RBC units. One objective is to develop or the saline solutions (for deglycerolization), the outdating robust procedures that could possibly inactivate all period of thawed RBCs stored at 1°C to 6°C has been pathogens that may be present, including new and emergent 24 hours.23 Generally, RBCs in CPD or CPDA-1 anticoag- viruses. Amustaline (S-303) pathogen reduction system is ulant preservatives or additive solutions are glycerolized currently being studied and has demonstrated adequate post- and frozen within 6 days of whole blood collection.23 transfusion viability according to FDA criteria.28 This nucleic Closed-system devices have been developed that allow acid-targeted pathogen inactivation technology was devel- the glycerolization and deglycerolization processes to be oped to reduce the risk of transfusion-transmitted infectious performed under sterile conditions.27 RBCs prepared from disease with RBC transfusions.29 Areas of concern that must 450-mL collections and frozen within 6 days of blood col- be addressed before pathogen inactivation technologies are lection with CPDA-1 can be stored after thawing at 1°C to approved for use with RBCs in the United States are potential 6°C for up to two weeks when prepared in a closed toxicity, immunogenicity, cellular function, and cost. Cur- system.23 rently, the FDA has not approved any Pathogen Reduction Technology (PRT) for use with RBCs.29 RBC Rejuvenation Rejuvenation of RBCs is the process by which ATP and 2,3- Formation of O Type RBCs DPG levels are restored or enhanced by metabolic alter- The inadequate supply of O type RBC units that is periodi- ations. Currently, FDA-approved rejuvenation solution cally encountered can hinder blood centers and hospital contains phosphate, inosine, and adenine.22 Rejuvenated blood banks in providing RBCs for specific patients. Re- RBCs may be prepared up to three days after expiration search over the last 30 years has been evaluating how A and when stored in CPD, CPDA-1, and AS-1 storage solu- B type RBCs can be converted to O type RBCs, the universal tions.22 Currently, rejuvenated RBCs must be washed be- donor.30 The use of enzymes that remove the carbohydrate fore infusion to remove the inosine (which may be toxic) moieties of the A and B antigens is the mechanism for form- and transfused within 24 hours or frozen for long-term ing O type RBCs.30 The enzymes are removed by washing storage.22 The rejuvenation process is expensive and time- after completion of the reaction time. consuming, thus it is not used often; however, the process is invaluable for preserving selected autologous and rare units of blood for later use. Blood Pharming Creating RBCs in the laboratory (blood pharming) is an- Current Trends in RBC Preservation other area of research that has the potential to increase the Research amount of blood available for transfusion. In 2008, the De- fense Advanced Research Projects Agency (DARPA) awarded Arteriocyte, a bioengineering company, a contract to develop Advanced Concepts a system for producing O-negative RBCs on the battlefield.31 Research and development in RBC preparation and preser- The company, which uses proprietary technology (NANEX) vation is being pursued in five areas: to turn hematopoietic stem cells (HSCs) from umbilical 1. Development of improved additive solutions cords into type O, Rh-negative RBCs, sent its first shipment 2. Development of procedures to reduce and inactivate the of the engineered blood to the FDA for evaluation in 2010.31 level of pathogens that may be in RBC units FDA approval is required before human trials can begin. Cultured RBCs generated from in vitro hematopoietic stem 12 PART I Fundamental Concepts cells has been reported as well.32 However, this has not Table 1–9 Phases of Testing proven practical for routine transfusion. The challenges associated with blood pharming are scalability or large-scale Phase Description of Testing production and cost-effectiveness. Preclinical In vivo and animal testing. RBC Substitutes Phase I Researchers test drug in a small group of people (20 to 80) for the first time to evaluate its safety, Scientists have been searching for a substitute for blood for determine a safe dosage range, and identify side effects. over 150 years. Blood substitutes continue to be of interest because of their potential to alleviate shortages of donated Phase II The drug is given to a larger group of people blood. In the 1980s, safety concerns about HIV led to re- (100 to 300) to see if it is effective and to further newed interest in finding a substitute for human blood; and evaluate its safety. more recently, the need for blood on remote battlefields has Phase III The drug is given to large groups of people heightened that interest.33 The U.S. military is one of the (1,000 to 3,000) to confirm its effectiveness, monitor strongest advocates for the development of blood substitutes, side effects, compare it to commonly used treat- which it supports through its own research and partnerships ments, and collect information that will allow the drug to be used safely. with private-sector companies.33 Today the search continues for a safe and effective oxygen carrier that could eliminate Phase IV Postmarketing studies to gather additional informa- many of the problems associated with blood transfusion, such tion about the drug’s risks, benefits, and optimal use. as the need for refrigeration, limited shelf-life, compatibility, immunogenicity, transmission of infectious agents, and short- ages. Box 1–3 lists the potential benefits of artificial oxygen carriers. Since RBC substitutes are drugs, they must go Hemoglobin-Based Oxygen Carriers through extensive testing in order to obtain FDA approval. HBOC commercial development focused on “oxygen thera- Safety and efficacy must be demonstrated through clinical peutic” indications to provide immediate oxygenation until trials. Table 1–9 outlines the different phases of testing. medical or surgical interventions could be initiated. Early Current research on blood substitutes is focused on two trauma trials with HemAssist® (BAXTER), Hemopure® areas: hemoglobin-based oxygen carriers (HBOCs) and per- (HbO2Therapeutics), and PolyHeme® (NORTHFIELD fluorocarbons (PFCs).34,35 Originally developed to be used Laboratories) for resuscitating hypotensive shock all failed in trauma situations such as accidents, combat, and surgery, due to the safety concerns of cardiac issues and increased RBC substitutes have, until recently, fallen short of meeting mortality. requirements for these applications.34 Despite years of re- Although several HBOCs have progressed to phase II and search, RBC substitutes are still not in routine use today. III clinical trials, currently none have been approved for South Africa, Mexico, and Russia are the only countries clinical use in humans in the United States.36,37 A 2008 meta- in which blood substitutes are approved for clinical use. analysis of 16 clinical trials involving 3,711 patients and five None have received FDA approval for clinical use in the different HBOCs found a significantly increased risk of death United States, although specific products are still in phase and myocardial infarction associated with the use of III clinical trials. HBOCs.37 As a result, in 2008, the Food and Drug Adminis- tration (FDA) put all HBOC trials in the United States on clinical hold due to the unfavorable outcomes.35 However, BOX 1–3 Hemopure (HBOC-201) and PolyHeme are still in phase III Potential Benefits of Artificial Oxygen Carriers clinical trials in the United States and Europe.38 Hemopure was approved for clinical use in South Africa in 2001 to treat Abundant supply adult surgical patients who are anemic, and in Russia for Readily available for use in prehospital settings, battlefields, and remote locations acute anemia.34 Many HBOCs have been researched; how- Can be stockpiled for emergencies and warfare ever, the majority have been discontinued due to complica- No need for typing and crossmatching tions of cardiac toxicity, gastrointestinal distress, neurotoxicity, Available for immediate infusion renal failure, and increased mortality.34 Table 1–10 summa- Extended shelf-life (1 to 3 years) rizes some of the many HBOCs developed. Can be stored at room temperature However, some experts believe that HBOCs hold more Free of bloodborne pathogens promise than PFCs.33,39 At full oxygen capacity immediately Table 1–11 lists the advantages and disadvantages of Do not prime circulating neutrophils, reducing the incidence of HBOCs. multiorgan failure Perfluorocarbons Can deliver oxygen to tissue that is inaccessible to RBCs Have been accepted by Jehovah’s Witnesses Perfluorocarbons are synthetic hydrocarbon structures in Could eventually cost less than units of blood which all hydrogen atoms have been replaced with fluorine. They are chemically inert, are excellent gas solvents, and  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 13 Table 1–10 Hemoglobin-Based Oxygen Carriers Product Manufacturer Chemistry/Source History/Status HemAssist (DCLHb) Baxter Diaspirin cross-linked Hgb from First HBOC to advance to phase III clini- outdated human RBCs cal trials in United States. Removed from production because of increased mortality rates. PolyHeme (SFH-P) Northfield Laboratories Polymerized and pyridoxalated Underwent phase II/III clinical trials in human Hgb United States. Did not obtain FDA approval. Hemopure (HBOC-201) HbO2 Polymerized bovine Hgb Still in phase II/III clinical trials in [hemoglobin glutamer – 250 (bovine)] United States and Europe. Approved Therapeutics for use in South Africa (2001) to treat adult surgical patients who are anemic, and in Russia for acute anemia. Oxyglobin HbO2 Polymerized bovine Hgb Approved by the FDA and the European Medicines Agency (EMA) to treat Therapeutics canine anemia in veterinary use. MP4OX Sangart Polyethylene glycol (PEG) attached to In phase II trials in United States; the surface of Hgb from human RBCs phase III in Europe. Hemospan (MP4) Terminated development and opera- tions in December 2013. HemoLink Hemosol Purified human Hgb from outdated Abandoned due to cardiac toxicity. RBCs, cross-linked and polymerized HemoTech HemoBioTech Derived from bovine Hgb Limited clinical trial outside the United States. Table 1–11 Advantages and Disadvantages treatment of traumatic brain injury in Switzerand and Israel.39 Refer to Table 1–12 for further details and review of of Hemoglobin-Based Oxygen PFCs, and Table 1–13 for the advantages and disadvantages Carriers of perfluorochemicals. Advantages Disadvantages Tissue Engineering of RBCs Long shelf-life Short intravascular half-life Very stable Possible toxicity Research into large-scale production of RBCs from stem cells (blood pharming) seems to have more promise and is receiving No antigenicity (unless bovine) Increased O2 affinity more attention and funding than are blood substitutes. RBCs No requirement for blood typing Increased oncotic effect have been cultured in-vitro for many years and have been suc- procedures cessfully tested in animal models. However, there are limita- tions in the number of RBCs that can be cultured from one unit of blood and the associated costs of these expensive cultures. By culturing stem cells in the presence of the essential carry O2 and CO2 by dissolving them. Because of their small cytokines, stem cell factor, and erythropoietin, unilineage size (about 0.2 µm in diameter), they are able to pass production of erythroblasts has been achieved.39 Culture of through areas of vasoconstriction and deliver oxygen to tis- cells in expansion medium and subsequently in maturation sues that are inaccessible to RBCs.39 PFCs have been under medium has shown progress of in-vitro erythroid expansion.39 investigation as possible RBC substitutes since the 1970s. The general consensus for producing RBCs in-vitro is a precul- Fluosol (Green Cross Corp.) was approved by the FDA in ture of hematopoietic stem cells (HSC) for erythroid progenitor 1989 but was removed from the market in 1994 due to clini- cells, with a subsequent generation of high numbers of ery- cal shortcomings and poor sales. Other PFCs have proceeded throblasts. This is followed by a erythroid maturation phase in to clinical trials. Perftoran West Ltd is in clinical use in the presence of a feeder layer to facilitate progression to a ma- Russia and Mexico.39 Two others are no longer under devel- ture RBC.39 Maturation without a feeder layer has also been opment, and one (Oxycyte, Oxygen Biotherapeutics Inc.) is reported and this development is necessary if tissue engineer- currently being investigated as an oxygen therapeutic for ing RBCs is to become used for transfusion therapy.39 14 PART I Fundamental Concepts Table 1–12 Perfluorocarbons Fluosol-DA Green Cross Corporation of Japan The first and only oxygen-carrying blood substitute ever to receive approval from the FDA for human clinical use in the United States. Approved in 1989; discontinued in 1994 because of clinical shortcomings and poor sales. Oxygent Alliance Pharmaceutical Corporation Phase III trial in Europe completed; phase III trial in United States terminated due to adverse effects. Development stopped due to lack of funding. Oxycyte Originally Synthetic Blood International; Shift in research from use as RBC substitute to other medical applications. Cur- name changed to Oxygen Biotherapeutics rently in phase II trials in Switzerland for treatment of traumatic brain injury. in 2008 Perftoran Perftoran Approved for use in Russia and Mexico. PHER-O2 Sanguine Corporation Under evaluation for transfusion, therapy for heart attack and stroke. Table 1–13 Advantages and Disadvantages Maintaining pH was determined to be a key parameter for retaining platelet viability in vivo when platelets were stored of Perfluorochemicals at 20°C to 24°C.41 The loss of platelet quality during storage Advantages Disadvantages is known as the platelet storage lesion. During storage, a varying degree of platelet activation occurs that results in Biological inertness Adverse clinical effects release of some intracellular granules and a decline in ATP Lack of immunogenicity High O2 affinity and ADP. 41 The reduced oxygen tension (pO2) in the plastic platelet Easily synthesized Retention in tissues storage container results in an increase in the rate of glycol- Requirement for O2 administration ysis by platelets to compensate for the decrease in ATP re- when infused generation from the oxidative (TCA) metabolism. This Deep-freeze storage temperatures increases glucose consumption and causes an increase in lac- tic acid that must be buffered. This results in a fall in pH. During the storage of platelet concentrates (PCs) in plasma, the principal buffer is bicarbonate. When the bicarbonate Platelet Preservation buffers are depleted during platelet concentrate storage, the Approximately 2.4 million platelet units are distributed and pH rapidly falls to less than 6.2, which is associated with a 2.2 million platelet transfusions are administered yearly in loss of platelet viability. In addition, when pH falls below 6.2, the United States.3 Platelets are involved in the blood coag- the platelets swell and there is a disk-to-sphere transforma- ulation process and are given to treat or prevent bleeding. tion in morphology that is associated with a loss of mem- They are given either therapeutically to stop bleeding or pro- brane integrity.40 The platelets then become irreversibly phylactically to prevent bleeding. Better availability and swollen, aggregate together, or lyse, and when infused, will management of platelet inventory has been a goal of blood not circulate or function. This change is irreversible when banks for many years. The financial impact of outdated and the pH falls to less than 6.2.40 During storage of platelet con- returned platelet units is the primary reason to find a way to centrates, the pH will remain stable as long as the production improve inventory management. Increasing the storage time of lactic acid does not exceed the buffering capacity of the during platelet preservation is one way to reduce the number plasma or other storage solution. Table 1–14 summarizes of outdated platelet units. With the limit of five days of stor- platelet changes during storage (the platelet storage lesion). age for platelet concentrates, approximately 20% to 30% of It should be noted that except for change in pH, the effect of the platelet inventory is discarded either by the blood sup- in vitro changes on post-transfusion platelet survival and plier or the hospital blood bank.4 function is unknown, and some of the changes may be re- versible upon transfusion.42 The Platelet Storage Lesion Generally, the quality-control measurements required by various accreditation organizations for platelet concentrates Platelet storage still presents one of the major challenges to include platelet concentrate volume, platelet count, pH of the the blood bank because of the limitations of storing platelets. unit, and residual leukocyte count if claims of leukoreduction In the United States, platelets are stored at 20°C to 24°C with are made.43 In addition, immediately before distribution to maintaining continuous gentle agitation throughout the stor- hospitals, a visual inspection is made that often includes an age period of 5 days. Agitation has been shown to facilitate assessment of platelet swirl (no visible aggregation).43 The oxygen transfer into the platelet bag and oxygen consump- absence of platelet swirling is associated with the loss of tion by the platelets. The positive role for oxygen has been membrane integrity during storage, resulting in the loss of associated with the maintenance of platelet component pH.40 discoid shape with irreversible sphering.44 Box 1–4 lists the  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 15 Table 1–14 The Platelet Storage Lesion apheresis (apheresis platelets). Currently, greater than 92% of platelet transfusions are from apheresed platelets and Characteristic Change Observed about 8% are pools of whole blood-derived platelets (WBD).3 Lactate Increased Platelets still remain the primary means of treating throm- bocytopenia, even though therapeutic responsiveness varies pH Decreased according to patient status and platelet storage conditions.43 ATP Decreased (See Chapter 15 for the methods for preparing platelet con- centrates.) One unit of whole blood-derived platelet concen- Morphology scores change Decreased trate contains ≥5.5 × 1010 platelets suspended in 40 to from discoid to spherical 70 mL of plasma.45 These platelets may be provided as a (loss of swirling effect) single unit or as pooled units; however, pooled units only Degranulation Increased have a shelf life of 4 hours. Apheresis platelets contain ((β-thromboglobulin, ≥3.0 × 1011 in one unit which is the therapeutic equivalent platelet factor 4) of 4 to 6 units of whole blood-derived platelets.45 There are Platelet activation markers Increased a number of containers used for 5-day storage of whole (P-selectin [CD62P] or CD63) blood–derived (WBD) and apheresis platelets. Box 1-5 lists the factors to be considered when using 5-day plastic Platelet aggregation Drop in responses to some agonists storage bags.