MLS 323 Module 1 PDF - Immunohematology - AY 2021-2022

MODULE IN IMMUNOHEMATOLOGY MLS 323 Department of Medical...

MODULE IN IMMUNOHEMATOLOGY MLS 323 Department of Medical Laboratory Science SCHOOL of NATURAL SCIENCES Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited.  MLS 323 COURSE LEARNING OUTCOMES At the end of the course, you should be able to: 1. Explain the structure of the red cell membrane with the occurrence or presence of blood group antigens and its impact in blood storage. 2. Apply the concepts of genetics and molecular biology in the inheritance of blood groups and be able to use these principles and laws of inheritance in resolving medico-legal cases. 3. Differentiate the characteristics, structures, functions, and significance of antigens, antibodies, and complement. 4. Assess the characteristics of the immune response with the principles of the antigen – antibody reactions in blood group studies. 5. Discuss the different blood group systems and blood collections in terms of their inheritance, formation, frequencies, types, antigens, antibodies, serologic properties, manner of identification, and clinical significance. 6. Relate blood protocols in the selection of donors, collection, and processing of blood preparation and therapy in terms of principles, purpose, methods, kinds, preparations, storage, issuance, transport, and disposal. 7. Elaborate on the types of transfusion processes and be able to describe the classification, signs and symptoms, and investigations of transfusion reactions. 8. Describe donor phlebotomy accurately taking into consideration proper blood donor care as well as the criteria for a quality blood unit. 9. Explain immunohematological procedures and tests adhering to standards and appropriate safety measures and apply corrective actions to ensure the validity of all immunohematological tests may it be a traditional or a nontraditional method. 10. Explain proper biosafety and waste management when handling specimens in the laboratory. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 1  COURSE INTRODUCTION Dear future Registered Medical Technologists, The COVID-19 pandemic might have limited us physically in terms of being able to go to places, but this should never limit our minds into continuously learning. As we remember our slogan, “Med Lab Sci, Mahusay na tunay!” put this to heart, and if you do, you will surely finish this course with a renewed passion for the program that you have chosen to take. This part of the course introduces the student to the basics of immunohematology starting from the history of how its practice came about up to the topics that a student would need to review in order to relate Immunohematological concepts to the actual practice. From the history of Immunohematology, Module 1 would also cover the general functions of blood paying particular attention to the red blood cell and how it is being used in the practice of Blood Banking. This part of the course would also introduce the students to Laboratory Safety and Quality Control in the Blood Bank as these are important as one starts to perform procedures in the Blood Bank. By the end of this module, the student should be able to identify important contributors and historical events that led to the development of immunohematology and blood transfusion practice, identify the functions of blood giving emphasis on its components and how these could be used for blood transfusion during disease states, relate the structure of the red cell membrane with the occurrence or presence of blood group antigens and its impact in blood storage, differentiate the characteristics, structures, functions, and significance of antigens, antibodies, and complement, and be able to apply these knowledge in characterizing the immune response as well as in illustrating the principles of antigen – antibody reactions in blood group studies. MLS mahusay na tunay! Welcome to the Immunohematology course! Best regards, MLS 323 and MLS 323L Facilitators, AY 2021 – 2022 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 2  To guide you in this module, the following icons are used: This icon “ENGAGES” you to the topic at hand. These means that you are to think/relate/do some activities to get started. This icon “ELABORATES” on the concepts included in the model. This could be in the form of tables, algorithms, and the like. This icon indicates an “EXPLANATION” about the topics in the modules. This icon would indicate that there are questions that you are to answer to provoke critical thinking and to check on your understanding by letting you “ELABORATE” on what you have learned. This is the “EXPLORE” icon. This icon indicates a graded activity. It implies that you are going to perform a lecture or laboratory activity. This icon refers to a reading task. Refer to the exact pages of the prescribed reference when you see this icon. We can give elaboration on some of the concepts which are not explicitly discussed in the module. Also, make it a habit to write your own notes during a reading activity. It helps in retaining concepts and facilitates organization of mental processes. If you see this icon, it means you are required to watch a lecture/ video prepared for you by your instructors. You can always ask questions or clarifications about the contents and discussions. Also, don’t just watch. You also need to take down notes as you read and repeat the video as necessary. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 3  FAIR USE STATEMENT AND DISCLAIMER The modules contained in this course may contain copyrighted material, the use of which may not have been specifically authorized by the owner/s of the copyright. The faculty members have made these materials available in their effort to advance the knowledge of their students in the field of IMMUNOHEMATOLOGY. We believe this constitutes a fair use of any such copyrighted material as provided for in section 107 of the US Copyright Law and the Four Factors of Fair Use which is also observed in the Philippine judicial system. Fair use refers to the right to reproduce, use and share the copyrighted materials without direct permission from or payment to the original copyright holders. The maintenance of fair use protections is used by many non-profit and education projects particularly those operating in digital and online platforms. To emphasize, the materials on these modules are distributed without profit to our students solely for educational purposes only. Thus, if you intend to use any or a part of the modules and the copyrighted materials for personal purposes that go beyond fair use, you must first obtain permission from the owner of the copyright. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 4  Introduction to Immunohematology MODULE OBJECTIVES After finishing this module, you should be able to: 1. List the major developments in the history of transfusion medicine. 2. Identify the functions of blood giving emphasis on its components and how these could be used for blood transfusion during disease states 3. Relate the structure of the red cell membrane with the occurrence or presence of blood group antigens and its impact in blood storage. 4. Describe several biological properties of red blood cells (RBCs) that can affect post- transfusion survival. 5. Define storage lesion (RBC and platelet) and list the associated biochemical changes. 6. Explain the mechanics of red cell and platelet preservation as well as the current trends in preservation research for these two components. MODULE STUDY SCHEDULE: Weeks 1 & 2 (January 17 – 29, 2022) Module Contents Introduction to Immunohematology...................................................................................................................5 MODULE STUDY SCHEDULE: Weeks 1 & 2 (January 17 – 29, 2021)............................................................5 UNIT 1: History of Immunohematology and Blood Transfusion Practice and Future trends..............................6 ENGAGE:...........................................................................................................................................................6 EXPLORE: IMMUNOHEMATOLOGY HISTORY TIMELINE...................................................................................7 EXPLAIN:........................................................................................................................................................ 11 UNIT 2 GENERAL FUNCTIONS OF BLOOD, THE RED BLOOD CELL MEMBRANE, AND RED CELL PRESERVATION.......................................................................................................................................................................... 11 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 5  CHARACTERISTICS AND FUNCTIONS OF EACH COMPONENT....................................................................... 12 RED BLOOD CELL BIOLOGY AND PRESERVATION.......................................................................................... 13 RBC MEMBRANE........................................................................................................................................... 13 HEMOGLOBIN STRUCTURE AND FUNCTION................................................................................................. 16 METABOLIC PATHWAYS................................................................................................................................ 16 HEMOGLOBIN- OXYGEN DISOCIATION CURVE............................................................................................. 17 BLOOD GROUP ANTIGENS AND THE RED CELL MEMBRANE........................................................................ 18 RBC PRESERVATION...................................................................................................................................... 20 ANTICOAGULANT PRESERVATIVE SOLUTIONS.............................................................................................. 21 ADDITIVE SOLUTIONS................................................................................................................................... 21 RBC FREEZING............................................................................................................................................... 22 RBC REJUVENATION...................................................................................................................................... 23 CURRENT TRENDS IN RBC PRESERVATION RESEARCH.................................................................................. 23 UNIT 3: PLATELET PRESERVATION.................................................................................................................... 26 PLATELET STORAGE LESION.......................................................................................................................... 26 CLINICAL USE OF PLATELETS......................................................................................................................... 28 Platelet Testing and Quality Control Monitoring.......................................................................................... 29 Measurement of Viability and Functional Properties of Stored Platelets.................................................... 30 Platelet Storage and Bacterial Contamination.............................................................................................. 30 Pathogen Reduction for Platelets................................................................................................................. 32 Current Trends in Platelet Preservation Research........................................................................................ 32 ELABORATE:.................................................................................................................................................. 33 EVALUATE:.................................................................................................................................................... 33 MAIN REFERENCES/SOURCE MATERIALS......................................................................................................... 33 UNIT 1: History of Immunohematology and Blood Transfusion Practice and Future trends ENGAGE: Before beginning the activity, WRITE THREE WORDS that you can relate with IMMUNOHEMATOLOGY. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 6  Immunohematology refers to the SEROLOGIC, GENETIC, BIOCHEMICAL, and MOLECULAR STUDY of antigens associated with membrane structures on the cellular constituents of blood, as well as the immunologic properties and reactions of blood components and constituents. It is intimately related to transfusion medicine, which represents a section of clinical pathology that involves the transfusion of blood, its components and constituents. Fundamental discoveries in the area of immunohematology have played an integral role in the development of transfusion medicine. Immunohematologists perform and interpret a wide variety of serologic and molecular assays to aid in the diagnosis, prevention, and management of immunization associated with transfusion, pregnancy, and organ transplantation. Over the years, research in the field of immunohematology has contributed significantly to the fundamental understanding of human genetics and immunology, with broad applications to membrane physiology and function, epidemiology, anthropology, and forensic science. EXPLORE: IMMUNOHEMATOLOGY HISTORY TIMELINE 1492: First time blood transfusion was recorded in history: Pope Innocent VIII. In 1492, blood was taken from three young men and given to the stricken Pope Innocent VIII in the hope of curing him; unfortunately, all four died. Although the outcome of this event was unsatisfactory, it is the first time a blood transfusion was recorded in history. 1628: British physician WILLIAM HARVEY discovers the circulation of blood. Shortly afterward, the earliest known blood transfusion was attempted. 1658: JAN SWAMMERDAM observes and describes RED BLOOD CELLS. 1665: First recorded successful blood transfusion occurs in England: Physician Richard Lower keeps dogs alive by transfusion of blood from other dogs. 1667: Jean-Baptiste Denis in France and Richard Lower in England separately report successful transfusions from lambs to humans. Within 10 years, transfusing the blood of animals to humans becomes prohibited by law because of reactions. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 7  1795: In Philadelphia, American physician Philip Syng Physick, performs the first human blood transfusion, although he does not publish this information. 1818: (In Hilyer, 1825) British obstetrician James Blundell performs the first successful transfusion of human blood to a patient for the treatment of POSTPARTUM HEMORRHAGE. Using the patient’s husband as a donor, he extracts approximately four ounces of blood from the husband’s arm, and, using a syringe, successfully transfuses the wife. Between 1825 and 1830, he performs 10 transfusions, 5 of which prove beneficial to his patients, and publishes these results. He also devises various instruments (impellor and gravitator) for performing transfusions and proposed ration indications. Dr. James Blundell (1791-1878) was an English (British) physician. In 1818 James Blundell determined that a blood transfusion would be appropriate to treat a severe hemorrhage. He also discovered the importance of letting all the air out of a syringe prior to the transfusion. 1840: Samuel Armstrong Lane, aided by consultant Dr. Blundell, performs the first successful whole blood transfusion to treat hemophilia 1867: English physician Joseph Lister uses antiseptics to control infection during transfusions 1869: Attempts to find a nontoxic anticoagulant began; BRAXTON HICKS recommended sodium phosphate, a nontoxic anticoagulant- FIRST EXAMPLE OF BLOOD PRESERVATION RESEARCH 1901: Karl Landsteiner, an Austrian physician, discovers the first three human blood groups, A, B, and C. Blood type C was later changed to O. His colleagues Alfred Decastello and Adriano Sturli add AB, the fourth type, Landsteiner receives the Nobel Prize for Medicine for this discovery in 1930. 1907: LUDVIG HEKTOEN suggests that the safety of transfusion might be improved by CROSS MATCHING blood between donors and patients to exclude incompatible mixtures Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 8  REUBEN OTTENBERG performs the first blood transfusion using blood typing and cross-matching 1908: French surgeon Alexis Carrel devises a way to prevent clotting by sewing the vein of the recipient directly to the artery of the donor, vein-to-vein or direct method, known as anastomosis Carlos Moreschi describes the antiglobulin reaction 1912: Roger Lee, a visiting physician at the Massachusetts General Hospital, along with Paul Dudley White, develops the Lee-White clotting time. Lee demonstrates that it is safe to give group O blood to patients of any blood group, and that blood from all groups can be given to group AB patients. The terms "universal donor" and "universal recipient" are coined. 1913: EDWARD LINDEMAN: carried out vein-to-vein transfusion of blood by using multiple syringes and a special cannula for puncturing the vein through the skin. LESTER J. UNGER: Designed the syringe – valve apparatus 1914: ALBERT HUSTIN reported the use of sodium citrate as an anticoagulant 1915: RICHARD LEWISOHN determined the minimum amount of citrate needed for anticoagulation 1916: FRANCIS ROUS AND J.R. TURNER introduced a citrate-glucose solution that permits storage of blood for several days after collection OSWALD ROBERTSON, an American Army officer, is credited with creating the blood depots. Robertson received the AABB Landsteiner Award in 1958 as developer of the first blood bank. 1927-1947: The MNSs and P systems are discovered. MNSs and P are two more blood group antigen systems — just as ABO is one system and Rh is another. 1937: BERNARD FANTUS, director of therapeutics at the Cook County Hospital in Chicago, establishes the first hospital blood bank in the United States. In creating a hospital laboratory that can preserve and store donor blood, Fantus originates the term "blood bank." 1939-1940: The Rh Blood group system is discovered by Karl Landsteiner, Alexander Wiener, Philip Levine and R.E. Stetson Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 9  1940: EDWIN COHN develops cold ethanol fractionation (Albumin, gamma globulin and fibrinogen are isolated and become available for clinical use) JOHN ELLIOTT develops the first blood container 1941: DR. CHARLES DREW: appointed as director of the first AMERICAN Red Cross Blood bank at the Presbyterian Hospital ISODOR RAVDIN, a prominent surgeon from Philadelphia, effectively treats victims of the Pearl Harbor attack with Cohn's albumin for shock 1943: LOUTIT AND MOLLISON of England introduced the formula for the preservative Acid- Citrate Dextrose (ACD) 1943: P. Beeson publishes the classic description of transfusion-transmitted hepatitis. 1945: COOMBS, MOURANT AND RACE describe the use of ANTI-HUMAN GLOBULIN to identify incomplete antibodies 1947: ABO blood-typing and syphilis testing is performed on each unit of blood 1950: AUDREY SMITH reports the use of glycerol CRYOPROTECTANT for red blood cells 1950: Carl Walter and W.P. Murphy, Jr., introduced the plastic bag for blood collection 1953: Development of the refrigerated centrifuge 1957: GIBSON introduced an improved preservative solution called citrate-phosphate- dextrose (CPD) 1961: PLATELET CONCENTRATES are recognized for reducing the mortality from hemorrhage in cancer patients 1964: PLASMAPHERESIS is introduced as a means of collecting plasma for fractionation. 1969: S. Murphy and F. Gardner demonstrate the feasibility of storing platelets at ROOM TEMPERATURE, revolutionizing platelet transfusion therapy. 1970: U.S. blood banks move toward an ALL-VOLUNTEER blood donor system. 1971: Hepatitis B surface antigen (HBsAg) testing of donated blood begins 1972: Apheresis is used to extract one cellular component, returning the rest of the blood to the donor Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 10  1978: FDA requires blood bags to be labeled “paid” or “volunteer” 1981: First Acquired Immune Deficiency Syndrome (AIDS) case reported. 1983: Additive solutions extend the shelf life of red blood cells to 42 days. 1984: Human Immunodeficiency Virus (HIV) identified as cause of AIDS 1985: The first blood-screening test to detect HIV is licensed and quickly implemented by blood banks to protect the blood supply. 1992: Testing of donor blood for HIV-1 and HIV-2 antibodies (anti-HIV-1 and anti-HIV-2) is implemented. 2002: Nucleic acid amplification test (NAT) for HIV and hepatitis C virus (HCV) licensed by the Food and Drug Administration PHILIPPINE LAWS RELATED TO BLOOD BANKING: RA 7719: The National Blood Services Act of 1994 DOH A.O. Series of 1995: Implementing Rules of RA 7719 DOH A.O. No. 2005-002: Rules and Regulations for the Establishment of the Philippine National Blood Services (Amends pertinent provisions of AO No. 9 s. 1995) DOH A.O. No. 2008 – 0008: Rules and Regulations Governing the Regulation of Blood Services Facilities EXPLAIN: UNIT 2 GENERAL FUNCTIONS OF BLOOD, THE RED BLOOD CELL MEMBRANE, AND RED CELL PRESERVATION Knowing the functions of blood is important as this would help you understand how each blood cell is being utilized in Immunohematology. Blood in the body has a volume of four (4) to six (6) liters. It has a pH of 7.35 to 7.45. Functions of blood include: (1) transport of oxygen, carbon dioxide, nutrients, hormones, heat, and metabolic wastes (2) Regulation of pH, body temperature, and water content of cells (3) protection against blood loss through clotting and (4) protection against diseases through phagocytic white blood cells and antibodies. Figure 1 shows the components of a normal adult blood. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 11  CHARACTERISTICS AND FUNCTIONS OF EACH COMPONENT 1. Plasma components a. Plasma proteins i. Albumin: partly responsible for blood viscosity and osmotic pressure; acts as a buffer ii. Globulins: transport of lipids, carbohydrates, hormones and ions such as iron and copper; antibodies and complement are involved in immunity iii. Fibrinogen: functions in blood clotting b. Ions: (Na+, K+, Ca++, Mg++, Cl–, Fe++, PO4, H, OH–, HCO3): involved in osmosis, membrane potentials and acid-base balance c. Nutrients include glucose, amino acids, triacyl glycerol, cholesterol, and vitamins ▪ Promote enzyme activity and serve as sources of energy and basic building blocks of more complex molecules d. Waste products i. Urea, uric acid, creatinine, ammonia salts: breakdown products of protein metabolism, excreted by kidneys ii. Bilirubin: breakdown product of erythrocytes, excreted as part of the bile from the liver into the intestines e. Gases i. O2: necessary for aerobic respiration; terminal electron acceptor in electron transport chain ii. CO2: waste product of aerobic respiration, as HCO3– : helps buffer blood iii. Nitrogen: inert f. Regulatory substances: enzymes catalyze chemical reactions; Hormones stimulate or inhibit many bod functions g. Water: acts as a solvent and suspending medium for blood components Figure 1.Components of Normal Adult Blood Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 12  2. Formed Elements a. Erythrocytes: transport oxygen and carbon dioxide b. Leukocytes i. Neutrophils: phagocytizes microorganisms and other substances ii. Basophils: release histamine which promotes inflammation and heparin which prevents clot formation iii. Lymphocytes: produces antibodies and other chemicals responsible for destroying microorganisms, contribute to allergic reactions, graft rejection, tumor control, and regulation of the immune system iv. Monocytes: phagocytic cells in the blood; they leave the blood and become macrophages which phagocytizes bacteria and dead cells, cell fragments and other debris within tissues v. Platelets: form platelet plugs; release chemicals necessary for blood clotting These components of blood can be used individually or in combination by a blood recipient depending on his/her need. Understanding the blood cells’ functions as to its application in Immunohematology could benefit the millions of blood recipients. READING TASK: Read Pages 1 - 23 of your Textbook: Modern blood banking and transfusion practices (Seventh Edition) RED BLOOD CELL BIOLOGY AND PRESERVATION Three areas of RBC biology are crucial for normal erythrocyte survival and function: 1. Normal chemical composition and structure of the RBC membrane 2. Hemoglobin structure and function 3. RBC metabolism ***Defects in any or all these areas will result in RBCs surviving fewer than the normal 120 days in circulation. RBC MEMBRANE The RBC membrane represents a semipermeable lipid bilayer supported by a mesh-like protein cytoskeleton structure (Figure 2). Both proteins and lipids are organized asymmetrically within the RBC membrane. Lipids are not equally distributed in the two layers of the membrane. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 13  The external layer is rich in glycolipids and choline phospholipids (sphingomyelin and phosphatidylcholine). The internal cytoplasmic layer of the membrane is rich in amino phospholipids (phophatidylethanolamine and phosphatidylserine) o Exposure of phosphatidylserine in the outer leaflet of the erythrocyte plasma membrane increases the cell’s vascular adherence and is a signal for macrophage recognition and phagocytosis. The biochemical composition of the RBC membrane is approximately 52% protein, 40% lipid, and 8% carbohydrate. The normal chemical composition and the structural arrangement and molecular interactions of the erythrocyte membrane are crucial to the normal length of RBC survival of 120 days in circulation. In addition, they maintain a critical role in two important RBC characteristics: deformability and permeability. Figure 2. Schematic Illustration of the RBC membrane Phospholipids, 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 [Examples of integral proteins: Glycophorins A,B,C; Anion exchange channel protein (Band 3)] 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 cytoskeleton. (Examples of peripheral proteins: Spectrin, Actin (band 5), Ankyrin (Band 2.1), Band 4.1 and 4.2, Band 6, Adducin) Name the different integral membrane proteins and the peripheral proteins found in the red cell and state the function of each. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 14  o DEFORMABILITY ▪ To remain viable, normal RBCs must also remain flexible, deformable, and permeable ▪ The loss of adenosine triphosphate (ATP) (energy) levels leads to a decrease in the phosphorylation of spectrin and, in turn, a loss of membrane deformability. ▪ An accumulation or increase in deposition of membrane calcium also results, causing an increase in membrane rigidity and loss of pliability. ▪ The loss of RBC membrane is exemplified by the formation of “spherocytes” (cells with a reduced surface-to-volume ratio) and “bite cells,” in which the removal of a portion of membrane has left a permanent indentation in the remaining cell membrane. The survival of these forms is also shortened. o PERMEABILITY ▪ The permeability properties of the RBC membrane and the active RBC cation transport prevent colloid hemolysis and control the volume of the RBC. ▪ Any abnormality that increases permeability or alters cationic transport may decrease RBC survival. ▪ The RBC membrane is freely permeable to water and anions, also to chloride and bicarbonate; it is relatively 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. The erythrocyte intracellular-to-extracellular ratios for Na+ and K+ are 1:12 and 25:1, respectively. ▪ The active transport of sodium out of the cell and potassium into the cell is an energy-requiring process. ▪ Calcium is also actively pumped out of the red blood cell through energy- dependent calcium-ATPase pumps. Calmodulin is speculated to control these pumps and to prevent excessive intracellular Ca2+ buildup, which changes the shape and makes it more rigid. ▪ When RBCs are ATP- depleted, Ca2+ and Na+ can accumulate intracellularly, and K+ and water are lost, resulting in a dehydrated rigid cell subsequently sequestered by the spleen, resulting in a decrease in RBC survival. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 15  HEMOGLOBIN STRUCTURE AND FUNCTION Hemoglobin comprises 95% of the rbc’s dry weight and 33% of the rbc weight by volume Consists of: a. Globin –Tetramer of 2 pairs of unlike globin polypeptide chains ✓ Embryonic Hemoglobins o Gower I: 22 o Gower II: 22 o Portland: 22 ✓ Normal/ adult Hemoglobins o HbA : 22 chains (95- 97%) o HbA2: 22chains (2 – 3%) o HbF: 22 chains (1 – 2%) b. 4 heme (ferroprotoporphyrin IX) groups –Heme = Protoporphyrin ring + Ferrous iron (Fe2+) FUNCTIONS: (1) Delivery and release of oxygen to tissues (2) Facilitates excretion of carbon dioxide One of the most important controls of hemoglobin affinity for oxygen is the RBC organic phosphate 2,3-DPG. METABOLIC PATHWAYS Considered essential if the erythrocyte is to transport oxygen and to maintain critical physical characteristics for its survival. 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 has no nucleus and there is no mitochondrial apparatus for oxidative metabolism, energy must be generated almost exclusively through the breakdown of glucose Pathways: 1. Embden Meyerhof Glycolytic Pathway/ Glycolysis ✓ generates about 90% of the ATP needed by the RBC. 2. Pentose phosphate pathway (Hexose Monophosphate Shunt)/ Phosphogluconate Pathway ✓ Provides approximately 10% of the ATP needed by the RBC ✓ Produces pyridine nucleotide: NADPH from NADP+ ✓ Reduced glutathione + NADPH → main line of defense against oxidative injury 3. Methemoglobin reductase pathway ✓ Maintain iron in the reduced functional state: ferrous form Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 16  4. Luebering-Rapoport shunt ✓ This pathway permits the accumulation of an important RBC organic phosphate, 2,3-diphosphoglycerate (2,3-DPG). ✓ The amount of 2,3-DPG found within RBCs has a significant effect on the affinity of hemoglobin for oxygen and therefore affects how well RBCs function post- transfusion. Figure 3. Red Cell Metabolism HEMOGLOBIN- OXYGEN DISOCIATION CURVE The unloading of oxygen by hemoglobin is accompanied by widening of a space between β chains and the binding of 2,3-DPG on a mole-for-mole basis, with the formation of anionic salt bridges between the chains. The resulting conformation of the deoxyhemoglobin molecule is known as the tense (T) form, which has a lower affinity for oxygen. When hemoglobin loads oxygen and becomes oxyhemoglobin, the established salt bridges are broken, and β chains are pulled together, expelling 2,3-DPG. This is the relaxed (R) form of the hemoglobin molecule, which has a higher affinity for oxygen. Allosteric changes that occur as the hemoglobin loads and unloads oxygen are referred to as the respiratory movement. The dissociation and binding of oxygen by hemoglobin are not directly proportional to the partial pressure of oxygen (pO2) in its environment but instead exhibit a sigmoid-curve relationship, known as the hemoglobin-oxygen dissociation curve. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 17  Explains the relationship of partial pressure of oxygen and hemoglobin’s affinity for oxygen. Normal shape of oxyhemoglobin dissociation curve: SIGMOIDAL ▪ Low hemoglobin affinity for oxygen at low oxygen tension and high affinity for oxygen at high oxygen tension p50 ▪ Partial pressure of oxygen needed to saturate 50% of hemoglobin ▪ Normal value: 27 mmHg KEY POINTS: ▪ Normal basal state: 25% of the oxygen is released into the tissues ▪ Shift to the right: Results in DECREASED affinity of Hb for oxygen thus leading to an increase in oxygen delivery to the tissues ▪ Shift to the left: Results in INCREASED affinity of Hb for oxygen thus leading to a decrease in oxygen delivery to the tissues. RBCs are much less efficient because only 12% of the oxygen can be released to the tissues. Figure 4. Hemoglobin-Oxygen Dissociation Curve BLOOD GROUP ANTIGENS AND THE RED CELL MEMBRANE Structure and Function of Red Cell Antigens 1. Membrane Transporters o Transfer of biologically important molecules in and out of the cell o Examples: ▪ Diego – anion exchanger ▪ Kidd glycoprotein – urea transporter ▪ Colton glycoprotein – water channel ▪ Gill glycoprotein – water and glycerol ▪ Lan and Junior – ATP-fueled transporters of porphyrin and uric acid ▪ Band 3 – CO2 transporter Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 18  2. Receptors and adhesion molecules o Duffy glycoprotein – chemokine receptor o Members of the immunoglobulin superfamily (IgSF) – glycoprotein antigens of Lutheran, Scianna, Ok o Indian antigen – member of the link module superfamily (adhesion molecule in many tissues) o Raph – may associate with integrin in red cell progenitors 3. Complement regulatory proteins o Belonging to complement control protein superfamily: ▪ Cromer glycoprotein (DAF) ▪ Knops (CR1) 4. Enzymes o Yt glycoprotein – acetylcholinesterase o Kell glycoprotein – endopeptidase o Dombrock glycoprotein – belong to a family of ADP-ribosylransferases 5. Structural components o Maintain shape and integrity of the red cell membrane: part of the cytoskeleton ▪ Band 3 ▪ Glycophorin C ▪ Gerbich ▪ Others: Lutheran, Kx, RHAG o Mutations in the genes involving these can lead to abnormally shaped red cells Figure 5. Model of the RBC membrane components that carry the blood group antigens 6. Components of the glycocalyx o Protects cells from mechanical damage and microbial attack o Band 3 and Glycophorin A (MN antigen) Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 19  RBC PRESERVATION The 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. The U.S. Food and Drug Administration (FDA) requires an average 24-hour post- transfusion RBC survival of more than 75%. In addition, the FDA mandates that red blood cell integrity be maintained throughout the shelf-life of the stored RBCs. This is assessed as free hemoglobin less than 1% of total hemoglobin. To maintain optimum viability, blood is stored in the liquid state between 1°C and 6°C for a specific number of days, as determined by the preservative solution(s) used. The loss of RBC viability has been correlated with the storage lesion, which is associated with various biochemical changes (see table). As RBCs are stored, 2,3-DPG levels decrease, with a shift to the left of the hemoglobin- oxygen dissociation curve, and less oxygen is delivered to the tissues. It is well accepted, however, that 2,3-DPG is re-formed in stored RBCs after in vivo circulation, resulting in restored oxygen delivery. The rate of restoration of 2,3-DPG is influenced by the acid-base status of the recipient, the phosphorus metabolism, the degree of anemia, and the overall severity of the disorder. Despite the biochemical, structural, and functional changes that occur to RBCs during storage, there is no significant difference in rates of death between patients who were transfused with only fresh blood versus those patients who were transfused with the oldest blood available. Table 1. RBC Storage Lesion % CHARACTERISTIC CHANGE OBSERVED % Viable cells Decreased Glucose Decreased ATP Decreased Lactic acid Increased pH Decreased 2,3-DPG Decreased Oxygen dissociation curve Shift to the left (increase in hemoglobin and oxygen affinity; less oxygen delivered to tissues) Plasma K+ Increased Plasma hemoglobin Increased Source: Modern Blood Banking and Transfusion Practices by Harmening (7th Edition). Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 20  ANTICOAGULANT PRESERVATIVE SOLUTIONS Table 2 lists the approved anticoagulant preservative solutions for whole blood and RBC storage at 1°C to 6°C. Table 2. Approved Anticoagulant Preservative Solutions Name Abbreviation Storage Time (Days) Acid-citrare-dextrose (formula A) ACD-A 21 Citrate-phosphate dextrose CPD 21 Citrate-phosphate-double-dextrose CP2D 21 Citrate-phosphate-dextrose-adenine CPDA-1 35 Source: Modern Blood Banking and Transfusion Practices by Harmening (7th Edition). Chemicals in Anticoagulant Solutions 1. Citrate (sodium citrate/citric acid): Chelates calcium; prevents clotting. 2. Monobasic sodium phosphate: Maintains pH during storage; necessary for maintenance of adequate levels of 2,3-DPG. 3. Dextrose: Substrate for ATP production (cellular energy). 4. Adenine: Production of ATP (extends shelf-life from 21 to 35 days). *NOTE: First three items are all found in the anticoagulant preservative solutions except adenine, which is only found in CPDA-1. ADDITIVE SOLUTIONS preserving solutions that are added to the RBCs after removal of the plasma with or without platelets. Additive solutions are now widely used. Currently, four additive solutions are licensed in the United States (all with a storage time of 42 days): 1. Adsol (AS-1; Fenwal Inc.) 2. Nutricel (AS-3; Haemonetics Corporation) 3. Optisol (AS-5; Terumo Corporation) 4. SOLX (AS-7; Haemonetics Corporation) The additive solution is contained in a satellite bag and is added to the RBCs after most of the plasma has been expressed. All three additives contain saline, adenine, and glucose. AS-1, AS-5, and AS-7 also contain mannitol, which protects against storage-related hemolysis, whereas AS-3 contains citrate and phosphate for the same purpose. All of the additive solutions are approved for 42 days of storage for packed RBCs. Benefits of RBC Additive Solutions: o Extends the shelf-life of RBCs to 42 days by adding nutrients o Allows for the harvesting of more plasma and platelets from the unit o Produces a packed RBC of lower viscosity that is easier to infuse Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 21  RBC FREEZING RBC 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 to meet their needs for blood transfusion. It involves the addition of a cryoprotective agent to RBCs that are less than 6 days old. o Glycerol is used most commonly and is added to the RBCs slowly with vigorous shaking, thereby enabling the glycerol to permeate the RBCs. o The cells are then rapidly frozen and stored in a freezer. o The usual storage temperature is below –65°C, although storage (and freezing) temperature depends on the concentration of glycerol used. o Two concentrations of glycerol have been used to freeze RBCs: ▪ high-concentration glycerol (40% weight in volume [wt/vol]) ▪ low-concentration glycerol (20% wt/vol) in the final concentration of the cryopreservative. Currently, the FDA licenses frozen RBCs for a period of 10 years from the date of freezing; that is, frozen RBCs may be stored up to 10 years before thawing and transfusion. Once thawed, these RBCs demonstrate function and viability near those of fresh blood. Table 3 lists the advantages of the high-concentration glycerol technique in comparison with the low-concentration glycerol technique. Table 3: Advantages of High-Concentration Glycerol Technique Over Low Concentration Glycerol Technique Advantages 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 Can be thawed and Critical temperature refrozen Source: Modern Blood Banking and Transfusion Practices by Harmening (7th Edition). Transfusion of frozen cells must be preceded by a deglycerolization process; otherwise, the thawed cells would be accompanied by hypertonic glycerol when infused, and RBC lysis would result. o Removal of glycerol is achieved by systematically replacing the cryoprotectant with decreasing concentrations of saline. o The usual protocol involves washing with 12% saline, followed by 1.6% saline, with a final wash of 0.2% dextrose in normal saline. Advantages of RBC Freezing: o Long-term storage (10 years) o Maintenance of RBC viability and function o Low residual leukocytes and platelets o Removal of significant amounts of plasma proteins Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 22  Disadvantages of RBC Freezing: o A time-consuming process o Higher cost of equipment and materials o Storage requirements (–65°C) o Higher cost of product RBC REJUVENATION Rejuvenation of RBCs is the process by which ATP and 2,3- DPG levels are restored or enhanced by metabolic alterations. Currently, FDA-approved rejuvenation solution contains phosphate, inosine, and adenine. Rejuvenated RBCs may be prepared up to three days after expiration when stored in CPD, CPDA-1, and AS-1 storage solutions. Currently, rejuvenated RBCs must be washed before infusion to remove the inosine (which may be toxic) and transfused within 24 hours or frozen for long-term storage. The rejuvenation process is expensive and 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. CURRENT TRENDS IN RBC PRESERVATION RESEARCH Research and development in RBC preparation and preservation is being pursued in five areas: 1. Development of improved additive solutions ✓ Research is being conducted to develop improved additive solutions for RBC preservation. ✓ One reason for this is because longer storage periods could improve the logistics of providing RBCs for clinical use. 2. Development of procedures to reduce and inactivate the level of pathogens that may be in RBC units ✓ Research is being conducted to develop procedures that would reduce the level of or inactivate residual viruses, bacteria, and parasites in RBC units. ✓ Areas of concern that must be addressed before pathogen inactivation technologies are approved for use with RBCs in the United States are potential toxicity, immunogenicity, cellular function, and cost. ✓ Currently, the FDA has not approved any Pathogen Reduction Technology (PRT) for use with RBCs 3. Development of procedures to convert A, B, and AB type RBCs to O type RBCs ✓ The use of enzymes that remove the carbohydrate moieties of the A and B antigens is the mechanism for forming O type RBCs ✓ The enzymes are removed by washing after completion of the reaction time. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 23  4. Development of methods to produce RBCs through bioengineering (blood pharming) ✓ Creating RBCs in the laboratory (blood pharming) is another area of research that has the potential to increase the amount of blood available for transfusion. ✓ In 2008, the Defense Advanced Research Projects Agency (DARPA) awarded Arteriocyte, a bioengineering company, a contract to develop a system for producing O-negative RBCs on the battlefield. ▪ The company, which uses proprietary technology (NANEX) to turn hematopoietic stem cells (HSCs) from umbilical cords into type O, Rh-negative RBCs, sent its first shipment of the engineered blood to the FDA for evaluation in 2010. ✓ FDA approval is required before human trials can begin. ✓ Cultured RBCs generated from in vitro hematopoietic stem cells has been reported as well. ✓ However, this has not proven practical for routine transfusion. ✓ The challenges associated with blood pharming are scalability or large-scale production and cost-effectiveness. 5. Development of RBC substitutes ✓ Scientists have been searching for a substitute for blood for over 150 years. ✓ Blood substitutes continue to be of interest because of their potential to alleviate shortages of donated blood. ✓ Today the search continues for a safe and effective oxygen carrier that could eliminate many of the problems associated with blood transfusion, such as the need for refrigeration, limited shelf-life, compatibility, immunogenicity, transmission of infectious agents, and shortages. ✓ Potential Benefits of Artificial Oxygen Carriers ▪ Abundant supply ▪ Readily available for use in prehospital settings, battlefields, and remote locations ▪ Can be stockpiled for emergencies and warfare ▪ No need for typing and crossmatching ▪ Available for immediate infusion ▪ Extended shelf-life (1 to 3 years) ▪ Can be stored at room temperature ▪ Free of bloodborne pathogens ▪ At full oxygen capacity immediately ▪ Do not prime circulating neutrophils, reducing the incidence of multiorgan failure ▪ Can deliver oxygen to tissue that is inaccessible to RBCs ▪ Have been accepted by Jehovah’s Witnesses ▪ Could eventually cost less than units of blood ✓ Current research on blood substitutes is focused on two areas: hemoglobin-based oxygen carriers (HBOCs) and perfluorocarbons (PFCs). ✓ Despite years of research, RBC substitutes are still not in routine use today. ▪ South Africa, Mexico, and Russia are the only countries in which blood substitutes are approved for clinical use. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 24  ✓ Hemoglobin-Based Oxygen Carriers (HBOC) ▪ Early trauma trials with HemAssist® (BAXTER), Hemopure® (HbO2Therapeutics), and PolyHeme® (NORTHFIELD Laboratories) for resuscitating hypotensive shock all failed due to the safety concerns of cardiac issues and increased mortality. ▪ Many HBOCs have been researched; however, the majority have been discontinued due to complications of cardiac toxicity, gastrointestinal distress, neurotoxicity, renal failure, and increased mortality. ▪ Advantages of HBOCs Long shelf-life Very stable No antigenicity (unless bovine) No requirement for blood typing procedures ▪ Disadvantage of HBOCs Short intravascular half-life Possible toxicity Increased O2 affinity Increased oncotic effect ✓ Perfluorocarbons ▪ Perfluorocarbons are synthetic hydrocarbon structures in which all hydrogen atoms have been replaced with fluorine. ▪ They are chemically inert, are excellent gas solvents, and carry O2 and CO2 by dissolving them. ▪ Because of their small size (about 0.2 μm in diameter), they are able to pass through areas of vasoconstriction and deliver oxygen to tissues that are inaccessible to RBCs. ▪ Advantages of Perfluorocarbons Biological inertness Lack of immunogenicity Easily synthesized ▪ Disadvantages of Perfluorocarbons Adverse clinical effects High O2 affinity Retention in tissues Requirement for O2 administration when infused Deep-freeze storage temperatures ✓ Tissue Engineering of RBCs ▪ Research into large-scale production of RBCs from stem cells (blood pharming) seems to have more promise and is receiving more attention and funding than are blood substitutes. ▪ RBCs have been cultured in-vitro for many years and have been successfully tested in animal models. ▪ However, there are limitations in the number of RBCs that can be cultured from one unit of blood and the associated costs of these expensive cultures. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 25  UNIT 3: PLATELET PRESERVATION Approximately 2.4 million platelet units are distributed, and 2.2 million platelet transfusions are administered yearly in the United States. Platelets are involved in the blood coagulation process and are given to treat or prevent bleeding. They are given either therapeutically to stop bleeding or prophylactically to prevent bleeding. Better availability and management of platelet inventory has been a goal of blood banks for many years. The financial impact of outdated and returned platelet units is the primary reason to find a way to improve inventory management. Increasing the storage time during platelet preservation is one way to reduce the number of outdated platelet units. With the limit of five days of storage for platelet concentrates, approximately 20% to 30% of the platelet inventory is discarded either by the blood supplier or the hospital blood bank. PLATELET STORAGE LESION Platelet storage still presents one of the major challenges to the blood bank because of the limitations of storing platelets. In the United States, platelets are stored at 20°C to 24°C with maintaining continuous gentle agitation throughout the storage period of 5 days. 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 20°C to 24°C. The loss of platelet quality during storage is known as the platelet storage lesion. During storage, a varying degree of platelet activation occurs that results in release of some intracellular granules and a decline in ATP and ADP. The reduced oxygen tension (pO2) in the plastic platelet storage container results in an increase in the rate of glycolysis by platelets to compensate for the decrease in ATP regeneration from the oxidative (TCA) metabolism. This increases glucose consumption and causes an increase in lactic 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 buffers are depleted during platelet concentrate storage, the pH rapidly falls to less than 6.2, which is associated with a loss of platelet viability. In addition, when pH falls below 6.2, the platelets swell and there is a disk-to-sphere transformation in morphology that is associated with a loss of membrane integrity. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 26  The platelets then become irreversibly swollen, aggregate together, or lyse, and when infused, will not circulate or function. This change is irreversible when the pH falls to less than 6.2. During storage of platelet concentrates, the pH will remain stable as long as the production of lactic acid does not exceed the buffering capacity of the plasma or other storage solution. Table 4 summarizes platelet changes during storage (the platelet storage lesion). Table 4. The Platelet Storage Lesion Characteristic Change Observed Lactate Increased pH Decreased ATP Decreased Morphology scores change from discoid to spherical (loss of Decreased swirling effect) Degranulation [(β-thromboglobulin, platelet factor 4)] Increased Platelet activation markers (P-selectin [CD62P] or CD63) Increased Platelet aggregation Drop in responses to some agonists Source: Modern Blood Banking and Transfusion Practices by Harmening (7th Edition). It should be noted that except for change in pH, the effect of in vitro changes on post- transfusion platelet survival and function is unknown, and some of the changes may be reversible upon transfusion. Generally, the quality-control measurements required by various accreditation organizations for platelet concentrates include platelet concentrate volume, platelet count, pH of the unit, and residual leukocyte count if claims of leukoreduction are made. In addition, immediately before distribution to hospitals, a visual inspection is made that often includes an assessment of platelet swirl (no visible aggregation). The absence of platelet swirling is associated with the loss of membrane integrity during storage, resulting in the loss of discoid shape with irreversible sphering. In vitro platelet assays that have been correlated with in vivo survival: o pH o Shape change o Hypotonic shock response o Lactate production o pO2 Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 27  CLINICAL USE OF PLATELETS Platelet components are effectively used to treat bleeding associated with thrombocytopenia, a marked decrease in platelet number, or dysfunctional platelets. That is, platelets are transfused when there is a quantitative or qualitative defect with the patient’s platelets. The efficacy of the platelet transfusion is usually estimated from the corrected count increment (CCI) of platelets measured after transfusion. ✓ The corrected count increment (CCI) is a calculated measure of patient response to platelet transfusion that adjusts for the number of platelets infused and the size of the recipient, based upon body surface area (BSA). (Detailed discussion of the CCI will be done during the Final Term). Today, platelets are prepared as concentrates from whole blood (whole blood-derived platelet concentrates) and by apheresis (apheresis platelets). Currently, greater than 92% of platelet transfusions are from apheresed platelets and about 8% are pools of whole blood-derived platelets (WBD). Platelets still remain the primary means of treating thrombocytopenia, even though therapeutic responsiveness varies according to patient status and platelet storage conditions. One unit of whole blood-derived platelet concentrate contains ≥ 5.5 × 1010 platelets suspended in 40 to 70 mL of plasma. These platelets may be provided as a single unit or as pooled units; however, pooled units only have a shelf life of 4 hours. Apheresis platelets contain ≥ 3.0 × 1011 in one unit which is the therapeutic equivalent of 4 to 6 units of whole blood-derived platelets. There are a number of containers used for 5-day storage of whole blood–derived (WBD) and apheresis platelets. Factors to be considered when using 5-day plastic storage bags: ✓ Temperature control of 20°C to 24°C is critical during platelet preparation and storage. ✓ Careful handling of plastic bags during expression of platelet-poor plasma helps prevent the platelet button from being distributed and prevents removal of excess platelets with the platelet-poor plasma. ✓ Residual plasma volumes recommended for the storage of platelet concentrates from whole blood (45 to 65 mL). ✓ For apheresis platelets, the surface area of the storage bags needs to allow for the number of platelets that will be stored. Additive solutions may be used for storage of apheresis platelets. In the United States, platelets are being stored in a 100% plasma medium, unless a platelet additive solution is used. Two platelet additive solutions (PAS) are FDA approved, PAS-C (Intersol) and PAS-F (Isoplate), for the storage of apheresis platelets for 5 days. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 28  The PASs are designed to support platelets during storage in reduced amounts of residual plasma. With the addition of a PAS, residual plasma is reduced to 35% with both InterSol and Isoplate. One advantage is that this approach provides more plasma for fractionation. In addition, there are data indicating that optimal additive solutions may improve the quality of platelets during storage, reduce adverse effects associated with transfusion of plasma, and promote earlier detection of bacteria. Advantages of using a platelet additive solution for platelet storage ✓ Optimizes platelet storage in vitro ✓ Saves plasma for other purposes (e.g., transfusion or fractionation) ✓ Facilitates ABO-incompatible platelet transfusions ✓ Reduces plasma-associated transfusion side effects, such as febrile and allergic reactions, and may reduce risk of transfusion-related acute lung injury (TRALI) ✓ Improves effectiveness of photochemical pathogen reduction technologies ✓ Potentially improves bacterial detection Platelet Testing and Quality Control Monitoring For component testing, the FDA Guidance document recommends: 1. Actual platelet yield (volume × platelet count) must be determined after each platelet collection 2. Weight/ volume conversion is necessary to determine the volume of each platelet collection 3. Bacterial contamination testing as specified by the storage container manufacturer.45 In terms of Quality Control (QC), the FDA recommend the following as a part of your QC: o “Define a plan for nonselectively identifying collections to be tested. ▪ This should ensure testing of components collected on each individual automated blood cell separator device, each collection type, and each location. o “Define sampling schemes for actual platelet yield (including volume determination) and pH, and residual WBC.” ▪ The platelet yield of the collection and designation of single, double, or triple PC should be made prior to performing the residual WBC count QC. o “Test actual platelet yield (platelet count times the volume) and pH at the maximum allowable storage time for the container system used (or representing the dating period).” ▪ “In addition, actual platelet yield and pH testing may be conducted on one storage container of a double or triple collection.” o “Include the residual WBC count for leukocyte-reduced collections, if manufacturing leukocyte-reduced products.” o “Describe the criteria for investigation of failures during QC and have a method to document all calculations and test results. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 29  Table 5 lists the Performance Criteria for Platelet Concentrate Collections. Table 5. Performance Criteria for Platelet Concentrate Collections. TEST RECOMMENDED RESULT Actual platelet yield of transfusable component ≥ 3.0 × 1011 pH ≥ 6.2 Percent component retention ≥ 85% component retention if performed Residual WBC count Single collection: < 5.0 × 106 Double collection: Collection: < 8.0 × 106 or Components: < 5.0 × 106 Triple collection: Collection: < 1.2 × 107 or Components: < 5.0 × 106 Source: Modern Blood Banking and Transfusion Practices by Harmening (7th Edition). Measurement of Viability and Functional Properties of Stored Platelets Viability indicates the capacity of platelets to circulate after infusion without premature removal or destruction. Platelets have a life span of 8 to 10 days after release from megakaryocytes. Storage causes a reduction in this parameter, even when pH is maintained. Viability of stored platelets is determined by measuring pretransfusion and post- transfusion platelet counts and expressing the difference based on the number of platelets transfused (CCI). The observation of the swirling phenomenon (absence of aggregation) caused by discoid platelets when placed in front of a light source has been used to obtain a semiqualitative evaluation of the retention of platelet viability properties in stored units. The extent of shape change and the hypotonic shock response in in vitro tests appears to provide some indication about the retention of platelet viability properties. The maintenance of pH during storage at 20°C to 24°C has been associated with the retention of posttransfusion platelet viability and has been the key issue that has been addressed to improve conditions for storage at this temperature. Retaining platelet function during storage is also an issue. Function is defined as the ability of viable platelets to respond to vascular damage in promoting hemostasis. Platelet Storage and Bacterial Contamination The major concern associated with storage of platelets at 20°C to 24°C is the potential for bacterial growth if the prepared platelets contain bacteria because of contamination at the phlebotomy site or if the donor has an unrecognized bacterial infection. Property of and for the exclusive use of SLU. Reproduction, storing in a retrieval system, distributing, uploading or posting online, or transmitting in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise of any part of this document, without the prior written permission of SLU, is strictly prohibited. 30  Environmental contamination during processing and storage is another potential, though less common, source of bacteria. Room temperature storage and the presence of oxygen provide a good environment for bacterial proliferation. Sepsis due to contaminated platelets is the most common infectious complication of transfusion. Commercial systems such as BacT/ALERT (bioMérieux) and eBDS (Pall Corp.) have been approved by the FDA for screening platelets for bacterial contamination. o These are both culture-based systems. o As the level of bacteria in the platelets at the time of collection can be low, samples are not taken until after at least 24 hours of storage. o This provides time for any bacteria present to replicate to detectable levels. o BacT/ALERT measures bacteria by detecting a change in carbon dioxide levels associated with bacterial growth. ▪ This system provides continuous monitoring of the platelet sample– containing culture bottles, which are held for the shelf-life of the platelet unit or until a positive re