L01 - RBC and Platelet Preservation PDF

9/20/2024 Chapter 1: Red Blood Cell and Platelet Preservation Preamble ▪ PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. – PowerPoints DO NOT cover the details needed for the Unit exam ▪ Eac...

9/20/2024 Chapter 1: Red Blood Cell and Platelet Preservation Preamble ▪ PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. – PowerPoints DO NOT cover the details needed for the Unit exam ▪ Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES ▪ Unit Objectives are your study guide (not this PowerPoint) ▪ Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  9/20/2024 Introduction ▪ The fascination with blood and historical events leading to the current status of how blood is stored will be presented. ▪ A review of red blood cell (RBC) biology serves as a building block for the discussion of red blood cell preservation. ▪ A brief description of platelet metabolism sets the stage for reviewing the platelet storage lesion. Historical Overview ▪ In 1492, first recorded blood transfusion involving Pope Innocent VII ▪ Obstacles to overcome in transfusion therapy – A nontoxic anticoagulant – Appropriate devices to perform the transfusion – Appropriate preservative solutions – Avoiding circulatory overload – Component therapy 2  9/20/2024 Current Status ▪ Efforts and standards of the American Association of Blood Banks (AABB) ▪ General requirements for collection of blood from volunteer donors ▪ Components prepared from donated whole blood The Donation Process 1. Educational materials 2. Donor health history questionnaire 3. Abbreviated physical examination 3  9/20/2024 RBC Biology and Preservation ▪ Three areas of red blood cell (RBC) biology are crucial for normal survival and function. – RBC membrane – Hemoglobin structure and function – RBC metabolism ▪ Defects in any or all of these areas will result in RBC survival of fewer than the normal 120 days in circulation. RBC Membrane ▪ Represents a semipermeable lipid bilayer supported by a protein meshlike cytoskeleton structure ▪ Phospholipids and their orientation ▪ Integral and peripheral proteins ▪ Membrane deformability 4  9/20/2024 RBC Membrane (cont’d) ▪ Asymmetrical organization – External layer - glycolipids and choline phospholipids. – Internal cytoplasmic layer - amino phospholipids. ▪ Biochemical composition of the RBC – 52% protein – 40% lipid – 8% carbohydrate Deformability ▪ Loss of ATP = decreased phosphorylation of spectrin required for membrane deformability. ▪ Increases membrane calcium = membrane rigidity. ▪ Cells are sequestered by the spleen. 5  9/20/2024 Deformability (cont’d) The loss of RBC membrane, as is seen in spherocytes and bite cells, shortens the survival of these forms. Permeability ▪ Properties of the RBC membrane and the active RBC cation transport prevent colloid hemolysis and control the volume of the RBCs. ▪ Permeable to water and anions (Cl-; HCO3-) ▪ Impermeable to cations (Na+ ; K+) ▪ Calcium (Ca2+) is also actively pumped from the interior of the RBC ▪ ATP’s are needed to keep Na+ and Ca+ out of cell ▪ Storage depletes ATP’s; Na+ and Ca+ are allowed to accumulate intracellularly, and K+ and water are lost ▪ Cell becomes rigid 6  9/20/2024 Metabolic Pathways ▪ The RBC’s metabolic pathways that produce ATP are mainly anaerobic. – RBCs—anucleate and have no mitochondrial apparatus for oxidative metabolism – Importance of the anaerobic glycolytic pathway - 90% of ATP – Three ancillary pathways that serve to maintain the structure and function of hemoglobin 1. Pentose Pathway -10% of ATP 2. Methemoglobin Pathway – Affects RBC survival and function after transfusion 3. Luebering-Rapaport Pathway – permits accumulation of 2,3-DPG Hemoglobin Oxygen Dissociation Curve ▪ Hemoglobin’s role in oxygen delivery to the tissues and carbon dioxide excretion ▪ A sigmoid-curve relationship 7  9/20/2024 Hemoglobin Oxygen Dissociation Curve (cont’d) ▪ Allosteric changes occur as the hemoglobin loads and unloads oxygen ▪ Role of the RBC organic phosphate 2,3-DPG ▪ Shift to the right – Hypoxia – Increase in 2,3-DPG (organic phosphate) which increases the Hgb to release more O2 ▪ Shift to the left - Alkalosis – Decrease in 2,3-DPG causes less O2 released RBC Preservation ▪ The goal of blood preservation is to provide viable and functional blood components for patients requiring blood transfusion. ▪ Viability of RBCs must be maintained during the storage time. ▪ Food and Drug Administration requirements – Average 24-hour posttransfusion RBC survival of more than 75% – Free hemoglobin less than 1% of total hemoglobin 8  9/20/2024 RBC Preservation ▪ RBC viability – Assessment of posttransfusion RBC survival – The loss of RBC viability has been correlated with the “lesion of storage,” which is associated with various biochemical changes – Influence of 2,3-DPG levels ▪ As RBCs are stored, 2,3-DPG levels decreases. ▪ DPG-depleted RBCs may have an impaired capacity to deliver oxygen to the tissues. RBC Preservation ▪ 2,3-DPG is re-formed in stored RBCs after in vivo circulation depending on the recipeints acid-base status, phosphorus metabolism, degree of anemia, and the overall severity of the disorder. ▪ Pathophysiologic effects of the transfusion of RBCs with low 2,3-DPG levels and increased affinity for oxygen – Increase in cardiac output – Decrease in mixed venous (pO2) tension – Combination of these 9  9/20/2024 Anticoagulant Preservative Solutions ▪ Incorporation of adenine and its effects on glycolysis and ATP levels – Increases ADP levels which in turn increases ATP levels – Allows for blood storage at 1-6° C for up to 21 days – See Table 1-3 for approved anticoagulant preservative solutions ▪ Plastic material used for storage bags – Must allow for permeability of CO2 in order to maintain higher pH levels during storage. ▪ Effects of the PVC bags relates to the plasticizer, di(ethylhexyl)-phthalate (DEHP), which is used in the manufacture of the bags. – DEHP leaches from the plastic into the lipids of the plasma medium and RBC membranes – Seems to stabilize the RBC membrane and reduces hemolysis in storage Additive Solutions ▪ Additive solutions (AS) are preserving solutions that are added to the RBCs after removal of the plasma with/without platelets. – Effects on RBC viability – Effects on viscosity of RBC concentrates ▪ Currently, three additive solutions are licensed in the United States. – Adsol (AS-1) (Baxter Healthcare) – Nutricel (AS-3) (Pall Corporation) – Optisol (AS-5) (Terumo Corporation) ▪ All of the additive solutions are approved for 42 days of storage for packed RBCs. ▪ None of the additive solutions maintain 2,3-DPG throughout the storage time. ▪ As with RBCs stored only with primary anticoagulant preservatives, 2,3-DPG is depleted by the second week of storage. 10  9/20/2024 RBC Freezing ▪ Primarily used for autologous units and the storage of rare blood types – A cryoprotective agent is added to RBCs that are less than 6 days old. – Glycerol (40% or 20% w/v) is used most commonly and is added to the RBCs slowly with vigorous shaking, enabling the glycerol to permeate the RBCs. ▪ RBCs are then rapidly frozen and stored at -65°C. ▪ Currently, the FDA licenses frozen RBCs for a period of 10 years from the date of freezing. RBC Freezing ▪ Transfusion of frozen cells must be preceded by a deglycerolization process. ▪ Removal of glycerol is achieved by systematically replacing the cryoprotectant with decreasing concentrations of saline. ▪ Excessive hemolysis is monitored with the hemoglobin concentration of the wash supernatant. ▪ Osmolality of the unit should also be monitored to ensure adequate deglycerolization. 11  9/20/2024 RBC Rejuvenation ▪ Rejuvenation of RBCs is the process by which ATP and 2,3-DPG levels are restored or enhanced by metabolic alterations. ▪ RBCs stored in the liquid state can be rejuvenated at outdate or up to 3 days after outdate, depending on RBC preservative solutions used. Research and Development in RBC Preparation and Preservation ▪ Improved additive solutions ▪ Procedures to reduce and inactivate pathogens ▪ Procedures to convert A-, B-, and AB-type RBCs to O-type RBCs ▪ Methods to produce RBCs through bioengineering (blood pharming) 12  9/20/2024 Research and Development in RBC Preparation and Preservation (2 of 3) ▪ RBC substitutes – Will provide safe and effective oxygen carrier that could eliminate many of the problems associated with blood transfusion. ▪ Ensuring product safety ▪ RBC substitutes – Hemoglobin-Based Oxygen Carriers ▪ RBC substitutes – Perfluorocarbons Research and Development in RBC Preparation and Preservation (3 of 3) ▪ Tissue engineering of RBCs – Receiving more attention and funding than are blood substitutes. – Produced by culturing stem cells in the presence of the essential cytokines stem cell factor and erythropoietin. 13  9/20/2024 Platelet Preservation ▪ Annually in the U.S., millions of platelet units are distributed and transfused. ▪ The financial impact of outdated and returned platelet units is the primary reason to find a way to improve inventory management. ▪ Platelet preservation is one way to reduce the number of outdated, discarded platelet units. Platelet Preservation (2 of 3) ▪ Platelet storage is a major challenge to the blood bank because of storage limitations. – 5-day shelf life in the United States – Bacterial contamination at incubation of 22°C – A varying degree of platelet activation/aggregation – Release of intracellular granules – A decline in ATP and ADP levels ▪ Platelet storage lesion 14  9/20/2024 Platelet Preservation (3 of 3) ▪ Quality control measurements typically required include – Platelet concentrate volume – Platelet count – pH of the unit – Residual leukocyte count if claims of leukoreduction are made – Platelet swirl assessment Clinical Use of Platelets ▪ Treatment of bleeding associated with thrombocytopenia ▪ Prophylactic treatment for hematology-oncology patients with thrombocytopenia ▪ Today, platelets are prepared as concentrates from whole blood and increasingly by apheresis. ▪ The efficacy of the transfused platelet concentrates is usually estimated from the corrected count increment (CCI) of platelets measured after transfusion. 15  9/20/2024 Clinical Use of Platelets ▪ Platelet concentrates prepared from whole blood and apheresis components are routinely stored at 20° to 24°C, with continuous agitation for up to 5 days. ▪ FDA standards define the expiration time as midnight of day 5. ▪ In the United States, platelets are being stored in a 100% plasma medium, unless a platelet additive solution is used Clinical Use of Platelets ▪ Maintaining pH was a key parameter for retaining platelet viability in vivo when platelets were stored at 20° to 24°C. ▪ pH 6.2 is the current standard for maintaining satisfactory platelet viability. ▪ Second generation storage containers have increased gas transport properties. 16  9/20/2024 Platelet Testing and Quality Control Monitoring ▪ Actual platelet yield ▪ Weight/volume conversion is necessary to determine the volume of each platelet collection ▪ Bacterial contamination testing Measurement of Viability and Functional Properties of Stored Platelets ▪ Pretransfusion and posttransfusion platelet counts at 1 hour and/or 24 hours and expressing the difference based on the number of platelets transfused (corrected count increment) to assess platelet viability ▪ Room temperature–stored vs. cold-stored platelets 17  9/20/2024 Platelet Storage and Bacterial Contamination ▪ Major concerns associated with storage of platelets at 20° to 24°C is the potential for bacterial growth – Room temperature storage and the presence of oxygen can encourage bacterial proliferation – Sepsis due to contaminated platelets is the most common infectious complication of transfusion – An estimated 10% to 40% of patients transfused with a bacterially contaminated platelet unit develop life-threatening sepsis ▪ In 2002, the CAP added a requirement for laboratories to have a method to screen platelets for bacterial contamination. ▪ AABB introduced a similar requirement in 2004. Platelet Storage and Bacterial Contamination ▪ Three FDA-approved commercial systems for screening platelets for bacterial contamination – BacT/ALERT (bioMerieux, Durham, NC) – eBDS (Pall Corp., East Hills, NY) – Scansystem (Hemosystem, Marseilles, France) ▪ Samples are not taken until after at least 24 hours of storage to allow bacterial replication to detectable levels ▪ Potentially effects availability of platelets 18  9/20/2024 Pathogen Reduction for Platelets ▪ Pathogen inactivation (PI) is the process of treating the blood component. ▪ Components are referred to as being pathogen reduced (PR). ▪ Pathogen inactivation technology reduces the risk of transfusion-transmitted infections. Current Trends in Platelet Preservation Research ▪ Methods that would allow platelets to be stored for 7 days ▪ Additive solutions, also termed synthetic media ▪ Procedures to reduce and inactivate the level of pathogens that may be in platelet units 19  9/20/2024 Current Trends in Platelet Preservation Research ▪ Better platelet additive solutions ▪ Procedures to reduce and inactivate the level of pathogens that may be in platelet units ▪ Platelet substitutes ▪ New approaches for storage of platelets at 1°C to 6°C ▪ Cryopreservation Current Trends in Platelet Preservation Research ▪ Platelet Substitutes – Lyophilization – freeze-drying of trehalose-loaded platelets ▪ Frozen Platelets – cryopreservative dimethyl sulfoxide (DMSO) – stored for up to 2 years 20  9/20/2024 Postamble ▪ READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. ▪ USE THE UNIT OBJECTIVES AS A STUDY GUIDE ▪ All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 21