Biopreservation: Extension of the ex vivo Life Span of Stored Human Erythrocytes by the Addition of Ascorbic Acid to Additive Solutions in Modern Blood Banking DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jorge Andrés Fontes Graduate Program in Chemical and Biomolecular Engineering The Ohio State University 2014 Dissertation Committee: Andre Francis Palmer, Advisor Jeffery J. Chalmers David W. Wood Copyright by Jorge Andrés Fontes 2014 Abstract The main goal of the research discussed in this dissertation is to extend the life span of erythrocytes in hypothermic storage. This dissertation focuses on the modification of the composition of modern additive solutions by the addition of an antioxidant, ascorbic acid (AA), to extend both the absolute and effective shelf life of red blood cells (RBCs). The research focuses on the analysis of the biochemical and biophysical changes that occur during hypothermic storage of RBCs (i.e. storage lesion). It is focused on the protecting effect of the addition of AA against the slow oxidative damage incurred by reactive oxygen species (ROS) formed during hypothermic storage. This dissertation presents in vitro analyses of both human (Chapter 3) and canine (Chapter 4) erythrocytes stored in AA-supplemented additive solutions. The analyses described (Chapter 2) are quantifications of the functional status of stored RBCs during hypothermic storage. The methods described demonstrate that important in vitro results that could translate to increased shelf life, viability of RBCs after transfusion, and most importantly, safety for the recipient of stored RBCs. This cost effective chemical is used broadly and approved by the United States Food and Drug Administration (FDA). The incorporation of this small molecule antioxidant into modern additive solutions involves essentially no change in blood banking procedure or infrastructure. Alternative methodology for the extension of RBC shelf life include desiccation, cryopreservation, and rejuvenation where the extension of shelf life is iii associated with a high degree of manipulation, technical knowledge, increased losses, change in blood banking infrastructure, and economic cost. The knowledge gained from this work provides a push into the use of antioxidants as method of RBC biopreservation that has been seldom studied. Overall, this dissertation presents an additive solution modified with ascorbic acid with the potential of extending the life span of stored erythrocytes for use in transfusion medicine. iv Dedication Dedicated to my wife, Verónica Elizabeth Pereira, my parents, Jorge Enrique Fontes and Myriam Fontes, my brother, Adrián Gabriel Fontes and the rest of my extended family v Acknowledgments I would like to express my most sincere gratitude to my advisor, Professor Andre F. Palmer, for his guidance and support throughout my graduate studies at The Ohio State University. His invaluable insight helped me develop my ability to approach and solve problems throughout my research. I would like to thank Dr. Jeffery J. Chalmers and Dr. David W. Wood not only for serving on my Qualifying Exam, Candidacy Exam, and Dissertation Committee, but also for their encouragement and suggestions on my graduate research. I would also like to thank my collaborators Dr. Jay S. Raval, Dr. Mark H. Yazer, Dr. James C. Zimring, and Dr. Paul W. Buehler for their support in this research. I would like to sincerely thank both Dr. Maria Cristina Iazbik and Dr. C. Guillermo Couto at the Veterinary Medical Center for their professional support and for their genuine interest in my work. I would like to thank my colleagues Dr. Guo Chen, David R. Harris, Dr. Ning Zhang, Dr. Yipin Zhou, Dr. Jacob Elmer, Kristopher Richardson, and Dr. Kitty Agarwal for their support and expertise in experiments. I would like to especially thank Dr. Shahid Rameez for his guidance and mentoring. I would also like to thank Brian Belcik, Alexander Roth, and Uddyalok Banerjee for their immense support, camaraderie, and genuine friendship. vi I would like to take this opportunity to thank my entire family including Miguel Pereira, Mónica Cejas, Victoria Pereira, and especially my parents, Jorge E. and Myriam Fontes, and my brother, Adrián G. Fontes, for their tremendous support and love. Lastly, I would like to especially thank my wife, Verónica E. Pereira, for her unwavering support, love, and inspiration. vii Vita July 12, 1987………...……….……….Born Maspeth, Queens, New York City, NY, USA 2005.………………………………………………………………….……….High School Gateway Senior High School, Monroeville, PA, USA 2009……………………………………………………………B.S. Chemical Engineering Michigan State University, East Lansing, MI, USA 2009 – 2014 …………………………………………………Graduate Research Associate The Ohio State University, Columbus, OH, USA Publications Raval JS‡, Fontes J‡, Banerjee U‡, Yazer MH, Mank E, Palmer AF. Ascorbic acid improves membrane fragility and decreases haemolysis during red blood cell storage. Transfus Med. 2013;23(2):87-93. (‡ - co-first author) Rameez S, Banerjee U, Fontes J, Roth A, Palmer AF. Reactivity of Polymersome Encapsulated Hemoglobin with Physiologically Important Gaseous Ligands: Oxygen, Carbon Monoxide, and Nitric Oxide. Macromolecules. 2012;45(5):2385-9. Rameez S, Guzman N, Banerjee U, Fontes J, Paulaitis ME, Palmer AF, et al. Encapsulation of hemoglobin inside liposomes surface conjugated with poly(ethylene viii glycol) attenuates their reactions with gaseous ligands and regulates nitric oxide dependent vasodilation. Biotechnology Progress. 2012;28(3):636-45. Stowell SR, Smith NH, Zimring JC, Fu X, Palmer AF, Fontes J, et al. Addition of ascorbic acid solution to stored murine red blood cells increases posttransfusion recovery and decreases microparticles and alloimmunization. Transfusion. 2013;53(10):2248-57. Fields of Study Major Field: Chemical and Biomolecular Engineering ix Table of Contents Abstract ............................................................................................................................. iii Dedication .......................................................................................................................... v Acknowledgments ............................................................................................................ vi Vita .................................................................................................................................. viii List of Tables .................................................................................................................. xvi List of Figures ................................................................................................................. xvi Chapter 1: Blood banking: Modern storage solutions and human Erythrocytes ....... 1 1.1 Introduction ................................................................................................................. 1 1.2 Background ................................................................................................................. 1 1.2.1 Hemoglobin................................................................................................................ 1 1.2.2 Red Blood Cells ......................................................................................................... 3 1.2.3 Clearance of RBCs, Hemoglobin, and Heme ............................................................ 5 1.3 Motivation for Blood Storage .................................................................................... 5 1.3.1 Supply of RBCs in the U.S. and around the world .................................................... 5 x 1.3.2 Clinical Safety ............................................................................................................ 7 1.3.3 Clinical Concerns ....................................................................................................... 8 1.3.4 Impaired Oxygen Delivery ........................................................................................ 9 1.3.5 Iron Overload ........................................................................................................... 10 1.3.6 Microvesicle Complications .................................................................................... 11 1.4 Blood and Red Blood Cell Storage .......................................................................... 12 1.4.1 History of Anticoagulants and Preservatives for Whole Blood ............................... 12 1.4.2 Modern Additive Solutions ...................................................................................... 15 1.5 Hypothermic Storage Lesion ................................................................................... 17 1.5.1 Biochemical Changes............................................................................................... 17 1.5.2 Biophysical Changes ................................................................................................ 18 1.5.3 Oxidative Damage ................................................................................................... 20 1.5.3.1 Hemoglobin Oxidation.......................................................................................... 21 1.5.3.2 Lipid Oxidation and Loss...................................................................................... 22 1.5.3.3 Cytoskeleton Oxidation .......................................................................................