Extension of the Ex Vivo Life Span of Stored Human Erythrocytes by the Addition of Ascorbic Acid to Additive Solutions in Modern Blood Banking

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

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

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.

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

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.

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

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

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 ...... 22

1.5.4 Post Transfusion Changes ...... 25

1.6 Recent advances ...... 26

1.6.1 Modification of pH, metabolites, or osmotic pressure of additive solution components ...... 26

1.6.2 Protection from Oxidative Damage ...... 28

1.7 Other Factors Affecting Shelf Life ...... 29

1.8 Other Methods of Preservation ...... 30

Chapter 2: Quantification of Biochemical and Biophysical Properties of Stored

Erythrocytes ...... 32

2.1 Introduction ...... 32

2.2 General Methods ...... 32

2.2.1 Sample Timeline ...... 32

2.2.2 Complete Blood Count ...... 32

2.2.3 Blood Gas Analysis...... 33

2.2.4 Separation of RBCs, Lysate, and Supernatant ...... 33

2.2.5 Measurement of Hematocrit, Hemoglobin and Methemoglobin Concentration, and

Percent Hemolysis ...... 34

2.2.6 P50 and Cooperativity Coefficient ...... 34

2.2.7 ATP Concentration ...... 36

2.2.8 2,3-DPG Concentration...... 36

2.2.9 Total Protein Concentration in Supernatant and RBC Lysate ...... 36

2.2.10 RBC Gaseous Ligand Binding/Release Kinetics ...... 37

2.2.11 Statistical Analysis ...... 41

Chapter 3: Extension of the ex vivo life span of stored human erythrocytes by the addition of ascorbic acid to modern storage solutions ...... 42

3.1 Introduction ...... 42

3.1.1 Links to Oxidation ...... 42

3.1.2 Protection from Oxidation in Erythrocytes and Plasma ...... 42

3.1.2.1 Enzymatic Protection ...... 43 xii

3.1.2.2 Glutathione ...... 44

3.1.2.3 Uric Acid ...... 45

3.1.2.4 Vitamin E ...... 45

3.1.2.5 Ascorbic Acid ...... 46

3.1.3 Natural Antioxidant Defense in Storage ...... 49

3.1.4 Previous Attempts at the Use of Antioxidants in Storage...... 50

3.1.5 Ascorbic Acid as a U.S. FDA Additive ...... 54

3.1.6 Proposed Use of AA in Modern Storage Solutions ...... 55

3.2 Materials and Methods ...... 56

3.2.1 Donor Selection, RBC Collection, Processing and Storage ...... 56

3.2.2 Treatment of hRBCs with 5.86 mM AA ...... 57

3.2.3 Treatment of hRBCs with 2.93 and 8.78 mM AA ...... 57

3.2.4 Transport of Units ...... 58

3.2.5 Mechanical Fragility Test and Percent Hemolysis ...... 58

3.3 Results ...... 59

3.3.1 Mechanical Fragility ...... 59

3.3.2 Percent Hemolysis ...... 61

3.3.3 Blood Gases and pH...... 63

3.3.4 Methemoglobin Level in Lysate and Supernatant ...... 65

3.3.5 Hb Concentration ...... 66

3.3.6 Oxygen Equilibrium Curve ...... 67

3.3.7 RBC Gaseous Ligand Binding/Release Kinetics ...... 69

3.3.8 ATP and 2,3-DPG Content ...... 71

xiii

3.3.9 Total Protein...... 72

3.4 Discussion...... 75

3.5 Conclusion ...... 78

Chapter 4: Canine blood banking: Extension of the ex vivo life span of stored

Greyhound erythrocytes by the addition of ascorbic acid to modern storage solutions ...... 79

4.1 Introduction ...... 79

4.1.1 Storage Solutions ...... 79

4.1.2 Greyhounds as Donors ...... 80

4.1.3 Greyhound Hemoglobin...... 82

4.1.4 Ascorbic Acid in Canines ...... 83

4.1.5 Ascorbic Acid as a Supplement in Additive Solutions ...... 84

4.2 Materials and Methods ...... 84

4.2.1 Donor Selection, RBC Collection and Processing ...... 84

4.2.2 Addition of Stock Solutions ...... 85

4.2.3 Sampling ...... 86

4.3 Results ...... 86

4.3.1 Complete Blood Count and Blood Gas Analysis comparison between Day 0 and 7

Values ...... 86

4.3.2 Temporal Changes between Day 7 and Day 35 ...... 87

4.3.2.1 Blood Gas Analysis and CBC ...... 87

4.3.2.2 Percent Hemolysis and Methemoglobin Levels ...... 89

xiv

4.3.2.3 O2 Affinity and Cooperativity Coefficient ...... 91

4.3.2.4 2,3-DPG Concentration...... 93

4.3.2.5 RBC Gaseous Ligand Binding/Release Kinetics ...... 94

4.3.3 The Effect of 7.1mM AA on stored Greyhound RBCs ...... 98

4.3.3.1 Blood Gas Analysis and CBC ...... 98

4.3.3.2 Biochemical Assays ...... 98

4. 4 Discussion...... 98

4.4.1 Changes between Day 0 and Day 7 ...... 99

4.4.2 Changes between Day 7 and Day 35 ...... 101

4.5 Conclusions ...... 103

Chapter 5: Conclusions and Future Studies ...... 105

5.1 Lipid Composition and Oxidation ...... 106

5.2 Cytoskeleton Oxidation ...... 107

5.3 Erythrocyte Deformability ...... 107

5.4 Microvesicle Formation ...... 108

5.5 Antioxidant Capacity of Stored Erythrocytes ...... 109

5.6 In vivo studies of Post Transfusion Viability ...... 109

Bibliography ...... 111

List of Tables

Table 1.1 - Anticoagulant Solution Composition ------14

Table 1.2 - Red Cell Additive Solution Composition ------16

Table 2.1 - Oxygen Affinity and Cooperativity Coefficient of a Single Human Donor during Storage Compared to Human Hemoglobin ------35

Table 3.1 - Linear regressions of MFI data for RBCs ------76

Table 3.2- Linear regressions of percent hemolysis data for RBC ------76

Table 4.1 - Canine blood groups ------81

Table 4.2 - Frequency of ‘universal’ and positive donors ------81

Table 4.3 - Oxygen Affinity and Cooperativity Coefficients of Greyhound and Human

Hb and RBCs ------83

Table 4.4 - Complete Blood Count and Blood Gas Parameters for Greyhound Whole

Blood. ------87

Table 4.5 - Complete Blood Count Parameters During Storage (Mean ± SD). ------88

Table 4.6 - Blood Gas Analysis Parameters During Storage (Mean ± SD). ------89

xvi

List of Figures

Figure 1.1 - Tetrameric deoxyHb...... 2

Figure 1.2 - Ferrous heme in associated with O2 ...... 2

Figure 1.3 – Simplified Metabolic Pathways of the Erythrocyte ...... 4

Figure 1.4 - Organization of membrane protein complexes in the hRBC membrane ...... 23

Figure 2.1 - Oxygen Equilibrium Curve for a Single Human Donor During Storage...... 36

Figure 2.2 - Sample Time Course for the Deoxygenation of hRBCs ...... 37

Figure 2.3 - Sample Time Course for CO Association of hRBCs at 464 and 232 μM CO

...... 378

Figure 2.4 - Plot of Pseudo-First Order Rate Constants Against CO Concentrations ...... 39

Figure 2.5 - Sample Time Course for NO Dioxygenation of hRBCs at 25 and 12.5 μM

NO ...... 40

Figure 2.6 - Plot of Pseudo-First Order Rate Constants against NO Concentrations ...... 40

Figure 3.1 –Ascorbic acid recycling scheme with vitamin E ...... 46

Figure 3.2 - Ascorbic acid (AA) recycling in RBCs\...... 48

Figure 3.3 - The effect of 5.86 mM AA on MFI of RBCs during storage period ...... 60

Figure 3.4 - The effect of different AA concentrations on MFI of RBCs during storage period ...... 60

Figure 3.5 - The effect of 5.86 mM AA on percent hemolysis of RBCs during storage period ...... 62

Figure 3.6 - The effect of different AA concentrations on percent hemolysis of male

RBCs during storage period ...... 62

Figure 3.7 - The effect of 5.86 mM AA on pCO2 during storage period ...... 63

xvii

Figure 3.8 - The effect of 5.86 mM AA on pO2 during storage period ...... 64

Figure 3.9 - The effect of 5.86 mM AA on pH during storage period...... 64

Figure 3.10 – The effect of 5.86 mM AA on metHb level in RBC lysates during storage period ...... 66

Figure 3.11 – The effect of 5.86 mM AA on metHb level in supernatant of packed RBCs during storage period ...... 66

Figure 3.12 - The effect of 5.86 mM AA on Hb concentration in RBC lysate during storage period ...... 67

Figure 3.13 - The effect of 5.86mM AA on P50 during storage period ...... 68

Figure 3.14 - The effect of 5.86mM AA on O2-Hb cooperativity coefficient during storage period ...... 68

Figure 3.15 - The effect of 5.86 mM AA on O2 dissociation rate constants during storage period ...... 69

Figure 3.16 - The effect of 5.86 mM AA on CO association rate constants during storage period ...... 70

Figure 3.17 - The effect of 5.86 mM AA on NO dioxygenation rate constants during storage period ...... 70

Figure 3.18 - The effect of 5.86 mM AA on ATP concentrations in RBC lysate during storage period ...... 71

Figure 3.19 - The effect of 5.86 mM AA on 2,3-DPG concentrations in RBCs during storage period...... 72

Figure 3.20 - The effect of 5.86 mM AA on total protein concentration in RBC lysate during storage period ...... 73

xviii

Figure 3.21 - The effect of 5.86 mM AA on total protein concentration in supernatant during storage period ...... 74

Figure 3.22 - The effect of 5.86 mM AA on total protein concentration in vesicles during storage period ...... 74

Figure 4.1 - RBC oxygen affinity values of healthy Greyhounds and non-Greyhounds .. 82

Figure 4.2 - Oxygen Dissociation Curves for human and Greyhound Hb and RBCs ...... 83

Figure 4.3 – Percent hemolysis in stored RBCs for RBCs stored in AA or saline supplemented storage solution ...... 90

Figure 4.4 – MetHb level in supernatant and lysate for RBCs stored in AA or saline supplemented storage solution ...... 91

Figure 4.5 – Hemoglobin-Oxygen affinity for RBCs stored in AA or saline supplemented storage solution ...... 92

Figure 4.6 – Cooperativity coefficient for RBCs stored in AA or saline supplemented storage solution ...... 93

Figure 4.7 – 2,3-DPG content in RBCs for RBCs stored in AA or saline supplemented storage solution ...... 94

Figure 4.8 –Deoxygenation kinetic rate constants for RBCs stored in AA or saline supplemented storage solution ...... 95

Figure 4.9 – CO association kinetic rate constants for RBCs stored in AA or saline supplemented storage solution ...... 96

Figure 4.10 –NO dioxygenation kinetic rate constants for RBCs stored in AA or saline supplemented storage solution ...... 97

xix

Chapter 1: Blood banking: Modern storage solutions and human Erythrocytes

1.1 Introduction Currently, modern storage solutions (i.e. CPDA-1, SAGM, AS-1, AS-3 and AS-5) coupled with hypothermic storage can preserve human red blood cells (RBC) for up to six weeks (i.e. 42 days). This relatively short ex vivo storage shelf life of stored human

RBCs (hRBC) is set by the United States Food and Drug Administration (U.S. FDA) based on the post transfusion viability (PTV) at 24 hours, which must be greater than or equal to 75 ± 9% [1], and low levels of hemolysis in the unit (< 1%) [2]. There are new concerns regarding the safety of blood transfusions following extended durations of storage (i.e. the storage lesion) [3-6]. The time during which the product is not potentially dangerous marks the ‘effective’ life span of the RBCs. By extending both the absolute life span and the effective life span of stored RBCs, the supply of RBCs for transfusion could begin to match worldwide demand by reducing the loss of units due to outdating and could make older stored units safer for transfusion. Also, it could extend the reach and availability of packed RBCs to remote locations and also minimize seasonal fluctuations in supply.

1.2 Background 1.2.1 Hemoglobin Hemoglobin (Hb) is composed of two alpha (α) and two beta (β) subunits/globin chains (141 and 146 amino acids, respectively), each containing a heme porphyrin ring to which gaseous ligands may bind and release (Figure 1.1). The ferrous iron atom (Fe+2) at

1 the center of the heme ring can bind oxygen (O2) at elevated O2 levels in the lungs, and release it at lower O2 tensions in the tissues [7].

Figure 1.1 - Tetrameric deoxyHb composed of Figure 1.2 - Ferrous heme in associated alpha (light green) and beta (dark green) with O2 (oxyHb) [9]. subunits with heme rings visible [8].

The heme moiety in the heme pocket of Hb can become spontaneously oxidized when a dissociating O2 molecule sequesters an additional electron from the iron atom. This process is referred to as Hb autoxidation [10, 11]. Autoxidation yields two major

- products, namely superoxide (O2· ) and methemoglobin (metHb), the oxidize form of Hb which has a ferric iron (Fe+3) in the heme group [10]. The heme pocket of metHb is

- occupied by either water (H2O) or a hydroxide ion (OH ), and it is incapable of transporting O2, rendering it useless for blood transfusion purposes [12]. This process usually occurs at a rate of about 3%/day [13], but can be accelerated at lower partial pressures of O2 [14].

When a Hb molecule is completely bound to O2 (oxyHb) it is, by convention, in the relaxed state (R-state), whereas in the tense state (T-state) it is not bound to O2 and is in the deoxygenated state (deoxyHb) (Figure 1.2) [7]. The O2 affinity of Hb is described by the Hill Equation, which has two coefficients; the P50 is the partial pressure of O2 (pO2) at which the Hb molecule is 50% saturated with O2, and the cooperativity coefficient (n) describes the cooperative binding of O2 to the four subunits. A high P50 value indicates a low O2 affinity while the opposite is true for a low P50 value [15]. The affinity of human

Hb is also modified by several factors including pH (i.e. hydrogen ions), carbon dioxide

(CO2) concentrations, and 2,3-diphosphoglycerate (2,3-DPG) [16, 17].

1.2.2 Red Blood Cells Hemoglobin is the most abundant protein in the RBC (~95% dry weight). Aside from carrying O2, the RBC also removes CO2 back to the lungs [18]. The RBC is a biconcave disk approximately 8 μm in diameter and about 2 μm think. To be able to navigate the narrow capillaries (2-3 μm), it must be able to resist large forces as it deforms through the vasculature [19]. It is highly deformable and supported by a complex meshwork of proteins known as the cytoskeleton.

Maintenance of the RBC includes four pathways: Embden-Meyerhof (glycolytic) pathway, the pentose phosphate pathway (PPP) (i.e. hexose monophosphate shunt

(HMP)), the Rapoport-Luebering shunt, and lastly, the metHb reduction pathway

(Figure 1.3). The first pathway is the primary pathway for adenosine-5'-triphosphate

(ATP) production; the second produces nicotinamide adenine dinucleotide phosphate

(NADPH); the third produces the aforementioned 2,3-DPG, and the last maintains Hb is its reduced state [18].

Figure 1.3 – Simplified Metabolic Pathways of the Erythrocyte. Some substances (shaded) that can be added to the storage medium to influence metabolism, and some factors promoting metabolic steps (-->). ADP, adenosine diphosphate; AK, adenylate kinase; AMP, adenosine monophosphate; A- PRT, adenine phosphoribosyl transferase; ATP, adenosine triphosphate; DHA (DHA-P), dihydroxyacetone (dihydroxyacetone phosphate); DPG (1,3-DPG and 2,3-DPG), 1,3- diphosphoglycerate and 2,3-diphosphoglycerate, respectively; DPGM, diphosphoglycerate mutase; DPGP, diphosphoglycerate phosphatase; F-1,6-P, fructose 1,6-diphosphate; F-6-P, fructose 6- phosphate; GAPD, glyceraldehydephosphate dehydrogenase; GP, glutathione peroxidase; GR, glutathione reductase; G-6-P, glucose 6-phosphate; G-6-PD, glucose 6-phosphate deydrogenase; HK, hexokinase; LDH, lactate dehydrogenase; NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide (reduced);P-, phosphorylated compound; PFK, phosphofructokinase; Pi, inorganic phosphate; PK, pyruvate kinase; PNP, purine nucleoside phosphorylase; SOD, superoxide dismutase; TPI, triosephosphate isomerase. [20].

1.2.3 Clearance of RBCs, Hemoglobin, and Heme Normally, human erythrocytes undergo senescence after their normal life span of approximately 110-120 days [21-23]. Senescent RBCs are pulled out of circulation at a high turnover rate (0.83%/day) (~2.4 million RBC/s) [24]. The mononuclear phagocyte system (MPS) (e.g. reticuloendothelial system (RES)) clears erythrocytes, and most of this work is done by the spleen [25] although some is attributed to the liver (i.e. Kupffer cells) and macrophages in the lymph nodes, especially in the case of a splenectomy [26].

In the spleen, RBCs are physically separated from the bloodstream by nearly 90° arterial branches, which skim off the plasma, concentrating the RBCs in the red pulp area of the spleen. In this area, the 8 μm diameter RBC is squeezed through a 1 to 3 μm slit, forcing the cell to deform and reducing the velocity to a few millimeters per minute [25]. Splenic macrophages cull RBCs marked for phagocytosis by drawing it into the phagolysosome.

The macrophage is charged with degrading the globin chain, breaking down the heme ring, and recycling the iron atom. A significant portion of erythrocytes, however, succumb to injury in the vasculature and rupture, releasing its contents, most of which is

Hb, in the bloodstream [27]. Cell-free Hb is identified by haptoglobin (Hp), a high affinity glycoprotein which binds Hb dimers. Hemopexin (Hpx) scavenges free heme from the plasma, attenuating its damaging effects. The Hpx:heme and Hp:Hb complexes are recognized by macrophage endocytosis receptors CD91/LRP and CD163, respectively [28].

1.3 Motivation for Blood Storage 1.3.1 Supply of RBCs in the U.S. and around the world Blood transfusion is an important treatment for ailments such as anemia, hemoglobinopathies, thalassemias, blood loss during traumatic events, cancer treatments,

5 as well as surgical procedures, including organ transplantation [29-32]. Recent data has shown that the need for blood is steadily rising at an average rate of 6% per year [33].

Unfortunately, the availability of human blood components is shrinking due to an aging population base and the increasing number of patients who need a blood transfusion [29,

34, 35]. To highlight the problem, a recent study predicts that there will be a shortage of 4 million units of blood in the U.S. by 2030 [35], with similar predictions made in

Germany [29]. The situation is critical, since the blood supply can be even more limited in emergency situations such as wars or natural disasters [36, 37].

The most recent statistics in the 2011 National Blood Collection and Utilization

Survey reported that 15.7 million whole blood and RBC units were collected [38]. This is a significant 9.1% decline from the 2009 report where 17.3 million units were collected

[39]. In 2011, only 14.6 million units were available for transfusion and of the available allogeneic units, 94.8% were used in allogeneic transfusions, compared to 95.5% in 2004,

93.4% in 2006, and 87.9% in 2008, suggesting an slight surplus in 2011 [38, 39]. During

2011, blood shortages were suggested to be rare and significant only for very few U.S. hospitals that were geographically isolated [38]. Ultimately, the surplus of total units of blood components in 2011 was 752,000, a decrease from the surplus recorded in 2008

[38, 39]. In this report, although the mean age of an RBC unit at the time of transfusion was 17.9 days, 375,986 whole blood/RBC units were outdated before use. This accounts for 2.4% of the total processed units, and although the number seems small, it represents a source of waste and inefficiency [38].

Worldwide supply, however, is a very different issue. According the World Health

Organization (WHO), 91.8 million annual blood donations were collected globally in

2008. Unfortunately, there is a great worldwide imbalance in blood supply and donations marked by economic and geopolitical lines. Nearly half (48%) of donations in that year occurred in high income countries; high income countries have only 15% of the world’s population, and even more staggering is that 10 countries (United States, China, India,

Japan, Germany, Russia, Italy, France, Republic of Korea, and United Kingdom, in decreasing order) account for 65% of worldwide blood collection [40].

Due to the limited shelf life, fluctuations in supply are largely inevitable [41].

Therefore, it has been a long-term goal of scientists to extend the storage shelf life of blood components. Extension of the life span of RBCs can help overcome local and seasonal shortages by making better use of available resources, allowing the accumulation of these components for better availability during times of greater need.

Overall, there is a need for a larger supply of safe RBC units to shorten the gap between supply and demand.

1.3.2 Clinical Safety General complications of RBCs as transfusion products include but are not limited to the transmission of infectious disease, pathogen contamination (i.e. sepsis), hemolytic reaction (i.e. blood typing mismatch), alloimmunization, and febrile non-hemolytic transfusion reactions [41]. Testing for infectious disease has greatly minimized the risk of several diseases in the U.S. [42, 43], but unfortunately, this is not the case in all countries

[44, 45]. The integration of closed systems for collection and processing, along with irradiation of components and pathogen inactivation techniques, has minimized pathogen contamination, although new pathogens are of considerable concern [43]. Blood typing has become universal and cost effective, but human error is always present, and blood type mismatch is among the leading causes of transfusion-related mortality [45]. 7

Alloimmunization and febrile non-hemolytic transfusion reactions, likely caused by the presence of leukocytes, can be minimized by leukoreduction of blood components either at the point of collection or before transfusion at the bedside [45, 46]. However, these issues in blood processing and banking are not in the scope of this dissertation; the focus will be on the biochemical and biophysical changes that occur to human erythrocytes during storage, which is collectively called the ‘storage lesion’. These changes are likely the cause of transfusion complications including hyperkalemia, transfusion-related acute lung injury (TRALI), iron overload, and hemolysis-associated pulmonary hypertension.

1.3.3 Clinical Concerns Several studies and reviews have shown the possible dangers of transfusion of RBCs of any age [47-52]. Generally, reviews of retrospective studies [3-6] have suggested that transfusion of older RBCs to various subgroups of critically ill patients may produce adverse effects and increased mortality, although contradictory results are also presented.

One study found a weak association between mortality and stored red cell age in patients with cardiovascular disease [53]. Robinson et al. found an association of 30-day mortality with the age of RBCs used in transfusion in patients undergoing percutaneous coronary intervention [54]. Another study found a higher risk of graft failure and mortality in orthotopic liver transplant patients transfused with RBCs older than 15 days [55]. Still another study found a tendency for higher risk of death when transfused with 30-42 day old RBCs when compared to 10-19 day old RBCs [56]. Both in-hospital and out-of- hospital mortality were found to be associated with duration of storage of transfused

RBCs in yet another study [57]. Even in less severely injured patients, RBCs older than 2 weeks were associated with increased mortality, renal failure, and pneumonia [58].

A recent study showed that transfusion of RBCs stored for longer times (i.e. 14+ days) were associated with a significantly increased risk of complications, and significantly reduced survival after cardiac surgery [59]. These findings may have been confounded by other variables (i.e. transfusion volume) and, after adjustments were made, the significance was no longer present [5]. Still other studies [60] found no correlation at all.

These studies point to the possibility that short term adverse and sometimes fatal outcomes are the result of the transfusion of RBCs that have been stored for longer amounts of time. However, some variables are difficult to separate, and confounding is present in almost all studies; retrospective studies usually have difficulty separating the effects of the age of RBCs from the number of units transfused [6]. Moreover, long term and non-lethal effects after transfusion make causation more difficult to demonstrate.

Although these reviews and studies demonstrate that this subject is not straightforward, the potential for these clinical issues warrants further research.

1.3.4 Impaired Oxygen Delivery

The main causations for these clinical results have focused on impaired O2 delivery, iron overload, complications due to microparticle formation, and an imbalance in nitric oxide (NO) homeostasis. The ability of stored RBCs to deliver oxygen is of importance due to the decline in 2,3-DPG content and subsequent increased O2 affinity. This increase in affinity should reduce O2 release when compared to fresh RBCs at the same pO2. In a study using an animal model, storage of rat RBCs reduces O2 delivery by 15% [61].

Similarly, when stored hRBCs were transfused in a rat model, older cells produced lower microvascular O2 concentrations [62]. However, in a larger, similar study, a significant effect of storage time on O2 delivery was not found [63]. In another rat model,

Eichelbronner et al. could not demonstrate an increase in O2 uptake when the Hb used 9 was modified with an allosteric effector to increase the oxygen affinity, as occurs in stored RBCs [64]. The small decrease in O2 delivery seen and those not seen may not have a large clinical impact. Furthermore, the animal studies mentioned may not be applicable to clinical outcomes in humans due to the differences in O2 affinity of the Hbs, the different rate of aging of RBCs from these other species, and the different rate of post transfusion 2,3-DPG regeneration between humans and mice or rats [62, 65]. Decreases in 2,3-DPG are temporary in humans due to its regeneration after transfusion, and thus may make O2 delivery not clinically relevant for most transfusions [20, 66].

1.3.5 Iron Overload Since the minimum PTV of a standard RBC unit is 75% at expiration, a maximum of

25% of the cells in the bag will be cleared within 24 hours of transfusion, although much of the clearance occurs within the first hour. Once transfused, extravascular clearance of

RBCs damaged during storage has been shown to induce inflammation and deliver large amounts of iron to tissues involved in the clearance of older RBCs in a mouse model

[67], while also increasing serum iron, transferrin saturation, and non-transferrin bound iron in humans [68]. In a guinea pig model, transfusion of older RBCs, which also had decreased deformability, resulted in intravascular hemolysis leading to acute hypertension, vascular injury, and renal failure [69]. Normally, humans clear one milliliter of RBCs or one milligram of iron every hour, but transfusion of a single unit of aged cells can introduce nearly 60 milligrams of cell free iron at once, stressing the clearance mechanisms and causing tissue toxicity [68].

In the plasma, Hb is prone to dissociation into αβ dimers [70, 71], which are easily oxidized [72]. The released Hb can exacerbate the complications by generating reactive oxygen species (ROS), activating the oxidative cascade, and causing oxidative damage 10

[73, 74]. If the clearance mechanisms are saturated, cell-free Hb and heme in the blood stream can have further clinical consequences including inflammation and prooxidant and cytotoxic effects [70, 71, 75-78].

1.3.6 Microvesicle Complications It was suggested that microvesicles formed during storage of RBCs can be directly culpable for adverse effects including TRALI, and postoperative thrombosis [79].TRALI is a leading cause of transfusion related mortality [45] and although the underlying mechanism is not established, neutrophil priming and activation by bioactive lipids

(oxidation-induced lysophospholipids) in microvesicles formed during RBC hypothermic storage is likely a contributing factor [5, 80-83].

Nitric oxide (NO) is a small molecule involved in physiologic signaling and is responsible for controlling blood flow by activating a pathway which regulates the tone of blood vessels, controlling blood vessel diameter and hence, blood pressure and flow rate [84-88]. It is produced by nitric oxide synthase in endothelial cells and RBCs [89].

The reaction of both deoxyHb and oxyHb with NO is very fast and provides a mechanism for NO scavenging in vivo [90] and in sickle cell disease [91]. Additionally, αβ dimers can extravasate between the endothelial cells and increase NO scavenging by their proximity to NO production [91]. Even low levels of cell-free Hb can directly induce increases in blood pressure by NO scavenging [92].

As a result of storage, vesiculation ejects small amounts of encapsulated Hb from the

RBC. These Hb microvesicles have been suggested to be more deleterious than cell-free

Hb because they are unable to be cleared by the serum Hp or other mechanisms [87, 92].

Microvesicles have lower internal Hb concentration than RBCs and will therefore react with NO much like cell-free Hb [93], which reacts with NO nearly 1,000 times faster 11 than do RBCs [94-96]. Retardation of the NO reaction rate is seen in artificial vesicles where a high concentration of Hb is encapsulated inside liposomes [97, 98] and polymer vesicles [99] due to these same high intracellular concentrations of Hb [95]. Due to their reduce size, Hb microvesicles and cell-free Hb can, unlike RBCs, travel near the blood vessel wall and rapidly scavenge NO due to their proximity to endothelial cells and aforementioned increased reaction rate [96, 100, 101]. Once near the blood vessel wall, cell-free Hb and Hb microvesicles can react with NO, producing both metHb and nitrite ions [101-104], inducing further complications. Supernatant from stored RBCs elicits vasoconstriction that correlates with extent of ex vivo hemolysis [92]. These NO scavenging issues have been quantified, and it is proposed that transfusion of stored

RBCs with Hb microvesicles and cell-free Hb from both ex vivo hemolysis and in vivo intravascular hemolysis after transfusion can induce an imbalance in the NO equilibrium

[87, 91, 93], triggering hypertension [90].

1.4 Blood and Red Blood Cell Storage 1.4.1 History of Anticoagulants and Preservatives for Whole Blood The first ex vivo stored human blood component came in 1914 by Hustin with the clinical use of citrate as an anticoagulant to preserve whole blood [18], removing the need for the simultaneous presence of both donor and recipient, and allowing up to 6 days of storage [3, 105]. Two advances came rapidly: the addition of glucose (dextrose), and cold storage. Glucose and other hexoses were used to inhibit hemolysis for up to 4 weeks in a solution called the Rous-Turner solution [106, 107]. Glucose, although originally added to preserve cell shape since the membrane was considered impermeable to sugars

[106], was present for metabolism through (90%) glycolysis and (10%) PPP [20], directly producing ATP and NADPH [18]. Cold storage was later introduced by O.H. Robertson

12 who packed glass bottles of blood in Rous-Turner solution with ice and sawdust in 1917

[105]. Hypothermic storage was implemented so that low temperatures would inhibit depletion of essential metabolites, but its effect on biological systems is more complicated [18].

The use of this citrate-glucose solution drastically increased the shelf life of whole blood to 10 to 15 days [3]. Unfortunately, the sterilization of this citrate-glucose solution required the two chemical components to be sterilized separately to avoid caramelization and discoloration. Mixing these two solutions was also time consuming and increased the potential for contamination [108]. The introduction of citric acid to sodium citrate- glucose solutions solved this problem by lowering the pH, preventing caramelization while also boasting higher PTV and lower hemolysis than previous citrate-glucose solutions [109]. This eventually led to the advancement of these acid-citrate-dextrose

(ACD) solutions that allowed whole blood to be stored for three weeks [110].

A new solution containing phosphate called citrate-phosphate-dextrose (CPD) showed adequate PTV after an additional week of storage (i.e. 28 days), and it was implemented for the preservation of RBCs [111-116]. Phosphate salts were included to support the production phosphorylated molecules including ATP [117] and 2,3-DPG

[118, 119], as well as to buffer the mixture since glycolysis produces two protons and two lactate molecules for every glucose molecule consumed [20, 120, 121].

Since RBCs cannot de novo synthesize adenine, it was added as an additional metabolite for the production of ATP [122]. This additional metabolite also increased the

PTV of RBCs stored for extended periods of time; adenine was added to RBCs stored for

28 days in citrate-phosphate-double dextrose (CP2D) [123], and to RBCs stored for 35

13 days in both ACD to become ACD-adenine [124-126] and CPD to become CPD-adenine

(CPDA) [127, 128]. CP2D is an anticoagulant with double the concentration of glucose designed for the preparation of packed RBCs (i.e. separated from plasma) with more metabolites [129]. Studies with adenine showed that ATP levels were better maintained in these solutions [130]. Later in vivo tests showed that CPD modified with adenine and additional glucose was suitable for the storage of whole blood and red cell concentrates for 35 days, licensing citrate-phosphate-dextrose-adenine (CPDA-1) as a new preservative for both whole blood and packed RBCs [127, 131].

Table 1.1 - Anticoagulant Solution Composition (adapted from [31])

Constituent (g/L) ACD-A CPD CPDA-1 CP2D

Trisodium citrate 22.0 26.3 26.3 26.3 Citric acid 8.0 3.27 3.27 3.27 Dextrose 24.5 25.5 31.9 51.5 Monobasic sodium - 2.22 2.22 2.22 phosphate Adenine - - 0.28 -

These solutions (Table 1.1) evolved from a simple sodium citrate anticoagulant to include some metabolites necessary for cellular maintenance so that ATP could be preserved as well as maintain an acceptable 24-hour PTV.

1.4.2 Modern Additive Solutions With the newly adopted separation of components, increased pressure was put on producing plasma. When plasma was separated from RBCs, the liquid phase, containing the soluble metabolites in the preservative solution, moved with the plasma and left

RBCs packed to a high packing density or hematocrit (HCT). Erythrocytes in these solutions were excessively viscous and lacked sufficient metabolites for prolonged storage, pushing for the creation of modern additive solutions to add to packed RBCs

[129, 132].

For the separation of components, 450 mL of whole blood is usually collected into 63 mL of an anticoagulant like CPD, CP2D, or CPDA-1which is then centrifuged to separate

RBCs from plasma. Manual or semi-automatic pressing separates RBCs and plasma into separate collection bags within a closed system [43]. The removal of leukocytes or leukoreduction via filtration is widely used to reduce complications in storage [2, 133-

136] and subsequent transfusion [45]. An additive solution in another compartment is added to the remaining RBCs to preserve them for up to 42 days [31, 131]. The first additive solution contained saline, adenine, and glucose and was therefore named SAG

[133]. This additive solution not only reduced viscosity, but also delivered metabolites for red cell storage [2, 131].

Although PTV at the time of expiration was sufficient, there was a concern with increased hemolysis near the end of storage. Mannitol was suggested earlier to reduce hemolysis in stored RBCs [122], but was implemented later in an additive solution named saline-adenine-glucose-mannitol (SAGM) due to its protecting effects [121, 131, 137,

138], and although the mechanism is not clear [139], it has the ability to act as a

15 membrane stabilizer [2]. SAGM is currently approved for packed RBC storage for 35 days, but can be held for 42 days in extreme cases [138].

Six week storage has been approved for similar additive solutions that have glucose, adenine, and sodium chloride, and additionally a combination of phosphate, mannitol, citrate, and citric acid [31, 140]. Adsol (AS-1), Nutricel (AS-3), and Optisol (AS-5), along with SAGM, are widely used in the U.S., Canada, and Europe for the extended storage of packed hRBCs [2, 121, 141].

These advances in simple composition have drastically improved the shelf life of

RBCs from hours (without anticoagulant) to 6 weeks. Table 1.2 shows the composition of the most widely used current additive solutions. It should be noted that none of the approved solutions mentioned in this section preserve the levels of 2,3-DPG; most of the

RBCs in these additive solutions will lose a significant quantity during the first few weeks of storage [131].

Table 1.2 - Red Cell Additive Solution Composition (adapted from [31])

Adsol Nutricel O ptisol Constituent (mM) SAGM AS-1 AS-3 AS-5 Dextrose 45.40 111.00 55.50 45.40 Adenine 1.26 2.00 2.22 2.22 Monobasic sodium phosphate - - 23.00 - Mannitol 29.00 41.20 - 45.40 Sodium chloride 150.00 154.00 70.00 150.00 Citric acid - - 2.00 - Sodium citrate - - 23.00 -

1.5 Hypothermic Storage Lesion Erythrocytes in these additive solutions under hypothermic storage conditions are not in suspended animation. Instead, most processes continue, but at slower rates, slowly aging the cell. As the RBC ages ex vivo, it incurs several biochemical and biophysical changes which are considered by most as the ‘storage lesion’. The hypothermic storage lesion (HSL) has been extensively quantified in human blood component storage studies and reviews [18, 142-147]. The accumulated changes that occur in RBCs during storage are numerous and although some changes can be reversible, others are permanent.

The cells that are most irreversibly damaged during storage likely are the ones removed from the blood stream within 24 hours after transfusion. One study demonstrated that older RBCs exhibited a 13% lower PTV than more fresh RBCs [148].

The clearance of these deteriorated cells limits the efficacy of the transfused unit and could potentially cause complications after transfusion. If this gradual deterioration due to the HSL can be reduced, the effective life span of RBCs could be extended, making them safer for use in clinical settings.

1.5.1 Biochemical Changes Hypothermic storage was introduced so that cellular processes could be slowed.

Lower temperatures reduce the overall rates of degenerative processes, but do create several other difficulties. Importantly, glycolysis is slowed by about 40 times [20, 121,

149] so that the use of metabolites like glucose is reduced. However, since glycolysis is the only pathway for making energy, glycolysis is necessary to form ATP and NADH for downstream processes. In glycolysis, lactic acid is formed decreasing pH during six weeks of storage [142] further diminishing the rate of glycolysis [145]. Since ATP production is reduced by this acidosis and ATP itself is used for other processes, ATP

17 levels decrease during storage [146]. The loss of ATP occurs not only through metabolism but also through irreversible deamination of adenosine monophosphate to produce inosine monophosphate and subsequently hypoxanthine [131].

The glycolytic pathway acidifies the storage solution and since higher pH favors synthesis of 2,3-DPG and low pH (< 7.3) favors consumption, there is a reduction in the internal concentration of 2,3-DPG in the RBC [131]. In turn, the reduced concentration of this allosteric effector shifts the O2–Hb equilibrium curve to the left, effectively increasing the O2 affinity of Hb (i.e. lowering the P50) [142]. The loss of 2,3-DPG is progressive and in its absence, RBCs have a decreased ability to deliver O2 [18].

The progressive loss of potassium ions into the extracellular space is associated with the HSL and is caused by the gradual diffusion through the membrane. At cold temperatures, the sodium/potassium ion (Na+/K+) pump is slowed drastically, permitting a K+ efflux and a Na+ influx [144, 145, 147]; the K+ leak is compounded by the loss of

ATP for this ATP-dependent process.

1.5.2 Biophysical Changes The asymmetry of the RBC membrane has been well documented in the literature.

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) preferentially exist on the cytosolic leaflet of the RBC bilayer, while phosphatidylcholine (PC) and sphingomyelin

(SM) preferentially exist on the outer leaflet of the RBC bilayer [150]. Intracellular ATP sustains translocase activity, a protein responsible for phospholipid asymmetry in the

RBC bilayer [151, 152]. As the RBC ages during storage, the distribution of lipids in the membrane changes and exposes PS [153] and PE [154] to the outer leaflet of the membrane. Translocase activity during storage is altered, but it cannot be fully explained by ATP loss [152].The loss of translocase activity is important to the viability of RBCs 18 after transfusion because, as mentioned previously, the exposure of PS has been linked to

RBC recognition by the RES, signaling their clearance and uptake by macrophages [155-

159].

RBCs change shape during storage, slowly becoming more spherical over time and changing from their normal biconcave disc to more round spheroechinocytes [160-162].

This occurs due to a significant loss of cholesterol and phospholipids that has been documented during extended hypothermic storage [154, 163-165]. Vesiculation of the membrane during storage whereby RBCs lose surface area relative to volume and change shape has been quantified [2, 161, 166-170]. Vesiculation also occurs as the RBC ages in vivo [171] and in vitro with the loss of intracellular ATP [166]. Both nanovesicles and microvesicles (50-200nm in diameter) are present in the supernatant of stored cells [167,

170, 171].

The membrane changes mentioned are associated with changes in deformability, filterability, and aggregability. Increased aggregability during storage is partially caused by a reduction in surface charge [162]. This negative surface charge is associated with the presence of the carboxyl group of sialic acid [172, 173], and as the cells lose sialic acid on their surface during storage, aggregation between RBCs and adherence to endothelial cells increase [162, 174].

Measurements of cell deformability show gradual but significant loss of RBC deformability during hypothermic storage [142, 161, 174-177]. Changes in deformability are easily measured and diagnosed by ektacytometry [19, 178], and deformability loss during storage has been shown to be caused by reduced ratio of surface area to volume, but is not a result of membrane stiffening or increased internal viscosity (i.e. changes in

19 cell volume) [176]. These changes in deformability were of significance in predicting the effectiveness of storage solutions because they were correlated to PTV [175-177].

The RBC membrane also progressively becomes more susceptible to damage from external mechanical forces. This susceptibility to mechanical damage is referred to as sublethal injury and is quantified by the mechanical fragility index (MFI) [179, 180]. The

RBCs that are have high MFI values will presumably be the first to be lethally damaged after transfusion. The most susceptible or most weakened cells are lethally damage during storage (i.e. hemolysis) which increases with the progression of storage [142]. The subsequent release of Hb from hemolyzed both ex vivo and in vivo cells will be discussed.

1.5.3 Oxidative Damage At reduced temperatures, biological processes that maintain a reduced state in the cell are compromised and enzymatic protection from oxidative damage is hampered [181,

182]. The reduced rate of glycolysis (due to low temperatures and reduced pH) leads to reduced rates of nicotinamide adenine dinucleotide (NADH) formation, hindering

NADH-dependent processes including metHb reductase. At reduced temperatures, glucose flux through the PPP is decreased also, limiting the concentration of another reducing agent, NADPH and its associated processes. The loss of erythrocyte antioxidant activity during RBC storage has been demonstrated in the literature [165, 183-185] and will be discussed further in Chapter 3. This loss of this activity during storage may expose the stored RBCs to ROS [186] from Hb autoxidation [187, 188], agitation [189], or the exposure to light [190]. The presence of metabolically active leukocytes consumes glucose, exposes cells to increased metabolic waste, cytokines, enzymes, and also ROS, and results in increased hemolysis and vesicle formation during storage [18, 121, 136,

181]. 20

1.5.3.1 Hemoglobin Oxidation Even though lower temperatures reduce the rate of Hb autoxidation [16], the pathways for reduction of metHb are also compromised. At lower temperatures, the rate of metHb reduction is diminished and metHb itself may be less stable [191]. To compound the issue, the solubility of O2 at 4°C is higher, providing more O2 to the RBCs

[191]; this increases the possibility of autoxidation since partial oxygenation gives the highest rate of metHb formation [14].

Oxidized Hb will denature and aggregate on the cytosolic side of the RBC membrane and form Heinz bodies [192]. They are known to form in ATP-depleted cells [193], cells with a decreased reducing power [194], and cells with unstable Hbs [195] like sickle cell disease RBCs [196] where Hb has accelerated autoxidation rates [197]. It was subsequently found that denatured Hb associates specifically with the cytosolic portion of an integral membrane protein band 3 [192]. Other Hb degradation products like hemin will associate to the cytoskeleton [198] and membrane, damaging it further since its proximity to the cell wall inhibits antioxidant protection [199]. Heme stabilization by the addition of carbon monoxide (CO) prevented these changes from occurring, indicating heme as the site of this oxidative modification [200].

Incubation with hydrogen peroxide (H2O2) expedites this oxidation in vitro and promotes an increase in metHb levels. This oxidative stress caused metHb to become increasingly associated with spectrin, a cytoskeletal protein. Modification of the cytoskeleton created functional alterations in shape (i.e. increased percentage of echinocytes) and biophysical properties by decreasing membrane deformability.

Autoxidation of Hb still occurs in ex vivo storage and begins the process of oxidative damage from within the cell. During hypothermic storage, Hb and its degradation

21 products accumulate on the membrane protein band 3, as in the in vitro tests [201].

Importantly, a portion of this bound Hb consisted of non-reducible crosslinked modifications that were quantifiable early in storage [202, 203]. Similarly, high molecular weight Hb species were found crosslinked to spectrin during storage, even in the first two weeks [204].

1.5.3.2 Lipid Oxidation and Loss As previously mentioned, the loss of lipid via vesiculation and the loss of phospholipid asymmetry have been documented [152-154, 163-165]. Permanent geometric changes stem from loss of phospholipid asymmetry, the loss of phospholipids, and lipid oxidation. It was suggested that the progressive loss of lipid asymmetry may be caused by oxidation of the translocase protein that is partially responsible for phospholipid asymmetry in the RBC [152]. Furthermore, normal RBCs treated with oxidants and thalassemic RBCs, which are already under increased oxidative stress [205], shed more PS than normal RBCs [206].

Further studies suggest that a decrease in antioxidant capacity causes similar irreversible changes to lipids in stored RBCs. Vesiculation and general lipid loss were postulated to be the result of lipid peroxidation which was quantified by the buildup of a byproduct of lipid peroxidation, malondialdehyde (MDA) [165]. Hb autoxidation in the presence of unsaturated lipids, as in the RBC, can produce increased levels of oxidation products and result in lipid peroxidation [207].

1.5.3.3 Cytoskeleton Oxidation The cytoskeleton is of great importance in the mechanical properties of the RBC as it provides shape, structure, and stability, but also allows for deformability. The combination of a lipid bilayer, integral membrane proteins, and a hexagonal lattice of

22 proteins on the cytosolic leaflet of the membrane allows for tremendous properties, allowing an 8μm cell to pass through the microvasculature openings of 2-3μm [19] and elongations of 2.3 times the erythrocyte diameter [208].

Figure 1.4 - Organization of major membrane protein complexes in the hRBC membrane. GLUT1, glucose transporter 1; 3, Band 3; GPA, glycophorin A; GPC, glycophorin C; Hb, hemoglobin; 4.1, protein 4.1; 4.2, protein 4.2; RhAG, Rh-associated glycoprotein [209]

Both α and β dimers of spectrin (band 1 and 2, respectively) compose the network that interacts with actin (band 5) and protein 4.1 (band 4.1). Ankyrin (band 2.1) links the hexagonal network of cytoskeletal proteins with the integral band 3 protein. Band 3 is an anion transport channel that also acts as a major anchor point for the spectrin-protein 4.1- akyrin complex [176]. Several other adaptor proteins including protein 4.2, p55, adducin,

23 dematin, tropomyosin, and tropomodulin also provide important functions (Figure 1.4)

[202, 208, 209].

Hereditary diseases that affect these proteins significantly change the biophysical properties of the affected RBCs, altering morphology and deformability [210]. Similarly, modifications to these protein components during hypothermic storage results in changes in deformability and vesiculation, and ultimately determine the survival of these cells in vivo after transfusion [160].

There is a significant decrease in the ability of spectrin extracted from stored RBCs to associate with actin after extended storage. This loss of activity includes changes in spectrin-protein 4.1-actin interactions that significantly decreased during storage [201].

Since it was previously shown that both hereditary spectrin abnormalities [210, 211] and the presence of hemin [212] would disrupt the association of spectrin with protein 4.1, it was hypothesized that spectrin oxidation during storage contributed to this loss of activity. This hypothesis was further supported when the proposed spectrin oxidation was reversed in vitro by the addition of a reducing agent [201]. Spectrin oxidation was later correlated to vesiculation under blood banking conditions and localized interactions with integral proteins were disrupted, allowing small areas of the membrane to dissociate from the cytoskeleton and form spectrin-free vesicles [213].

As mentioned previously, denatured Hb [192] and hemin [198] will both associate to the cytosolic portion of the integral membrane protein band 3. In doing so, the band 3 proteins will aggregate and crosslink, constituting a biomarker for recognition by antibodies [195, 214]. These in vitro tests artificially produced denatured Hb, but more recently, similar results were seen in ex vivo storage. Membrane band 3 protein

24 aggregation increased in a time-dependent manner [165, 203, 204] and preceded lipid loss (i.e. vesicle formation) [215]. Band 3 aggregation increased binding of autologous immunoglobin G (IgG) and anti-band 3 autoantibody which also increased with time

[203, 216]. The resistance of band 3 aggregates to reduction suggest that these adducts are stabilized by non-reducible linkages, usually free radical generated adducts [203].

The oxidation of other cytoskeletal proteins adds to the evidence of oxidative damage as part of the HSL. An increase in carbonylation of band 4.1 protein was found in stored hRBCs with decreasing antioxidant capacity [165]. Further evidence of carbonyl group formation identifies the cytoskeleton as a target of oxidative damage [202, 204, 217].

The evidence of Hb oxidation and denaturation, oxidative damage to membrane phospholipids and cytoskeletal proteins combined with the evidence of decreased antioxidant capacity in stored cells highlights a mechanism for the HSL. The current additive solutions do not mitigate this damage due to ROS formation, and therefore there is a need for the supplementation of additive solutions with an antioxidant.

1.5.4 Post Transfusion Changes Erythrocytes also undergo changes after transfusion that could further complicate clinical outcomes. These changes include increased potassium leakage, hemolysis, outward exposure of normally inward-facing phospholipids (i.e. PS), and vesicle formation after transfusion of stored RBCs; these changes were elucidated by a simple overnight incubation at 37°C [218]. Extracellular potassium is not only accumulated during hypothermic storage, but also after a brief incubation at physiological temperatures. This leakage, even after transfusion, makes RBCs more likely to expose PS on the extracellular layer of the membrane; a lower intracellular concentration of

25 potassium inhibits flippase activity, which maintains the cell membrane asymmetry. The

PS exposure makes cells more prone to vesiculation [218].

1.6 Recent advances Several advances came more recently and other experimental additive solutions have been suggested and tested, although only two have been approved for the storage of human blood components. Most of these advances involve the supplementation of additional metabolites, the manipulation of the pH, or the modification of the osmotic balance, while only a select few actively attempt to prevent oxidative damage [219].

1.6.1 Modification of pH, metabolites, or osmotic pressure of additive solution components Meryman and colleagues showed up to 14 week storage with 75% 24 hour PTV and

18 week storage with 70% PTV. However, these solutions would not be acceptable for transfusion due to excessively high hemolysis. This study could not specify which factor was responsible for increase in viability: hypotonicity or the presence of ammonium salts

[220, 221]. This preservative was studied further, but hypotonicity was not the cause of increased ATP levels; instead, it was suggested to be a reduction in deamination of adenosine by ammonium ions and/or inorganic phosphate [222]. Further studies showed that ammonium ions were not necessary and that mannitol and chloride ions (Cl-) were not the major factor, but instead initial pH controlled ATP levels [223, 224].

Optimization of this solution provided a solution that could maintain both ATP and 2,3-

DPG levels, and provide acceptable PTV values at 9 weeks, but would ultimately still have to be washed before transfusion, limiting its usefulness [225, 226].

Concerns about the capability of transfused RBCs to deliver O2 drew attention back to 2,3-DPG quantities within stored units. Since a pH below 7.3 promotes depletion of

2,3-DPG by activation of bisphosphoglycerate phosphatase [227], maintaining physiological pH during storage became increasingly important. Improvements in 2,3-

DPG concentrations were achieved by introducing a solution without chloride for RBC storage [228]. The ‘chloride shift’ is produced by a wash with a chloride free solution.

With the outward movement of this negative ion, the charge equilibrium drives hydroxide ions (OH-) inwards, increasing the intracellular pH and promoting both ATP and 2,3-

DPG production. Unfortunately, large amounts of wash solution are necessary, making this method unfeasible for blood banking [229, 230].

Erythro-Sol, a solution containing citrate, phosphate, mannitol, adenine, and dextrose, in combination with 0.5CPD (i.e. citrate concentration is halved), was also developed to prevent the loss of 2,3-DPG and ATP for the preservation of low O2 affinity [231]. This combination 0.5CPD/Erythro-Sol was shown to have very low hemolysis levels while maintaining high 2,3-DPG and ATP levels and high PTV values after 49 days of storage

[231-234] [235]. A push to maintain CPD (over 0.5CPD) for plasma preparation led to the development of Erythro-Sol 2. This newer additive solution has a higher pH (8.8) than its predecessor and is used in larger volumes with the anticoagulant CPD [236].

Red blood cells have been stored for 9 [237], 10 [238], 11[239], and 12 weeks [240] with acceptable levels of hemolysis, ATP, and PTV. These hypotonic solutions work on the principle of inducing cell swelling, reducing membrane loss via vesiculation. They also have a high pH (~8.4) to counteract the progressive decrease by lactate formation and also to promote glycolysis [241]. Although phosphate salts were included in anticoagulants, they were not used in the first generations of additive solutions, but were reconsidered later since they promoted ATP production [221]. The first of these solutions

27 optimized the amounts of adenine, dextrose, mannitol, sodium chloride, and disodium phosphate in EAS 61 [237] and EAS 64 [238]. The use of sodium bicarbonate was shown to increase ATP production [241], and it was later added to EAS 76 [239] and optimized in EAS 76v6 [240]. However, none of these solutions maintained 2,3-DPG for extended amount of time [237, 238, 240]. Importantly, these extended life spans were only possible by the addition of large volumes of additive solution, and they are not currently approved.

Several other additive solutions have been approved for RBC storage worldwide, including phosphate-adenine-guanosine-glucose-saline-mannitol (PAGGS-M) [242],

PAGGS-Sorbitol [243], and mannitol-adenine-phosphate (MAP) [244]. Additionally, expired units of rare blood types can be modified with a solution containing pyruvate, inosine, phosphate, and adenine for the regeneration of ATP and 2,3-DPG, and the normalization of O2 affinity before transfusion [245]. A return to the notion that maintenance of both ATP and 2,3-DPG levels is vital, PAGGS-M replaced sodium chloride with sodium gluconate, now PAGGG-M, resulted in better maintenance of these in vitro parameters, but in vivo parameters remain to be quantified [246].

1.6.2 Protection from Oxidative Damage Some of the damage seen during hypothermic storage is attributed to the presence of

ROS. Attempts to reduce the oxidation of Hb have included its stabilization with CO in vitro [200] and in ex vivo storage [181]. Yoshida and colleagues proposed that the oxidative damage seen in stored RBCs could be prevented if O2 was not present in the blood unit. Deoxygenation in a double volume of additive solution resulted in less hemolysis, reduced membrane vesiculation, superior ATP, and exceptional PTV at 9 weeks of storage [191]. In a subsequent study, 2,3-DPG maintenance was bettered and PS exposure was reduced by anaerobic storage [247]. Although it was concluded that 28 anaerobic conditions could extend the storage time significantly, gas exchange systems are impractical in modern blood banking and these non-traditional methods require additional handling [219]. Studies that have involved the addition of antioxidants to protect RBCs from oxidative damage will be discussed in further detail in Chapter 3.

1.7 Other Factors Affecting Shelf Life Processing varies between blood banks and changes in HCT and methodology are common. First variability is introduced by the donor, whose HCT varies between 38% and 50%, giving different volumes of RBCs per unit [2]. The blood is collected into a bag with 63 mL of anticoagulant, but can have 450 ± 45 mL of whole blood, varying the ratio of anticoagulant to blood [140, 248]. Furthermore, when packed RBCs are produced, some banks manually separate the liquid phase (plasma/anticoagulant) from centrifuged

RBCs before the addition of the additive solution, creating additional variability [121].

The time that it takes to separate and the temperature at which whole blood is held also directly affects the glucose consumption, pH, and cytokine levels in packed RBC units

[249]. When anticoagulants and additive solutions are added, the final concentration of metabolites and HCT fluctuate greatly, and it is estimated that most units are between

250 to 300 mL with a HCT between 65 and 80% [140], making the variability in dose of

Hb per unit very high (49 to 87 g Hb) [20, 121]. It has been found that a higher HCT in storage leads to progressively lower ATP and glucose levels, and reduced PTV [250].

The final volume of the unit is important also since the cell metabolizes glucose. In this metabolism, it produces protons and lactate, or CO2 from the PPP. The volume of the

- liquid fraction will determine the balance of bicarbonate (HCO3 ) and CO2, and ultimately acidity [20].

Mixing during refrigerated storage increases PTV and ATP concentrations and lowers plasma Hb levels [251]. It prevents the quick depletion of 2,3-DPG levels by preventing acid buildup and glucose consumption in the immediate surroundings of settled RBCs

[122]. Lastly, it was shown to reduce microvesiculation, hemolysis, and fluidity in stored

RBCs [252].

Also important is the material from which the RBC bag is made. This factor was first documented when comparing PTV of RBCs stored in glass containers and plastic containers [125, 251]. Permeability to CO2 and O2 will determine the acidity and balance

- of HCO3 /CO2 and O2 saturation levels of Hb [20, 121, 122]. Today, polyvinylchloride

(PVC) bags are produced with plasticizers like di-2-ethylhexylphthalate (DEHP) or butyryl trihexyl citrate (BTHC) which can change the permeability of the container. For example, bags made from PL-2209 (containing BTHC) are more gas permeable than those made of PL-146 (containing DEHP) [20, 121]. Also, DEHP has been found to leech into solution from PVC bags [253]; when compared to other methods of storage, it significantly reduces hemolysis [254, 255], significantly increases PTV [256], and is still used in FDA approved bags.

1.8 Other Methods of Preservation Briefly, other methods of RBC preservation will be described as will the advantages and disadvantages inherent in these processes. Cryopreservation, like hypothermic storage, depends on slowed metabolic rates to preserve biological components. At very low temperatures (-150°C) these processes can be slowed enough to conserve biologic function. Cells stored at these temperatures can be held in ‘suspended animation’ for extended amount of time (> 10 years). Although damage during storage is minimized or negligible, damage during freezing and thawing is of significance. To protect from low 30 temperature injury, the only additive approved is glycerol, but its removal is necessary before transfusion. The freeze-thaw-wash procedure accepted by the AABB requires 80% recovery before transfusion, and additionally must have the same 75% 24-hour PTV as liquid stored RBCs. This process is labor-intensive, expensive, and not feasible for large scale blood banking, but is used for some rare blood types. Lyophilization, or freeze- drying, of RBCs has also been proposed, but previous attempts have been stifled.

Excessive hemolysis and cellular damage was seen in recent attempts of lyophilization, and no current method or product is approved. The economic viability of these methods has also been a major concern [18, 144].

Chapter 2: Quantification of Biochemical and Biophysical Properties of Stored Erythrocytes

2.1 Introduction The studies presented in Chapter 3 and Chapter 4 have several methods that are similar in nature and will be described in this chapter for brevity. Blood from both species was collected normally into FDA approved anticoagulants and separated normally into plasma and packed RBCs stored in FDA approved additive solutions.

These units were separated into smaller transfer bags to which the AA and appropriate control solutions were added. These daughter units were stored for extended periods of time as describe by blood banking procedures. The human (Chapter 3) and canine

(Chapter 4) studies however, differ importantly in other aspects which will be described in their individual chapters.

2.2 General Methods 2.2.1 Sample Timeline Samples were taken from stored RBC blood bags on several occasions during storage to monitor biochemical and biophysical changes. For Greyhound RBCs, samples were taken on day 7, 21, and the date of expiration (day 35). For hRBCs, samples were taken on day 7, 21, the date of expiration (day 42), and two weeks after expiration, day 56.

2.2.2 Complete Blood Count Samples from stored units were collected in sodium ethylenediaminetetraacetic acid

(EDTA) and used for a complete blood count (CBC) which analyzed for RBC count

(RBCC), hematocrit (HCT), Hb concentration (HGB), mean corpuscular volume (MCV),

32 mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), and red cell distribution width (RDW) using a hematology analyzer i with the appropriate software settings.

2.2.3 Blood Gas Analysis Samples taken from the units at the several time points during storage were immediately analyzed for pH, pO2 and partial pressure of carbon dioxide (pCO2), percent

- + + - saturation of Hb, and the concentration of HCO3 , Na , K , and Cl using a blood gas and electrolyte analyzer with respiratory/blood gas cassettes ii.

2.2.4 Separation of RBCs, Lysate, and Supernatant Stored units were gently manually rocked before samples were taken. Red blood cells suspended in storage solution were sterilely removed from each daughter unit at each time point and centrifuged at 2,000 rpm (860×g) for 30 min at 4°C. The supernatant was aspirated manually and centrifuged again at 3,500 rpm (1300×g) to ensure no RBCs were present. The supernatant was then removed for further analysis.

The packed RBCs from the first spin were resuspended in a double volume of chilled isotonic (0.9% w/v) saline solution. This suspension was centrifuged again at 2,000 rpm

(860×g) for 30 min at 4°C, and the supernatant was discarded. This wash procedure was performed twice or until the supernatant became visibly clear of cell-free Hb. The packed

RBCs were then lysed in three volumes of chilled 3.75 mM phosphate buffer (PB, pH

7.2). The lysate was centrifuged at 4,150 rpm (3,716×g) for 10 minutes to remove any large cell debris. An aliquot of this lysate was removed for further analysis.

2.2.5 Measurement of Hematocrit, Hemoglobin and Methemoglobin Concentration, and Percent Hemolysis For each sample, the packed cell volume (PCV) was measured by spinning the samples drawn from the storage bags in a HCT centrifuge iii at 11,700 rpm (13,700×g) for three minutes. The concentration of Hb and metHb in both the RBC lysate and supernatant partitions was assayed via UV–visible spectroscopy iv using the Winterbourn equation [257]. The Hb concentrations were then used to calculate the percent hemolysis according to Equation 2.1.

𝐻푏 푆 ∙ 푉푡 ∙ 1 − 𝑃퐶푉 %𝐿𝑦푠𝑖푠 = 𝐻푏 𝐿 ∙ 퐷 ∙ 푉푡 ∙ 𝑃퐶푉 + 𝐻푏 푆 ∙ 푉푡 ∙ 1 − 𝑃퐶푉 (2.1)

𝐻푏 = Hb concentration in supernatant, 𝐻푏 = Hb concentration in RBC lysate, 푉 = total sample volume, 𝑃퐶푉 = packed cell volume of sample taken, 퐷 = dilution factor in

PB.

2.2.6 P50 and Cooperativity Coefficient Oxygen-RBC equilibrium curves derived from 50 µL of stored RBCs in storage solution were measured via dual wavelength spectroscopy v at 37°C as previously described [258]. Briefly, samples were prepared by thoroughly mixing 50 µL of RBCs in storage solution with 5 mL of buffer (pH 7.4), 20 µL of Additive-A, 10 µL of Additive-

vi B, and 10 µL of antifoaming agent . The samples were allowed to saturate to a pO2 of

147 mm Hg using compressed air. The RBC samples were then deoxygenated using a compressed nitrogen stream. The absorbance of oxygenated and deoxygenated RBC samples was recorded as a function of pO2 via dual wavelength spectroscopy. Oxygen-

RBC equilibrium curves were fit to a four-parameter (A0, A∞, P50, n) Hill model 34

(Equation 2.2). In this model, A0 and A∞ represent the absorbance at 0 mm Hg and full

O2 saturation, respectively. The pO2 represents the measured partial pressure of O2, and

P50 represents the pO2 where the Hb is 50% saturated with O2. Lastly, ‘n’ represents the cooperativity coefficient of Hb.

(2.2)

Oxygen equilibrium curves were measured routinely throughout the storage of both human and canine RBCs. It is important to note that these curves change drastically during storage. Figure 2.1 and Table 2.1 demonstrate typical curves for a single human donor during storage. A similar trend is seen for canine donor RBCs stored in storage solutions like AS-1.

Table 2.1 - Oxygen Affinity and Cooperativity Coefficient of a Single Human Donor during Storage Compared to Human Hemoglobin

Duration of Storage P50 Cooperativity (days) (mm Hg) Coefficient 7 31.23 2.34 21 17.29 2.33 42 14.70 2.28 56 12.04 2.11 hHb 12.00 2.80

Oxygen Dissociation Curve for Stored Human RBCs 1.0

Day 7 0.8 Day 21 Day 42 Day 56 0.6

0.4

0.2 Fractional Oxygen Saturation Oxygen Fractional 0.0 20 40 60 80 100 120 140 pO (mm Hg) 2

Figure 2.1 - Oxygen Equilibrium Curve for a Single Human Donor During Storage.

2.2.7 ATP Concentration Red blood cell lysate was assayed for ATP concentration with an ATP bioluminescence assay kit vii and a luminometer viii. The results were normalized by the

Hb content of the cell lysate sample taken.

2.2.8 2,3-DPG Concentration A sample taken directly from the RBC satellite bag was tested for 2,3-DPG concentration utilizing a 2,3-DPG assay kit ix and a UV-visible spectrophotometer iv as per the manufacturer’s protocol. The results were normalized by the Hb content of the sample.

2.2.9 Total Protein Concentration in Supernatant and RBC Lysate The protein concentration in each RBC lysate and supernatant sample was quantified according to the Bradford method using a protein assay kit x.

2.2.10 RBC Gaseous Ligand Binding/Release Kinetics For all measurements, one milliliter of stored RBCs was sterilely removed from each daughter unit at each time point and centrifuged at 3,500 rpm for 5 min (1300×g) at 4°C.

The supernatant was aspirated and discarded, and the packed RBCs were resuspended in a double volume of chilled isotonic (0.9% w/v) saline solution and centrifuged again.

This wash procedure was performed twice or until the supernatant became visibly clear of cell-free Hb. The kinetic rate constants of these washed RBCs were measured.

All RBC gaseous ligand binding/release kinetic measurements were performed using a stopped-flow spectrophotometer xi as previously described in the literature [98]. In all cases, five to ten individual kinetic time course measurements were averaged. The averaged curve was then fit to a first order exponential equation to acquire rate constants.

Time Course of Deoxygenation of hRBCs 0.32

0.30

0.28

0.26

Absorbance(AU) 0.24

0.22 Data Fit 0.20 0 1 2 3 4 Time (s)

Figure 2.2 - Sample Time Course for the Deoxygenation of hRBCs

The O2 dissociation time courses of RBCs (koff,O2) were obtained by rapidly mixing an oxygenated RBC solution (30 M heme) with a deoxygenated solution containing 1.5 mg/mL sodium dithionite. Both the RBC and deoxygenation solution were made in 0.1 M phosphate buffered saline (PBS, pH 7.4). The absorbance from the deoxygenation reaction was observed at 437.5 nm at 20°C. Figure 2.2 depicts a sample time course for the deoxygenation reaction, and it was fit to give the zero-order rate constant (koff,O2) in the case of O2 dissociation.

Time Course for CO Association (464 µM CO)

A Data 0.30 Fit

0.25

0.20 Absorbance (AU)Absorbance

0.15

0.0 0.2 0.4 0.6 0.8 1.0 Time (s)

Time Course for CO Association (232 µM CO)

B Data 0.30 Fit

0.25

0.20 Absorbance (AU)Absorbance

0.15

0.0 0.2 0.4 0.6 0.8 1.0 Time (s)

Figure 2.3 - Sample Time Course for CO Association of hRBCs at A) 464 μM CO and B) 232 μM CO

CO Association Rate Constant 50

40 )

-1 30 (s

on,CO ' k 10 0 0 100 200 300 400 500 Concentration (µM)

Figure 2.4 - Plot of Pseudo-First Order Rate Constants Against CO Concentrations

By measuring kinetic time courses at different gaseous ligand concentrations (Figure

2.3 for CO and Figure 2.5 for NO), the pseudo-first order apparent rate constants were acquired. These were plotted against their corresponding gaseous ligand concentrations, and the slope of the fitted line yielded the second order rate constant for CO association

(kon,CO) (Figure 2.4) and NO dioxygenation (kox,NO) (Figure 2.6).

The CO association time courses of RBCs were observed at 437.5 nm and 20°C by instantly mixing deoxygenated RBCs (30 M heme) with a saturated CO solution. Both deoxygenated RBCs and CO solutions were prepared in the presence of 1.5 mg/mL sodium dithionite solution in 0.1 M PBS (pH 7.4). The apparent reaction rate constants

′ (k on,CO) were measured at two CO concentrations (464 and 232 M).

The NO dioxygenation time courses of RBCs were acquired based on absorbance changes at 420 nm and 20°C following rapid mixing of oxygenated RBCs (7.5 M heme) and appropriate dilutions of the NO stock solution as previously described in the

′ literature [98]. The apparent reaction rate constants (k ox,NO) were measured at different

NO concentrations (12.5 and 20 M).

Time Course for NO Dioxygenation (25µM) -6.7 A Fit -6.8 Data

-6.9

Absorbance (AU) -7.0

-3 -7.1x10 0.5 1.0 1.5 2.0 Time (s)

Time Course for NO Dioxygenation (12.5µM) -7.4 B Fit -7.5 Data -7.6 -7.7 -7.8 Absorbance (AU) -7.9 -3 -8.0x10 0.5 1.0 1.5 2.0 Time (s)

Figure 2.5 - Sample Time Course for NO Dioxygenation of hRBCs at A) 25 μM NO and B) 12.5 μM NO

NO Association Rate Constant

6 )

-1 4 (s

ox,NO 2

' k

0 0 5 10 15 20 25 30 Concentration (µM)

Figure 2.6 - Plot of Pseudo-First Order Rate Constants against NO Concentrations

2.2.11 Statistical Analysis Two-way ANOVA with a Bonferroni post-hoc test was utilized to compare differences between continuous variables in both groups at each time point for all measurements in donor RBCs with statistical software xii. Statistical significance was defined as p < 0.05.

i Procyte-Dx, IDEXX Laboratories, Westbrook, ME ii VetStat Electrolyte and Blood Gas Analyzer, IDEXX Laboratories, Westbrook, ME iii Autocrit Ultra 3, Beckton Dickson, Franklin Lakes, NJ iv UV-vis Spectrophotometer, Hewlett-Packard, Palo Alto, CA, and Olis Globalworks, Bogart, GA v Hemox Analyzer, TCS Scientific Corp., New Hope, PA vi Hemox buffer and additives, TCS Scientific Corp., New Hope, PA vii ATP Bioluminescence Kit, Sigma-Aldrich, St. Louis, MO viii Lumat LB 9507, Berthold Technologies, Bad Wildbad, Germany ix 2,3-DPG Kit, Roche Diagnostics, Indianapolis, IN x Coomassie Plus protein assay kit, Pierce Biotechnology, Rockford, IL xi SX-20 stopped flow apparatus, Applied Photophysics Ltd., Surrey, United Kingdom xii GraphPad Prism, GraphPad Software, Inc., La Jolla, CA

Chapter 3: Extension of the ex vivo life span of stored human erythrocytes by the addition of ascorbic acid to modern storage solutions

3.1 Introduction 3.1.1 Links to Oxidation As detailed in Chapter 1, during routine storage, biochemical and biophysical changes occur to stored RBCs. Outside of the body, stored RBCs are exposed to increasing extracellular and intracellular oxidant levels. Agitation of the RBC unit [189], exposure to light [190], and the breakdown of leukocytes [181] can increase the presence of ROS in storage solutions. To further compound the problem, increasing levels of hemolysis of

RBCs adds both ferric (Fe+3) and ferrous iron (Fe+2) to the extracellular medium. This free iron, cell free Hb, and metHb from lysed RBCs can promote further damage by ROS formation as mentioned before [188]. Several membrane perturbations, including increased vesiculation [170, 213], aggregation of structural proteins [203], and the association of degraded/oxidized heme products [170, 202] have been associated to increasing oxidative damage during storage of RBCs. This oxidative damage to vital components of the RBC leads to increased lethal damage (i.e. hemolysis), and increased sublethal injury [180].

3.1.2 Protection from Oxidation in Erythrocytes and Plasma Naturally, there are many protective reducing molecules and enzymes inside hRBCs and in the plasma that continuously maintain a reduced state inside and outside the cell.

These reducing molecules and enzymes are codependent and keep Hb in its reduced

42 form. To prevent permanent damage, these reducing molecules maintain metHb levels in erythrocytes below 1% [41].

3.1.2.1 Enzymatic Protection Intracellular enzymes like superoxide dismutase (SOD) will scavenge the superoxide

- radical (O2· ), while glutathione peroxidase (GSHPx) and catalase (CAT) will scavenge

H2O2 [144]. Superoxide and H2O2 produced from Hb autoxidation can initiate further radical chemistry, but both are neutralized by SOD (Reaction 1), CAT (Reaction 2), or

GSHPx (Reaction 3), which oxidizes two GSH molecules to GSSG. With this network of reactions, the total hydrogen peroxide concentration is kept at approximately 0.2 nM within the RBC [259]. The other product of autoxidation, metHb, is directly reduced by a specific enzyme called NADH-cytochrome b5-metHb reductase (MetHbr) with reducing power coming from another heme-protein cytochrome b5 (cytb5) (Reaction 4) [260,

261]. Alternatively, metHb can be reduced via an NADPH-dependent MetHb reductase

[261]. Both GSSG and cytob5 must be returned to their reduced forms by GSH reductase

(GSHr) (Reaction 5) and cytochrome-b5-reductase (cytb5r) (Reaction 6). These reactions are mechanisms for removing two deleterious species stemming from Hb autoxidation.

𝐻 𝐻 [144] (1)

𝐻 𝐻 [144] (2)

𝐻 푆𝐻 푆푆 𝐻 [144] (3)

푡𝐻푏 퐶𝑦푡푏 𝐻푏 퐶𝑦푡푏 [261] (4)

푆푆 퐷𝑃𝐻 𝐻 푆𝐻 퐷𝑃 [144] (5)

퐷𝐻 퐶𝑦푡푏 퐷 퐶𝑦푡푏 [261] (6)

3.1.2.2 Glutathione To maintain low intracellular and extracellular levels of oxidants, small protective molecules including glutathione (GSH), ascorbic acid (AA), uric acid (UA) and α- tocopherol (vitamin E) create a reducing environment both in the plasma and the cytosol of RBCs.

In the blood stream, GSH is primarily (>99%) found in erythrocytes with an average concentration of approximately 1 mM [262] to 3 mM [183, 263] in human subjects, and it is also maintained at a high ratio of reduced to oxidized glutathione (GSH/GSSG) [264].

Intracellular GSH/GSSG ratios are kept high due to the presence of GSHr [265] and the use of NADPH [266]. In the plasma, glutathione has a concentration of only ~ 6 μM

[264, 266], possibly up to 25 μM [265], and is normally 55% [266] to 85% [265] reduced. Since GSHr is not present in the plasma, it was concluded that reduced GSH is actively translocated out of cells [265]. Under oxidative stress, glutathione appears to be removed from the erythrocyte [267] by a family of transporters formerly called ATP binding cassette (ABC) which is now known as the multidrug resistance protein (MRP1) and cystic fibrosis transmembrane conductance regulator (CFTR) [268, 269]. These transporters have not been shown to provide an influx of glutathione, so synthesis within the RBC seems to the only method of creating millimolar amounts within the cell.

Purification of glutamylcysteine synthase (GCs) and glutathione synthetase (GSHs) from human erythrocytes provides proof of intracellular synthesis [270]. In RBCs, de novo synthesis requires three amino acid metabolites (cysteine, glutamate, and glycine) and two ATP molecules for peptide formation [15, 183]. Since in storage solutions, the only constituents normally present are glucose, sodium chloride, mannitol, and adenine [140],

GSH synthesis depends on the stores of free amino acids in the RBC. Instead of

44 synthesis, the RBC can maintain GSH levels by enzymatic recycling (i.e. via GSHr

(Reaction 4)), but requires NADPH for GSH regeneration.

The role of GSH in ROS scavenging has already been emphasized. Apart from its importance in H2O2 scavenging, glutathione has been shown to scavenge hemin [263], and the stability of GSH during storage has been positively correlated with PTV [182].

3.1.2.3 Uric Acid Urate, the sodium salt of UA, is present in the plasma in approximately 300 μM concentrations [271], and has been shown to be transported into erythrocytes via facilitated transport, much like glucose [272]. In the blood stream, UA can chelate iron and copper ions, preventing further free radical reactions [273-276]. By donating an electron, UA becomes an oxidizable substrate for heme protein/H2O2 systems, like those found in erythrocytes [277]. Uric acid has also been shown to directly scavenge free radicals [271], and even protects RBCs from hemolysis by minimizing lipid peroxidation and metHb formation in the presence of oxidant stress [278].

3.1.2.4 Vitamin E Vitamin E, or α-tocopherol, is present in the lipid membrane of RBCs and is an important antioxidant that acts by donating a hydrogen atom to a peroxyl radical

(PUFAOO·). This peroxyl radical was formed from a polyunsaturated fatty acid (PUFA) in the membrane that was first converted into a radical (PUFA·) by a free hydroxyl

- radical (OH· ); this PUFA radical was later modified by O2 to form the peroxyl radical. In neutralizing the peroxyl radical, α-tocopherol itself becomes a radical, but is quite unreactive because the unpaired electron is delocalized in the aromatic ring [279]. Its protective effect via this method has been demonstrated in in vivo studies [280].

Interestingly, vitamin E can be regenerated from its radical form by reduction by another

45 vital reducing agent, AA (vitamin C) (Figure 3.1) and also the aforementioned NADH- dependent cytob5r [281, 282].

Figure 3.1 –Ascorbic acid recycling scheme with vitamin E (α-tocopherol) [281].

3.1.2.5 Ascorbic Acid Ascorbic acid is present in the plasma in ~ 50 µM quantities [283, 284] and is usually found in the reduced state [285]. In humans, this concentration of AA is maintained by the contents of the diet since humans are unable to produce AA via de novo synthesis

[286]. Ascorbic acid acts as a reducing agent and can be oxidized twice, donating two electrons (first, ascorbate free radical (AFR) is formed, then dehydroascorbate (DHA))

(Figure 3.2) [287, 288]. Once AA is oxidized outside of the hRBC, DHA is taken up by the hRBC where it is reduced back to AA and can accumulate to levels of 1-2 mM inside the cell [289], acting as a reducing agent inside the RBC [290]. Because of its negative

46 charge at physiological pH and its hydrophilic properties, reduced AA only slowly crosses the RBC membrane into the extracellular space, thus allowing an accumulation in the intracellular space [289]. Although it was originally thought that DHA was taken up by cells through an unknown hexose transporter protein and transported across the membrane by facilitated diffusion [291], more recent studies show otherwise. Contrary to nucleated cells that transport AA via the sodium-dependent vitamin C transporter

(SVCT2), a transporter which is lost during maturation of RBCs [186, 289], AA and

DHA are both taken up by the GLUT1 glucose transporter (as well as other transporters of the family) on the hRBC membrane [290]. The apparent Km for DHA is 1.1 mM for

GLUT1, 1.7 mM for GLUT3, and 0.98 mM for GLUT4 [292, 293], making the rate of

DHA uptake much greater than that of AA [290]. The hRBC contains more than 200,000 copies of the GLUT1 transporter, which is more than any other cell line [286]; it is uniquely expressed in non AA-synthesizing species to import DHA [286, 293].

Ascorbic acid provides protection from oxidative species as it has been shown to

- - scavenge not only O2· , OH· , and H2O2, but also water soluble peroxyl (RO2·),

• hydroperoxyl (HO2 ), nitroxide, thiyl, and sulphenyl radicals [276, 277, 294, 295]. Even

- in its oxidized form, DHA has also been shown to directly scavenge H2O2 and O2· molecules and undergo peroxidative decarboxylation [294, 295]. Specifically, AA in the plasma has been shown to protect RBCs from peroxyl radical-initiated lipid peroxidation in vitro by direct interaction with radical species [296]. It can inhibit lipid peroxidation by Hb-H2O2 mixtures, preventing the release of iron during heme breakdown [276]. It can protect the cell from oxidant stresses in the lipid portion by its interaction with α- tocopherol as mentioned before. By interaction with oxidoreductase (OR), AA can even

47 transfer an electron to an extracellular oxidant [287]. It can mitigate damage caused by oxidizing agents from free Hb, metHb, ferrylHb and globin radicals [74] and inhibit the apoptotic-like degeneration of RBCs [297]. Vitamin C can decrease the extent of oxidative injury, as measured by PS externalization, of thalassemic RBCs [206], which are known to be under increased oxidative stress compared to RBCs without a hemoglobinopathy [205].

Figure 3.2 - Ascorbic acid (AA) recycling in RBCs [287]. AFR, ascorbate free radical; ASC, ascorbate; DHA, dehydroascorbate; FC (Fe+3), ferricyanide; FC (Fe+2), ferrocyanide; GLUT1, glucose transporter type 1; GR, glutaredoxin; GSH, glutathione; GSSG, oxidized glutathione; PDI, protein-disulfide isomerase; TR, thioredoxin reductase.

Since AA is not produced de novo in humans, erythrocytes must replenish the available plasma AA levels by recycling DHA. Specifically, AA is recycled by GSH- dependent enzymatic reduction of DHA via glutaredoxin (GR), protein-disulfide isomerase (PDI) and dehydroascorbate reductase (DHAr) [277, 287, 298-300]. The use of

NADH with ascorbate free radical reductase (AFRr) [277, 301, 302] or NADPH with thioredoxin reductase (TR) [289] can also regenerate AA from either of its two oxidized states [287]. Importantly, two AFR molecules can directly dismutate to produce AA and

DHA [301], although DHA is unstable and can undergo irreversible ring opening due to its instability at physiological pH [298]. This complex series of pathways provides two mechanisms for AA recycling: a high affinity, low capacity AFR reduction which will dominate in cases of low oxidative pressure while a low affinity, high capacity DHA reduction will use both NADPH and GSH to recycle AA in the case of higher oxidative stress [287]. Remarkably, RBCs can regenerate up to 35 μM ascorbate every three minutes, with adequate amounts of glucose and GSH present [289, 290].

3.1.3 Natural Antioxidant Defense in Storage Even at reduced blood banking temperatures, autoxidation of Hb still occurs in ex vivo storage and begins the process of oxidative damage from within the cell. Reduction in temperature also diminishes enzymatic protection, and the loss of ATP that occurs during storage hampers those processes that depend on it [160, 181, 182].

Plasma AA concentrations were shown to be maintained for 28 days for whole fresh blood stored in CPD; however, these measurements occurred in non-leukoreduced blood where leukocyte AA levels decreased, and erythrocyte AA was not measured [303].

Specifically, the loss of erythrocyte antioxidant activity during RBC storage has been demonstrated in the literature [165, 183-185]. It was proposed that a decrease in this 49 antioxidant defense may permit oxidative damage to protein and lipid components of erythrocytes [165]. Activity of GSHPx decreased significantly by the 42 days of storage in AS-1 [165, 185]. According to several studies, a decrease in the levels of GSH (-30%) was seen in storage of RBCs [165, 183, 184]; a decline in activity of GSHr (-8%) and

SOD (-10%) was documented during storage of blood for 25 days [184]. The decrease in

SOD and CAT activity was shown recently [185], although CAT activity was previously not shown to change significantly with time, even under increasing oxidative stress as in storage [165]. Glutathione depletion in storage is due to its short half life, reduced synthesis at low temperatures and its release from erythrocytes in response to oxidative stress [267] and was demonstrated in storage [165]. Glutathione stability in storage has also been shown to diminish and has been correlated to ATP loss. Both are significantly correlated to PTV in stored RBCs [182].

3.1.4 Previous Attempts at the Use of Antioxidants in Storage Modern storage solutions contain only sodium chloride, adenine, glucose, and mannitol and lack the natural antioxidants found in plasma [140]. According to Wolfe et al., an ideal storage solution would include an anticoagulant to maintain ATP levels and also an antioxidant capable of penetrating into the cytoplasm [181, 201]. More recently, antioxidants have been suggested for use in stored RBC units as a result of evidence of oxidative damage to protein and lipid components of RBCs [202, 216].

Several attempts have been made to use AA and its salts before, but not for its antioxidant properties. New compositions of storage solutions were attempts to preserve the content of 2,3-DPG, an allosteric effector molecule responsible for regulating O2 binding properties of Hb [304]. Higher 2,3-DPG concentrations allows oxyHb to release bound O2 more readily [305], allowing better O2 delivery to the patient. The first attempt 50 of the addition of sodium ascorbate was by Wood and Beutler [306] where an improvement of 2,3-DPG concentration was found with 5.05 mM sodium ascorbate.

Similar maintenance of 2,3-DPG levels occurred when 5.05 mM AA was supplemented with metabolic precursors to 2,3-DPG, including dihydroxyacetone [307] or inosine

[308], which by themselves has maintained 2,3-DPG levels. The addition of D-ascorbate

(the unnatural isomer) [309] and ascorbate-2-phosphate (AsP) [310-312] provided similar maintenance of 2,3-DPG values, while the oxidized form of AA, DHA, showed better results [313].

Most of these studies, however, showed that the increase in 2,3-DPG content was associated with a decrease in ATP [306-310, 312, 314-316]. Although higher levels of

AA (20-80 mM) showed increasing maintenance of 2,3-DPG, ATP loses were substantial and progressively inferior. An optimum occurred when high levels of both 2,3-DPG and

ATP were found after six weeks of storage in 5 mM AA; another composition (10 mM

AA with 40 mM dihydroxyacetone) had similar results [314].

Several mechanisms were drawn from these studies, and it was proposed, but not shown, that ascorbate indirectly oxidized NADH [306]. This was not by oxidation of Hb and the subsequent reduction of metHb through NADH-dependent metHbr or the oxidation of GSH [317]. The oxidation of NADPH was suggested [318], and this was thought to cause a relative decrease in ATP concentrations [309]. Optimization techniques and computerized experimental design provided further support for the use of

AsP when both ATP and 2,3-DPG could be maintained for 42 days [315]. The addition of low doses of both AA (10 μM) and vitamin E (140 μM) decreased osmotic fragility and echinocyte formation [319] and was based on the premise that these two vitamins can

51 interact [320-322]. An additive solution (AS-4) containing AsP was later shown to maintain PTV at 74.1 ± 9.8% after 35 days of storage, providing definitive proof that the addition of AsP was vital in RBC ex vivo storage [312].

It is important to note however, that these studies differ from modern studies in that, instead of packed RBCs, whole blood was stored with the preservative media of the time

[323] including ACD, CPD, or modifications of these including ACD-adenine or CPD- adenine. The units in some studies were not leukoreduced [306, 307, 310, 311, 314, 315,

319], and some investigators also routinely agitated the bags during storage [306, 307] while others stored RBCs in glass containers [319].

Attempts at elucidating the mechanism for this ‘ascorbate effect’ failed [122, 306,

317]. Once AA was available in higher purities, the effect on 2,3-DPG vanished, and unfortunately, it was later found that oxalate, a contaminant of AA (and derivatives), was the cause of the maintenance of 2,3-DPG [312, 316, 324, 325]. Since oxalate increases

2,3-DPG levels during RBC storage [326], this artifact may have also influenced these previous results. This finding ended the interest in maintaining 2,3-DPG concentrations, and modern solutions do not attempt to minimize its loss [327] likely due to its regeneration after transfusion [328-330], or its perceived clinical insignificance [66].

A study on IgG binding during storage showed that 200 mM L-ascorbic acid and 200 mM erythorbic acid (separately) partially reduced IgG binding to Band 3 aggregates on the cell surface, indicating an oxidative mechanism for this aggregation [216]. A more recent study used AA to preserve 2,3-DPG, but no quantification of oxidative damage was performed, and the effects may be confounded since AA was added along with

52 nicotinic acid sodium citrate, and PBS (a source of phosphate) as well as a reduced adenine concentration and changed pH [331].

Although the interest in the use of AA has diminished, the use of antioxidants continued. Oral supplementation of vitamin C and E to blood donors decreased lipid peroxidation in RBCs that were subsequently donated [332]. However, direct addition of antioxidants to stored RBCs was found to have the potential to mitigate oxidative damage

[333, 334]. The addition of vitamin E reduced oxidative stress as measured by MDA, conjugated dienes, and GSH content [333]. Knight et al. supplemented blood samples with GSH (10 mM) or AA (10 mM), and found a protecting effect of GSH, but a prooxidant effect of AA. The prooxidant effect, interestingly, occurred at 37°C, but at storage temperatures, it had significantly lower lipid peroxidation levels at day 7 of storage and similar levels on day 14 when compared to the control. This experiment was not performed to mimic RBC storage; the whole blood was not leukoreduced, and small samples (7 mL) were stored in containers that are different from modern storage bags in several aspects (i.e. size and material properties) [334]. Others have used AA in conjunction with hRBCs, but have studied the effects at 25°C for only 6 days [335].

Alternatively, attempts at utilizing compounds that elicit changes in antioxidant levels have been successful in abrogating oxidative stress-induced changes in stored RBCs

[183, 334, 336, 337]. Four studies supported the levels of GSH via different pathways.

The first (Knight et al.) was mentioned previously, as the addition of GSH was found to protect the stored RBCs from oxidative damage [334]. Another used nicotinic acid, a precursor of NADPH and NADH, to support RBC antioxidant protection by supporting the reduction of oxidized GSH. This addition decreased oxidative damage as measured

53 by MDA, conjugated diene, and hemolysis [336]. The third study hypothesized that by storing RBCs with the amino acid precursors for GSH, GSH synthesis rates and GSH levels would be conserved during 6-week storage. However, this study failed to report markers of oxidative damage, so its overall effect on oxidative damage was not quantified

[183]. Lastly, the addition of glutathione precursors (glutamine, glycine, and N-acetyl-L- cysteine) showed increased GSH content and GSH/GSSG ratio as well as decreased metHb formation, loss of membrane proteins, and hemolysis [337].

3.1.5 Ascorbic Acid as a U.S. FDA Additive Importantly, AA is a small molecule that is considered by the U.S. FDA as a GRAS

(i.e. generally regarded as safe) chemical [338] and is generally considered to have the lowest toxicity of all vitamins [339, 340]. For rat subjects, the lethal dose was 11.9g/kg while for a mouse, it is lower at 3.367g/kg [341]. The general guideline in humans is that if a patient is properly screened for proper renal and glucose-6-phosphate dehydrogenase function, iron overload, and oxalate nephropathy, negative side effects should not be encountered [342, 343]. Only moderate effects have been shown to occur following high dosages: nausea, abdominal cramps, and diarrhea [344].

Early studies by Cameron and Pauling have subjected cancer patients to ten grams of

AA daily by injection for ten days, with oral doses of ten grams indefinitely afterwards, although plasma levels of AA were not quantified [339, 345, 346]. In early clinical studies, some authors have claimed 4.5 mg/mL (~0.250 mM) plasma levels to be the highest attainable via oral supplementation [347]. Recent studies support this theory and have noted that the oral route can only moderately increase plasma AA levels in mice to

0.2 mM, while parenteral supplementation (1 dose, 4g/kg wt) achieved 30 mM

54 concentrations [342]. Similar differences between the two routes of administration are seen in human studies [340, 348, 349].

Concentrations of plasma AA greater than 10 mM can be maintained for over four hours by a 1.5 g/kg dose and has been shown to increase plasma AA levels to 26.2 ± 4.9 mM. When administered three times a week, and the patient was properly screened, this level was shown to be safe, but was not considered the maximum dose [343]. In a more recent study that exposed patients to intravenous doses between 30 and 110 g/m2, the highest dosage levels maintained AA between 10-20 mM for 5-6 hours without significant complications in patients [350]. The half life and the clearance rate of AA in the plasma was unaffected by dosage and was 2.0 ± 0.6 hr and 21 ± 5 dL/hr m2, respectively [350]. Importantly, the highest intravenous dose (1 g/min) for four consecutive days/week for four weeks yielded a maximum of 49 mM plasma AA concentration and was well tolerated by patients [350].

3.1.6 Proposed Use of AA in Modern Storage Solutions During hypothermic storage, RBCs are exposed to increasing amount of free iron,

ROS, and products of leukocyte breakdown [181, 188]. These factors have been shown to cause oxidative modifications to lipids in the cell membrane, Hb in the cytoplasm, and proteins in the cytoskeleton [170, 202, 203, 213]. Throughout storage, erythrocytes lose the ability to protect cellular components from oxidative insult as their natural antioxidant capabilities decrease [165, 183, 184]. The supplements in modern storage solutions do not provide metabolites for maintenance of antioxidant activity and fail to protect RBCs from oxidative injury. In light of the storage-induced oxidative stress on human erythrocytes and the fact that they are exposed to antioxidants in vivo, it was hypothesized that the addition of AA to an additive solution would mitigate oxidative 55 damage, reducing both lethal and sublethal injury. This antioxidant would protect erythrocyte protein function (i.e. Hb) and protect cellular lipid and protein components from oxidative damage, extending the effective and absolute ex vivo shelf life.

For this study, low levels of ascorbate (2.93, 5.86, 8.78 mM) were added to a common additive solution soon after RBC processing. These are relatively low amounts of AA, and for a full-sized unit at the same concentration, this amounts to 135 mg of AA.

Transfusing one unit at this high concentration would amount to a fraction of the amounts of AA injected intravenously in other studies (>1000 mg/min).

The addition of AA to modern storage solutions could have a direct and immediate effect on the blood supply at the global level. The use of this common additive in current hRBC additive solutions would be completely unobtrusive to today’s clinical methods, and could allow even greater separation in time and space between donor and recipient, closing the ever-growing gap between supply and demand of lifesaving hRBCs worldwide.

3.2 Materials and Methods 3.2.1 Donor Selection, RBC Collection, Processing and Storage The RBC units were procured from a US FDA licensed collection facility (Central

Blood Bank, Pittsburgh, PA) where they were derived from whole blood (group A+), leukoreduced (pre-storage), collected into CPD, and stored in AS-5 solution in standard storage bags i. A total of 20 RBC units from male donors and 10 units from pre- menopausal female (age <50 years) were used. The protocol was approved by the

University of Pittsburgh Total Quality Council.

After initial testing and processing, on day 3 of storage, each RBC unit was divided into smaller bags ii forming four equal satellite units, each containing a total volume of 75

56 mL of RBCs using a sterile docking device iii. All satellite units were modified with a sampling port iv and stored under routine blood bank conditions in a refrigerator between

1 and 6°C.

3.2.2 Treatment of hRBCs with 5.86 mM AA Initially, a small pilot study was conducted where a range of AA concentrations (0 -

58.55 mM) was added to small aliquots of RBCs. The hRBCs were stored for the maximum 42 days, and the AA concentrations that yielded low hemolysis values (<1%) were determined. An AA concentration of 5.86 mM was selected because it was 100 times the normal human plasma concentration of AA and yielded <1% hemolysis on storage day 42. On storage day 3, half of all satellite RBC units produced from each of 10 male RBC units and 10 pre-menopausal female RBC units were each treated with 7.5 mL of a concentrated AA solution (23.42 mM AA in saline, adjusted to pH 7.1) to achieve an overall concentration in the storage solution of 5.86 mM. The other half were infused with an equivalent volume of a saline solution without AA (adjusted to pH 7.1) and served as controls. In this way, each donor acted as their own control. Testing was performed on the AA-treated and saline control RBCs on storage days 7, 21, 42 and 56.

The latter time point was selected to determine if any positive effect of adding AA would continue to be present beyond the normal storage time limit.

3.2.3 Treatment of hRBCs with 2.93 and 8.78 mM AA An AA dose response study was performed using AA concentration of 2.93 and 8.78 mM (representing 50 and 150 times the normal human plasma AA concentration, respectively) using RBCs from a total of 10 additional male donors. As before, on day 3 of storage, after separation and processing, 7.5 mL of a concentrated AA solution (11.71 mM and 35.13 mM AA in saline, adjusted to pH 7.1, respectively) was added to achieve

57 an overall concentration in the storage solution of either 2.93 mM (n=5) or 8.78 mM

(n=5). The paired bag was infused with an equivalent volume of saline solution without

AA (adjusted to pH 7.1) to serve as controls. In this way, as before, each donor acted as their own control. Testing was performed on the satellite units on days 7, 21 and 42 only.

3.2.4 Transport of Units For each RBC unit, one pair of satellite units to which AA and saline had been added was shipped by overnight courier from Pittsburgh, PA to Columbus, OH under routine

RBC component transport (temperature maintained between 1 and 10 °C). The other pair was kept in Pittsburgh, PA where the MF test and percent hemolysis were measured on all pairs of satellite units. Biochemical assays were performed on all pairs of satellite units in Columbus, OH on the same days testing in Pittsburgh, PA was performed.

3.2.5 Mechanical Fragility Test and Percent Hemolysis The mechanical fragility test and percent hemolysis were performed on samples from each pair of AA-treated and saline control satellite units using a method that has been previously described in the literature [179]. Brieﬂy, at each time point, 20 mL aliquots from each of the satellite units were removed using aseptic technique and adjusted to a standard HCT of 40% with Dulbecco’s phosphate buffered saline v as previously reported and the Hb concentration was determined spectrophotometrically vi. Three milliliter of each aliquot was then added to each of five tubes (7mL, 13×100-mm serum blood collection tubes vii), three of which contained ﬁve 3.2 mm steel ball bearings viii and two of which did not. The tubes with ball bearings were rocked on a rocker platform ix for one hour, while the remaining tubes without bearings were not rocked and served as controls to ascertain the initial concentration of free Hb in each aliquot. After rocking, all tubes were centrifuged twice, and the free Hb concentrations in the supernatants were

58 determined spectrophotometrically x by light absorbance at 540 nm. The MFI

(Equation 7) and percent hemolysis (Equation 8) were calculated as previously described

[179]:

(7)

𝑦푠𝑖푠 (8)

where fHbrocked is the average free Hb concentration in the supernatant of the rocked sample, fHbcontrol is the mean free Hb concentration in the supernatant of the unrocked control sample and Hbaliquot is the mean total Hb concentration of the RBC aliquot at a

HCT of 40%.

i Blood-pack unit; Fenwal Inc., Lake Zurich, IL ii Teruflex 150 mL Transfer Bags; Terumo Corp., Tokyo, Japan iii TSCD-II Sterile Tubing Welder; Terumo Corp., Tokyo, Japan iv Blood bag spike adapter; Baxter, Deerfield, IL v Lonza Bio Whittaker DPBS with Calcium and Magnesium; Fisher Scientiﬁc, Pittsburgh, PA vi ABX Micro S60; Horiba Ltd., Kyoto, Japan vii BD Vacutainer; Becton Dickinson and Co., Franklin Lakes, NJ viii BNMX-2, Type 316 balls; Small Parts Inc., MiamiLakes, FL ix Type M79700 Platform Vari-Mix rocker; Barnstead Thermolyne Corp., Dubuque, IA x Spectronic Genesys 5 spectrophotometer; Spectronic Instruments Inc., Columbus, OH

3.3 Results 3.3.1 Mechanical Fragility The addition of AA at these concentrations was found to have a significant effect on

MFI during storage. As seen in Figure 3.3, the addition of AA at 5.86 mM significantly reduced the MFI values compared to their paired saline control on both day 42 (1.08 ±

0.17 vs 1.28 ± 0.21) and after expiration on day 56 (1.24 ± 0.18 vs 1.46 ± 0.21). The

MFI of both AA and saline-treated RBCs increased significantly (p < 0.001) from day 7 vs day 42 (AA, 0.63 ± 0.11 vs 1.08 ± 0.17; saline, 0.66 ± 0.10 vs 1.28 ± 0.21) and vs day

56 (AA, 1.24 ± 0.18; saline, 1.46 ± 0.21) which was consistent with previous results [179,

351].

For the dose-dependent response, the 5.86 and 8.78 mM AA concentrations used for male RBCs demonstrated lower MFI values (1.22 ± 0.11 and 0.93 ± 0.10) when compared to the saline control RBCs on day 42 (1.41 ± 0.20) (Figure 3.4). The lowest concentration of AA (2.93 mM) did not demonstrate significantly different MFI values compared to the saline controls at any time point.

Figure 3.3 - The effect of 5.86 mM AA on MFI of RBCs during storage period. AA-treated RBCs (n=20) were compared to the saline control RBCs (n=20). **p < 0.01; ***p < 0.001. Error bars represent 1 SD [352].

Figure 3.4 - The effect of different AA concentrations on MFI of male RBCs during storage period. RBCs from male donors treated with 2.93 mM AA (n=5), 5.86 mM AA (n=10), or 8.78 mM AA (n=5) were compared to the saline control RBCs (n=20). *p < 0.05; ****p < 0.0001. Error bars represent 1 SD [352].

3.3.2 Percent Hemolysis Consistent with previous studies [179, 351], percent hemolysis (Figure 3.5) increased significantly (p < 0.001) for both AA and saline-treated RBCs from day 7 vs day 42 (AA,

0.20 ± 0.10% vs 0.58 ± 0.16%; saline, 0.20 ± 0.09% vs 0.69 ± 0.21%) and vs day 56

(AA, 0.94 ± 0.14%; saline, 1.16 ± 0.18%), although the level never exceeded 1.0% on storage day 42. On storage days 21 and 42, AA-treated RBCs demonstrated a trend towards lower hemolysis compared to the control RBC units. It was only on day 56 that hemolysis was significantly decreased by the addition of 5.86 mM AA to both male and female RBC units (AA, 0.94 ± 0.14%; saline, 1.16 ± 0.18%).

Figure 3.5 - The effect of 5.86 mM AA on percent hemolysis of RBCs during storage period. AA- treated RBCs (n=20) were compared to the saline control RBCs (n=20). ***p < 0.001. Error bars represent 1 SD [352].

Figure 3.6 - The effect of different AA concentrations on percent hemolysis of male RBCs during storage period. RBCs from male donors treated with 2.93 mM AA (n=5), 5.86 mM AA (n=10), or 8.78 mM AA (n=5) were compared to saline control RBCs (n=20). **p < 0.01; ****p < 0.0001. Error bars represent 1 SD [352].

For male RBC units, 8.78 mM AA was found to significantly reduce hemolysis on day 21 (AA, 0.26 ± 0.05% vs saline, 0.47 ± 0.10%) and day 42 (AA, 0.40 ± 0.07% vs saline, 0.79 ± 0.15%) (Figure 3.6). The percent hemolysis for the RBCs stored in 2.93 and 5.86 mM AA were not significantly different from the saline control RBCs at any time point.

3.3.3 Blood Gases and pH For the remainder of these analyses, data from the male and female RBCs were combined because significant differences between the male and female units were not observed during storage (data stratified by gender not shown). The pCO2 (Figure 3.7) of the AA-treated RBCs declined significantly (p < 0.0001) between day 7 and 42 (AA,

43.87 ± 12.71 vs 22.31 ± 5.63) and between day 7 and 56 (AA, 16.68 ± 5.48). The pO2

(Figure 3.8) increased significantly (p < 0.0001) for AA-treated RBCs between day 7 and

42 (AA, 66.29 ± 21.66 vs 147.72 ± 8.59) and vs day 56 (AA, 156.35 ± 25.16). The pH

(Figure 3.9) of the AA-treated RBCs, however, remained constant (p > 0.05) between day

7 and 56 (AA, 7.22 ± 0.17 vs 7.18 ± 0.27). The pCO2, pO2, and pH of RBCs treated with

2.93, 5.86, and 8.78 mM AA did not show statistically significant differences at any time point when compared to saline treatment controls (p > 0.05).

80 5.86 mmol/L AA Saline

(mm (mm Hg)

2 pCO 20

0 7 21 42 56 Length of Storage (days)

Figure 3.7 - The effect of 5.86 mM AA on pCO2 during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

250 5.86 mmol/L AA Saline

200

150

(mm (mm Hg)

2 100 pO

0 7 21 42 56 Length of Storage (days)

Figure 3.8 - The effect of 5.86 mM AA on pO2 during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1SD.

10 5.86 mmol/L AA Saline

6 pH 4

0 7 21 42 56 Length of Storage (days)

Figure 3.9 - The effect of 5.86 mM AA on pH during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

3.3.4 Methemoglobin Level in Lysate and Supernatant The metHb levels in the lysates (Figure 3.10) and supernatants (Figure 3.11) of AA- treated RBCs declined significantly (p < 0.01 for both) between day 7 and day 21

(lysates, 0.29 ± 0.29 vs 0.07 ± 0.13; supernatants, 0.55 ± 0.38 vs 0.26 ± 0.29), but remained unchanged (p > 0.05) for the remainder of storage. The metHb levels in the lysates and supernatants of RBCs treated with 2.93, 5.86, and 8.78 mM AA did not demonstrate significantly different values at any point during storage compared to saline control RBCs (p > 0.05).

1.0 5.86 mmol/L AA Saline

0.8

0.6

0.4 metHbLevel (%)

0.2

0.0 7 21 42 56 Length of Storage (days)

Figure 3.10 – The effect of 5.86 mM AA on metHb level in RBC lysates during storage period. The lysates of saline control RBCs (n=20) were compared to the lysates of RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

1.0 5.86 mmol/L AA Saline

0.8

0.6

0.4 metHbLevel (%)

0.2

0.0 7 21 42 56 Length of Storage (days)

Figure 3.11 – The effect of 5.86 mM AA on metHb level in supernatant of packed RBCs during storage period. The supernatant of saline control RBCs (n=20) was compared to the supernatant of RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

3.3.5 Hb Concentration Although significantly more Hb was present in the lysate of male saline-treated RBCs when compared to female saline-treated RBCs at day 7 (p < 0.01), no significant difference between gender groups were observed at any other time point (p > 0.05).

Hb concentrations in the lysate (Figure 3.12) decreased significantly throughout the storage period for both 5.86 mM AA-treated and saline-treated male RBCs (p < 0.0001 and p < 0.01, respectively, for day 7 vs. 56), but not for female RBCs (p > 0.05).

Treatment of RBCs with AA had no significant effect on Hb concentration in the lysate compared to the saline-treated RBCs at any time point (p > 0.05). Compared to RBCs treated with 5.86 mM AA, treatment with either 2.93 mM AA or 8.78 mM AA yielded statistically identical results (p > 0.05; data not shown).

Figure 3.12 - The effect of 5.86 mM AA on Hb concentration in RBC lysate during storage period. The saline control RBCs (male, n=10; female, n=10) were compared to RBCs treated with 5.86 mM AA (male, n=10; female, n=10). **p < 0.01; ****p < 0.001). Error bars represent 1 SD.

3.3.6 Oxygen Equilibrium Curve

The P50 (Figure 3.13) of saline-treated RBCs demonstrated a statistically significant decrease between day 7 and 21 (p < 0.0001), after which levels remained unchanged over the remainder of the storage interval (p > 0.05). The RBCs treated with 5.86 mM AA did not demonstrate significantly different values at any point during storage compared to the saline-treated RBCs (p > 0.05). Compared to RBCs treated with 5.86 mM AA, treatment with 2.93 mM AA or 8.78 mM AA yielded statistically identical results (p > 0.05; data not shown). The cooperativity coefficient (Figure 3.14) remained unchanged over 56 days (p > 0.05). The RBCs treated with 5.86 mM AA did not demonstrate significantly different values (p > 0.05) at any point during storage compared to the saline-treated

RBCs or the RBCs treated with 2.93 mM AA or 8.78 mM AA (data not shown).

40 5.86 mmol/L AA Saline

(mm (mm Hg)

50 P

0 7 21 42 56 Length of Storage (days)

Figure 3.13 - The effect of 5.86 mM AA on P50 during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

3.5 5.86 mmol/L AA Saline 3.0

2.5

2.0

1.5

1.0 Cooperativity Coefficient 0.5

0.0 7 21 42 56 Length of Storage (days)

Figure 3.14 - The effect of 5.86mM AA on O2-Hb cooperativity coefficient during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

3.3.7 RBC Gaseous Ligand Binding/Release Kinetics

The O2 dissociation rate constants (Figure 3.15), CO association rate constants

(Figure 3.16), and the NO dioxygenation rate constants (Figure 3.17) did not change significantly throughout the storage period (p > 0.05).

Additionally, the RBCs treated with 5.86 mM AA did not demonstrate significantly different values for O2 dissociation, CO association, or NO dioxygenation at any point during storage compared to the saline-treated RBCs (p > 0.05). Compared to RBCs treated with 5.86 mM AA, RBCs treated with 2.93 mM AA or 8.78 mM AA yielded statistically identical results for all measurements (p > 0.05; data not shown).

25 5.86 mmol/L AA Saline

20 )

-1 15 (s (s

off, off, O2 10 k k

0 7 21 42 56 Length of Storage (days)

Figure 3.15 - The effect of 5.86 mM AA on O2 dissociation rate constants during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

0.16 5.86 mmol/L AA Saline 0.14

0.12 )

-1 0.10

s s -1

0.08 (µM

0.06

on, CO k k 0.04

0.02

0.00 7 21 42 56 Length of Storage (days)

Figure 3.16 - The effect of 5.86 mM AA on CO association rate constants during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

0.8 5.86 mmol/L AA Saline

0.6

)

-1

s s -1

0.4

(µM

ox, NO k k 0.2

0.0 7 21 42 56 Length of Storage (days)

Figure 3.17 - The effect of 5.86 mM AA on NO dioxygenation rate constants during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

3.3.8 ATP and 2,3-DPG Content The Hb-normalized ATP concentration (Figure 3.18) decreased significantly between day 7 and day 56 of storage for both RBCs treated with 5.86 mM AA and saline-treated

RBCs (p < 0.0001 for both). However, AA treatment did not significantly change ATP concentrations at any time point throughout the storage period compared to saline treatment (p > 0.05). Compared to RBCs treated with 5.86 mM AA, treatment with 2.93 mM AA or 8.78 mM AA yielded statistically identical results (p > 0.05; data not shown).

15 5.86 mmol/L AA Saline

10 (µM ATP g / Hb)

5 ATP ATP Content

0 7 21 42 56 Length of Storage (days)

Figure 3.18 - The effect of 5.86 mM AA on ATP concentrations in RBC lysate during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

Concentrations of 2,3-DPG (Figure 3.19) decreased significantly between day 7 and day 56 of storage for both RBCs treated with 5.86 mM AA and saline controls (p <

0.0001; p < 0.001, respectively). Interestingly, AA treatment resulted in a statistically

71 significant increase in 2,3-DPG concentrations at day 7 compared to saline treatment (p <

0.01). Significant differences were not observed with AA treatment compared to saline treatment at all subsequent time points (p > 0.05). Compared to RBCs treated with 5.86 mM AA, treatment with 2.93 mM AA or 8.78 mM AA yielded statistically identical results (p > 0.05; data not shown).

15 5.86 mmol/L AA Saline **

10 (µmol2,3-DPG g / Hb)

2,3-DPG Content 0 7 21 42 56 Length of Storage (days)

Figure 3.19 - The effect of 5.86 mM AA on 2,3-DPG concentrations in RBCs during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). **p < 0.01. Error bars represent 1 SD.

3.3.9 Total Protein The protein concentrations of RBC lysates (Figure 3.20) were not found to increase significantly by the end of the storage period (p > 0.05).The protein concentration in the supernatant (Figure 3.21) was, however, found to increase significantly for both RBCs treated with 5.86 mM AA and saline controls between day 7 and day 56 (p < 0.001; p <

0.0001, respectively). Lastly, no significant increases in vesicle protein content (Figure

3.22) were observed for either RBCs treated with 5.86 mM AA or saline-treated RBCs over the storage period (p > 0.05).

The 5.86 mM AA treatment did not significantly affect the total protein concentration of RBC lysates, supernatants, or vesicles compared to saline treatment at any given time point (p > 0.05). Compared to RBCs treated with 5.86 mM AA, treatment with either

2.93 mM AA or 8.78 mM AA yielded statistically identical results in all three total assays

(p > 0.05; data not shown).

500 5.86 mmol/L AA Saline

400 (mg mL) /

300

200

100

TotalProtein Concentration 0 7 21 42 56 Length of Storage (days)

Figure 3.20 - The effect of 5.86 mM AA on total protein concentration in RBC lysate during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

25 5.86 mmol/L AA Saline

20 (mg mL) /

TotalProtein Concentration 0 7 21 42 56 Length of Storage (days)

Figure 3.21 - The effect of 5.86 mM AA on total protein concentration in supernatant during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

5 5.86 mmol/L AA Saline

4 (mg mL) /

TotalProtein Concentration 0 7 21 42 56 Length of Storage (days)

Figure 3.22 - The effect of 5.86 mM AA on total protein concentration in vesicles during storage period. The saline control RBCs (n=20) were compared to RBCs treated with 5.86 mM AA (n=20). Error bars represent 1 SD.

3.4 Discussion This study demonstrates that the addition of the antioxidant AA to the RBC units at the onset of storage reduced both hemolysis and sublethal injury after 42 days of storage and up to day 56 (2 weeks after expiration) without significantly altering the RBCs’ biochemical parameters. The MFI, a measure of RBC membrane sublethal injury, was found to be significantly lower in RBCs treated with as little as 5.86 mM AA compared to the saline control RBCs by day 42 of storage. The percent hemolysis of AA-treated

RBCs was also significantly lower compared to the saline controls by storage day 21 when a higher concentration of AA (8.78 mM) was used. These results demonstrate that both sublethal injury (measured by MFI) and lethal injury (measured by hemolysis) can be mitigated by the addition of AA at certain concentrations. In Table 3.1, a linear regression was calculated for each concentration of AA and for saline controls. With 8.78 mM AA, 42 day old AA-treated RBCs demonstrated MFI values equivalent to those of

19 day old saline-treated RBCs. Similarly in Table 3.2, RBCs stored with 8.78 mM AA demonstrated the same percent hemolysis values as 17 day old saline-stored RBCs.

Importantly, there were no significant changes in biochemical properties when comparing

RBCs stored in AS-5 modified with saline to those stored in AS-5 modified with AA.

Importantly, metHb levels remained low, and were not oxidized by the addition of AA.

On the basis of these data, there may be post-transfusion in vivo RBC survival advantages with AA treatment, although this assumption must be formally demonstrated in clinical studies. It has been previously shown that upon reinfusion, older stored RBCs demonstrate a mean of 13% lower 24-hr PTV compared to that of freshly stored RBCs

[148], along with elevations in some of the markers of extravascular hemolysis [353].

Since the RBCs treated with 8.78 mM AA demonstrated MFI and percent hemolysis

75 profiles on storage day 42 equivalent to that of saline control RBCs which were less than

20 days old, AA treatment may improve in vivo RBC survival after transfusion due to their improved rheological profile which is similar to younger control RBCs.

Table 3.1 - Linear regressions of MFI data for RBCs at various AA concentrations during storage period were calculated from the mean MFI values from male donors at each time point during the dosage study [352].

RBC unit Percent Hemolysis Equation R2 value 2.93 mM AA (n=5) MFI = 0.014×days + 0.666 0.994 5.86 mM AA (n=10) MFI = 0.015×days + 0.596 0.996 8.78 mM AA (n=5) MFI = 0.008×days + 0.606 0.987 Saline control (n=20) MFI = 0.020×days + 0.554 0.997

Table 3.2- Linear regressions of percent hemolysis data for RBCs at various AA concentrations during storage period were calculated from the mean percent hemolysis values from male donors at each time point during the dosage study [352]. RBC unit Percent Hemolysis Equation R2 value 2.93 mM AA (n=5) Percent hemolysis = 0.011×days + 0.154 0.999 5.86 mM AA (n=10) Percent hemolysis = 0.012×days + 0.139 0.949 8.78 mM AA (n=5) Percent hemolysis = 0.007×days + 0.095 0.995 Saline control (n=20) Percent hemolysis = 0.016×days + 0.125 0.999

The in vitro biochemical parameters of stored RBC units analyzed in this study were uniformly unaffected by AA treatment at any concentration compared to the saline control RBCs, demonstrating that AA exposure did not adversely alter the biochemistry of RBCs at the doses utilized in these experiments. It has been previously shown that AA

76 added to CPD-adenine preserved RBCs does not considerably alter the pH throughout storage [314], a conclusion supported by the findings in this study. Additionally, although

AA in high concentrations has been shown to have pro-oxidant activity [354, 355], oxidize Hb [356], and mediate oxidative injury to RBCs [165, 334, 357, 358], this was not observed in this study as evidenced by the statistically identical supernatant and lysate metHb levels in AA-treated RBCs and saline controls over the duration of storage.

This suggests that RBCs in additive solution are capable of maintaining Hb in a reduced state in the presence of at least 8.78 mM AA. Additionally, the experiments in this study were performed at 1-6°C, not 25 or 37°C, and with AA concentrations lower than those reported to cause oxidative damage [334, 358].

The P50 of stored RBCs correlates well with previous studies [359]. The O2 affinity of saline-treated RBCs decreased significantly between day 7 and 21, but remained constant until day 56. This is most likely due to the significant drop in 2,3-DPG concentration inside the RBCs [140], which was also seen in this study. It has been shown that the concentration of 2,3-DPG is rapidly returned to the baseline range upon transfusion of stored RBCs in humans [328-330, 360], although it has been considered as a concern for large transfusions [360]. As mentioned before, AA treatment resulted in a statistically significant increase in 2,3-DPG concentrations at day 7 compared to saline treatment which was also a finding previously demonstrated in CPD-adenine preserved RBCs

[314]. It was found that an oxalate contaminant was the cause of the increase in 2,3-DPG in several studies [312, 316, 324, 325], but was ruled out in this particular study by analysis of our AA by HPLC (results not shown).

3.5 Conclusion In conclusion, AA treatment of AS-5 preserved, leukoreduced RBCs significantly decreased the MFI and percent hemolysis compared to saline control RBCs over 56 days of storage. Additionally, the higher concentrations of AA (5.86 mM and 8.78 mM) demonstrated reductions in MFI and hemolysis in a dose-dependent manner, with the maximal effects seen at the highest AA concentration. The addition of AA to hRBCs at the onset of storage produced RBCs with a longer ‘effective’ life span (i.e. less sublethal and lethal injury). The MFI and percent hemolysis levels in these RBCs at 42 days of storage were the equivalent of RBCs stored in saline for less than half the time. This was achieved without significant changes to RBC biochemical parameters, including metHb levels, O2 affinity, and kinetic rate constants, over the entire duration of storage. These findings suggest that storing RBCs with AA levels of 5.86 - 8.78 mM may confer upon them a rheological benefit, possibly through decreased oxidative stress.

The proposed work is significant since it uses a naturally occurring vitamin to extend the shelf life of stored hRBCs without incurring any significant changes in current blood banking practices. Other options of extending the shelf life of RBCs include cryopreservation and deoxygenation of stored RBCs, but these require non-standard blood banking equipment [18, 191, 247, 248]. Its addition to current hRBC additive solutions would not require changes in blood banking infrastructure; hRBC storage would still occur in the same polymer bags at 1-6°C in the presence of a storage solution and anticoagulant, and component separation techniques would not have to change.

Chapter 4: Canine blood banking: Extension of the ex vivo life span of stored Greyhound erythrocytes by the addition of ascorbic acid to modern storage solutions

4.1 Introduction 4.1.1 Storage Solutions Canine blood banking is an indispensable part of veterinary medicine, used in several clinical procedures [361], and its use has rapidly expanded and is primarily modeled after human blood banking [362]. Erythrocytes from both canines and humans are stored in chemically defined storage solutions (like those mentioned in Chapter 1) that maximize

24 hr PTV and minimize RBC hemolysis or lethal injury [20, 361]. According to Palmer et al., canine and human erythrocytes exhibit similar resistances to hemolysis, and therefore storage solutions should be similar between the two species [363]. According to this assumption, canine whole fresh blood was stored in ACD [364-368], CPD [365,

366], ACD or CPD modified with chemical additives [364, 365], and a novel storage solution referred to as SMB [367].

As the importance of ex vivo blood component shelf life became more obvious, canine whole fresh blood was leukoreduced [369], partitioned, and stored as important components like fresh frozen plasma and packed RBCs, just like human components

[362]. Optimum additive solutions for packed RBCS were investigated for canines as they were for humans. Anticoagulants or preservatives like CPDA-1 and additive solutions based on SAGM were produced for human component storage [370]. Canine packed RBCs were stored in these solutions, although they were originally designed for

79 human component preparation. Canine RBCs have been stored in CPDA-1 [362, 371,

372], Nutricel (AS-3) [371, 372], Nutricel supplemented with additives [372], and Adsol

(AS-1) [369, 372, 373]. Currently, whole fresh blood or packed RBC from dogs are stored for up to 35 days in storage solutions such as Adsol or Nutricel [140, 361, 374].

4.1.2 Greyhounds as Donors At The Ohio State University Veterinary Medical Center Blood Bank, common blood donors are retired racing Greyhounds which possess ideal characteristics for blood donations, but differ in some aspects from other breeds [375]. These large animals

(average ~32 kg (70 lbs)) [376, 377] can safely donate the full 450 mL of whole fresh blood for canine transfusion [378-380] every three weeks for up to two years without any adverse effects [374]. This breed has a gentle temperament and easily accessible jugular veins [374, 381].

Breeding for racing purposes has resulted in Greyhounds having different hematological values when compared to other dog breeds. Previous studies have found higher HCT, HGB, and MCHC in Greyhounds compared to other breeds [382-386].

Their high resting HCT increases significantly during exercise conditions due to splenic contraction, bone marrow erythrocyte release, or changes in liquid volume in the vascular space, or some combination [376, 387-389]. Due to the high HCT, it has been shown that

Greyhounds have higher total Hb concentrations, and therefore higher pO2, oxygen saturation (SO2), oxygen content (O2Ct), and oxygen capacity (O2Cap). These changes in biochemical properties result in a breed of canine with higher total O2 carrying capacity versus other breeds [377]. Although it has been stated that Greyhound RBCs (gRBCs) have a shorter life span than other breeds [382], other have found that the life span is relatively similar in all canine species [378, 390]. 80

Table 4.1 - Canine blood groups (adapted from [374]) Current Previous Population Species nomenclature nomenclature incidence (%)

Canine DEA-1.1 A1, CEA-1 40 DEA-1.2 A2, CEA-2 20 DEA-3 B, CEA-3 5 DEA-4 C, CEA-4 98 DEA-5 D, CEA-5 25 DEA-6 F, CEA-6 98 DEA-7 Tr, CEA-7 45 DEA-8 He, CEA-8 40

Table 4.2 - Frequency of 'universal' (negative for all DEA except DEA 4) and positive (negative for all DEA except DEA 1.1 and 4) donors (adapted from [391]) Short Panel Full Panel Combined % (number tested) % (number tested) % (number tested) Greyhounds Universal 63.4 (93) 52.2 (113) 57.3 (206) Positive 6.5 (93) 7.1 (113) 6.8 (206) Non-Greyhounds Universal 18.2 (22) 37.5 (24) 28 (46) Positive 45.5 (22) (Not available)* * – Non-Greyhound dogs positive for DEA 1.1 did not have full panels

Like humans, RBC surface antigens mark compatibility of RBC transfusions, and therefore donors and recipients must match to avoid clinical manifestations of incompatibility reactions [391]. Table 4.1 demonstrates the prevalence of dog erythrocyte antigens in the general canine population. As defined by Iazbik et al., a ‘universal donor’ must be at least dog erythrocyte antigen (DEA) 1.1, 1.2, 3, 5, and 7 negative, but DEA-4 positive [374, 391, 392]. In this study, it was shown that there were more ‘universal donors’ in the Greyhound group than in the non-Greyhound group [391], making them

81 ideal donors since they are compatible with a larger pool of whole fresh blood or packed

RBC recipients (Table 4.2).

4.1.3 Greyhound Hemoglobin

Greyhound RBCs have been found to have a higher O2 affinity (Figure 4.1) than non-

Greyhound canines [377]. The O2 affinity of Greyhound Hb (gHb), like most other Hbs, is affected by the pH, pCO2, temperature, and 2,3-DPG concentrations, and found to have a high O2 affinity under standard conditions (37°C, pH 7.4, pCO2 40 mm Hg) (Figure 4.2 and Table 4.3), similar to that of human Hb [393]. It was proposed that this high O2 affinity could provide O2 to hypoxic muscle tissue during strenuous exercise in the

Greyhound by delivering O2 only to muscles with extremely low O2 tensions [394, 395].

Figure 4.2 shows the O2 affinity of gHb compared gRBC as well as both hHb and hRBCs.

Figure 4.1 - RBC oxygen affinity values of healthy Greyhounds and non-Greyhounds. Greyhounds group did not pass the normality test [377].

1.0

0.9

0.8

0.7

0.6 hHb 0.5 hRBC gHb 0.4 gRBC 0.3

Fractional Oxygen Saturation Oxygen Fractional 0.2

0.1

0.0 0 20 40 60 80 100 120 140 pO2 (mm Hg)

Figure 4.2 - Oxygen Dissociation Curves for human and Greyhound Hb and RBCs

Table 4.2 - Oxygen Affinity and Cooperativity Coefficients of Greyhound and Human Hb and RBCs hHb hRBC gHb gRBC

P50 (mm Hg) 12.307 28.935 10.132 30.901 n 2.6124 2.2709 2.2759 2.0266

4.1.4 Ascorbic Acid in Canines It is important to note that canines produce their own AA, unlike humans, primates, guinea pigs, and fruit bats [286, 396] and maintain plasma AA levels similar to human levels (40.2 ± 6.8 μM) [397-399]. Although AA is then not considered an essential vitamin, supplementation is used for enhanced performance and health, and was shown to

83 increase the plasma AA to 46.3 μM above baseline levels, accomplished by a dose of 50 mg/kg [397]. In canines, the overall elimination rate constant is 0.214 ± 0.16 hr-1 [397].

4.1.5 Ascorbic Acid as a Supplement in Additive Solutions The supplementation of additive and anticoagulant solutions with AA has occurred previously. In these first studies, ATP and 2,3-DPG concentrations, PTV, and pH were quantified. The addition of 4.54 mM AA, however, was not found to have a significant effect on these measurements, although it is likely due in part to small sample size (n=2)

[364]. In a subsequent study, higher levels of 2,3-DPG, but lower levels of ATP were found in both ACD and CPD modified with AA [365]. These results were previously also found in human studies [306, 307] and were explained in Chapter 3. Ascorbate phosphate

(AsP) was later used due to its stability, and similar results were seen. This new storage solution increased the shelf life of canine RBCs to 6 weeks, although the mechanism and the effect of AsP were unclear [367].

Importantly, all of these studies collected non-leukoreduced blood [364, 365, 367] directly in preservative solutions As the technology changed, blood was separated into components and was eventually more likely to be leukoreduced [369]. Recent studies quantified the changes of canine RBCs in more modern human storage solutions [362,

371-373, 400], but have not included ascorbate or its salts as possible additives.

4.2 Materials and Methods 4.2.1 Donor Selection, RBC Collection and Processing Seven retired racing Greyhounds (Canis lupus familiaris) were selected from a pool of blood and plasma donors (DEA 1.1-negative neutered males) at the Animal Blood

Bank in the Veterinary Medical Center at The Ohio State University (Columbus, OH,

U.S.) according to the current Animal Use Protocol in place. Venous whole blood

84 samples were taken from these subjects to determine donation eligibility. Eligible donors must have a minimum HCT of 50%, and from these donors, 450 mL of whole blood was collected into standard blood storage bags i containing 63 mL of CPD on Day 0, as per normal blood banking procedure. After centrifugation of the whole blood at 4,500 rpm

(5,895×g) for 20 minutes, the packed RBCs were manually separated from the fresh plasma in a closed transfer system, and stored in the blood bank for use in future patients.

A volume of 110 mL of ADSOL (AS-1) was added to the packed RBCs [361]. From each blood bag, three daughter bags were produced; two contained 90 mL of stored RBCs, and the remainder (200 mL) was stored in the blood bank for use in canine patients. The

RBCs in the storage solution had a final HCT of approximately 60%. On Day 0, sampling ports ii were aseptically attached to the two daughter bags used in this study.

4.2.2 Addition of Stock Solutions Two stock solutions were made to add to the daughter bags on storage Day 0. The

AA stock solution (6.45 mg/mL ≈ 36.6 mM) was made in saline (0.9% w/v) and the pH was adjusted to 7.1 by the addition of a small amount of sodium hydroxide. Another solution of saline (0.9% w/v), with pH adjusted to 7.1, served as the control. Both solutions were sterile filtered through a 0.22 µm filter before addition to the stored units.

The stock solution of AA was added to one set of RBC daughter units (n=7) to achieve an overall AA storage solution concentration equivalent to approximately 125× the normal human plasma concentration (0.01 mg/mL ≈ 0.057 mM, normal) in the storage solution

(1.25 mg/mL ≈ 7.1 mM, solution). This concentration of AA was chosen, because studies on hRBCs and murine RBCs stored in similar concentrations of AA yielded beneficial effects [352, 401]. The saline solution was added to the other paired daughter bags (n=7), which served as controls. These two additives were added so that the total volume of the 85 bags increased by 10%. The bags were stored at 1-6°C for the standard 35 days of storage.

4.2.3 Sampling Samples were taken after a week, three weeks, and at the expiration date (day 7, 21, and 35) to monitor changes in the biochemical and biophysical properties of the RBCs over the time course of storage, and to assess the effect of the addition of an additive solution composed of AA. This concentration of AA was chosen, because similar studies on hRBCs stored in 3 to 9 mM AA yielded significant differences in biochemical and biophysical properties between AA-treated RBCs and saline-treated RBCs [352]

i Blood-pack unit, Fenwal Inc., Lake Zurich, IL ii Blood bag spike adapter, Baxter, Deerfield, IL

4.3 Results 4.3.1 Complete Blood Count and Blood Gas Analysis comparison between Day 0 and Day 7 Values The values for CBC and blood gas for initial venous (Day 0) samples (Table 4.4) in this study correlate well with previous findings [375, 377, 382-385, 402]. Between Day 0 and Day 7, Greyhound RBCs demonstrated a decrease in PCV, HCT, and MCV, and an increase in MCHC. The concentration of Na+, Cl-, and K+, decreased slightly, and the

- concentration of HCO3 decreased drastically. There was an increase in the pO2 and pCO2, as well as Sat O2. A large decrease in pH was also quantified between Day 0 and

Day 7. RBCC, HGB, MCH, RDW, and WBCC did not change between Day 0 and Day 7.

Table 4.3 - Complete Blood Count and Blood Gas Parameters for Greyhound Whole Blood. Greyhound Whole Blood PCV (%) 57 ± 4 HCT (%) 58.5 ± 3.7 RBCC (M/µL) 8.14 ± 0.57 HGB (g/dL) 19.9 ± 1.3 MCV (fL) 71.9 ± 1.7 MCH (pg) 24.5 ± 0.5 MCHC (g/dL) 34.1 ± 0.6 RDW (%) 17.8 ± 1.5 WBCC (K/µL) 130 ± 28 pH 7.44 ± 0.01

pCO2 (mmHg) 37 ± 2

pO2 (mmHg) 50 ± 7

Sat O2 (%) 84 ± 5 [Na+] (mM) 159 ± 1 [K+] (mM) 4.0 ± 0.2 [Cl-] (mM) 118 ± 1 - [HCO3 ] (mM) 23.8 ± 1.0 PCV, packed cell volume; HCT, hematocrit; RBCC, RBC count; HGB, hemoglobin concentration; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; WBCC, white blood cell count; pCO2, partial pressure of carbon dioxide; pO2, partial pressure of oxygen; Sat O2, percent saturation of Hb; Na+, sodium ion concentration; K+, potassium ion concentration; Cl-, chloride ion concentration; - HCO3 , bicarbonate ion concentration.

4.3.2 Temporal Changes between Day 7 and Day 35 4.3.2.1 Blood Gas Analysis and CBC Between Day 7 and Day 35, there was a significant effect of storage time on all CBC

+ and blood gas measurements made on stored RBCs except MCH, pO2, Sat O2, Na and

Cl-. The results of CBC analysis on stored RBCs are listed in Table 4.5. Although significant temporal changes occurred, there was no general trend in the changes of HCT,

PCV, RBCC, and HGB. Between Day 7 and Day 35, a significant increase was observed in MCV and a significant decrease was also observed in MCHC for both treatment

87 groups. A significant increase in RDW was detected between Day 7 and Day 35 for both

AA and saline stored RBCs. WBCC was significantly affected by storage time.

Table 4.4 - Complete Blood Count Parameters During Storage (Mean ± SD).

Day 7 Day 7 Day 21 Day 21 Day 35 Day 35 Statistical AA Saline AA Saline AA Saline Difference PCV (%)* 51 ± 5 49 ± 4 47 ± 6 46 ± 7 50 ± 4 49 ± 5 † (p<0.01) HCT (%)* 56.3 ± 5.2 53.7 ± 4.8 53.1 ± 7.0 50.1 ± 7.9 56.2 ± 4.2 54.3 ± 4.7 || (p<0.05) RBCC (M/µL)* 8.39 ± 0.84 8.02 ± 0.57 7.90 ± 1.05 7.47 ± 1.00 8.29 ± 0.61 8.00 ± 0.58 - HGB (g/dL)* 20.7 ± 1.9 19.8 ± 1.5 19.4 ± 2.4 18.3 ± 2.5 20.3 ± 1.3 19.7 ± 1.5 - § ,|| (p<0.05), MCV (fL)* 67.1 ± 1.0 66.9 ± 1.6 67.2 ± 0.6 66.9 ± 1.8 67.8 ± 0.8 67.9 ± 1.5 ¶ (p<0.01) MCH (pg) 24.7 ± 0.5 24.7 ± 0.4 24.6 ± 0.5 24.6 ± 0.5 24.5 ± 0.3 24.6 ± 0.5 - MCHC (g/dL)* 36.8 ± 0.6 37.0 ± 0.7 36.6 ± 0.6 36.7 ± 0.9 36.1 ± 0.6 36.3 ± 0.7 § ,¶ (p<0.05) ||,‡ (p<0.05), RDW (%)* 17.9 ± 1.6 17.3 ± 1.4 18.0 ± 2.0 17.7 ± 1.5 19.5 ± 1.0 19.0 ± 1.1 ¶,§ (p<0.01) WBCC (K/µL)* 130 ± 47 121 ± 42 127 ± 28 111 ± 43 138 ± 33 115 ± 42 -

*: measured value was significantly affected by storage time †: statistically significant difference between AA measurement on Day 7 and Day 21 ‡: statistically significant difference between AA measurement on Day 21 and Day 35 §: statistically significant difference between AA measurement on Day 7 and Day 35 ||: statistically significant difference between X measurement on Day 21 and Day 35 ¶: statistically significant difference between X measurement on Day 7 and Day 35 PCV, packed cell volume; HCT, hematocrit; RBCC, RBC count; HGB, hemoglobin concentration; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; WBCC, White blood cell count.

The results of blood gas analysis on stored RBCs are listed in Table 4.6. The pH of both AA and saline stored RBCs showed a significant downward change, reaching an acidic pH of 6.79. The pCO2 levels of Day 35 were significantly decreased to nearly half of the Day 7 values for both treatments of RBCs. For both AA stored RBCs and the controls, potassium ion levels increased significantly to 5.7 and 4.9 mM, respectively.

Bicarbonate ion levels in AA and saline stored RBCs also decreased significantly between these two time points.

Table 4.5 - Blood Gas Analysis Parameters During Storage (Mean ± SD).

Day 7 Day 7 Day 21* Day 21* Day 35* Day 35* Statistical AA Saline AA Saline AA Saline Difference pH† 6.95 ± 0.02 6.94 ± 0.01 6.85 ± 0.03 6.85 ± 0.01 6.79 ± 0.04 6.79 ± 0.03 §,|| (p<0.05)

pCO2 (mmHg)† 45 ± 3 42 ± 2 37 ± 3 33 ± 1 24 ± 1 21 ± 1 §,|| (p<0.05)

pO2 (mmHg) 258 ± 31 260 ± 24 289 ± 6 273 ± 47 292 ± 22 312 ± 10 -

Sat O2 (%) 99 ± 1 100 ± 1 99 ± 1 100 ± 1 99 ± 1 100 ± 0 - [Na+] (mM) 155 ± 1 154 ± 1 157 ± 2 154 ± 2 156 ± 2 154 ± 2 - §,|| (p<0.05), [K+] (mM)†‡ 4.2 ± 0.2 3.4 ± 0.3 5.3 ± 0.1 4.5 ± 0.2 5.7 ± 0.2 4.9 ± 0.3 ¶, #, ** (p<0.01) [Cl-] (mM) 116 ± 1 113 ± 1 115 ± 2 114 ± 2 115 ± 1 114 ± 1 ¶ (p<0.05) [HCO3-] (mM)† 9.1 ± 0.5 8.4 ± 0.4 6.0 ± 0.4 5.3 ± 0.1 3.4 ± 0.3 3.0 ± 0.1 §,|| (p<0.05)

*: denotes 3 measurements, all other time points have 7 measurements †: measured value was significantly affected by storage time ‡: measured value was significantly affected by additive solution §: statistically significant difference between AA measurement on Day 7 and Day 35 ||: statistically significant difference between X measurement on Day 7 and Day 35 ¶: statistically significant difference between measurement of AA and X sample on Day 7 #: statistically significant difference between measurement of AA and X sample on Day 21 **: statistically significant difference between measurement of AA and X sample on Day 35 pCO2, partial pressure of carbon dioxide; pO2, partial pressure of oxygen; Sat O2, percent saturation of hemoglobin; Na+, sodium ion concentration; K+, potassium ion concentration; Cl-, chloride ion - concentration; HCO3 , bicarbonate ion concentration.

4.3.2.2 Percent Hemolysis and Methemoglobin Levels Percent hemolysis (Figure 4.3) increased significantly with time, reaching an average level of about 0.8% by the date of expiration (Day 35). On Day 35, hemolysis in three of the saline-treated bags and in one AA-treated bag surpassed the 1% threshold set by the

U.S. FDA. The metHb levels in the supernatant of stored RBCs (Figure 4.4) showed no significant change between Day 7 and Day 35. The metHb values in the supernatant remained low at approximately 0.5%. MetHb levels in the RBC lysate (Figure 4.4) were lower (≈ 0.1%), and although they changed significantly over time, there was no obvious trend.

*** *** **** * ***

1.0

0.8

0.6 Lysis (%)

0.4

0.2 AA Saline

0.0 7 21 35 Length of Storage (days)

Figure 4.3 - Percent hemolysis in stored RBCs (n=7) for RBCs stored in AA or saline supplemented storage solution. Asterisks denote different p-values (* - 0.05, *** - 0.001, **** - <0.001) in comparison to values between days or treatments. Error bars represent 1 SD.

1.0 AA Lysate Saline Lysate AA Supernatant Saline Supernatant 0.8

0.6

(%) C

[MetHb] 0.4

0.2

0.0 7 21 35 Length of Storage (days)

Figure 4.4 - MetHb level in supernatant (n=7) and metHb level in lysate (n=7) for RBCs stored in AA or saline supplemented additive solution. Asterisks denote different p-values (* - 0.05, ** - 0.01, *** - 0.001, **** - <0.001) in comparison to values between days or treatments.

4.3.2.3 O2 Affinity and Cooperativity Coefficient

The O2 affinity (Figure 4.5) and cooperativity coefficient (Figure 4.6) of RBCs in additive solution changed significantly over time. The O2 affinity (i.e. P50) for the AA- and saline-treated RBCs were 34.11 mm Hg and 30.90 mm Hg, respectively on Day 7; this value remained virtually unchanged through Day 21. On Day 35, however this value was significantly lower. The cooperativity coefficient changed significantly throughout storage, although a trend was not observed.

** ** *** *** 40

(mm (mm Hg)

P 20

10 AA Saline

0 7 21 35 Length of Storage (days)

Figure 4.5 - Hb-O2 affinity (n=7) for RBCs stored in AA or saline supplemented additive solution. Asterisks denote different p-values (* - 0.05, ** - 0.01, *** - 0.001, **** - <0.001) in comparison to values between days or treatments.

* 2.4 ***

2.2 (n)

2.0

1.8 Cooperativity CoefficientCooperativity 1.6 AA Saline

1.4 7 21 35 Length of Storage (days)

Figure 4.6 - Cooperativity coefficient (n=7) for RBCs stored in AA or saline supplemented additive solution. Asterisks denote different p-values (* - 0.05, ** - 0.01, *** - 0.001, **** - <0.001) in comparison to values between days or treatments.

4.3.2.4 2,3-DPG Concentration On Day 7, the 2,3-DPG content of stored RBCs (Figure 4.7) was higher than the Day

0 values observed by others [371-373], but similar to the initial values of other studies

[364, 365, 403]. The 2,3-DPG content decreased over time, and demonstrated a significant decrease from Day 7 values to Day 35 with both treatment groups. For saline- treated RBCs, a significant decrease was also observed between Day 7 and 21, whereas

AA-treated RBCs demonstrated a decrease between Day 21 and Day 35.

** ****

30 **

* 25

20 (µmol/g Hb)

10 2,3-DPG Content 2,3-DPG

5 AA Saline

0 7 21 35 Length of Storage (days)

Figure 4.7 - 2,3-DPG content in RBCs (n=7) for RBCs stored in AA or saline supplemented additive solution. Asterisks denote different p-values (* - 0.05, ** - 0.01, *** - 0.001, **** - <0.001) in comparison to values between days or treatments.

4.3.2.5 RBC Gaseous Ligand Binding/Release Kinetics The kinetic rate constant for RBC deoxygenation (Figure 4.8) increased significantly for saline stored RBCs between Day 7 and Day 35. The kinetic rate constant for CO association (Figure 4.9) increased significantly for AA stored RBCs between Day 7 and

Day 35. Lastly, NO dioxygenation rate constants (Figure 4.10) increased significantly between Day 7 and Day 35 for both treatments of RBCs. For saline-stored RBCs, there was a significant increase in NO dioxygenation rate between Day 7 and 21, and then subsequently between Day 21 and Day 35.

AA Saline * *

)

-1 (s

2 40

off,O k

30 7 21 35

Length of Storage (days)

Figure 4.8 - Deoxygenation kinetic rate constants for RBCs (n=7) stored in AA or saline supplemented additive solution. Asterisks denote different p-values (* - 0.05, ** - 0.01, *** - 0.001, **** - <0.001) in comparison to values between days or treatments.

0.14

) 0.13

-1

(µM

0.12

on,CO k

0.11 AA Saline

0.10 7 21 35 Length of Storage (days)

Figure 4.9 - CO association kinetic rate constants for RBCs (n=7) stored in AA or saline supplemented additive solution. Asterisks denote different p-values (* - 0.05, ** - 0.01, *** - 0.001, **** - <0.001) in comparison to values between days or treatments.

* ****

* **

1.5

)

-1

s -1

1.0

(µM

ox,NO k

0.5

AA Saline

0.0 7 21 35

Length of Storage (days)

Figure 4.10 - NO dioxygenation kinetic rate constants for RBCs (n=7) stored in AA or saline supplemented additive solution. Asterisks denote different p-values (* - 0.05, ** - 0.01, *** - 0.001, **** - <0.001) in comparison to values between days or treatments.

4.3.3 The Effect of 7.1mM AA on stored Greyhound RBCs 4.3.3.1 Blood Gas Analysis and CBC RBCs treated with 7.1mM AA were not statistically different when compared to the saline treated RBCs in any of the CBC measurements (Table 4.5). Blood gas analysis

(Table 4.6) demonstrated a significant effect of AA supplementation on potassium ion concentration. Saline and AA supplemented RBCs demonstrated significantly different levels of K+ on Day 7, Day 21, and Day 35. Chloride ion levels differed between these two treatments on Day 7, but there was no overall effect of AA. There was a significant difference in pH on Day 21 between the AA- and saline-treated RBCs.

4.3.3.2 Biochemical Assays The percent of hemolysis (Figure 4.3), metHb levels in the supernatant and lysate

(Figure 4.4), O2 affinity (Figure 4.5), cooperativity coefficient (Figure 4.6) demonstrated no effect of AA supplementation. The concentration of 2,3-DPG (Figure 4.7) was not significantly affected by the supplementation of AA to the additive solution. Lastly, kinetic rate constants of O2 dissociation (Figure 4.8), CO association (Figure 4.9), and

NO dioxygenation (Figure 4.10) for AA-stored RBCs and saline-stored RBCs were not significantly different at any time point.

4. 4 Discussion Previous studies in the storage of canine RBCs [364, 365] have used AA as an additive, and had not found a beneficial effect. Although similar AA concentrations were used, samples sizes were small, and RBC storage methods differ. Although ACD and

CPD were commonly used at the time of publication, they were generally replaced by

CPDA-1 for FWB storage. With component separation, additive solutions are now used to store pRBCs. In the first study, two of the six units were modified with a solution of

AA, changing the HCT of these two units, but not the controls. In the second study, the control units used for comparison have various pHs that may confound the results of the study. Differences in both HCT and pH can, individually, have a significant effect on the outcome of RBC storage [161, 221, 224, 229, 241, 247]. Although 2,3-DPG, ATP, pH, and PTV were measured in previous studies, there are new aspects of the HSL that must be quantified.

We evaluated the biochemical and biophysical properties of stored RBCs over the standard five-week storage time for canine blood. Most of these changes had not been previously reported in canines. Overall, it was concluded that the addition of 7.1 mM AA did not reduce hemolysis nor did it significantly affect most of the biochemical and biophysical properties of the stored RBCs.

4.4.1 Changes between Day 0 and Day 7 In this study, between the Day 0 venous blood measurement and the Day 7 stored

RBC measurement, RBCs were extracted from the canine donor into an anticoagulant solution, allowed to cool to room temperature, centrifuged and processed manually, separated from the plasma component, mixed with an additive solution, and stored at reduced temperatures for the following seven days.

During this time, several of the blood gas and CBC measurements change, however, not all of these changes are of importance in determining the changes in RBCs as a result of the HSL. Changes in PCV and HCT are likely artifacts of the manipulation that the

RBCs undergo as the liquid phase plasma is removed and replaced with a different volume of additive solution. The HGB, MCH, and RDW remain constant during that first week compared to Day 0 since the RBCs do not undergo lose much Hb either by

99 vesiculation or hemolysis; however, MCV is reduced in Greyhound RBCs, driving up the

MCHC in this first week, indicating that the additive solution is slightly hypertonic.

The changes in concentration of sodium, potassium, and chloride ions are likely also artifacts of changes in the volume of the liquid preservative during processing. The decrease in the concentration of bicarbonate ions is due to the buffering action of this ion as well as a dilution of the available bicarbonate since it is not a component of the additive solution. In neutralizing the free hydrogen atoms, a more drastic decrease in pH is prevented. The pH still decreases sharply between blood donation on Day 0 and Day7.

This initial drop in pH is likely due to the collection of whole blood into CPD, and subsequent storage in AS-1 (pH of 5.5), which has been shown to produce packed RBCs with a pH of approximately 7.0 [221]. The increase in pCO2 is partially due to the neutralizing action of the bicarbonate, and the production of carbon dioxide via the pentose phosphate pathway (PPP) during the first week of storage [20]. The difference in pO2 values between Day 0 and Day 7 is caused by the exposure of deoxygenated RBCs

(50 mm Hg) from the veins to oxygen in the additive solution (~145 mm Hg). During the first week of storage, additional gaseous oxygen permeates the blood bag [404], and, at colder temperatures, has an increased solubility in the additive solution [191], increasing pO2. Leukoreduction was not performed on these units, and WBCC on Day 0 is similar to that of Day 7. Although most of these changes are likely artifacts of the RBC processing, since we did not make any measurements immediately after processing the canine RBCs, we cannot state whether or not these changes occur due to RBC processing or if they occur as a result of the HSL during the first week of cold storage.

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4.4.2 Changes between Day 7 and Day 35 The biochemical and biophysical properties of the stored RBCs changed significantly over the 35 days of storage. The MCV of Greyhound RBCs increases significantly between Day 7 and Day 35, causing a significant decrease in MCHC. This change in cell volume could be the result of an electrolyte imbalance, drawing water into the cell, effectively reducing MCHC by diluting the hemoglobin inside the RBC. The RDW increased due to the volume increase in a portion of the RBC population. MCH remains constant due to low levels of Hb loss via vesiculation and hemolysis. A significant decrease in WBCC occurs during storage as leukocytes degrade during cold storage.

The constant decrease in pH is seen in stored RBCs due to lactate formation and the decreased availability of bicarbonate in the RBC additive solution. When CO2 is formed by the combination of hydrogen and bicarbonate ions or via the PPP, it freely permeates the blood bag, hence reducing the pCO2 in the liquid phase of the storage system continuously. The pO2 and Sat O2 remain high since the polymer blood bag is also permeable to O2,which has increased solubility at reduced temperatures. Sodium and chloride ion concentrations remain constant throughout, since additive solutions are composed of these ions in amounts equal to that inside the RBC. Supernatant K+ levels increase significantly by the expiration date, but these levels are still very low when compared to potassium levels in hRBCs stored until expiration [218], and are similar to those in other studies [362, 372]. The potassium level increases are likely due to the disintegrating leukocytes (i.e. decrease in WBCC), which have high intracellular K+ levels [405], and not necessarily due to the release of intracellular K+ from RBC hemolysis or RBC ATPase pump failure [362, 372]. Although it is common for hRBCs to

101 be leukoreduced [406, 407], these canine units were not, although it may be beneficial to the canine recipient [369].

During the last weeks of storage, the O2 affinity of stored RBCs rose markedly, as seen by a sharp decrease in P50. This is most likely due to the progressive decrease in 2,3-

DPG concentration inside the RBCs [304]. Upon transfusion of stored RBCs in humans, the concentration of this key allosteric effecter is rapidly regenerated [328, 330] and in one study there was a 40% regeneration of 2,3-DPG levels in 3 hours after transfusion

[360]. Hemolysis during ex vivo storage could also cause an increase in oxygen affinity of the stored RBC unit, since it is a mixture of intact RBCs, Hb microvesicles, and cell- free Hb, which has a higher O2 affinity.

The rate of O2 dissociation from Hb-O2 (i.e. oxygenated Hb), the rate of CO binding to deoxygenated Hb, and the rate of NO dioxygenation of Hb-O2 increased significantly over the storage period. NO dioxygenation rate constants more than doubled, while deoxygenation and CO binding increased by 5 to 10% in five weeks of storage. The changes in O2 dissociation rates differ from those in our unpublished results, and a study on hRBCs that revealed no change in these kinetic rate constants during storage in AS-5 additive solution for 42 days [359]. The causes of these differences are undetermined, but we can speculate that they are inherent to the species RBCs.

An elevated O2 dissociation rate constant could lead to vasoconstriction according to

‘autoregulation’ theory, which hypothesizes that excess O2 delivery to the tissues will be counteracted by a reduction in blood vessel diameter (i.e. vasoconstriction) in order to reduce O2 transport to the surrounding tissues [408]. In this case, however, stored RBCs may not deliver enough O2 due to the increased O2 affinity that comes with decreased

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2,3-DPG concentrations at the end of storage. The clinical outcome and relevance of these two changes is unclear, and may only be significant in a recipient requiring a large transfusion volume.

The increase in the magnitude of the NO dioxygenation rate constant could lead to scavenging of endothelial-derived NO by RBC encapsulated Hb-O2. In the absence of a putative vasodilator like NO, localized vasoconstriction occurs [409]. However, vasoconstriction is not a likely result of this increase in NO dioxygenation rate since there are multiple barriers to endothelial NO diffusion to the circulating RBCs [93, 96, 100].

Any changes in NO homeostasis will be dominated by the presence of cell-free Hb and

Hb microvesicles from stored RBC units, both of which have been found to scavenge NO at a much higher rate than intact RBCs [92].

4.5 Conclusions Some of these biochemical and biophysical changes have been previously documented in hRBCs during routine storage for 42 days. The addition of AA at 5.86 and

8.78 mM concentrations to an additive solution for hRBCs has demonstrated reduced hemolysis and mechanical fragility. The MFI of hRBCs decreased in a dose-dependent manner for various AA concentrations [352]. Supplementation of AA in stored hRBCs also significantly reduced hemolysis after 42 and 56 days of storage in AS-5 [352]. The addition of AA to the RBC additive solution may have yielded better results with hRBCs compared to canine RBCs, because of the lack of leukoreduction in canine pRBC units.

The presence of metabolically active leukocytes consumes glucose, exposes cells to increased metabolic waste, cytokines, enzymes, and ROS, and results in increased hemolysis and vesicle formation during storage [18, 121, 136, 181]. Also, the difference in the number of GLUT1 and GLUT4 transporters on the surface of RBCs in these two 103 mammalian species may have affected the outcome [286, 396]. Humans, higher primates, guinea pigs, and fruit bats express higher levels of GLUT1 on the RBC surface compared to other species, since these species are unable to synthesize AA de novo. Canines, and other mammalian species that produce their own endogenous AA, have high levels of

GLUT1 transporters during the perinatal period. As adults, the expression of GLUT1 is replaced with the expression of another glucose transporter, GLUT4. This isoform of the glucose transporter does not transport AA as efficiently as GLUT1 [293]. Therefore, a higher dose of AA could potentially provide a stronger driving force for AA uptake to better preserve canine RBCs during hypothermal storage. Increasing the flux of AA into the RBC could provide adequate protection from oxidative damage during storage. In conclusion, Greyhound RBCs stored for 35 days in standard preservative solution undergo specific biophysical and biochemical changes that are not prevented by the addition of 7.1 mM AA.

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Chapter 5: Conclusions and Future Studies

In this dissertation, our goal was to develop an additive solution for the ex vivo storage of human and canine erythrocytes to extend their effective and absolute life span.

We supplemented an FDA approved storage solution with AA and found promising results in hRBCs. The addition of this antioxidant reduced the mechanical fragility of stored hRBCs and reduced hemolysis while maintaining hRBC function as quantified by its reactions with gaseous ligands [352]. Also, AA was shown to reduce the amount of shed Hb vesicles produced during storage of murine RBCs under similar conditions

[401]. These promising results, however, require further evaluation in order to elucidate the mechanism by which this effect occurs, and eventually to develop a new additive solution for clinical use.

As discussed in Chapter 1, it is evident that RBC membrane lipids, cytoskeletal proteins, and Hb undergo oxidative damage ex vivo. In our work on hRBC storage, the intracellular and extracellular Hb exists predominantly in the reduced state (less than 1% metHb), the kinetic reaction rates with O2, CO, and NO do not change substantially during storage, and the O2 equilibrium curve left shifts due to decreasing 2,3-DPG concentration. Hence, oxidative damage to the Hb may not be significantly affecting the oxygenation function of the RBC. Critical oxidative damage must be centralized on the

RBC lipid membrane and/or cytoskeleton, affecting the biophysical properties of the

RBC and limiting the PTV of RBCs stored in additive solutions.

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5.1 Lipid Composition and Oxidation

We propose to expand our investigation on the use of AA supplemented RBC additive solutions by quantifying the changes in RBC membrane phospholipid composition, asymmetry, and oxidation that they endure as part of the HSL. We hypothesize that the addition of AA will preserve membrane phospholipids in a reduced state, reducing changes in lipid asymmetry and reducing microvesicle formation.

Identification and quantification of the major lipid components in the RBC membrane will help us determine the distribution of lipids in the RBC membrane as well as those ejected from the RBC as microvesicles. Studying the partition of lipids between RBCs and shed microvesicles should also give insight into the mechanism of microvesicle formation.

Briefly, shed vesicles will be separated from the stored RBC supernatant (Chapter 2) via ultracentrifugation (50,000×g, 60 min, 4°C) and shed microvesicles will be obtained as pellets. These will be collected in PBS and analyzed as is or stored at -80°C for lipid and protein extraction and analysis.

Lipids from samples of hRBCs and microvesicles derived from stored hRBCs will be extracted and analyzed via electron spray ionization (ESI) as described in the literature

[410]. The exposure of PS will be quantified by exposure of RBCs to fluorescently labeled fluorescein isothiocyanate (FITC) annexin V and subsequent detection by flow cytometry [153, 411, 412]. Lastly, the oxidation of membrane lipids will be quantified by the presence of MDA via thiobarbituric acid reactive substances (TBARS) method as described previously [165, 207, 413].

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5.2 Cytoskeleton Oxidation

In continuation of the analysis of lipid oxidation, we propose further studies on the effects of AA supplementation on the cytoskeleton oxidative state. We hypothesize that the addition of AA will preserve the cytoskeleton components in a reduced state and maintaining important membrane protein associations, hence preserving the structural integrity of the RBC and reducing Hb vesicle formation.

The cytoskeleton of the RBC will be isolated from intact RBCs by hypotonic lysis with ice-cold PB. The majority of intracellular proteins (i.e. Hb, catalase, SOD) will be removed by subsequent centrifugation of RBC lysate at 14,000 rpm (20,817×g) for 10 minutes and decanting. To remove the lipids, a Triton X-100 solution will be added to the

RBC membrane pellet and will then be centrifuged at 14,000 rpm (20,817×g) for 10 minutes to produce the RBC membrane cytoskeleton.

Global protein oxidation will be quantified by assessing overall protein carbonylation i in stored RBC cytoskeletons and RBC-derived vesicle proteins as described previously [170, 202, 217].

5.3 Erythrocyte Deformability

The function of the cytoskeleton and membrane will be ultimately tested by the measurement of cellular deformability during storage. Deformability of RBCs stored in an additive solution supplemented with AA will be measured by ektacytometry during the time course of storage. Deformability as been previously been shown to decrease during storage [161, 175] and is similar to those changes seen when RBCs are exposed to oxidants in vitro [411]. Therefore the addition of an antioxidant may improve cellular deformability. We hypothesize that the addition of AA to the additive solution will

107 preserve RBC deformability during storage for longer by preserving the reduced state of critical membrane phospholipids and cytoskeletal proteins.

This technique measures the response of RBCs to shear forces, while removing the confounding effects of cell-cell collision and rouleaux formation [414]. The ektacytometer will gradually change the osmolarity of the solution around the RBC population, and measure the deformability index (DI). The local minimum on the left side of a standard curve (Omin) measures the surface area to volume ratio of the RBC. The point where the DI crosses the x-axis (O’) is linear with the inverse of mean corpuscular

Hb concentration (1/MCHC), consistent with dehydration of the RBC [414]. This method will enable us to probe the effect of AA on RBC membrane mechanics, and specify the cause of the changes in deformability (i.e. changes in RBC surface area to volume ratio or increase in intracellular viscosity) [178]. Specifically, RBC deformability will be measured using laser diffraction in PBS at a solution osmolarity of 300 mOsm/L and subjected to several rotational speeds at 37°C with final shear stresses ranging from 0.3 to

30 Pa as stated in the literature [69]. Elongation index (EI) will be calculated from the major (A) and minor (B) axes lengths as EI = (A-B) / (A+B) over the range of shear stresses. These data points will be plotted on a linear/log scale from 0.5 to 20 Pa.

5.4 Microvesicle Formation We have previously measured microvesicle formation for murine RBCs stored in additive solutions supplemented with similar concentrations of AA [401]. We hypothesize that the equivalent study in human RBCs will give similar results. The addition of AA at similar concentrations will likely maintain the intracellular components in a reduced state, preserving cytoskeleton/membrane protein interactions, and limit the formation of microvesicles. 108

Microvesicle formation will be monitored by staining stored RBCs and microvesicle populations with Ter119 conjugated to allophycocyanin. Flow cytometry will be used to examine the Ter119 positive fragments as describe previously [401].

5.5 Antioxidant Capacity of Stored Erythrocytes

The antioxidant status of the RBC is paramount in the storage of erythrocytes. The supplementation of AA into modern storage solutions should provide a reducing environment both inside and outside of the cells.

Since GLUT-1 is the major transporter present on the surface of hRBCs, its function should be quantified during the course of storage. A standard assay for the detection of

GLUT-1 deficiency syndrome involves the uptake of radiolabeled 3-O-methyl-D-Glucose

(3-OMG) and will be used to quantify GLUT-1 activity [415]. The antioxidant status of the RBC can be monitored by quantifying the concentration of intracellular GSH during storage. Intracellular GSH will be measured in 96-well cell-culture microplates by using the luminescence ii. Luminescence can be measured with a multi-detection microplate reader [416].

5.6 In vivo studies of Post Transfusion Viability

Hemolysis and MFI were significantly decreased during storage by the addition of

AA to an additive solution. These ex vivo markers demonstrate that both lethal and sublethal damage to the RBC were reduced during storage. The direct measurement of

PTV, however, is critical and irreplaceable. Before the use of human studies, it would be beneficial to create an animal model to predict human PTV. Recently, Gilson et al. were able to use a mouse model to measure PTV of murine RBCs stored in CPDA-1 [417].

This experiment was an attempt at modeling human PTV with that of murine PTV by

109 storing erythrocytes from mice under very similar conditions to those used in human

RBC storage. Stowell et al. had a similar argument and stored murine RBCs for 14 days; this is equivalent to storing human cells for 42 days when taken as a percent of the total

RBC life span in the body. Measurements of PTV have been performed for murine RBCs stored with AA-supplemented additive solutions with promising results [401]. Other reliable methods used in several animal species include the use of radio-labeled [362,

364, 367, 373, 417, 418], antibody-labeled [148], biotin-labeled [382, 419, 420], or green fluorescence protein (GFP)-labeled [417, 420] RBCs.

i Oxyblot Protein Oxidation Detection Kit, Millipore, Billerica, MA, USA ii GSH-Glo assay, Promega, Madison, WI, USA

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