Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
DSpace
UBCV
Islamzada, Emel
Doctor of Philosophy - PhD
University of British Columbia
Red blood cell (RBC) transfusions are a critical component of patient care, with around 1.2 million units utilized annually in Canada alone. Patients with acute blood loss require temporary RBC transfusions until their erythropoiesis restores RBC levels. Those with chronic disorders, such as hemoglobinopathies and bone marrow cancers, rely on transfused RBCs for entire RBC circulatory lifespan. For these chronic recipients, selecting long-circulating RBC units could minimize transfusion-associated morbidity like iron overload and lung damage, yet established methods to identify these units are lacking. This dissertation studied the potential to use RBC deformability as a surrogate marker for the transfused RBC circulatory clearance time. Deformability is vital for RBC function and has been considered a physical biomarker of circulation time in transfusion recipients. Our team has developed a unique microfluidic ratchet device to analyze RBC deformability. This device operates by directing individual RBCs to deform through a series of micrometer-scale constrictions much smaller than RBC diameter. A consistent force gradient is applied to each cell as it deforms through constrictions, enabling measurement of RBC deformability with high sensitivity and repeatability. Using the microfluidic ratchet device, we show that RBC deformability varies across healthy donors, but is consistent for each donor over multiple donations. Furthermore, RBC deformability is preserved during the standard 42-day cold storage in blood bags but degrades significantly beyond this timeframe. Additionally, deformability can be estimated by sampling from the blood bag tubing segment instead of puncturing the bag. Finally, we use a mouse transfusion model to investigate whether low-deformability RBCs are preferentially cleared from circulation. We show the rigid fraction of transfused RBCs are rapidly cleared, while transfused RBCs remaining in circulation had a deformability profile closely matching that of endogenous RBCs from the recipient mouse. Our findings provide strong evidence that RBC deformability, measured using the microfluidic ratchet device, may be used to predict RBC circulation time in transfusion recipients. If validated using future clinical studies, this approach could be used to identify donors who can provide long-circulating RBC units and reserve these units for chronic transfusion recipients to reduce transfusion frequency and associated morbidity.
DEFORMABILITY BASED CELL SORTING ENABLING QUALITY CONTROL OF DONATED RED BLOOD CELLS by Emel Islamzada B.Sc., The University of British Columbia, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2024 © Emel Islamzada, 2024 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Deformability based cell sorting enabling quality control of donated red blood cells submitted by Emel Islamzada in partial fulfilment of the requirements for the degree of Doctor of Philosophy In Pathology and Laboratory Medicine Examining Committee: Dr. Hongshen Ma, Mechanical Engineering, UBC Supervisor Dr. Mark D. Scott, Pathology and Laboratory Medicine, UBC Co-supervisor Dr. Cedric J. Carter, Pathology and Laboratory Medicine, UBC Supervisory Committee Member Dr. James Feng, Chemical and Biological Engineering, UBC University Examiner Dr. Edward Conway, Medicine, UBC University Examiner Additional Supervisory Committee Members: Dr. Karen Cheung, Electrical and Computer Engineering, UBC Supervisory Committee Member iii Abstract Red blood cell (RBC) transfusions are a critical component of patient care, with around 1.2 million units utilized annually in Canada alone. Patients with acute blood loss require temporary RBC transfusions until their erythropoiesis restores RBC levels. Those with chronic disorders, such as hemoglobinopathies and bone marrow cancers, rely on transfused RBCs for entire RBC circulatory lifespan. For these chronic recipients, selecting long-circulating RBC units could minimize transfusion-associated morbidity like iron overload and lung damage, yet established methods to identify these units are lacking. This dissertation studied the potential to use RBC deformability as a surrogate marker for the transfused RBC circulatory clearance time. Deformability is vital for RBC function and has been considered a physical biomarker of circulation time in transfusion recipients. Our team has developed a unique microfluidic ratchet device to analyze RBC deformability. This device operates by directing individual RBCs to deform through a series of micrometer-scale constrictions much smaller than RBC diameter. A consistent force gradient is applied to each cell as it deforms through constrictions, enabling measurement of RBC deformability with high sensitivity and repeatability. Using the microfluidic ratchet device, we show that RBC deformability varies across healthy donors, but is consistent for each donor over multiple donations. Furthermore, RBC deformability is preserved during the standard 42-day cold storage in blood bags but degrades significantly beyond this timeframe. Additionally, deformability can be estimated by sampling from the blood bag tubing segment instead of puncturing the bag. Finally, we use a mouse transfusion model to investigate whether low-deformability RBCs are preferentially cleared from circulation. We show the rigid fraction of transfused RBCs are rapidly cleared, while transfused RBCs remaining in iv circulation had a deformability profile closely matching that of endogenous RBCs from the recipient mouse. Our findings provide strong evidence that RBC deformability, measured using the microfluidic ratchet device, may be used to predict RBC circulation time in transfusion recipients. If validated using future clinical studies, this approach could be used to identify donors who can provide long-circulating RBC units and reserve these units for chronic transfusion recipients to reduce transfusion frequency and associated morbidity. v Lay Summary Donated red blood cells (RBCs) are a vital component of patient care. For some patients, like those with inherited blood disorders or severe anemia, dependency on blood transfusions may be lifelong. Predicting longevity of donor blood in circulation is currently challenging. Our research investigated whether we can estimate blood's circulation time by assessing RBC deformability. RBCs must be able to fold and deform through openings much smaller than their own size to transport oxygen. Less deformable cells are inefficient and get cleared quickly after transfusion. We developed a microfluidic ratchet device that sensitively and accurately measures RBC deformability. Through our studies, we demonstrate donor-specific differences in RBC deformability that remain consistent over time. Our findings strongly imply that non-deformable RBCs are rapidly eliminated upon transfusion. If deformability predicts circulation time, this discovery may allow blood banks to reserve long-lasting units for vulnerable patients, reducing transfusion frequency and associated adverse effects. vi Preface I am pleased to present this thesis, which represents the culmination of several years of dedicated research and investigation into the field of blood storage and red blood cell (RBC) deformability. As per the requirements of the University of British Columbia (UBC), I provide the following information regarding my contributions, collaborations, and relevant ethics approvals. Ethics Approvals: This research study received ethical approval from the University of British Columbia's Clinical Research Ethics Board (UBC REB# H19-01121), the Canadian Blood Services Research Ethics Board (CBS REB# 2019-029), and the University of British Columbia's Animal Research Ethics Board (UBC REB #A18-0362). Supervision: The research conducted for this thesis was carried out under the guidance and supervision of Dr. Hongshen Ma, with co-supervision provided by Dr. Mark D. Scott, both from the Centre for Blood Research at UBC. Dr. Ma was instrumental in proposing the concept of investigating RBC deformability using the microfluidic ratchet device developed by our team. Chapter Contributions: Chapter 3: This chapter presents the results of experiments validating the use of the microfluidic ratchet device. I designed and conducted these experiments under the supervision of Dr. Hongshen Ma and co-supervision of Dr. Scott. Subsequently, I analyzed the data and compiled the results into graphical representations. A version of Chapter 3 has been published in: vii Islamzada E, Matthews K, Guo Q, Santoso AT, Duffy SP, Scott MD, Ma H. Deformability based sorting of stored red blood cells reveals donor-dependent aging curves. Lab on a Chip. 2020;20(2):226-35. Chapter 4: This section contains the findings from our blood bag storage study exploring how the RBC deformability changes throughout the storage in standard RBC units. Dr. Ma initially conceived the idea for this study. I was responsible for the experimental design and execution, conducted under the guidance of Dr. Hongshen Ma and Dr. Scott. The results have been published in: Islamzada E, Matthews K, Lamoureux ES, Duffy SP, Scott MD, Ma H. Degradation of red blood cell deformability during cold storage in blood bags. EJHaem. 2022 Feb;3(1):63-71. Islamzada E, Matthews K, Lamoureux E, Duffy SP, Scott MD, Ma H. Blood unit segments accurately represent the biophysical properties of red blood cells in blood bags but not hemolysis. Transfusion. 2022 Feb;62(2):448-56 Chapter 5: this section describes our study on the impact of RBC Gamma irradiation on RBC deformability. The results in this chapter are the product of a collaborative effort with Dr. Dana Devine and Dr. Katherine Serrano from the Devine Lab. The initial idea to investigate the impact of RBC irradiation on RBC deformability came from Dr. Ma and Dr. Devine. Dr. Brana Culibrk was instrumental in providing training and Dr. Katherine Serrano was instrumental in arranging the irradiation of RBC units. I designed and executed the experiments, analyzed the data, and created graphical representations under the guidance of Dr. Ma and Dr. Devine. A manuscript related to this work is currently in preparation. viii Chapter 6: This chapter delves into a murine transfusion study exploring the role of RBC deformability in circulation longevity. The original concept was conceived by Dr. Ma. I designed and conducted the experiments under Dr. Ma and Dr. Scott's supervision, and a manuscript related to this research is also in preparation. In closing, I would like to emphasize the substantial contributions of my co-authors, collaborators, and advisors throughout the research process. Their unwavering support, intellectual input, and countless hours of editing and discussion have been invaluable in producing the publications and research presented in this thesis. ix Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ......................................................................................................................... ix List of Tables .............................................................................................................................. xiv List of Figures ...............................................................................................................................xv List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................... xix Dedication ................................................................................................................................... xxi Chapter 1: Introduction ................................................................................................................1 1.1. The function, lifespan, and clearance of a normal RBC ................................................. 1 1.1.1 The crucial role of RBCs in circulation ...................................................................1 1.1.2 The role of RBC deformability in circulation ..........................................................2 1.1.3 Factors affecting RBC deformability .......................................................................3 1.2 The role of RBC transfusions in patient care .................................................................. 5 1.2.1 Necessity and impact of RBC transfusions: vital component of patient care ..........5 1.2.2 Complications associated with RBC transfusions ...................................................6 1.3 RBC unit production and storage. Factors impacting RBC unit quality in storage ........ 7 1.3.1 RBC unit manufacture and storage ..........................................................................7 1.3.2 Post-production modifications to RBC units: RBC irradiation ...............................8 1.3.3 RBC storage lesion during cold storage of RBC units ............................................9 x 1.3.4 Impact of donor-to-donor differences on RBC storage quality .............................10 1.3.5 The impact of RBC storage duration on transfusion outcomes .............................11 1.4 Existing methods of characterization of RBC deformability ........................................ 12 1.4.1 Flow-based techniques ...........................................................................................12 1.4.2 Deformation-based techniques ..............................................................................13 1.4.3 Microfluidic techniques .........................................................................................14 1.4.3.1 Sorting RBCs based on deformability .............................................................. 15 1.5 Motivation for this work ............................................................................................... 16 1.5.1 Chapters .................................................................................................................17 Chapter 2: Materials and Methods ............................................................................................20 2.1 The Microfluidic Ratchet Device Design, Production, and Operation ......................... 20 2.1.1 Microfluidic ratchet device design and operation..................................................20 2.1.2 Microfluidic ratchet device production..................................................................21 2.1.3 Microfluidic ratchet device operation ....................................................................23 2.1.4 Microbead sorting experiments for device validation ...........................................24 2.1.5 RBC sorting experiments for device validation .....................................................25 2.1.6 RBC chemical degradation (glutaraldehyde fixation) sorting experiments...........25 2.1.7 RBC storage pilot study: storing and sampling RBCs in an accelerated storage model ............................................................................................................................... 25 2.2 Assessment of RBC deformability in cold stored standard blood bag units and their corresponding segments ............................................................................................................ 26 2.2.1 Packed RBC bags and segments ............................................................................26 2.2.2 RBC storage and processing for deformability assessment ...................................27 xi 2.2.3 Assessment of hematological parameters ..............................................................27 2.2.4 Assessment of hemolysis .......................................................................................28 2.2.5 Statistical analysis ..................................................................................................28 2.3 Processing, storage, and assessment of RBC deformability for the RBC irradiation study ....................................................................................................................................... 29 2.3.1 Blood bags .............................................................................................................29 2.3.2 RBC deformability and hematological parameters assessment .............................30 2.3.3 Assessment of RBC hemolysis ..............................................................................30 2.3.4 Statistical analysis ..................................................................................................31 Chapter 3: Deformability based sorting of stored RBCs reveals donor-dependent aging curves ............................................................................................................................................32 3.1 Introduction ................................................................................................................... 32 3.2 Results ........................................................................................................................... 33 3.2.1 Process characterization and validation .................................................................33 3.2.2 Donor-based variability in RBC deformability .....................................................35 3.2.3 Donor-based variability in RBC deformability aging curves ................................37 3.2.4 Hematological parameters do not affect RBC sorting ...........................................39 3.3 Discussion ..................................................................................................................... 40 Chapter 4: RBC deformability during cold storage in standard blood bags and corresponding segments ..............................................................................................................45 4.1 Introduction ................................................................................................................... 45 4.2 Results ........................................................................................................................... 48 4.2.1 RBC deformability profiles at time of collection ..................................................48 xii 4.2.2 Measurement repeatability .....................................................................................49 4.2.3 RBC deformability loss during cold storage .........................................................49 4.2.4 Differences in RBC stability during storage between Male and Female donors ...52 4.2.5 Hematological parameters over 6 weeks of storage ..............................................53 4.2.6 Hemolysis levels in blood bags and segments .......................................................53 4.3 Discussion ..................................................................................................................... 55 Chapter 5: Effect of RBC Gamma Irradiation on RBC Deformability .................................59 5.1 Introduction ................................................................................................................... 59 5.2 Results ........................................................................................................................... 60 5.2.1 Irradiation-induced differences in RBC deformability between control and irradiated RBC units immediately post-irradiation and 2 weeks post-irradiation .................60 5.2.2 Hematological parameters during storage .............................................................64 5.3 Discussion ..................................................................................................................... 65 Chapter 6: Deformability-Driven Circulatory Clearance of Red Blood Cells in a Murine Transfusion Model .......................................................................................................................71 6.1 Introduction ................................................................................................................... 71 6.2 Methods......................................................................................................................... 73 6.2.1 Mouse transfusion model .......................................................................................73 6.2.2 Chemical treatment of RBCs .................................................................................74 6.2.3 Deformability assessment using the Microfluidic Ratchet Device ........................75 6.2.4 Data analysis ..........................................................................................................75 6.3 Results ........................................................................................................................... 76 6.3.1 Approach ................................................................................................................76 xiii 6.3.2 AMT treatment of murine RBCs leads to loss of deformability ...........................77 6.3.3 Less deformable RBCs are selectively removed from circulation within 24 hours post-transfusion. .....................................................................................................................78 6.3.4 Deformability of transfused RBCs over five weeks in circulation. .......................80 6.3.5 Changes in RBC deformability correspond with cell loss in circulation. ..............81 6.4 Discussion ..................................................................................................................... 82 Chapter 7: Conclusion .................................................................................................................85 References .....................................................................................................................................88 Appendix: Supplementary Data for Chapter 6 .......................................................................112 xiv List of Tables Table 2-1. Outlet numbers and their corresponding constriction sizes. ........................................ 23 Table 3-1. Hematological parameters of individual donors.......................................................... 43 Table 5-1. RS values of RBCs in control and irradiated RBC unit pairs. ..................................... 68 Table 5-2. Hematological parameters of RBC in control and irradiated RBC unit pairs. (Numbers in bold indicate outside of healthy range) .................................................................... 69 xv List of Figures Figure 2.1. The microfluidic ratchet device for cell sorting. ........................................................ 22 Figure 3.1. Analysis of deformability-based cell sorting data. ..................................................... 34 Figure 3.2. RBC deformability aging curves. ............................................................................... 36 Figure 3.3. Inter- and Intra-donor variability of fresh RBC deformability. .................................. 36 Figure 3.4. Intra-donor variability of RBC deformability aging profiles. .................................... 38 Figure 3.5. Summary of RBC deformability aging curves. .......................................................... 38 Figure 3.6. Linear regression analysis of the Rigidity Score and each of the hematological indices. .......................................................................................................................................... 39 Figure 4.1. Inter-donor variability of packed RBC units on the day of collection and measurement repeatability. ........................................................................................................... 48 Figure 4.2.Deformability profiles of RBCs from blood bags and segments during storage. ....... 50 Figure 4.3.Degradation of RBC deformability during cold storage. ............................................ 51 Figure 4.4. Summary of RBC Rigidity Score during storage. ...................................................... 51 Figure 4.5. Comparison of RBC deformability loss profiles between male and female donors. . 52 Figure 4.6. Correlation between hematological parameters and rigidity score (RS). ................... 54 Figure 4.7. Hemolysis during storage in bags and corresponding segments. ............................... 55 Figure 5.1. Study overview. .......................................................................................................... 61 Figure 5.2. Inter-donor variability of irradiated and control packed RBC units on the day of unit irradiation and 2 weeks post irradiation. ....................................................................................... 62 Figure 5.3. Degradation of RBC deformability during cold storage in control and irradiated RBC samples. ......................................................................................................................................... 63 Figure 5.4. Hemolysis during storage. .......................................................................................... 64 xvi Figure 5.5. Correlation between hematological parameters and RS in irradiated and control RBC units. .............................................................................................................................................. 66 Figure 6.1. Study overview. .......................................................................................................... 76 Figure 6.2. AMT treatment effectively reduces murine RBC deformability. ............................... 78 Figure 6.3. Deformability of donor and recipient RBCs pre- and 24 hours post-transfusion. ...... 79 Figure 6.4. Correlation between transfused and control RS scores. ............................................. 80 Figure 6.5. Percent remaining of transfused RBCs in circulation in recipients. ........................... 81 xvii List of Abbreviations AMT Aminotriazole ANOVA Two-way analysis of variance ATP Adenosine triphosphate CBS The Canadian Blood Services CO2 Carbon dioxide DEHP Di(2-ethylhexyl) phthalate DMSO Dimethyl sulfoxide G6PD Glucose-6-phosphate dehydrogenase GSH Glutathione GTA Glutaraldehyde Gy Gray H2O2 Hydrogen peroxide Hb Hemoglobin HBSS Hank’s Balanced Salt Solution HCT Hematocrit MCH mean corpuscular hemoglobin MCHC mean corpuscular hemoglobin content MCV mean corpuscular volume metHb Methemoglobin NADPH or NADP+ Nicotinamide-adenine dinucleotide phosphate NetCAD Network Centre for Applied Development PKH-26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling xviii PKH-67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labeling PBS Phosphate Buffered Saline PMS Phenazine Methosulfate QC Quality control RBC(s) Red blood cell(s) RCC(s) Red cell concentrate(s) RDW-CV Red cell distribution width ROS Reactive oxygen species SAGM Saline-adenine-glucose-mannitol UBC University of British Columbia xix Acknowledgements I would like to extend my heartfelt gratitude to the following individuals and organizations, without whom this research and thesis would not have been possible: Dr. Hongshen Ma, my supervisor, whose guidance, teachings, and unwavering support have been invaluable throughout this journey. Your expertise and mentorship have shaped not only my research but also my growth as a scientist. Dr. Mark D. Scott, my co-supervisor, for his invaluable insights, contributions, guidance, and wisdom through this research. Your guidance and collaboration have enriched the depth of this study. Dr. Kerryn Matthews from our team, for her exceptional training, guidance, and unwavering support in all aspects of our studies. Your expertise and kindness have been a guiding light in my research endeavors. I extend my appreciation to the members of our team, whose support have created a nurturing and welcoming environment. Special thanks to Dr. Quan Guo, a PhD graduate from our lab, and Aline Santoso, a master’s graduate from our lab, for their invaluable training and involvement in the projects I later embarked on. To our collaborators, Dr. Dana Devine, and her esteemed team members, including Dr. Katherine Serrano and Dr. Brana Culibrk, I express my sincere gratitude for the collaborative efforts that enriched this research. I would like to acknowledge the members of my research committee, Dr. Cedric Carter, Dr. Helene Cote, and Dr. Karen Chung, for their insightful advice, unwavering guidance, and contributions to the development of this thesis. xx My sincere thanks to Canadian Blood Services (CBS) for granting me the Graduate Fellowship in support of my study. Additionally, I am grateful to the UBC Centre for Blood Research for providing valuable opportunities for outreach, participation in annual research symposia, travel grants, and financial support. I extend my appreciation to the dedicated individuals at the CBS NetCAD Blood for Research Facility, especially Dr. Peter Schubert and CBS for their support in providing the necessary blood products for my research project. I also thank the Centre for Blood Research for creating a collaborative and welcoming environment to work and grow in. In conclusion, I would like to express my deepest gratitude to all those mentioned above, as well as to my friends, family, and colleagues who have been part of this journey. Your support, encouragement, and dedication have been instrumental in the successful completion of this thesis. xxi Dedication This dissertation is dedicated to my mom and dad, Samima Garamammadli and Arif Islamov. Thank you for your love, support, and for being an inspiration to me to venture into this field. 1 Chapter 1: Introduction 1.1. The function, lifespan, and clearance of a normal RBC 1.1.1 The crucial role of RBCs in circulation Red blood cells (RBCs), or erythrocytes, are the most abundant blood cell type in the circulation1–3. They are small cells (~8 µm in diameter on average), with a specific biconcave discoid shape. Their unusual shape is attributed partly to the lack of nucleus. The lack of nucleus contributes to high deformability of the RBC, allowing it to transit through narrow capillary beds and vessel openings smaller than their diameter4,5. RBCs are responsible for multiple aspects of maintaining homeostasis. They primarily function as oxygen carriers, facilitating the transport of oxygen from the lungs to peripheral tissues via hemoglobin6. Additionally, RBCs participate in carbon dioxide removal, delivering it back to the lungs for exhalation. RBCs originate in bone marrow, produced as a response to low oxygen levels6. After undergoing multiple stages of maturation, RBCs eventually expel their nucleus and enter the bloodstream. In the bloodstream, mature RBCs have a circulation span of about 120 days, at the end of which the aged cells are removed, primarily by sequestration in spleen and liver and subsequent phagocytosis by macrophages7. During its relatively short lifespan of 120 days, RBCs travel through an equivalent of 500 km of total distance8, and go through the entire circulation in only about 1 minute3. To be able to function the full life cycle of 120 days, RBCs have developed a number of specialized antioxidant pathways that help it alleviate the oxidative damage and stress during its function9. Overall, RBCs play a vital role in maintaining homeostasis through transport of respiratory gases and contributing to optimal blood flow dynamics through their unique deformability. 2 1.1.2 The role of RBC deformability in circulation Deformability is an important parameter that impacts the rheological properties of RBCs, affecting their lifespan and their ability to effectively transport oxygen10,11. RBC deformability is vital for its proper function, as RBCs have to deform and pass through openings much smaller than their diameter in circulation. Although the human RBC is ~8 µm in diameter on average, it has to transverse through synaptic clefts and capillary openings around ~3 µm in size while traveling through the microvasculature in order to perform its function. This deformability is dependent on the mechanical properties of the RBC membrane, the interactions between membrane proteins, and the metabolic state of the cell10,12. Cellular factors that control deformability include cellular dehydration, phosphorylation of membrane proteins, the structural integrity of the cytoskeleton, metabolic processes, and the condition of hemoglobin9,13–15. The interplay of these factors can either enhance or reduce RBC deformability. The premature loss of RBC deformability leads to shortened cell lifespan and is a hallmark of a series of diseases including hereditary conditions such as sickle cell disease, thalassemias, hereditary spherocytosis and hemolytic anemia, as well as other conditions such as malaria infection. Additionally, a series of other conditions, such as G6PD deficiency, can affect RBC deformability as well2,16,17. Since RBC deformability is vital for its proper function, and loss of deformability due to natural ageing or disease can lead to clearance from circulation, deformability has long been considered a potential biophysical marker for RBC quality and longevity in circulation. 3 1.1.3 Factors affecting RBC deformability Factors that impact RBC deformability include dehydration, phosphorylation of membrane proteins, the structural integrity of the cytoskeleton, metabolic processes, and the overall integrity of hemoglobin. The interplay between these factors can enhance or diminish the deformability of RBCs. 1.1.3.1.Structural factors affecting RBC deformability The unique structural assembly of RBCs provides its membrane with stability while allowing it to deform10. Structurally, the RBC membrane is constructed from a triangular network of α- and β-spectrin molecules connected to transmembrane domain of band 3 protein, ankyrin, and proteins 4.1 and 4.218,19. RBC storage related breakdown as well as aggregation of band 3 protein, ankyrin, and spectrin, as well as membrane accumulation of Hb can alter and affect the membrane integrity, thus reducing RBC deformability10,18,19. Any abnormalities or mutations in the structural proteins that constitute the RBC membrane can lead to decreased RBC deformability, and in some cases, to hemolytic anemias10,20,21. Another mechanical factor influencing RBC deformability is the surface-to-volume ratio, which is usually measured as the mean corpuscular volume (MCV)10. RBC has a specific discoid shape, that maximizes its surface-to-volume ratio, and facilitates efficient gas exchange. The cell volume is intricately regulated by ion pumps and channels22. The RBC membrane loss through vesiculation, altered transport of water and ions through RBC membrane, such as one occurring during natural ageing or storage, affect RBC deformability. 1.1.3.2. Metabolic and biochemical factors affecting RBC deformability Metabolic and biochemical factors significantly impact RBC membrane flexibility, and the ability of the RBC to efficiently perform oxygen exchange10,23. RBC deformability is heavily dependent 4 on the viscosity of its cytoplasm. of RBC cytoplasm is a dynamic trait that is affected by both the biochemical environment and inherent cellular properties. Cytoplasm viscosity is controlled by hemoglobin concentration (measured in MCHC), hemoglobin polymerization, intracellular ion levels, cell hydration level, and membrane loss. Increase of Hb concentration of hemoglobin in the RBC can increase RBC viscosity and reduce deformability22,24. Metabolically, RBC deformability is affected by the ATP availability and the redox state of the cell23,25. ATP serves as a critical cellular regulator by influencing multiple biochemical cascades that involve phosphorylation and activity of ion transport, particularly through Na, K-ATPase and Ca-ATPase. ATP also plays a crucial role in maintaining ion gradients, which have a direct impact on RBC shape and cytoskeletal structure. Cellular redox state impacts RBC deformability; this state is dictated by levels of NADH and glutathione (GSH), as well as cellular antioxidants, which counterbalance the oxidative pressure that RBCs continuously experience in executing their physiological functions. Overall, all of the structural and metabolic systems work in unison in a healthy RBC to ensure its deformability. Age and storage related changes will slowly affect the RBC membrane structure, Hb integrity, and redox state, all of which will lead to rigidification of RBCs and their consequent clearance in the spleen. 1.1.4. Clearance route of normal RBCs Overall, RBC deformability is rapidly lost as the RBCs age10,26,27. This reduced deformability plays a vital role in RBC removal from circulation. During the homeostatic RBC turnover, RBCs are filtered through the red pulp of the spleen28. The spleen is specifically designed to filter senescent and rigid RBCs by forcing them to travel through the cords of red pulp and through a network of sinuses with slits as narrow as 2-3 µm28. 5 The red pulp consists of a unique open microcirculation system. It is comprised of splenic cords and splenic sinuses28–30. The unique feature of splenic cords is the absence of endothelial lining28. RBC clearance though the spleen is a two-step process. The RBCs are required to be deformable enough to pass through the endothelial splits of the sinus walls to re-enter circulation. RBCs that are healthy are able to deform through where the more rigid older or damaged cells are trapped in splenic cords28,28–30. The cords are rich in macrophages, with then encounter entrapped RBCs and phagocytose them. Phagocytosis of trapped RBCs is suggested to occur by several mechanisms, including the clustering of the anion channel Band 3, which upon clustering leads to C3B deposition and recognition by Fc-gamma and complement receptors on the macrophages28,30,31. PS exposure on RBC membrane and reduction of CD47 signal can also be recognized by macrophages28. It is still a matter of debate as to which of these mechanisms, and to what extent, plays the deciding role in macrophage phagocytosis of aged and damaged RBCs. What is indisputable though is that the loss of deformability plays a pivotal role in initial entrapment of RBCs in the splenic system. 1.2 The role of RBC transfusions in patient care 1.2.1 Necessity and impact of RBC transfusions: vital component of patient care RBC transfusions are a vital component of patient care. In Canada alone, approximately 1.2 million RBC concentrates are collected and transfused on yearly basis2,32. These transfusions benefit a broad spectrum of patients, from acute to chronic blood recipients. Acute patient groups include trauma, surgery, septic patients, and patients with acute coagulation deficiencies33. About 30% of critical care patients and 50% of cardiac surgery patients need at least one unit of RBC transfusion during their hospital stay3,34. In these acute cases, RBCs are only required to circulate for a short duration, hours to days, until the patient is able to naturally replenish their RBC population. On 6 the other hand, the longevity of transfused RBCs is crucial in the case of chronic and transfusion-dependent patients. Those with hemoglobinopathies, chronic anemia, thalassemias, bone marrow cancers, and acquired or congenital bone marrow failure require and depend on constant RBC transfusions35–38. For these patient groups, it is best if the transfused RBC unit circulates for as long as possible to provide maximum benefit. 1.2.2 Complications associated with RBC transfusions Although life-saving, chronic transfusions come with their own host of challenges14,29,35–40. Regular transfusion patients face a range of potential adverse reactions, such as immediate and delayed hemolytic reactions, iron overload, transfusion induced lung injury (TRALI), and sepsis32,14. Additionally, alloimmunization will make subsequent blood transfusions more challenging. Immediate reactions to transfusion can manifest as acute hemolytic events due to blood type incompatibility. Delayed hemolytic reactions, surfacing days to weeks post-transfusion, arise from slower immune responses against transfused RBCs. Over time, repetitive transfusions can lead to iron overload, a condition where excess iron deposits in vital organs, posing risks of organ damage. Additionally, availability of free iron in the bloodstream will render patients susceptible to bacterial infection and sepsis. Transfusion-Related Acute Lung Injury (TRALI) represents rare but severe pulmonary adverse reactions to transfusion. Furthermore, alloimmunization, the development of antibodies against antigens present on transfused RBCs, can complicate future transfusion efforts, and increase the risk of immune reactions to transfused RBC units. 7 1.3 RBC unit production and storage. Factors impacting RBC unit quality in storage 1.3.1 RBC unit manufacture and storage Every year, approximately 1.2 million of red cell concentrates are produced in Canada by Canadian Blood Services and Hema Quebec1,41. Each RBC unit is made from approximately 480 ml of donor whole blood collected into a collection bag with 70 ml CPD (citrate, phosphate, dextrose) anticoagulant solution.1 The blood unit then undergoes centrifugation to separate RBCs from other blood components. The resulting product is leukoreduced and mixed with additive solution SAGM (saline, adenine, glucose, and mannitol). An initial small amount of blood is diverted into a diversion pouch to reduce risk of contamination and to have a blood sample for quality control41. Each blood unit is tested for ABO, Rh, and undergoes pathogen testing, including HIV, hepatitis C virus (HCV), and hepatitis B virus (HBV). The average RBC unit issued by CBS has a volume of 285 ml and contains 55 g of Hb and an approximate hematocrit of 67 percent. RBC units are stored in specialized di-ethyl hexyl phalate (DEHP) plasticized bags1. The leftover tubing from RBC unit manufacture is used to make segments, which remain attached to the RBC unit and are used for further blood typing when necessary. RBCs are then stored in blood banks and designated distribution facilities at 1-6˚C for a maximum of 42 days. The Canadian Standards Association necessitates for stringent quality assessment of manufactured blood products, and at least 1% of manufactured blood units from each production facility are sent to testing at expiry42. Parameters such as unit volume, total Hb, hematocrit, residual WBC count, and others are assessed. Previous studies have shown that RBC unit quality at the time, such as hemolysis levels in the stored units, depends in part on donor-dependent factors42. 8 1.3.2 Post-production modifications to RBC units: RBC irradiation Transfusion-related graft-versus host disease (ta-GvHD) is a rare but serious complication with a mortality rate close to 90%43,44. It happens when the transfused RBC unit still has viable T-lymphocytes. These lymphocytes can engraft into the recipient’s bone marrow, proliferate, and attack the recipient’s tissues43,44. Patient groups including those with congenital or acquired immunodeficiencies, patients undergoing hematopoietic stem cell transplants (HSCT), and chemotherapy patients are particularly at risk. Irradiation is additionally required for transfusions originating from first or second-degree relatives, for every intrauterine transfusion, and for recipients of allogeneic stem cells, regardless of the recipient's immunocompetence3. Family donations increase the likelihood of the human leukocyte antigen (HLA) matches between the donor and the recipient45. This, in turn, may lead the recipient’s immune system to fail to recognize the transfused lymphocytes as foreign. To alleviate the risk of post-transfusion reactions in these patient groups, RBC units undergo irradiation prior to transfusion. Irradiation irreparably damages lymphocyte DNA, thus preventing their replication and population expansion in the recipient46. RBCs are irradiated using a gamma-ray source or an X-ray source. Canadian guidelines (cite CBS) recommend the irradiation dose of 25 Gy to the center of the RBC unit (with no parts receiving less than 15 and more than 50 Gy)47. Although life-saving, irradiation has been shown to negatively impact RBC quality43,44. It may induce structural, biochemical, and metabolic alterations to RBCs, thus leading to their reduced deformability and ability to efficiently perform gas exchange. Irradiated RBCs have been shown to have an increased rate of hemolysis, decreased ion balance (increased rate of K+ loss), changes to the membrane structure, and damage to the membrane and transmembrane proteins3,43,44,48. The current guidelines47 state that in order to decrease the impact on RBC unit quality, the irradiation 9 must take place within the first 28 days of RBC unit manufacture, and the irradiated unit can be stored for a maximum of 14 days. However, the impact of irradiation on RBC unit quality and the post-transfusion circulation time is still a matter of debate. Additionally, it is not clear how the irradiation impacts the ability of RBCs to deform and transverse capillary beds and small openings in circulation. 1.3.3 RBC storage lesion during cold storage of RBC units The RBC storage lesion is a collection of morphologic, biochemical, and metabolic changes that accumulate in RBCs throughout its 42-day storage window2,13,14,49–52. The storage lesion is characterized by a decline in the cellular ATP and 2,3-DPG levels and a diminished activity of antioxidant enzymes and proteins. As a result, there is an accumulation of reactive oxygen species (ROS), lipid peroxidation, and subsequent damage to the Hb structure. This leads to the release of free Hb and iron. 2,3-DPG is involved of hemoglobin-oxygen affinity and is central for successful oxygen delivery. Depletion of 2,3-DPG levels lead to poor oxygen delivery to organs. Additionally, RBCs undergo membrane vesiculation and a disruption in ion balance, highlighted by elevated plasma potassium levels and reduced calcium levels. RBC structural integrity also deteriorates, leading to damage to band-3 protein, increased exposure of phosphatidylserine (PS), and diminished presence of CD47, a marker that signifies "self" in the immune system. Some of the mentioned changes, such as decreased ATP, 2,3-DPG, and pH levels, are considered reversible. However, changes of Hb structure, accumulation of ROS, membrane damage and vesiculation are irreversible, and lead to shortened RBC lifespan and decreased efficacy in delivering oxygen. Moreover, these metabolic, structural, and biochemical changes will directly or indirectly reduce RBC deformability, leading to RBC sequestration and removal in the spleen. 10 1.3.4 Impact of donor-to-donor differences on RBC storage quality The ability of donated RBCs to withstand the deleterious effects of storage lesion is known to in part be impacted by donor-to-donor variations53,54,55. Effects of various factors on RBC storage quality and post-transfusion outcome have not been straightforward. However, there are several donor-specific factors known to contribute to donor-to-donor variability in the quality of stored RBCs. Genetic variations, such as hemoglobinopathies and enzymatic deficiencies like G6PD, can significantly alter RBC function and morphology. Individuals with Glucose-6-phosphate dehydrogenase (G6PD) deficiency exhibit diminished glutathione (GSH) levels, increasing their RBCs' susceptibility to oxidative stress and subsequently accelerating hemolysis rates56,16. Another enzymatic deficiency, pyruvate kinase (PKH) deficiency, hampers ATP production, inducing anemia in donors and resulting in RBC units that do not store well. Additionally, hemoglobin anomalies, such as alpha and beta thalassemias, can influence storage quality. Studies have shown that carriers of alpha and beta thalassemia exhibit reduced RBC deformability57,58. However, in most countries including Canada, UK, and others, individuals with minor types of thalassemia (such as alpha thalassemia minor) are allowed to donate if they meet the minimum hemoglobin requirement2. Other factors to take into account are sex, age, and ethnicity of donors. For example, storage hemolysis has been shown to be higher in males than females53, whereas RBCs donated by post-menopausal females have higher mechanical fragility, implying the importance of sex hormones in RBC health59. Storage hemolysis levels in female donors are affected by age, with less hemolysis in younger donors, which is not a trend observed in males60. Members of some ethnic groups have higher prevalence of hemoglobin variants or polymorphisms that impact the 11 biochemical or physical properties of RBCs. Lifestyle choices, such as the level of physical activity, consumption of alcohol and/or tobacco products, and diet can be tied to variability in antioxidant activity in RBCs, which in turn might affect hemolysis rate61,62. Higher BMI has been tied to higher storage hemolysis levels in both male and female donors60. Importantly, all of the abovementioned individual, genetic, and environmental factors can directly or indirectly impact RBC deformability. The extent and consequences of such impacts are still an area of active research. 1.3.5 The impact of RBC storage duration on transfusion outcomes The inherent quality of RBCs and the effects of storage-related RBC lesion are critical factors that may influence the functionality of RBCs, and consequently affect the post-transfusion outcomes in various patient groups. Numerous studies have been conducted to investigate the relationship between the storage duration of RBC units and their subsequent transfusion outcomes. For instance, the ARIPI study, which focused on pediatric intensive care prematurely born infants, found that the transfusion of newer RBC units did not improve outcomes63,64. Similarly, the ABLE study, which considered RBC transfusions to critically ill adults in intensive care, deduced that transfusing RBCs stored for less than 7 days did not ameliorate the 90-day mortality rate65. The RECESS study, evaluating cardiac surgery patients, concluded that RBC units stored for under 10 days did not enhance transfusion outcomes when assessing multiple organ dysfunction66. Additionally, both the TRALI and TRANSFUSE studies65,67, which delved into the transfusion outcomes in critically ill patients, including those on mechanical lung ventilation, and those in intensive care, respectively, revealed that the storage duration of the RBC units did not significantly affect outcomes, including pulmonary gas exchange and 90-day mortality rate. 12 It is worth noting that the focus of these studies was primarily on the duration of RBC storage and transfusion outcomes, with the majority of the research being carried out in acute patient populations, encompassing trauma, ICU, and surgical patients. However, current research has not yet extensively examined changes in RBC deformability and how it might influence transfusion outcomes. Given that RBC deformability is paramount for optimal functionality and longevity of RBCs in circulation, it's imperative to explore how changes in deformability over storage time might impact the quality of RBCs and subsequent transfusion outcomes. Moreover, there remains a gap in understanding the correlation of transfusion outcomes in patients who undergo chronic transfusions and would especially benefit from the transfused RBCs circulating for as long as possible to reduce the overall number of transfusions needed. 1.4 Existing methods of characterization of RBC deformability To date, there have been a number of technologies developed to measure RBC deformability, each with their benefits and drawbacks. Below is the overview of some of them. 1.4.1 Flow-based techniques Flow based methods measure the average RBC deformability of RBC sample68. The deformability assessment relies on fluid-induced shear stress to stretch RBCs, subsequently observing their elongation either through diffraction patterns or high-speed imaging. One of the more common methods used in clinical settings is ektacytometry69–71. An ektacytometer measures RBC deformability by subjecting red blood cells to fluid-induced shear stress, causing them to elongate70,71. The degree of cell elongation, typically inferred from diffraction patterns, provides a quantifiable metric of their deformability under the applied stress. Although it is a successful and sensitive method to measure the average changes in RBC deformability, this technique is unable to detect information on subpopulations of diseased cells, if they constitute a small percentage 13 (<10%) of the overall sample72,73. Micropore filtration measures the average deformability of the RBC sample by pushing the sample through a membrane filter with pores smaller than the RBC diameter74,75. The ease with which cells pass through these pores, often determined by the time taken or pressure needed, provides an indirect measure of their deformability. This technique has low sensitivity and a tendency for the filtration pores to clog if white blood cells and platelets are present in the sample76,77. Overall, the bulk flow techniques focus on measuring average deformability of a cell sample and rheological behavior of RBCs under rapid flow conditions, rather than examining how individual RBCs deform. These methods are also not sensitive enough to detect deformability changes in a small fraction of abnormal cells with potentially deleterious effects. 1.4.2 Deformation-based techniques The deformation-based methods on one hand include traditional single-cell manipulative techniques like micropipette aspiration, optical tweezers, and atomic force microscopy. Micropipette aspiration involves suctioning individual RBCs into a glass pipette with a radius smaller than the RBC diameter78–80. Deformability is calculated from the shear deformation of RBC membrane. This technique is sensitive but has a very low throughput of only 20-40 cells measured per test78. It also requires highly trained personnel and equipment to perform the delicate cell manipulations. Optical tweezers deploy focused laser beams to trap and exert forces on small particles, such as silica beads attached to the RBC membrane81,82. By monitoring the RBC's deformation in response to the forces applied via these trapped beads, the cell's deformability and mechanical properties can be deduced81,82. These assessments are intrinsically reliant on mathematical models, 14 incorporating parameters like RBC geometry and membrane viscoelasticity83. Such dependencies can engender variability, rendering cross-study comparisons challenging. Atomic force microscopy assesses RBC deformability by utilizing a sharp probe to apply localized forces on the RBC surface and measuring the resulting deformations84–86. The recorded force-distance curves are then analyzed to derive mechanical properties, such as elasticity and viscoelasticity, of the RBC membrane84,85. Atomic force microscopy measures RBC deformability with extremely high sensitivity. However, it only measures deformability at a portion of RBC membrane and not the overall cell87. Inherently, single-cell methodologies, though possessing granularity, are labor-intensive, necessitating advanced equipment and expertise. Their limited throughput further constrains their viability in clinical or point-of-care settings. 1.4.3 Microfluidic techniques The microfluidic techniques for RBC deformability assessment aim to simplify the deformability assessment while allowing for a higher sample throughput. Microfluidic techniques measure RBC deformability by directing cells through micro-scale channels or constrictions, assessing their ability to deform and pass through these constrictions. The deformability is then inferred from factors such as the cell's passage speed, pressure changes, and navigation behavior through these constrictions. One approach involves wedging RBCs in tapered constrictions using devices like the Human Erythrocyte Microchannel Analyzer (HEMA). This method utilizes applied pressure to squeeze RBCs through constrictions, providing information on their area-to-volume ratio88,89. However, it primarily assesses surface-to-volume ratio and may not capture subtle changes in overall RBC deformability88. 15 Another technique is elongation via fluid flow, where RBCs are deformed within narrow microchannels to simulate capillary conditions90. High-speed cameras capture RBC elongation, and the deformability index (DI) is calculated based on the ratio of deformed longitudinal length (L) to initial length (Lin)90,91. This method allows for comparisons between treated samples but requires substantial data processing due to high-speed image capture92. Transit time through constrictions measures the deformation time of individual RBCs in a test microchannel by assessing pressure drop changes93. This method mimics RBC deformation in microvasculature but may encounter issues such as potential cell clogging and the need for high-speed cameras. Another method, deformation through a series of constrictions involves determining the pressure required to deform RBCs through microchannel constrictions94–96. This technique can identify the threshold transit pressure of individual RBCs, although specific applications and benefits may vary depending on the context94–96. Overall, the microfluidic techniques for RBC deformability assessment provide distinct advantages, including significantly improved throughput. Unlike bulk methods, which average the properties of all cells in a sample, microfluidic devices can assess individual cell deformability within a heterogeneous population, thus offering insight into the distribution of cell mechanical properties. Overall, they offer a sensitive measurement of a high number of RBCs, and distinction between fractions of RBCs with different deformability profiles in the same sample. 1.4.3.1 Sorting RBCs based on deformability Our team’s microfluidic ratchet device, which is presented in this work, sorts RBCs using a ratchet transport. The microfluidic ratchet device sorts RBCs based on their ability to deform. RBCs are deformed through a matrix of asymmetric micrometer-sized constrictions using oscillatory flow, 16 which selectively transport cells based on their ability to squeeze through each constriction (Fig. 2.1)97–99. The constrictions are arranged in a matrix, where the openings of the constrictions are varied from 7.5 µm down to 1.5 µm along the rows of this matrix. RBCs move diagonally through the matrix until they encounter a constriction size that restricts further transit, after which they proceed horizontally along the limiting row of constrictions and exit through specific outlets100,101. The distribution of sorted RBCs can be determined through imaging or microscopic cell counting. Key advantages of this device are the ability to apply a consistent force to each RBC as it deforms as well as the ability to process a large number of cells in a timely manner99. Our studies have demonstrated the device's high sensitivity and reliability in measuring RBC deformability, making it capable of detecting significant and consistent differences between blood units from healthy donors101–103. 1.5 Motivation for this work This dissertation investigates the potential of RBC deformability as a surrogate marker for circulatory clearance time. To measure RBC deformability we employ the microfluidic ratchet device. The device forces RBCs to pass through a series of microscale constrictions, decreasing in size from 7.5 to 1.5 µm. The RBCs are sorted into distinct fractions based on their ability to deform though these constrictions. The relative size of the sorted fractions are then used to obtain a deformability profile of each sample. First, we conduct a study to assess the reliability and precision of the microfluidic ratchet device in measuring RBC deformability. Next, we investigate whether detectable differences exist in deformability profiles across various donors. Subsequently, we investigated RBC deformability changes in standard blood bags during cold storage. We then tested the correspondence between deformability of RBCs obtained from blood bags and their 17 tubing segments to establish a non-invasive sampling method. Additionally, we explore the consequences of gamma irradiation, a standard post-production modification of blood bags, on deformability of RBCs. Finally, we investigate the relationship between RBC deformability and post-transfusion circulation longevity in a mouse transfusion model. 1.5.1 Chapters Chapter 2 introduces the microfluidic ratchet device used in our studies. Specifically, we describe the device mechanism, operation, and data analysis. We also detail the manufacturing process for the device. The methods used to collect, process, and assess the RBC samples in subsequent chapters 3,4, and 5 are also detailed. Chapter 3 describes experiments performed to validate the microfluidic ratchet device for measuring RBC deformability. We first establish the sensitivity and repeatability of the device in deformability measurements, using silica calibration beads, as well as normal and chemically degraded RBCs. Subsequently, we assess whether there are distinct and measurable differences in RBC deformability profiles and aging curves within healthy donor population. To address this question, we measure changes in RBC deformability of RBCs stored in an accelerated RBC storge model using plastic test tubes. The results of this study have been published in: Islamzada E, Matthews K, Guo Q, Santoso AT, Duffy SP, Scott MD, Ma H. Deformability based sorting of stored red blood cells reveals donor-dependent aging curves. Lab on a Chip. 2020;20(2):226-35. 18 Chapter 4 measures changes in RBC deformability during cold storage in standard blood bag. We investigate whether these changes are donor-dependent, and whether some RBC units from sone donors exhibit a greater resilience to cold storage. This study involves biweekly assessments of deformability changes in 14 stored RBC units over an 8-week period. Additionally, we compare the deformability of RBCs obtained from blood bags with those from blood bag tubing segments to determine if the latter provides an alternate, non-destructive method for assessing RBC deformability. The results of this study were published in two papers below: Islamzada E, Matthews K, Lamoureux ES, Duffy SP, Scott MD, Ma H. Degradation of red blood cell deformability during cold storage in blood bags. EJHaem. 2022 Feb;3(1):63-71. Islamzada E, Matthews K, Lamoureux E, Duffy SP, Scott MD, Ma H. Blood unit segments accurately represent the biophysical properties of red blood cells in blood bags but not hemolysis. Transfusion. 2022 Feb;62(2):448-56 Chapter 5 assesses the impact of RBC irradiation on deformability. RBC irradiation is a post-production treatment performed before transfusion to certain immunocompromised transfusion recipients. We investigate the immediate impact of irradiation on RBC deformability and whether these effects compound during storage. To answer these questions, we collaborate with Dr. Dana Devine's research team and employ ABORh+ matched pairs of RBC units, pooling and splitting them to create identical units, one of which undergoes irradiation. The manuscript for this study is currently in preparation. Chapter 6 investigates the relationship between RBC deformability and circulation clearance time in a mouse transfusion model. We investigate the relationship between RBC deformability and 19 post-transfusion circulation time. We first develop a chemical degradation method to generate a sample of donor mouse RBCs that are stable but have a markedly reduced deformability. We then transfused the recipient mice with control and chemically rigidified donor RBCs and tracked their clearance rate and deformability over 5 weeks. The manuscript of this study is currently in preparation. 20 Chapter 2: Materials and Methods 2.1 The Microfluidic Ratchet Device Design, Production, and Operation 2.1.1 Microfluidic ratchet device design and operation The microfluidic ratchet device sorts RBCs based on deformability by deforming each cell through a series of tapered constrictions. (Fig. 2.1A). Due to the geometric asymmetry of the taper, the force required to deform cells through the constriction along the direction of taper is less than against the direction of taper104. Coupling this deformation asymmetry with an oscillatory flow creates a ratcheting effect that selectively transports cells based on their ability to squeeze through each microscopic constriction. Importantly, this oscillatory flow also minimizes the contact between cells and the filter microstructure to prevent clogging and fouling and to ensure that a consistent filtration force is applied to each cell105. To sort cells using this mechanism, a RBC sample is introduced at the bottom-left corner of a 2D array of micrometer-scale tapered constrictions. The openings of the constrictions are gradually decreased from the bottom row to the top row, forcing RBCs to deform as they transit through progressively smaller pores. The RBCs are transported through the array by a vertical oscillatory flow as well as a consistent horizontal cross flow, which combine to propel the cells in a zigzag diagonal path through the constriction matrix (Fig. 2.1B). As cells reach their limiting constriction that prevent their transport, they proceed horizontally along the terminal row until reaching a specific outlet (Fig. 2.1C). The microfluidic device used to sort RBCs from healthy donors is designed to sort RBCs into 12 outlets. The constriction matrix is housed in a 4 µm thick microchannel. The size of the constrictions ranges from 1.50 µm to 3.50 µm in steps of 0.25 µm 21 for outlets 1-9, and then from 3.50 µm to 7.50 µm in greater steps for outlets 10-12 (Table 2.1). The 4 µm thick microchannel largely exclude leukocytes, debris and cell aggregates. However, if these artefacts enter the sorting area, they are sorted into the least deformable outlets, e.g. 10-12. As a result, we have not found evidence for nucleated cells or cell clusters in outlets 1-9. This device is capable of sorting ~600 RBCs per minute. The distribution of cells after sorting could be determined by imaging the flow of cells into the outlets or by counting the cells in the outlet via microscopy (Fig. 2.2). 2.1.2 Microfluidic ratchet device production The microfluidic ratchet devices were prepared as described previously100,106. Briefly, the master device mold was created by photolithographic microfabrication. This mold was then used to create a secondary polyurethane mold, fabricated with Smooth-Cast urethane resin (Smooth-Cast ONYX SLOW, Smooth-On) as described previously107. Single-use microfluidic devices were molded from the secondary master using PDMS silicone (Sylgard-184, Ellsworth Adhesives) mixed at a 10:1 ratio with the curing agent (Sylgard-184, Ellsworth Adhesives). The PDMS devices were cured for two hours at 65°C. After removing the PDMS devices from the molds, the sample inlets and outlets were manually punched with 0.5 mm and 3.0 mm hole punches, respectively. (Technical Innovations). The bottom surface of the microfluidic devices was created using a thin layer of PDMS silicone (RTV 615, Momentive Performance Materials LLC). This thin layer of PDMS silicone was formed by spin coating the uncured PDMS on a 100 mm silicon wafer at 1500 RPM for 1 minute and then curing at 65°C for two hours. The Sylgard-184 PDMS microstructure was then bonded to the RTV 615 thin PDMS layer using air plasma (Model PDC-001, Harrick 22 Plasma). The composite structure was then bonded to a 75 x 50 mm glass slide (Corning) substrate using air plasma. Figure 2.1. The microfluidic ratchet device for cell sorting. (A) Tapered funnel constrictions enable unidirectional (upwards) transport of individual RBCs from oscillatory flow. Rows of constrictions decreasing in size (i.e. a<b) enable deformability based cell separation. (B) Deformability based cell sorting is performed using a matrix of funnel constrictions. The RBC sample is flowed into the lower left corner of the matrix and follows a diagonal trajectory resulting from vertical oscillatory flow and horizontal cross flow. RBCs travel diagonally through the matrix until reaching a limiting constriction size, which forces them to flow horizontally to a specific outlet. Deformable cells (blue shading) and rigid cells (red shading) follow distinct paths because of different limiting constrictions. (C) Micrograph of the microfluidic ratchet device. 23 Table 2-1. Outlet numbers and their corresponding constriction sizes. Outlet 1 2 3 4 5 6 7 8 9 10 11 12 Size (µm) 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 4.5 5.5 7.5 2.1.3 Microfluidic ratchet device operation The microfluidic ratchet device is operated using a horizontal crossflow and oscillatory (forward/reverse) pressure system. Prior to loading the RBC sample, the device is primed by infusion with HBSS with 0.2% Pluronic-F127 solution, through the forward and oscillatory pressure system inlets at high pressure (200-250 mBar) for 15 min. The RBC sample for each assay is suspended at 1% hematocrit and then infused into the constriction matrix using a pressure of 40-45 mBar through the sample inlet port. The sample is then flowed through the constriction matrix using a horizontal crossflow pressure of 55-60 mBar, and oscillatory pressures of 175 mBar forward and 162 mBar reverse (Fig. 2.1A). The forward oscillatory pressure is applied for 4 seconds, while the reverse oscillatory pressure is applied for 1 second. The crossflow pressure is adjusted visually within the pressure range to ensure the equal distribution of cells within the sample inlet as well as a consistent application of upward pressure to each cell. The throughput of the sorting process is approximately 600 cells per minute. From the constriction matrix, the RBC sample is flowed into 12 outlets. The distribution of cells after sorting can be counted via video analysis of the microchannels leading to the outlets, or by imaging the outlets. The sorted RBCs could also be collected by pipetting. 24 2.1.4 Microbead sorting experiments for device validation Polystyrene microbeads were sorted using the microfluidic ratchet device in order to establish the consistency across different microfluidic devices. Polystyrene beads with diameters of 1.53 µm and 3.42 µm (Cat # 17133, Polysciences Inc) were selected to coincide with approximate deformed sizes of deformable and rigid RBCs. Beads were vortexed prior to opening, and 25 µl of the bead solution was suspended in 650 µl of Hank’s Balanced Salt Solution (HBSS) with 0.2% Pluronic F127 and 0.2% TWEEN-20 (MilliporeSigma) solution to prevent aggregation, to a final concentration of 0.1 % beads in buffer. Each bead suspension was then sorted for at least 20 minutes. The number of beads in each outlet fraction was quantified using the TrackMate particle analysis plug-in with Image J73. 1 2 3 4 5 6 7020406080100Outlet numberFrequency (%) Rigid RBC sample Deformable RBC sample 1 2 3 4 5 6 7020406080100Outlet numberCumulative frequency (%)Rigid RBC sample Deformable RBC sample2.45 4.87A BFigure 2.2. Analysis of deformability-based cell sorting data. (A) Distribution of RBCs after sorting shown as histograms for deformable (blue) and rigid (red) RBC samples. (B) Distribution of RBCs after sorting shown as cumulative distributions. The rigidity score (2.45 and 4.87 for the deformable and rigid RBC samples) is defined as the 50% cross-over point for the cumulative distribution. 25 2.1.5 RBC sorting experiments for device validation Blood from a healthy donor was collected in a sodium citrate tube (BD Vacutainer), and RBCs were isolated as previously described above. The sample was then resuspended in HBSS and 0.2% Pluronic F127 solution to 1% hematocrit for further sorting with the microfluidic ratchet device. The sample from the same donor and suspension was run on 5 separate devices from different production batches. Sorted fractions were quantified using TrackMate particle analysis plug-in with Image J, as previously described. 2.1.6 RBC chemical degradation (glutaraldehyde fixation) sorting experiments In order to establish the sensitivity of the microfluidic ratchet device in measuring small changes in the deformability, RBCs from a healthy donor were collected in sodium citrate tubes (BD Vacutainer) and leuko-reduced as described above. The isolated RBCs were then washed three times and suspended at 10% hematocrit in HBSS with 0.02% Pluronic F127. These suspensions were fixed with either 0% (PBS control), 0.010%, 0.0125%, 0.015%, or 0.025% fresh glutaraldehyde (Cat # G5882, MilliporeSigma) for 30 minutes at room temperature. After fixation, the suspensions were washed three times in HBSS with 0.2% Pluronic F127 and then suspended at 1% Hct for the deformability experiment. The number of cells in each sorted fraction was quantified with the TrackMate particle analysis plug-in with Image J. 2.1.7 RBC storage pilot study: storing and sampling RBCs in an accelerated storage model Only donors self-identified as healthy and between the ages of 18 and 70 were included in this study. Following informed consent and in accordance with University of British Columbia Research Ethics Board guidelines (UBC REB H19-01121), whole blood was collected from 26 healthy donors via venipuncture into sodium citrate tubes (BD Vacutainer). The blood samples were leuko-reduced by first centrifuging at 3180 g for 10 minutes to decant plasma. The packed cells were then centrifuged at 677 g for 30 minutes with no brakes to remove the leukocytes. Each 1 mL of packed RBC was supplemented with 0.80 mL of saline-adenine-glucose-mannitol storage solution (SAGM) and 0.56 mL of plasma, and stored in a 15 mL plastic tube (Corning) upright at 4°C. On days 0, 3, 7, and 14, an aliquot of the sample was taken for RBC deformability measurement with the Microfluidic Ratchet device. The RBC pellet was resuspended in HBSS (Gibco) and 0.2% Pluronic solution (F127, MilliporeSigma) at 1% hematocrit for infusion into the microfluidic ratchet device. A second unwashed sample aliquot was used to measure standard hematological indices, such as RBC mean corpuscular volume (MCV), red blood cell distribution width (RDW-CV), mean cell hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), using the SYSMEX® system. 2.2 Assessment of RBC deformability in cold stored standard blood bag units and their corresponding segments 2.2.1 Packed RBC bags and segments This study was approved by the University of British Columbia’s Clinical Research Ethics Board (UBC REB# H19-01121) and Canadian Blood Services Research Ethics Board (CBS REB# 2019-029). Red Blood Cell (RBC) units were collected and processed by Canadian Blood Services between January 2020 and February 2021. For some tests, the tubing attached to blood bags was segmented into 4 - 8 sections as per standard practice. RBC units and, when applicable, their attached segments were stored according to Canadian Blood Services (CBS) standard operating 27 procedures, at 4 °C for a period of 8 weeks, 2 weeks longer than the CBS-approved storage period of 42 days (6 weeks). 2.2.2 RBC storage and processing for deformability assessment Samples were analyzed on the day of RBC unit collection and processing (referred as the week 0 data point), followed by analysis at weeks 2, 4, 6, and 8. RBCs were sampled from the blood bags and when available, segments, using a 27-gauge needle, while maintaining sterility with subsequent Parafilm sealing. The drawn sample was centrifuged at 1500 x g with no brakes for 10 min at room temperature, and supernatant was transferred to a fresh tube. The supernatant was centrifuged again at 1500 x g for 10 min to remove any remaining RBCs, transferred to a cryogenic vial and stored at -80°C for assessment of hemolysis at a later stage. Additionally, 100 µl of the blood drawn on day 1 was also stored for hemolysis assessment. The RBC pellet was suspended in Hank’s Balanced Salt Solution (HBSS, Gibco) and 0.2% Pluronic (F127, MilliporeSigma), and washed three more times, each time centrifuging for 5 minutes at 300 x g with brakes on. The final RBC sample was suspended at 1% hematocrit in HBSS+0.2% Pluronic solution and used for deformability assessment using the Microfluidic Ratchet Device. 2.2.3 Assessment of hematological parameters At each sampling timepoint, 100 µl of packed RBC sample (no washing) was used to monitor the hematological parameter changes. Mean corpuscular volume (MCV), red blood cell distribution width (RDW-CV), mean cell hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) was assessed using the Sysmex® Hematology Analyzer. 28 2.2.4 Assessment of hemolysis The hemolysis assessment was performed as previously described108. Briefly, frozen whole blood and supernatant samples were fully thawed. An aliquot of each supernatant sample was transferred to a fresh tube and centrifuged at 3000 x g for 3 minutes. Packed, unwashed blood samples from day 1 of storage were vortexed for 30 seconds and diluted 1:10 with DI water. Finally, 10 µl of each sample was transferred to a 96-well flat bottom plate (BioVision Inc) together with 100ul of Drabkin’s reagent (MilliporeSigma) containing Brij-35 solution (Thermofisher). The plate was then incubated at room temperature for 15 min on a plate shaker, and absorbance was read on a microplate reader (manufacturer) at 540 nm. Hemolysis was calculated using the following formula: % 𝐻𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 = 𝑂𝐷𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 × (1 − 𝐻𝑐𝑡)𝑂𝐷𝑤ℎ𝑜𝑙𝑒 𝑏𝑙𝑜𝑜𝑑× 100% where Hct is the hematocrit, and 𝑂𝐷𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 and 𝑂𝐷𝑤ℎ𝑜𝑙𝑒 𝑏𝑙𝑜𝑜𝑑 are the measured optical density from the supernatant and whole blood respectively. 2.2.5 Statistical analysis Statistical analysis was executed using GraphPad Prism (V8.0) software, with means and standard deviations (SD) graphically represented unless otherwise specified. Normality of data was assessed with the D'Agostino and Pearson omnibus test, and the Wilcoxon matched pairs signed rank test was employed for comparing data sets. Correlations between normally and non-normally distributed datasets were determined using Pearson r, with a 95% confidence interval. 29 Additionally, standard deviation for rigidity score obtained from doublet measurements was computed using the formula: 𝜎𝑅𝑆 = √1𝑁∑(𝑀1𝑖 − 𝑀2𝑖)2𝑁𝑖=1 where M1 and M2 symbolize the first and second RS measurements. 2.3 Processing, storage, and assessment of RBC deformability for the RBC irradiation study 2.3.1 Blood bags This study was conducted in accordance with University of British Columbia’s Clinical Research Ethics Board (UBC REB# H19-01121) and Canadian Blood Services Research Ethics Board (CBS REB#2019-029). ABO-matched pairs of leukoreduced packed RBC units (n=16) stored in saline-adenine-glucose-mannitol (SAGM) were collected and manufactured by the Canadian Blood Services Blood4Research facility between July and December 2021. The RBC unit pairs were pooled and divided using their segment lines to obtain 2 identical bags. To pool the units together, the segments from the two matched units were fused with sterile tubing fuser (TSCD-II, Terumo). The bags were then combined, mixed by gentle agitation, and divided into two units of equal volume, sealing the tubing back using sterile tube sealer (Hematron III, Baxter). The bags were stored for 14 days at 4˚C, at which point one bag in the pair was irradiated with 25 GY gamma rays. The bags were then stored for an additional 14 days post-irradiation. RBC deformability, 30 standard hematological parameters, and hemolysis rate of both control and irradiated units were measured on the day of RBC unit irradiation, followed by 14 days post-irradiation. 2.3.2 RBC deformability and hematological parameters assessment RBC bags at 14-day post-irradiation were manually agitated for 1 minute, and 3 ml RBC sample was drawn aseptically though the sampling site coupler using a 26-gauge needle (BD). The sampling site coupler was then covered with sealing film (Parafilm, SigmaAldrich) to ensure unit sterility. The RBC sample was centrifuged at 1500G for ten minutes at low brake to collect the supernatant. The supernatant was transferred to a clean tube and spun again to remove any remaining RBCs. This supernatant, along with a 1-ml aliquot of unaltered RBCs from the corresponding unit, were frozen at -80˚C until the hemolysis assessment. The remaining RBC pellet was washed 3x with Hank’s Balanced Salt Solution (HBSS, Gibco) with addition of non-ionic surfactant (0.2% Pluronic, Thermo Fisher). Surfactant use is important to ensure proper infusion and RBC sorting in the microfluidic ratchet device. The washed RBCs were resuspended at 1% hematocrit in HBSS+0.2% Pluronic solution to perform the RBC deformability assays. A 100 µl sample was set aside for measurement of hematological parameters (Sysmex). At each point tested, mean corpuscular volume (MCV), RBC distribution width (RDW), mean cell hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and hematocrit were measured. 2.3.3 Assessment of RBC hemolysis Assessment of RBC hemolysis of irradiated and non-irradiated stored RBC unit pairs was done as described in 2.4.5. 31 2.3.4 Statistical analysis Statistical analysis was performed using GraphPad Prism (V8.0) software. Means and standard deviations (SD) are plotted unless otherwise stated. The D'Agostino and Pearson omnibus test was used to determine the normality of all data. Trends in data were analyzed using RM one-way ANOVA with Sidak’s multiple comparisons, and adjusted P-values are reported. The P values were statistically significant if they were less than 0.05. Correlations between normally distributed datasets was calculated using linear regression analysis and displaying R2 and adjusted P values. 32 Chapter 3: Deformability based sorting of stored RBCs reveals donor-dependent aging curves 3.1 Introduction Donated RBC units are a vital component of critical care, with over 85 million RBC units transfused each year to different patient groups109. However, the transfusion of these RBCs presents a challenge since not all units offer equal benefits to their recipients110,111. Transfusion specialists have long recognized that while some RBC units persist in circulation for a long period of time, aiding in restoring oxygen delivery, other units are cleared quickly, necessitating additional transfusions112–116. Frequent transfusions are problematic for chronic recipients due to potential for adverse effects including iron accumulation, transfusion-associated acute lung injury (TRALI), hypervolemia, and a heightened infection risk117–120. As a result, there's an urgent need to identify reliable biomarkers that can help identify donors with longer-lasting RBCs121,122. RBC deformability is essential to RBCs proper function. RBCs must be able to deform and transit through narrow openings in capillary beds and splenic sinuses in order to deliver oxygen123. Loss of deformability leads to reduced ability and efficiency in maintaining homeostasis, and premature removal of RBCs from circulation124 by the splenic system. Premature removal of RBCs in circulation is especially concerning for transfused RBC units. A recognized consequence of cold storage is that donated RBCs undergo a loss in deformability as part of the storage lesion121,125–129. This lesion induces various cellular changes, such as 2,3-DPG depletion130, cytoskeletal remodeling131, lipid peroxidation132,133, and membrane vesiculation134,135, all of which directly or indirectly lead to reduction of RBC deformability. Given this, RBC deformability has been viewed as a potential biophysical marker for the quality control of donated and stored RBCs. Our lab has 33 developed a microfluidic device based assay designed to categorize RBCs based on deformability100,104,105,136. RBCs are sorted through a matrix of asymmetrical constrictions known as microfluidic ratchets, simulating cell transit through narrow openings in microvasculature99. Leveraging this capability, we show deformability based cell sorting using microfluidic ratchets provides a highly sensitive, repeatable, and consistent assessment of RBC deformability100,136. Specifically, this measurement is capable of detecting distinct and temporally consistent differences between blood units from healthy donors, which we observed to exhibit donor-specific deformability signatures as well as aging rates during cold storage. The ability to detect differences in RBC deformability between healthy donors, as well as the rates of loss of deformability during cold storage, provides a potential opportunity to identify donors who may provide RBCs that maintain their integrity during cold storage. This capability will be extremely important for chronic transfusion recipients that require long circulating RBC units in order to reduce the number of transfusions. Reserving longer-circulating RBC units for these patients will improve outcomes and increase the overall blood supply. 3.2 Results 3.2.1 Process characterization and validation We conducted a series of tests to evaluate the consistency, repeatability, and variability in our RBC sorting technique. To assess any device-to-device variability, we used a process to sort 1.54 and 3.43 µm polystyrene calibration beads. We sorted the beads on 8 different devices from different production batches. The 1.53 µm beads consistently sorted into outlets 2 and 3 (sorting region constriction sizes 1.75 and 2 µm), where the 3.43 µm beads consistently sorted into outlet 9 34 (constriction size 3.5). All 8 devices tested performed with similar results, where the mean rigidity score for the 1.53 µm beads were 2.60 ± 0.24 (Fig. 3.1A). Next, we assessed the consistency of RBC deformability sorting on human RBCs. We took a RBC sample from a single donor and sorted it simultaneously on 5 different devices from different production batches. The deformability curve of RBCs was highly consistent between the devices, where the rigidity score for this sample was 2.63 ± 0.17 (Fig. 3.1B). Collectively, these data show consistency and reliability of our device fabrication process. The fabrication is robust, and the experimental process shows consistent results. We then worked to establish whether the microfluidic ratchet-based device assay and the obtained sample rigidity score could discriminate between RBC populations from healthy donors. To establish whether we can detect a range of rigidity scores, we obtained fresh RBC sample and applied a range of very low glutaraldehyde (GTA) concentrations, ranging from 0.01% to 0.025%, in order to obtain controlled and mild rigidification of RBCs. After 30 minute incubation period, 12345678910Calibration beadsRigidity Score1.53 mm 3.43 mm 1 2 3 4 5 62.63 ± 0.17020406080100Outlet numberCumulative Frequency (%)A B1 2 3 4 5 6 7 8 9 10 11 12020406080100Outlet numberCumulative frequency (%)Control0.010%0.0125%0.015%0.025%CFigure 3.1. Characterization and validation of the cell sorting process. (A) Rigidity score from sorting 1.53 μm (8 repeats) and 3.43 μm (7 repeats) diameter polystyrene beads using devices from different manufacturing batches. Error bars indicate SD. (B) Cumulative distributions obtained from sorting the same RBC sample using 5 devices from different manufacturing batches shown with the mean rigidity score (solid line) and range (shading). (C) Cumulative distributions showing the loss of RBC deformability after mild glutaraldehyde fixation. 35 a dose-dependent rightward shift was observed in the distribution of RBCs within the device outlets, following sorting (Fig. 3.1C). Compared to the control sample at rigidity score of 2.63 ±0.17, GTA-fixed cells displayed rigidity scores of 3.6 (0.010% GTA), 8.0 (0.0125% GTA), 9.5 (0.015% GTA) and 10.7 (0.025% GTA). 3.2.2 Donor-based variability in RBC deformability After confirming the deformability assay’s sensitivity and consistency, we used our cell sorting technique to evaluate the variability in RBC deformability among 8 donors at the time of collection. Typically, freshly collected RBCs are sorted into outlets 2 and 3, though their distribution varies slightly. These donors exhibited an average rigidity score of 2.90 ± 0.4, with values spanning from 2.36 (for donor 1) to 3.59 (for donor 5) as shown in Fig. 3.3A. To determine if the deformability profiles of the donors remained steady over time, we gathered RBCs from donors 1 and 5 on two additional timepoints, ensuring a gap of at least 6 weeks between collections (Fig. 3.3B). As most RBCs are cleared from circulation in 120 days, a 6 week gap ensured a substantial renewal of circulating RBCs. Notably, the rigidity scores for each donor remained remarkably consistent across separate collection points. For instance, donor 1's scores fluctuated between 2.36 and 2.57, while donor 5's scores ranged from 3.19 to 3.59. 36 Figure 3.3. Inter- and Intra-donor variability of fresh RBC deformability. (A) Cumulative distributions from deformability based sorting of fresh RBC samples from 8 healthy donors. Mean rigidity score is shown as solid line and SD shown as shading. (B) Cumulative distributions of 3 repeat RBC samples from donors 1 (blue) and 5 (red) taken at least 6 weeks apart. 0 1 2 3 4 5 62.41 3.530255075100Outlet numberCumulative Frequency (%)Donor 1Donor 50 1 2 3 4 5 62.9 ± 0.40255075100Outlet numberCumulative Frequency (%)Donor 1Donor 2Donor 3Donor 4Donor 5Donor 6Donor 7Donor 8A BFigure 3.2. RBC deformability aging curves. Cumulative distributions curves from deformability based sorting of RBCs obtained from 8 donors after 0, 3, 7, and 14 days of accelerated aging. Rigidity scores (RS) are listed in the inset tables. Significant loss of RBC deformability is observed from most donors after 14 days with the notable exception of donors 5 and 8, which demonstrate little change after 14 days. 1 2 3 4 5 6 70255075100Outlet numberCumulative Frequency (%)Donor 11 2 3 4 5 6 70255075100Donor 2Outlet numberCumulative Frequency (%)1 2 3 4 5 6 70255075100Donor 3 Outlet numberCumulative Frequency (%)1 2 3 4 5 6 70255075100Outlet numberDonor 4Cumulative Frequency (%)1 2 3 4 5 6 70255075100Donor 5Outlet numberCumulative Frequency (%)1 2 3 4 5 6 70255075100Outlet numberDonor 6Cumulative Frequency (%)1 2 3 4 5 6 70255075100Donor 7Outlet numberCumulative Frequency (%)1 2 3 4 5 6 70255075100Donor 8Outlet numberCumulative Frequency (%)Day RS0 2.363 3.337 314 4.87Day RS0 3.273 3.467 3.6814 4.58Day RS0 2.633 3.177 3.5814 3.64Day RS0 2.543 2.577 3.7414 4.47Day RS0 3.593 3.737 3.6114 3.55Day RS0 2.783 2.787 3.6814 3.33Day RS0 2.663 2.527 3.7214 3.61Day RS0 3.353 3.417 3.1814 3.637 3.2.3 Donor-based variability in RBC deformability aging curves To determine if the microfluidic ratchet device-based cell sorting was a reliable technique for measuring RBC degradation during cold storage, we assessed the storage-related changes in RBC deformability profile in an accelerated storage model. RBCs were stored in standard plastic Falcon tubes in order to accelerate storage related lesion. These tubes offered a more uniform accelerated aging model over a span of 14 days compared to blood bags. Overall blood samples from 8 donors were collected and stored for 14 days at 4°C. Deformability assessments were conducted on days 0, 3, 7, and 14. Notably, while every sample displayed a decrease in RBC deformability during cold storage (average rigidity score increase = +1.0), there was a significant variability among donors (Fig. 3.3). In particular, donor 1 exhibited the most pronounced loss in deformability (a shift of +2.51 in rigidity score) despite having the most deformable RBCs initially (rigidity score = 2.36). On the contrary, RBCs from donors 5 and 8 remained consistent in their deformability signatures, with minor changes in their rigidity scores (-0.04 and +0.25 for donor 5 and 8 respectively, Fig 3.4). Having observed that RBC deformability of newly collected samples was consistent for each donor, we investigated whether the RBC aging curve was also donor-specific when blood was collected on different timepoints. RBC samples were collected from donors 1 and 5 at 6-week intervals and RBC deformability profiles were assessed over the course of accelerated storage to establish the RBC aging curves. Consistent with our observation that RBC rigidity was temporally consistent for each donor, we also observed that the aging curve was remarkably consistent when 38 sampled on different occasions (Fig. 3.4). Donor 1 consistently showed an accelerated RBC aging curve, while donor 5 consistently provided RBCs that retain their integrity during cold storage. Figure 3.5. Summary of RBC deformability aging curves. (A) Normalized rigidity score from donors 1-8 over 14 days of accelerated aging. (B) Change in rigidity score from day 0 to day 14. The shading indicates the mean change for all 8 donors. 0 1 2 3 4 5 6Donor 1Donor 2Donor 3Donor 4Donor 5Donor 6Donor 7Donor 8Rigidity Score0 3 7 14012345Days of storageNormalized Rigidity ScoreDonor 5Donor 1Donor 2Donor 3Donor 4Donor 6Donor 7Donor 8A BFigure 3.4. Intra-donor variability of RBC deformability aging profiles. Rigidity scores (dots) over 14 days of accelerated aging for 3 RBC samples obtained at least 6 apart from donors 1 and 5. Linear regression analysis (solid lines) showed that donor 1 (blue) had consistent loss of deformability during storage (R2=0.768, p=0.0002), while donor 5 (red) showed no differences between Day 0 and Day 14 of storage (R2=0.032, p=0.5799). 0 3 7 142345Days of storageRigidity Score0 3 7 142345Days of storageRigidity ScoreDonor 1 Donor 539 3.2.4 Hematological parameters do not affect RBC sorting We also measured standard hematological indices during storage (Fig. 3.6), including RBC mean corpuscular volume (MCV), red blood cell distribution width (RDW-CV), mean cell hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), using the SYSMEX® system. There was a trend of increasing RBC MCV and MCHC during storage in the plastic tubes over the 14 days, while the RDW-CV and MCH remained largely unchanged (Table 3.1). However, these parameters all remained within the normal range during storage. Furthermore, linear regression analysis of these parameters and RBC rigidity score, did not show any significant correlations. Figure 3.6. Linear regression analysis of the Rigidity Score and each of the hematological indices. (A) MCV, (B) RDW-CV, (C) MCH and (D) MCHC showed no correlation to the rigidity score during storage. 2.0 2.5 3.0 3.5 4.0 4.57580859095100105Rigidity ScoreMCV (fL)2.0 2.5 3.0 3.5 4.0 4.52628303234Rigidity ScoreMCH (pg)2.0 2.5 3.0 3.5 4.0 4.5101112131415Rigidity ScoreRDW-CV (%)2.0 2.5 3.0 3.5 4.0 4.5280300320340360380400Rigidity ScoreMCHC (g/L)ADCBR2=0.1044p=0.1235R2=0.0541p=0.2740R2=0.0007p=0.6966R2=0.0184p=0.527340 3.3 Discussion In this study, we utilized the microfluidic ratchet device-based RBC sorting assay to establish individual RBC deformability signatures from healthy donors. Once the RBCs are sorted based on deformability, we quantified the RBC distribution in each outlet, and used this distribution to calculate the rigidity score of each individual sample. Using our assay, we observe that base-level RBC deformability varies significantly across different donors. However, for repeated donations from the same donor, this deformability signature remained largely consistent. Crucially, we noted that the trajectory of RBC aging during cold storage, as marked by a decrease in RBC deformability, also showed variability across donors but remained stable for individual donor. Together, these findings imply that RBC deformability assessments using the microfluidic ratchet sorting method might aid in pinpointing donors whose RBCs uphold their structure during cold storage. RBC deformability plays a key role in the splenic clearance of senescent and damaged RBCs137,138, and has therefore been considered a potential biophysical marker for the clearance time of transfused RBCs139,140. Conventional techniques, like ektacytometry141–144, assess the rheological attributes of RBCs derived from bulk flow, but this doesn't show a strong correlation with actual circulatory clearance time145,146. Microfluidic methods offer the opportunity to improve this measurement process by employing microfabricated constructs that mimic the deformation dynamics seen in splenic red pulp/vessels147–149. However, challenges with current microfluidic tools include the speed and repeatability of the measurement procedures. By adopting the microfluidic ratchet method for ongoing deformability-centered cell classification, we have substantially accelerated the measurement process. Crucially, with a pronounced increase in 41 microstructure redundancy, we demonstrated our method's high repeatability and reliability. The capacity for such consistent high-speed assessment of RBC deformability lays the foundation for exploring the link between RBC deformability and circulation time, using strategies like the recovery speed of 51Cr- or biotin-tagged RBCs in transfusion experiments in animal models. Measurements of RBC deformability using the microfluidic ratchet highlighted considerable differences between donors in terms of freshly harvested RBCs, as well as variations in the RBC rigidity evolution during storage. Notably, past studies have documented the mechanical decline of RBCs during storage, however the deformability freshly collected RBCs has predominantly been depicted as consistent150,151. Together, the observed differences in RBC deformability among donors, both in fresh and stored cells, align with epidemiological data suggesting that the efficacy of RBC transfusion and storage is donor-dependent42,49,152–154. Although the data from our research are not extensive enough to draw firm links between RBC deformability and transfusion success, the observation that the individual donors exhibit distinct and consistent RBC deformability profile both prior and during storage paves the way for future research. This could help connect donor RBC deformability, aging profiles, and other genetic, biochemical, and hematological rheological factors to clinical results and the occurrence of complications after transfusion. Recognizing a link between donor RBC deformability and transfusion success might enable the categorization of donors based on their transfusion and RBC storage capacities. The microfluidic ratchet instrument offers a potential method for examining individual donor deformability aging trajectories. Those donors exhibiting minimal deformability changes over time could form a distinct donor group, with their contributions mainly set aside for patients requiring regular transfusions. 42 Identifying donors who can reliably offer longer-circulating RBC units could revolutionize the field of transfusion medicine. RBC units predicted to circulate for extended durations might be allocated to especially sensitive patients needing chronic transfusions. This would lessen transfusion frequency required and, consequently, lower transfusion-associated complications like iron accumulation, hypervolemia, TRALI, and potential infections. Moreover, being able to predict RBC circulation durations through cell deformability assay offers innovative quality control strategies to enhance blood quality control. For instance, while the standard protocol currently restricts blood storage to a maximum of 42 days, cell deformability might suggest certain units suitable for use beyond this conventional storage threshold. In conclusion, although all patients stand to gain from RBC units with extended circulation durations, these could be specially designated for vulnerable patients, such as those undergoing chronic transfusions, as they would most benefit from this limited resource. 43 Table 3-1. Hematological parameters of individual donors. MCV (fL) Day 0 Day 3 Day 7 Day 14 Donor 1 ii 85.7 86.3 88.3 85.2 Donor 1 iii 87.1 88 91.2 93 Donor 5 ii 86.9 88.6 89.7 94.1 Donor 5 iii 87.0 87.1 89.8 98.1 Donor 7 i 83.6 83 92.9 88.7 Donor 8 i 87.7 88.8 92.5 97.6 Mean 86.3 ± 1.5 87.0 ± 2.2 90.7 ± 1.8 92.8 ± 5.0 RDW-CV (%) Day 0 Day 3 Day 7 Day 14 Donor 1 ii 13.9 13.9 14.1 11.6 Donor 1 iii 13.4 13.8 14 13.6 Donor 5 ii 12.3 12.2 12.1 12.5 Donor 5 iii 12.0 11.8 11.9 12.5 Donor 7 i 11.4 11.4 13.9 11.7 Donor 8 i 12.1 12 11.9 11.7 Mean 12.5 ± 0.9 12.5 ± 1.1 13.0 ± 1.1 12.3 ± 0.8 MCH (pg) Day 0 Day 3 Day 7 Day 14 Donor 1 ii 28.4 28.4 28 29.9 Donor 1 iii 29.2 29.2 29 28.5 Donor 5 ii 30.0 30.6 30.1 30.1 Donor 5 iii 30.2 30.2 30.3 30.5 Donor 7 i 30.4 30.1 28.4 29.6 44 MCH (pg) Day 0 Day 3 Day 7 Day 14 Donor 8 i 30.5 30.8 31 30.8 Mean 29.9 ± 0.8 29.9 ± 0.9 29.5 ± 1.2 29.9 ± 0.8 MCHC (g/L) Day 0 Day 3 Day 7 Day 14 Donor 1 ii 331 329 317.5 350.5 Donor 1 iii 335.0 332 317.5 306 Donor 5 ii 345.5 345.5 335 319 Donor 5 iii 345.5 345.5 335 319 Donor 7 i 360.0 363 305.5 333.5 Donor 8 i 347.5 347 334 315.5 Mean 344.1 ± 10.2 343.7 ± 12.2 324.1 ± 12.4 323.9 ± 15.7 45 Chapter 4: RBC deformability during cold storage in standard blood bags and corresponding segments 4.1 Introduction Red blood cells (RBCs) collected for blood transfusions are stored at 4°C for a maximum of 42 days155,156. During this storage period, RBCs may undergo a storage lesion characterized by structural, metabolic, and morphologic transformations, including lipid peroxidation, Band 3 aggregation, membrane asymmetry, slowed metabolism due to ATP and 2,3-diphosphoglycerate depletion, and shape changes from discoid to echinocyte and spherocyte125,157–159. These changes may lead to a reduced circulation time of the transfused RBCs and their rapid uptake by reticuloendothelial macrophages after transfusions160, which in turn may necessitate more frequent transfusions. Interestingly, while RBC units are generally stored between 35-42 days depending on regulations of individual countries, the degradation rate of RBCs is known to be donor-dependent and variable from unit-to-unit125,158,161,162. Such variability in degradation is reflected in chronic transfusion recipients, with some RBC units sustaining durable hemoglobin levels, whereas others are rapidly cleared. Optimizing this could mean customizing expiration timelines based on the donor or selecting longer-lasting units for sensitive recipients, thereby improving clinical outcomes. A key challenge in transfusion medicine has therefore been the development of a simple biomarker to assess the quality of stored blood to optimally meet the needs of the transfusion recipient. Previous efforts to pinpoint cellular changes associated RBC storage lesions have yet to produce a reliable biomarker163. However, it has long been known that the collective effect of these changes affects RBC deformability. Thus, RBC deformability is an attractive potential parameter 46 as a biomarker candidate. Earlier research indicates that while deformability of cold-stored RBCs remains relatively stable for the first two weeks, it begins to degrade post the third week125,164–166. This change coincides with clinical evidence indicating that blood transfusion efficacy diminishes markedly after 30 days storage167,168, possibly due to the reduced deformability causing RBCs to be rapidly taken up by macrophages169 and occluding microvascular flow170. To study the impact of donor RBCs on transfusion outcomes, researchers have considered sampling from blood bag segments rather than the blood bag itself, due to practical challenges such as sterility requirements, limited time-windows, and a reluctance to deviate from established protocols. Blood bag segments are blood-filled tubing attached to the blood bag, heat-sealed at several points for serological and hemodynamic assays without compromising the unit. However, assessing RBC quality from segments is challenging, as standard parameters like hematocrit, hemoglobin content, and percent hemolysis diverge significantly between segment and blood bag by day 5 of storage171. Despite these challenges, RBC deformability, representing the cumulative effect of cellular transformations, may serve as a robust biomarker for blood quality. The consistency of time-dependent changes in RBC deformability between segment and blood bag during cold storage remains to be established. Recently, we developed a microfluidic technology to measure RBC deformability with sufficient sensitivity and repeatability to analyze differences between healthy donors172. We utilized an accelerated aging model of RBCs stored in plastic tubes and observed that donor RBCs had degradation profiles that were highly variable between donors but consistent for each donor. Notably, some donors exhibited a significant loss of RBC deformability during storage, while others showed little or no storage-induced loss of RBC deformability. In this study, we assessed 47 the degradation of RBC deformability during cold storage in blood bags over the 42-day storage window and for an additional 14 days, totaling 56 days. We found that, in most cases, blood bags preserved RBC deformability during the 42-day storage window. However, the degradation of RBC deformability in the subsequent 14 days was highly variable. Our results confirm that RBC deformability provides a potentially useful approach for donor-level screening to identify donors for whom the storage expiration window could be lengthened. We also conducted a comparative study to assess RBC deformability loss when samples are stored in blood bags versus blood bag segments. Our goal was to determine if RBC deformability in segments is predictive of the deformability of RBCs stored in the matching blood bag. Using the microfluidic technique, we performed deformability measurements on large numbers of single RBCs and observed distinct donor-specific RBC aging curves, revealing that the loss of RBC deformability proceeded at different rates for each donor. Importantly, the aging curves obtained from both segments and blood bags were well-matched. Therefore, RBCs sampled from the blood bag segments provide a reliable and non-destructive way to assess the biophysical degradation of RBCs stored in blood bags. 48 4.2 Results 4.2.1 RBC deformability profiles at time of collection We characterized the baseline RBC deformability profile of all RBC units at the time of collection. Packed RBC units in standard blood bags were collected from healthy donors (n=14), processed by Canadian Blood Services with standard protocol and provided to us by their NetCAD 1 2 3 4 5 6020406080100Outlet numberCumulative Frequency (%)3.00±0.18Rigidity Score1 2 3 4 5 6020406080100Outlet numberCumulative Frequency (%)3.29±0.48Rigidity ScoreA BC2.53.03.54.04.5Rigidity ScoreMale FemaleD2.0 2.5 3.0 3.5 4.0 4.5 5.02.53.03.54.04.55.0Device 2 (RS)Device 1 (RS)r = 0.9415sRS = 0.157Figure 4.1. Inter-donor variability of packed RBC units on the day of collection and measurement repeatability. Cumulative distribution of RBCs sorted based on deformability using the microfluidic ratchet device from male (A) and female (B) donors. A rigidity score (RS) is derived from the fractional outlet number at the 50% cross over point of the cumulative distribution. The mean RS for male and female donors are indicated using dashed lines. (C) RS for RBCs from the day of collection for male (n=8) and female (n=6) donors. (D) Repeatability of the RS from doublet measurements on the same samples, which showed a Pearson’s r = 0.9415 and a standard deviation 𝜎RS=0.157. 49 Blood for Research facility. Consistent with previous findings in literature, we observed significant variability in initial RBC deformability among donors, with a RS range from 2.71 to 4.13 with a mean of 3.13±0.37. The mean RS of male donor RBCs (n=8, 3.00±0.18, Fig. 4.1A) was slightly lower than female donor RBCs (n=6, 3.29±0.48, Fig. 4.1B), but this difference was not statistically significant (Fig. 4.1C). 4.2.2 Measurement repeatability Each RBC sample in this study was measured twice using different replicate microfluidic devices. We used this doublet data to confirm the repeatability of our measurement by plotting the RS from the first and second measurements against each other (Fig. 4.1D). These results suggest that the RBC deformability measurements were highly repeatable with a standard deviation of 0.157 in repeated measurements. 4.2.3 RBC deformability loss during cold storage To evaluate the RBC deformability loss during cold storage, we sampled RBCs from blood bags and corresponding segments every two weeks, extending to 8 weeks, which surpasses the current 42-day storage window (Fig. 4.2). Initial testing including a timepoint on day 7 showed no significant changes in deformability (N=6, Fig.4.7), hence we decided on the two-week sampling intervals. Between the storage weeks 0-4, we did not observe any significant deformability loss. However, by the 6-week expiration date, RBC units displayed a notable deformability loss, marked by an elevated RS of 0.35 (p<0.05; Fig. 4.3A). Between weeks 6-8, there was a dramatic loss of deformability with the average loss being more profound in this period than the entirety of the preceding weeks (ΔRS=0.42). While it should be noted that RBC deformability alone does not necessarily predict transfusion efficacy, this pronounced loss of RBC deformability after 6 weeks 50 strongly supports the current 42-day storage window. Interestingly, a few donated RBC units demonstrated an initial rise in deformability between weeks 0-2, which was subsequently followed by a steady decline in deformability. Doublet deformability measurements at each timepoint tested validate these findings. Our findings also align with previous studies which reported that RBC units can sometimes regain a portion of their deformability during the early stages of storage173,174. We further investigated if the deformability of freshly collected RBCs could predict the rate of RBC deformability loss during storage by comparing the RS at the time of collection with the RS after the 42-day storage period. No correlation was observed between the two RS values (Fig. 4.3B). In fact, irrespective of the initial RS, all RBC units settled to an RS of 3.48±0.16. These findings affirm that initial RBC deformability cannot forecast the RBC deformability loss 0 2 4 6 82345M010 2 4 6 82345M020 2 4 6 82345M060 2 4 6 82345F060 2 4 6 82345F010 2 4 6 82345M030 2 4 6 82345M080 2 4 6 82345F020 2 4 6 82345M040 2 4 6 82345F040 2 4 6 82345F030 2 4 6 82345M050 2 4 6 82345F05BagSegmentRigidity ScoreStorage Time (weeks)0 2 4 6 82345M07Figure 4.2.Deformability profiles of RBCs from blood bags and segments during storage. Rigidity Scores of RBCs measured from 14 donors (6 female and 8 male) during 8 weeks of storage in blood bags (solid lines). and their matching segments (dashed lines). Each data point is the mean of doublet measurements. F indicates female donors. M indicates male donors. 51 profile, and determining this for each donor necessitates multiple samplings across the storage duration. 0 2 4 6 8345Storage Time (Weeks)RS0.01640.00090.00270.0419 0.01640.00013.0 3.5 4.0 4.53.03.54.04.5RS at Week 0RS at Week 6A BFigure 4.4.Degradation of RBC deformability during cold storage. (A) Donor RBCs exhibited a progressive increase in RS over 6 weeks, with an accelerated increase in RS between weeks 6-8. The mean rigidity scores (RS) were 3.13±0.35 (week 0), 3.06±0.35 (week 2), 3.29±0.30 (week 4), 3.48±0.16 (week 6), and 3.91±0.42 (week 8). (B) Correlation between RS on the day of processing and day of expiration (week 6), r=0.0086. Figure 4.3. Summary of RBC Rigidity Score during storage. (A) Rigidity Scores for RBCs obtained from blood bags and segments during storage showing detectable deformability loss by 6 weeks (bag p=0.0164, segment p=0.0010), and significant deformability loss by 8 weeks (bag p=0.0009, segment p=0.0001) as determined by Wilcoxon matched pairs signed rank test. (B) Correlation (Pearson’s r with 95% confidence interval) between Rigidity Score obtained from RBCs from blood bags and RBCs from matching segments (r = 0.8863). 2345Storage Time (weeks)Rigidity Score0 2 4 6 8BagSegment*****A Br = 0.8863p < 0.00013 4 52345Bag (RS)Segment (RS)Week 0Week 2Week 4Week 6Week 852 In comparing RBCs from blood bags and segments, we saw a direct concordance between their deformability profiles. There was a pronounced loss in deformability at 8 weeks of storage, for both (bag p=0.0009, segment p=0.0001, Fig. 4.2, 4.3A). Moreover, a significant correlation between RS-values for blood bags and their segments was found (Pearson’s r = 0.8863), underscoring that assessing the deformability in segments offers an accurate, non-intrusive means to determine the RBC deformability in the associated blood bag. 4.2.4 Differences in RBC stability during storage between Male and Female donors We examined the differences in RBC deformability loss profiles among male (n = 8) and female donors (n = 6). During the initial six-week storage period, we detected no sex-specific differences in the loss of RBC deformability. However, between weeks 6 and 8, a significant decline in RBC deformability was evident in male donors, but not in female donors (p<0.05; Fig. 4). Specifically, the majority of male donors displayed a rapid decline in RBC deformability in these two weeks A B2 3 4 5Donor 1Donor 5Donor 6Donor 7Donor 8Donor 9Donor 10Donor 11Donor 2Donor 3Donor 4Donor 12Donor 13Donor 14RSWeek 0-6Week 6-8MaleFemale2345Storage TimeRigidity ScoreWeek 0 Week 2 Week 4 Week 6 Week 80.029Figure 4.5. Comparison of RBC deformability loss profiles between male and female donors. (A) Changes in rigidity scores (RS) from weeks 0-6 and weeks 6-8 of cold storage. Arrows indicate direction of change. (B) RS for male and female donors every 2 weeks of storage for 8 weeks. 53 (ΔRS=0.485), a trend not seen in female donors (ΔRS=0.172). These findings indicate that some donors could potentially provide more stable RBCs, and it is more likely for those donors to be from female donors. 4.2.5 Hematological parameters over 6 weeks of storage We monitored standard hematological parameters, including mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), and red cell distribution width (RDW) for each timepoints throughout the storage in both stored bags and their corresponding segments. Overall, the hematological parameters remained within the normal range (Fig. 5, grey shaded area) except for MCHC levels, which slightly dropped below the accepted range of 315-355 g/L (Medical Council of Canada reference values)175 by the 6-week expiration date. We correlated the general hematological data with the deformability of corresponding RBCs at Week 0 and Week 6 of storage. A mild positive correlation was observed between an increase in MCV and the RS for male donors (r = 0.7504), but not for female donors. Furthermore, male donors indicated a mild negative correlation between RS and MCHC (r = -0.6373). No correlations were found between other parameters and deformability changes. When comparing RBCs from bag and segments, we identified a robust correlation for MCV (r = 0.9419), MCHC (r = 0.9039), and MCH (r = 0.9878). The RDW correlation was more moderate (r = 0.6491), potentially due to the limited dynamic range of the RDW measurement. 4.2.6 Hemolysis levels in blood bags and segments We measured hemolysis levels in stored RBC units from both blood bags and segments at each time point during storage (Fig.4.7). In Canada, the maximum allowable hemolysis is 0.8% at the time of expiry. For blood bags, most RBC samples remained below the 0.8% hemolysis threshold 54 until week 8. Notably, Donor 9 exhibited 1.15% hemolysis at week 0 and Donor 13 had 1.11% by week 6. By the eighth week, 50% of the donor bags (n=3 male, n=2 female) surpassed the acceptable level. In contrast, hemolysis in the blood unit segments was significantly elevated. On the production day, over half (6/10) of the segments already exceeded the 0.8% hemolysis threshold. Hemolysis levels escalated dramatically in these segments, registering approximately 2% by week 6 and around 3.5% by week 8. For both bags and segments, hemolysis data did not correlate with RS, confirming that RS values are independent of the hemolysis levels (Fig. 4.6B). 2.5 3.0 3.5 4.0 4.58090100110120MCV (fL)r = 0.7504p = 0.00082.5 3.0 3.5 4.0 4.51214161820RDW (%)r = 0.4254p = 0.1005 2.5 3.0 3.5 4.0 4.524283236MCH (pg)r = 0.1218p = 0.67842.5 3.0 3.5 4.0 4.5280320360MCHC (g/L)r = -0.6373p = 0.0079 Rigidity ScoreMaleFemale2.5 3.0 3.5 4.0 4.51214161820RDW (%)r = -0.3456p = 0.40182.5 3.0 3.5 4.0 4.524283236MCH (pg)r = -0.5196p = 0.15162.5 3.0 3.5 4.0 4.5280320360MCHC (g/L) Day 1 Week 6r = 0.4045p = 0.3202 2.5 3.0 3.5 4.0 4.58090100110120MCV (fL)r = 0.5949p = 0.1198MCV RDW MCH MCHC80 100 120 1406080100120140Bag MCV (fL)Segment MCV (fL)r = 0.9419p < 0.0001200 250 300 350 400250300350400Bag MCHC (g/L)Segment MCHC (g/L)r = 0.9039p < 0.000110 12 14 16 18 20121416182022Bag RDW (%)Segment RDW (%)r = 0.6491p < 0.000120 25 30 35 4025303540Bag MCH (pg)Segment MCH (pg)r = 0.9878p < 0.0001Figure 4.6. Correlation between hematological parameters and rigidity score (RS). Male donors showed minor correlation between RS and MCV (r=0.7504; p=0.0008), as well as between RS and MCHC (r=-0.6373; p=0.0079). Female donors showed no correlations between RS and hematological parameter. Greyed areas indicate Medical Council of Canada reference values. (bag) 4. Correlations of RBC hematological parameters between blood bags and segments. (A) MCV (r = 0.9419), (B) MCHC (r = 0.9039), (C) MCH (r = 0.9878), and (D) RDW (r = 0.6491), as determined using Pearson’s r with 95% confidence interval. 55 4.3 Discussion In this study, we investigated the progressive loss of RBC deformability under standard cold storage conditions using a microfluidic ratchet device. We sorted RBCs into 12 fractions based on deformability and derived a RS based on the distribution of RBCs within these fractions. RS were obtained in freshly donated blood bags and over eight weeks of storage, two weeks longer than the 0123456Storage Time (weeks)Hemolysis (%)SegmentBag0 2 4 6 8******* ******0.5 1.5 2.50246Hemolysis (%) in BagHemolysis (%) in Segmentr = 0.5869p < 0.0001-10123Storage Time (weeks)Hemolysis (%)Male0 2 4 6 8FemaleACBFigure 4.7. Hemolysis during storage in bags and corresponding segments. (A) The majority of donor blood bags did not show hemolysis above the 0.8% threshold, except for one male donor at week 0 and female donor at week 6. Half of all the blood bags (n=3 male and n=2 female) showed hemolysis above the 0.8% threshold at 8 week. (B) Comparing hemolysis in blood bags (closed circles) and segments (open circles). ** week 0 p=0.0021, ** week 2 p=0.0029, ***week 4, 6 and 8 p<0.0001 (Wilcoxon matched pairs signed rank test). (C) Correlation between hemolysis in blood bags and segments (Pearson’s r with 95% confidence interval). 56 standard storage window. The consistent loss of RBC deformability during storage was observed, but the rate and magnitude of this loss were donor-specific. Importantly, the change in deformability over the storage period was not predictable based on the deformability of freshly donated RBCs. After six weeks of storage, RBCs from both male and female donors converged to similar deformability. However, from weeks 6-8, the RBC deformability loss accelerated dramatically for male donors, but not for female donors. The observed differences in RBC deformability loss profiles between male and female donors are consistent with other differences between male and female RBC properties42,176, such as male RBCs being smaller in size, as well as having greater hematocrit, MCV, hemoglobin concentration, viscosity, and RBC fragility compared to female RBCs2,34,42. These differences have been attributed to the female sex hormone estrogen, which has been shown to protect RBCs from deformability loss and also has a major impact on erythropoiesis regulation154. Differences in estrogen receptor distribution and function on RBCs, as well as differences in serum estradiol concentration, may affect intracellular signaling and better protect against oxidative stress in female RBCs. Profiling the loss of RBC deformability for individual donors could serve to guide blood unit selection prior to transfusion. It has been previously established that loss with the increased storage period of the RBC unit, the clinical efficacy may decline64–66,177,178. The loss of RBC deformability corresponds with this decline in clinical benefit106. Our study demonstrates that loss of RBC deformability can be profiled over the course of storage. RBC deformability was generally preserved during the 42-day storage expiration window. However, beyond this window, RBC deformability loss accelerated and varied significantly between donors, which may impact post-transfusion outcomes. 57 We also established that blood unit segments can be used to assess RBC deformability in blood bags without compromising their integrity. This analysis was made using the microfluidic ratchet device to compare RBC deformability in cells from blood bags and their corresponding segments. We observed a progressive loss of cell deformability and individual donor RBCs differed significantly in the magnitude of deformability loss. By sampling blood unit tubing segments, we found significant correlation between RBC deformability in both blood bag and segment across multiple donors and over the course of eight weeks of cold storage. RBC units can be stored for up to 42 days prior to transfusion at 4 °C in a preservative solution1,9,49. However, stored RBCs undergo structural, metabolic, and morphological changes that coincide with reduced transfusion efficacy,35,132. The loss of RBC deformability is a potential biomarker for RBC deterioration during cold storage because these cellular changes collectively contribute to a progressive loss in RBC deformability11,75,179,180. RBC deformability loss may facilitate uptake of transfused RBC by reticuloendothelial macrophages153 and can predict transfusion outcomes181. Our data support previous reports that the rate of RBC degradation differs between donors, suggesting that monitoring RBC deformability during storage can guide blood unit selection to optimize transfusion efficacy for sensitive recipients11,75,179,180. The ability to perform interim testing on RBC units during storage without compromising the blood product could have a significant impact on selecting RBC units for chronic transfusion recipients. The primary challenge when routinely monitoring RBC deformability in blood bags lies in the potential for contaminating the RBC unit while sampling cells. To address this, the tubing leading into the blood bag provides a viable source of RBCs that can be sealed into segments, allowing for sampling without jeopardizing the integrity of the RBC unit. However, previous attempts to assess the RBC unit by sampling segments have relied on hemolysis and hemoglobin assays, which have 58 shown inconsistencies between segments and blood bags. For instance, in a prior study by Kurach182 and colleagues examining the quality testing of blood bags during storage, it was observed that, after 42 days, MCH and MCHC values were notably higher in segments compared to blood bags when prepared using the same buffy coat method employed in our study, although MCV showed no such difference. Through direct linear comparison of all three RBC parameters, we found a strong correlation between the values obtained from blood bags and segments, whereas hemolysis exhibited a relatively poor correlation between the two. These variations may arise from the sensitivity of hemolysis to differences in the extracellular chemical environment, including the distinct plastics used in the construction of blood bags and segments, while changes in cell deformability are believed to stem from intracellular proteomic remodeling and oxidative changes. Collectively, these findings support the assessment of deformability, MCH, MCHC, and MCV in RBCs sampled from blood unit segments as a suitable alternative to using blood bags in studies investigating blood transfusion efficacy183. Current procedures in transfusion medicine mandate the storage of RBC units for a maximum of 42 days and their utilization based on a first-in-first-out principle. Nevertheless, these guidelines fail to account for donor-specific variations and, more specifically, disparities in the degradation of stored units. Identifying these distinctions is of particular significance for individuals requiring chronic transfusions, as the degradation of stored RBCs can curtail their circulation time, necessitating more frequent transfusions for these recipients. The ability to conduct intermediate testing of RBC units during storage, while preserving the quality of the blood product, could therefore have a substantial impact on the selection of RBC units for individuals requiring chronic transfusions. 59 Chapter 5: Effect of RBC Gamma Irradiation on RBC Deformability 5.1 Introduction Transfusion-associated graft-versus-host disease (TA-GVHD) is a potentially fatal event associated with therapies that employ cellular blood products, such as red blood cells (RBCs), platelets and granulocyte units44,48,184. TA-GVHD is rare, but this risk is cumulative for people who rely on repeated blood product therapies, such as blood or platelet transfusion during chemotherapy or to treat congenital blood disorders43,185,185,186. When TA-GVHD does occur, contaminating donor leukocytes in the blood product target recipient cells and may present as pancytopenia due to bone marrow dysfunction or as erythema, aplasia, hepatitis and diarrhea as a consequence of multiorgan failure of the skin, liver and gut. Leukoreduction of transfused blood products aims to reduce the incidence of TA-GVHD but is insufficient to eliminate all leukocytes44,46,187. For RBC units, gamma irradiation is an effective means of eliminating leukocytes by inducing DNA damage, while preserving the function of anucleate RBCs. The primary limitation of gamma irradiation of RBC units is that this process reduces the ability of RBCs to undergo cellular deformation. RBCs need to physically deform in order to perfuse capillaries that are smaller than the width of a single cell10. Rigid RBCs are trapped within splenic endothelial clefts and phagocytosed, such that less deformable transfused blood is presumed to have a shorter circulatory time28,31. RBCs stored in blood bags typically exhibit a storage lesion, characterized by structural (lipid peroxidation, Band 3, membrane asymmetry), metabolic (slowed metabolism due to ATP and 2,3-diphosphoglycerate depletion), and morphologic (discoid, echinocyte, and spherocyte) transformations188–190. The cumulative outcome of these storage effects is a progressive reduction of RBC deformability and reduced circulation time of stored blood112,191–193. Gamma irradiation can accelerate this process by 60 producing reactive oxygen species that contribute to lipid peroxidation and membrane damage194–197. The outcomes of cell damage include prolonged periods with excessive rates of hemolysis198, faster increase in potassium and lactate dehydrogenase activity199–201, increased hemoglobin oxidation202 and further changes to plasma membrane structure and permeability203. These effects exacerbate the existing RBC storage lesion and collectively reduce the effectiveness of the transfusion204. Although there have been some previous research186, it is currently not clear whether these effects of gamma irradiation directly correlate with changes in red cell deformability, and how significant the impact is. If RBC deformability could be used as an indicator for RBC stability post-irradiation, it would provide valuable insight into the effects of this treatment on the therapeutic value of transfusion blood. We developed the Microfluidic Ratchet Device for sensitive and high-throughput single cell measurement of RBC deformability. We have previously used this device to establish that RBCs from different donors vary in their susceptibility toward the storage lesion172,205. We therefore hypothesized that gamma irradiation of donor RBCs might similarly have inter-donor variability in regard to any degradation of those products. To test this hypothesis, we measured the deformability of control and irradiated RBCs at timepoints immediately after and 2 weeks post-gamma irradiation of 8 pairs of blood bag units. 5.2 Results 5.2.1 Irradiation-induced differences in RBC deformability between control and irradiated RBC units immediately post-irradiation and 2 weeks post-irradiation We aimed to examine the influence of gamma-irradiation on the deformability of RBC units immediately following irradiation. Additionally, we investigated how irradiation affects RBC 61 deformability during storage over a specified period. In blood banks, RBC units are typically stored before being transfused patients, and the duration of storage varies widely among centers due to factors such as demand and location. Some RBC units may undergo pre-emptive irradiation and storage before transfusion. Our study assessed the impact of irradiation on RBC rheological properties, focusing on their ability to deform, both immediately after irradiation and following a two-week storage period post-irradiation. Sixteen ABO-matched leukoreduced packed RBC units were utilized, comprising eight pairs. The RBC unit pairs were combined and then separated along their segment lines to create two identical Figure 5.1. Study overview. Eight pairs of ABORh+ matched RBC units were pooled and split to obtain 2 identical RBC units. The units were stored for 2 weeks, upon which one unit from each pair was Gamma-irradiated. The units were then stored for additional 2 weeks. RBC deformability, hemolysis, and hematological parameters were assessed immediately after irradiation as well as after 2 weeks of additional storage. We expect to see a reduction in RBC deformability post-irradiation, compounded by following storage for additional 2 weeks. 62 bags which were preserved at 4˚C under standard cold storage conditions for a duration of two weeks. Subsequently, the RBC units underwent gamma irradiation at 25GY, and the deformability of the RBCs was measured on the same day following irradiation. After an additional two weeks of storage (Week 4), RBC deformability was measured again (Fig. 5.3). A consistent observation among donors was that irradiated RBCs tended to be more rigid compared to their non-irradiated counterparts at both timepoints. Notably, Donors 3 and 5 displayed an increased deformability in non-irradiated control cells, deviating from the general trend. This variation in response to the storage lesion among donors contradicts the overall trend but has been reported previously 101,102. In the majority of irradiated units, RBCs exhibited reduced deformability within two weeks after storage when compared to non-irradiated controls. 1 2 3 4 5050100RBC unit pair 11 2 3 4 5050100RBC unit pair 51 2 3 4 5050100RBC unit pair 21 2 3 4 5050100RBC unit pair 61 2 3 4 5050100RBC unit pair 31 2 3 4 5050100RBC unit pair 71 2 3 4 5050100RBC unit pair 41 2 3 4 5050100RBC unit pair 8Irradiated (2 weeks post)IrradiatedControlControl (2 weeks post)Outlet numberCumulative frequency (%)Figure 5.2. Inter-donor variability of irradiated and control packed RBC units on the day of unit irradiation and 2 weeks post irradiation. Cumulative distribution of individual donor unit RBCs based on deformability using the microfluidic ratchet device assay. A rigidity score (RS) is derived from the fractional outlet number at the 50% cross over point of the cumulative distribution. 63 A Rigidity Score (RS) was calculated for each of the RBC unit pairs at every timepoint. The mean RS of non-irradiated RBCs varied between donors with a range of 2.42 to 2.92 over the two-week storage period, which is consistent with deformability changes linked to the storage lesion, as identified in our prior studies101,102. The RS of irradiated RBCs consistently exceeded that of non-irradiated controls (Fig. 5.4A). Interestingly, the degree of change in deformability in the control RBC unit did not correlate to the degree of change in the irradiated RBC unit. This implies that alterations in deformability were a response to irradiation rather than the cell's pre-existing sensitivity to the storage lesion. Immediately following gamma irradiation, RBCs had an RS of 3.10±0.20, significantly higher than control non-irradiated RBCs (Fig. 5.5; RS=2.66± 0.17, p=0.0271). Similarly, two weeks after gamma irradiation, irradiated RBCs (RS=3.17± 0.16) 2.0 2.5 3.0 3.5RBC unit pair 1RBC unit pair 2RBC unit pair 3RBC unit pair 4RBC unit pair 5RBC unit pair 6RBC unit pair 7RBC unit pair 8Rigidity ScoreControlIrradiated2.02.53.03.54.0Rigidity ScoreImmediately post-irradiation2 weeks post-irradiationControlIrradiated0.0271 <0.0001A BFigure 5.3. Degradation of RBC deformability during cold storage in control and irradiated RBC samples. A) Changes in RS over the 14-day period post-RBC irradiation. Solid lines correspond to control and dashed to irradiated RBC bags. The direction of change is indicated by the direction of the arrows. (B) Control RBCs show a mean ∆RS of 0.013, and irradiated RBCs show a mean ∆RS of 0.113 after 2 weeks of storage. The irradiated RBC units are significantly more rigid than control RBC units at each timepoint (adjusted p values: p=0.0271 immediately post-irradiation, p<0.0001 at 2 weeks post-irradiation). 64 exhibited significantly lower deformability than non-irradiated control cells (RS=2.67± 0.15, p<0.0001). 5.2.2 Hematological parameters during storage To determine whether gamma irradiation-mediated changes in RBC deformability were associated with generalized hematological dysfunction, we analyzed the hemolysis within each bag (Fig 5.3) as well as standard hematological parameters (Table 5.2) immediately post-irradiation as well as 2 weeks post-irradiation. Both control and irradiated RBC units demonstrate very low levels of hemolysis, with a mean 0.15% vs 0.14% respectively, immediately after irradiation, which is significantly lower than the acceptable level of 0.8%. There is a significant increase in hemolysis levels in irradiated RBC units (mean 0.24%) at 2 weeks post-irradiation compared to controls (mean 0.12%, p=0.0027), however, still lower than 0.8%. Next, we measured mean corpuscular volume (MCV), red cell distribution width (RDW-CV), mean corpuscular hemoglobin content (MCHC) and mean corpuscular hemoglobin (MCH). Using the Medical Council of Canada Reference Values as a standard, we observed that 7/8 donor control and irradiated units were within the normal range for MCV measured immediately post-irradiation and 6/8 at 2 weeks post irradiation. MCH values for all RBC units were within the 0.00.20.40.6Hemolysis (%)Immediately post-irradiation2 weeks post- irradiationIrradiatedControl0.0027Figure 5.4. Hemolysis during storage. Both control and irradiated RBC units demonstrate a low level of hemolysis immediately post irradiation (mean 0.15% vs 0.14%). There is a significant increase in hemolysis levels in irradiated RBC units at 2 weeks post-irradiation (mean 0.24%) compared to controls (mean 0.12%, P=0.0027). 65 normal range at both immediately as well as 2 weeks post-irradiation. Some control and irradiated RBCs had abnormally high RDW-CV and most of the control and irradiation RBC units had abnormally low MCHC at both timepoints. Gamma irradiation did not have a statistically significant effect on any of the hematological parameters of the matched blood bag unit pairs.We then explored the correlation between changes in deformability and hematological indices of the RBC within each unit (Fig 5.5). We found that immediately post-irradiation, there were no correlations between measurements of MCV, MCH, RDW and MCHC and RS in control or irradiated samples (Fig 5.5A-D). At 2 weeks post-irradiation, however, the control RBC units showed significant correlations between RBC rigidity score and MCV (Fig 4E, p=0.0404, R2 =0.5307), MCH (Fig 4F, p=0.0048, R2 = 0.7591), as well as MCHC (p=0.0091, R2 = 0.7055), but not in irradiated samples. 5.3 Discussion In this study, we used the microfluidic ratchet device to measure changes in RBC deformability as a consequence of gamma irradiation on the storage lesion. All RBCs subjected to gamma irradiation exhibited immediate rigidification, compared to non-irradiated controls. The acute rigidification of RBCs post-irradiation is significant because rigid RBCs may exhibit shorter circulation time and therefore show lower therapeutic effectiveness10,181,206. Cold storage had a marginal effect on the deformability of control samples, with a slightly more pronounced impact observed in irradiated RBC units (although not statistically significant). In line with our earlier investigations, each unit of donated RBCs exhibited a distinct pattern of deformability change102,103 , wherein certain donor RBCs displayed increased deformability during cold storage, while the collective group experienced an overall decrease in deformability. Interestingly, the degree of change in deformability of the control RBCs did not predict the degree of change of the irradiated 66 RBCs. These observations suggests that that some donor RBCs may be more resistant to the deleterious effects of irradiation than others. These changes were also not evident in other hematological parameters, making RBC deformability measurement a potentially sensitive biomarker for the effects of RBC irradiation. Previous studies showed RBC irradiation to lead to potassium loss, membrane lipid peroxidation, reduced ATP levels, and increased hemolysis185,186,207. Some studies also showed a reduced recovery of transfused irradiated RBCs that have been stored for a prolonged period of time after irradiation207. A general consensus is that RBC irradiation accelerates the storage lesion 2.0 2.5 3.0 3.5 4.08090100110120RSMCV (fL)2.0 2.5 3.0 3.5 4.024283236RSMCH (pg)Immediately post-irradition2 weeks post-irradiation 2.0 2.5 3.0 3.5 4.08090100110120RSMCV (fL)2.0 2.5 3.0 3.5 4.024283236RSMCH (pg)2.0 2.5 3.0 3.5 4.01214161820RSRDW-CV (%)2.0 2.5 3.0 3.5 4.0280320360RSMCHC (g/L)2.0 2.5 3.0 3.5 4.014161820RSRDW-CV (%)2.0 2.5 3.0 3.5 4.0280320360RSMCHC (g/L)12 A B C DIrradiatedControl p=0.0091, R2=0.7055 p=0.0404; R2=0.5307 p=0.0048, R2=0.7591E HGFFigure 5.5. Correlation between hematological parameters and RS in irradiated and control RBC units. Gray areas indicate the normal range of indices. (A-D) There is no correlations between control or irradiated samples and RS immediately post-irradiation. At 2 weeks post-irradiation, there is a significant correlation between changes in (E) MCV values and RS in control (p=0.0404, R2 =0.5307) but not irradiated samples (F) MCH and RS in control (p=0.0048, R2 = 0.7591) but not in irradiated samples and (H) MCHC levels and RS in control (p=0.0091, R2 = 0.7055) but not in irradiated units. (G) We observe no correlations between RDW and RS values. 67 in these cells and current Association for the Advancement of Blood and Biotherapies (AABB) guidelines have recommended that gamma irradiation can be performed during RBC storage so long as the maximum storage time is reduced from a standard of 42 days to 28 days185,208. However, our data show that RBCs exhibit significant biophysical changes immediately after irradiation and the magnitude of RBC rigidification by gamma irradiation is significantly greater than the effects of storage lesion. We previously observed inter-donor variability in RBC storage lesion during cold storage of blood101,102. Interestingly, we similarly observed donor-specific differences in RBC response to irradiation. All donor RBCs were rigidified over the course of cold storage and gamma irradiation, but the magnitude of rigidification varied between donors. This observation suggests that that some donor RBCs may be more resistant to the deleterious effects of irradiation. These changes were not evident in other hematological parameters, making RBC deformability a potentially sensitive biomarker for the effects of RBC irradiation. Together, these data suggest that gamma irradiation has an immediate and profound effect on the physical deformability of RBCs. These effects varied between donors but are significant because of the role of RBC deformation in perfusion of blood in the body. In light of this new evidence on the biophysical effects of gamma irradiation on RBC deformability, future work is needed to investigate the impact of RBC irradiation on transfusion efficacy. Furthermore, the inter-donor variability in response to irradiation suggests that RBC deformability could serve as a sensitive biomarker for donor sensitivity to RBC irradiation. 68 Table 5-1. RS values of RBCs in control and irradiated RBC unit pairs. Rigidity Score Immediately post-irradiation 2 Weeks post-irradiation Control Irradiated Control Irradiated RBC unit pair 1 2.66 3.04 2.78 3.32 RBC unit pair 2 2.54 3.14 2.57 3.18 RBC unit pair 3 2.73 2.87 2.51 3.15 RBC unit pair 4 2.48 3.29 2.67 3.11 RBC unit pair 5 2.86 2.79 2.61 2.99 RBC unit pair 6 2.88 3.34 2.92 3.46 RBC unit pair 7 2.71 3.04 2.8 3.06 RBC unit pair 8 2.42 2.91 2.52 3.05 Mean 2.66 3.05 2.67 3.17 Min 2.42 2.79 2.51 2.99 Max 2.88 3.34 2.92 3.46 69 Table 5-2. Hematological parameters of RBC in control and irradiated RBC unit pairs. (Numbers in bold indicate outside of healthy range) MCV (fL) Immediately post-irradiation 2 weeks post-irradiation Control Irradiated Control Irradiated RBC unit pair 1 92.5 93.9 94.6 96.5 RBC unit pair 2 100.5 101 103 103.5 RBC unit pair 3 96.4 97.5 97.3 97.7 RBC unit pair 4 96 97.5 97 97.8 RBC unit pair 5 97.9 97.1 100.3 100 RBC unit pair 6 91.6 91.3 93.4 94 RBC unit pair 7 89.7 86.3 90.1 88.4 RBC unit pair 8 96.8 96.5 97.9 98.9 Mean 95.2 95.1 96.7 97.1 MCH (pg) Immediately post-irradiation 2 weeks post-irradiation Control Irradiated Control Irradiated RBC unit pair 1 28.2 28.7 28.6 28.8 RBC unit pair 2 31.6 31.7 31.8 32.1 RBC unit pair 3 29.9 30.2 30.2 30 RBC unit pair 4 30 30.8 30 30.8 RBC unit pair 5 31.5 30.9 31.3 30.9 RBC unit pair 6 27.6 27.5 27.4 27.5 RBC unit pair 7 27.7 25.6 27.8 26.2 RBC unit pair 8 30.4 30.4 30.5 30.8 Mean 29.7 29.5 29.7 29.6 70 RDW-CV (%) Immediately post-irradiation 2 weeks post-irradiation Control Irradiated Control Irradiated RBC unit pair 1 17.8 17.3 18.1 17.7 RBC unit pair 2 12.6 12.7 12.8 12.8 RBC unit pair 3 14.4 13 14.1 13.5 RBC unit pair 4 15.3 13.7 15.6 13.5 RBC unit pair 5 14 14.7 13.6 15.7 RBC unit pair 6 16.1 16 16.4 16.5 RBC unit pair 7 14.9 17.2 14.9 17.3 RBC unit pair 8 18.4 18.4 18.3 18.8 Mean 15.4 15.4 15.5 15.7 MCHC (g/L) Immediately post-irradiation 2 weeks post-irradiation Control Irradiated Control Irradiated RBC unit pair 1 305 306 302 298 RBC unit pair 2 315 314 309 310 RBC unit pair 3 310 310 311 307 RBC unit pair 4 312 316 309 315 RBC unit pair 5 322 318 312 309 RBC unit pair 6 302 301 293 293 RBC unit pair 7 309 297 309 297 RBC unit pair 8 314 315 312 312 Mean 311 310 307 305 71 Chapter 6: Deformability-Driven Circulatory Clearance of Red Blood Cells in a Murine Transfusion Model 6.1 Introduction Red blood cell (RBC) transfusion is a life-saving therapy used to treat a wide range of anemias stemming from acute trauma as well as chronic blood disorders, such as thalassemia, sickle cell disease and bone marrow dysfunction. However, individuals with chronic anemia require repeated transfusions that place them at greater risk for iron-overload, transfusion-associated circulatory overload (TACO), transfusion-related acute lung injury (TRALI), as well as increased risk of infections117–119. As a consequence of the side effects of chronic transfusion, it is desirable to extend the interval between blood transfusions. This could be achieved through careful selection of donor blood, since it is well-established that there are potential differences between donors that can result in different RBC circulation times112,113. Various donor RBC selection criteria have been investigated, based on known structural, metabolic and morphological deterioration that has been described in stored RBCs, called the storage lesion14,49,51. These changes include lipid peroxidation209,210, membrane asymmetry and Band 3 clustering211, slowed metabolism due to ATP and 2,3-diphosphoglycerate depletion17, and they contribute to a visible morphological effect on the RBCs, causing them to transition from deformable biconcave discoid cells to rigid echinocytes and spherocytes188. However, despite the range of detectable cellular differences observed in donor RBCs no single factor has been established as a biomarker for RBC circulation time. RBC deformability has long been regarded as a potential biomarker for donor RBC quality189,190 because the biochemical changes within RBCs, such as lipid peroxidation and ATP-72 depletion, cumulatively contribute to progressive rigidification of RBCs212. Furthermore, RBC senescence is associated with membrane loss that causes densification and rigidification of the cell, and these biophysical changes correspond with the formation of echinocytes and spherocytes188. These changes are noticeable after 2 weeks of storage and accelerate over time126–128,193,213. Functional studies of post-transfusion efficacy also support the potential for deformability as a biomarker of transfusion outcomes. Specifically, RBCs with low cell deformability exhibit lower post-transfusion viability11 and transfusion of deformable RBCs are more effective at increasing blood hemoglobin than were rigid cells214. The relevance of RBC deformability also stems from the critical role cell deformability plays in the process of RBC clearance from circulation. RBCs are typically 6-8 µm in diameter and have the capacity to deform through blood vessels as small as 2-3 µm. However, as RBCs age both in storage and in circulation, these cells experience cytoskeletal crosslinking and membrane loss10,26,215 that contribute to altered morphology and progressive cellular rigidification. These rigidified RBCs may fail to deform and may bypass the smaller capillaries and instead become lodged in the capillaries or endothelial clefts of the spleen10,73,76. RBC surface modifications that occur during senescence, such as α-galactosyl carbohydrate and modified or clustered Band 3 antigen expression216,217 or physical rigidification of the cells cause splenic phagocytes to preferentially eliminate poorly deformable RBCs216,218. Here, we employ a sensitive microfluidic method for single-cell measurement of RBC deformability in order to assess the circulation time of both healthy and rigidified RBCs. Our findings were consistent with previous reports that RBC loss was most pronounced in the first 24 hours post transfusion. However, we added to this understanding by demonstrating that the degree of cell loss was significantly greater for chemically-rigidified RBCs and that there was a strong 73 bias toward elimination of the most rigid cells in the population. RBC aging continued over 5-weeks following transfusion, but we did not observe a significant difference in the rate of elimination for chemically rigidified RBCs as well as healthy transfused RBCs, compared to endogenous RBCs. This finding underscores the critical importance of measuring single-cell RBC deformability in quality assessment of transfusion blood, since it is the fraction of deformable cells, and not the mean RBC deformability, that determines the effectiveness of transfused units. 6.2 Methods 6.2.1 Mouse transfusion model This study was completed in accordance with University of British Columbia Animal Care Committee (A18-0362). A total of 20 female C57BL/6J mice between the ages of 6-10 weeks were purchased from Jackson Laboratories and represented both donors and recipients in the transfusion study. Blood donor mice (N=7) were euthanized with isoflurane and carbon dioxide method. A blood sample was then collected by terminal bleed via cardiac puncture in a heparinized syringe (BD) and 25-gauge needle (BD) and transferred into a EDTA tube. Transfusion recipient mice (n=7) were weighed and the volume of RBCs to be transfused was calculated according to the formula: blood volume = 25g /1000 * 72 ml/kg * 10% = 180 μl. Approximately 10% of circulating blood volume was transfused to the recipient mice. The transfusion was done in via tail vein injection. Following transfusion, the recipients were sampled via tail vein prick with a 27-gauge needle for RBCs within 24 hours post-transfusion, followed by weekly thereafter for the duration of 5 weeks. Animals were weighed and monitored daily for any adverse effects. None were observed post-transfusion. 74 At each sampling timepoint, about 30 µl of blood was drawn from recipient mice via tail vein poke into an EDTA-coated tube. The RBCs were then washed in HBSS and Pluronic buffer as described below and assessed for RBC deformability and percentage of remaining transfused RBCs. 6.2.2 Chemical treatment of RBCs We investigated the impact of RBC deformability, and specifically the loss of deformability, on RBC post-transfusion circulation longevity in a mouse model. To achieve a measurable rigidification of RBCs without inducing extensive cell damage and lysis, we used aminotriazole (AMT, MilliporeSigma) treatment219–221. Murine RBCs (n=7 donors pooled into 2 samples) were chemically rigidified using AMT. Donor mice were euthanized as described above, and blood was collected immediately after via cardiac puncture. About 500 µl of blood was collected from each donor mouse. Blood samples from donor mice were then pooled and washed 3x in HBSS and Pluronic F127 buffer (MilliporeSigma) at the spin speed of 400G for 10 minutes for the first wash, followed by 400G for 5 minutes for subsequent washes. The washed RBCs were then suspended to 20% hematocrit, either treated with PBS (control) or 25 mM AMT (MilliporeSigma) and incubated for 2.5 hours at 37˚C on a tube revolver (ThermoScientific). After incubation, the samples were washed 3x with HBSS and Pluronic F127 buffer to wash out the remaining AMT. After washing, the concentration of the RBC sample was determined with haemocytometer. The control RBC sample was stained with PKH-26 (red) and the AMT-treated sample was stained with PKH-67 (green) long-term general membrane stains (SigmaAlrich) as per manufacturer’s instructions, with 2 mL of final staining volume used for each 2 x106 cells. The stained control and AMT-treated blood samples were then mixed in 1:1 ratio and suspended to 50% hematocrit in sterile isotonic saline for transfusion into recipient mice. A sample from unstained and stained RBCs from both control and treatment groups were also assessed for 75 deformability prior to transfusion to ensure that the staining protocol did not alter the RBC deformability. We did not observe any significant changes in RBC deformability curve as a result of PKH staining. 6.2.3 Deformability assessment using the Microfluidic Ratchet Device Deformability is measured based on the ability of RBCs to deform and pass through constrictions much smaller than their diameter, ranging from 7.5 µm to 1.5 µm using constant forward and oscillatory upward pressure flows. RBCs traverse diagonally until reaching a limiting constriction size which they are unable to deform and pass through to one of 12 outlets. The murine RBC sample washed RBCs were suspended to 1% hematocrit in HBSS and Pluronic buffer and sorted based on their deformability into the 12 outlets. Each deformability fraction was then transferred from the corresponding outlet to a 96-well flat-bottom plate. RBCs from each outlet were imaged using an inverted microscope and Nikon software. The deformability of endogenous, control, and AMT-treated RBC sub-populations was calculated by quantifying the number of fluorescently labeled and unlabeled RBCs in each sorted fraction using ImageJ software. The cumulative distribution of the RBCs in the outlets is calculated and the outlet where 50% of most deformable cells are collected is described as the rigidity score, which is used to compare the RBC deformability between samples and timepoints. Similarly, the clearance rate of the control and AMT-treated RBC sub-populations was quantified at each time point. 6.2.4 Data analysis The deformability and rigidity score data were analyzed with GraphPad (Prism 10). Means and standard deviations are plotted unless otherwise stated. Linear regression with 95% confidence intervals were used to calculate the correlations between data sets. Data sets were compared using Anova with Geisser-Greenhouse correction and Tukey correction for multiple comparisons. 76 6.3 Results 6.3.1 Approach We used a sensitive microfluidic ratchet device to measure deformability of single RBCs both before and after transfusion in order to determine the impact of RBC rigidity on circulation time (Fig. 6.1.A-B). We generated RBCs with distinct deformability by chemical rigidification of cells using aminothiazole (AMT; Fig. 6.DA). We labelled AMT-treated RBCs with PKH-67 (green) and untreated RBCs with PKH-26 (red) membrane dye and transfused these cells at equal amounts into recipient mice. We then collected endogenous and labelled RBCs at 24 hours as well as over Figure 6.1. Study overview. (A-B): Potential models for circulatory clearance of donor RBCs as a function of cell deformability. If circulatory clearance is independent of deformability, the deformability profile of circulating cells will be unchanged (A). If circulatory clearance is dependent on cell deformability, the more rigid cells will be preferentially removed (B). (C): The microfluidic ratchet device sort RBCs based on deformability. Cells are typically distributed between outlets 2 and 5. (D-F): Experimental plan. Mouse RBCs are chemically-treated using aminothiazole (AMT) to alter their deformability distribution relative to control (D). Control and AMT-treated cells are labeled with long-lasting fluorescent dyes, and then transfused into recipient mice (D-E). RBCs sampled from recipients are sorted using the microfluidic ratchet device to concurrently measure the deformability profile of control donor RBCs, AMT-treated donor RBCs, and endogenous RBCs circulating in recipient mice (F). 77 5 weeks post-transfusion (Fig. 6.1F). The microfluidic ratchet device used to measure RBC deformability has been described previously222. Briefly, RBCs are flowed through a sorting matrix formed from rows of progressively smaller microconstrictions (Fig. 6.1C). When cells reach the terminal constriction through which they can no longer deform, they are flushed into an outlet, such that RBCs are fractionated based on cell deformability, where the most deformable cells accumulate in outlet #1 and the most rigid cells accumulate in outlet #6. The number of cells occupying each outlet is determined and used to model a distribution of cell deformability (Fig. 6.1F). Finally, this distribution is used to establish a rigidity score (RS) based on the 50% cross-over point in the distribution. Treatment of RBCs with AMT can be observed as a rightward shift in the deformability distribution, compared with transfused control RBCs or unlabeled endogenous RBCs. 6.3.2 AMT treatment of murine RBCs leads to loss of deformability To establish a reliable source of poorly deformable RBCs, we obtained RBCs from donor mice and treated murine RBC with AMT (25mM) ex vivo. AMT leads to increased reactive oxygen species formation in RBC by blocking catalase activity. In a healthy cell, catalase is one of the key antioxidant enzymes, converting large amounts of H2O2 to water and oxygen and thus preventing oxidation of hemoglobin and other RBC constituents25,223. AMT irreversibly binds to Catalase compound 1, preventing its function, leading to an accumulation of ROS and increased oxidative stress on the RBC, resulting in its premature removal from circulation. Control RBCs were treated with PBS. These RBCs were then evaluated by microfluidic ratchet and a distribution and RS-value was established for each sample. For both sample batches, AMT treatment affected the RBCs to cause a rightward shift in RBC deformability distribution (Fig. 6.2). This distribution is consistent with a greater proportion of RBCs collecting in outlets with more rigid cells. To quantify 78 these effects, we calculated the change in RS-value between AMT-treated and control RBCs. We observed ΔRS of 0.78 (Fig. 6.2A) and 0.59 (Fig. 6.2B), respectively. 6.3.3 Less deformable RBCs are selectively removed from circulation within 24 hours post-transfusion. To examine whether RBC deformability plays a role in post-transfusion circulation, PBS-treated (control) and AMT-treated RBCs were mixed at 1:1 ratio and transfused into seven recipient mice. For each mouse RBC deformability distributions were generated for AMT-treated and control RBCs both before and 24 hours following transfusion (Fig. 6.3). For control donor RBCs, the pre- and post-transfusion deformability profiles were nearly identical to each other and to endogenous recipient RBCs (Fig. 6.3A). For AMT-treated donor RBCs, the pre-transfusion RBCs were significantly more rigid than endogenous recipient RBCs, but at 24-hour post-transfusion, the deformability profile of AMT-treated donor RBCs in remaining in circulation was identical to that of endogenous recipient RBCs (Fig. 6.3B). This result suggests that the rigid donor RBCs, shown as green shade in Fig. 6.3B, were selectively eliminated from circulation. The observed selective clearance of rigid RBCs was consistent for all recipient mice in our study (Fig. 6.3C-I). 1 2 3 4 5 6 7 80204060Outlet numberFrequency (%) 1 2 3 4 5 6 7 80204060Outlet numberFrequency (%) CtrlAMTA BFigure 6.2. Aminothiazole (AMT) treatment effectively reduces murine RBC deformability. Deformability distribution of the mouse RBCs treated with DMSO control and aminothiazole treatment. Graphs A and B represent donor groups 1 and 2, n=3 each. 79 Importantly, we did not anticipate a change in deformability for individual RBCs since AMT treatment induces permanent inhibition of catalase and a corresponding permanent rigidification of RBCs219,220,223–225. Together, these results confirm that the murine circulatory system selectively clears RBCs based on loss of deformability. Figure 6.3. Deformability of donor and recipient RBCs pre- and 24 hours post-transfusion. (A): Deformability profiles of pre-transfusion donor (Pre-Tx Ctrl) RBCs, 24-hour post-transfusion donor (24h Ctrl) RBCs, and endogenous recipient RBCs. (B): Deformability profiles of pre-transfusion AMT-treated donor (Pre-Tx AMT) RBCs, 24-hour post-transfusion donor (24h AMT) RBCs, and endogenous recipient RBC. AMT-treated donor RBCs cleared from circulation are shown as shaded green area. Data averaged from n=7 recipients. (C-I): Deformability profiles of pre- and post-transfusion donor and endogenous recipient RBCs for individual mouse. 80 6.3.4 Deformability of transfused RBCs over five weeks in circulation. Following measurement of RBC deformability at 24-hour post-transfusion, we continued to sample RBCs over five subsequent weeks. This sampling period was selected because it captures the bulk of the expected 45-55 day lifespan of murine RBCs19,226,227. We observed fluctuations in RS for control and AMT-treated transfused RBCs, as well as endogenous RBCs (Fig. 6.4A). However, fluctuations in all three RBC populations were generally consistent and the RS of AMT-treated did not deviate significantly from the other RBC populations (Fig. 6.4B-D). Specifically, 0 1 2 3 4 512345Weeks post transfusionRigidity Score Ctrl AMT Endogenous 3 434RS control RBCsRS AMT treated RBCsR2=0.9304P<0.00013 434RS Endogenous RBCsRS AMT treated RBCsR2=0.7847P<0.00013 434RS Endogenous RBCsRS Control RBCsR2=0.7628P<0.0001AB C DFigure 6.4. Correlation between transfused and control RS scores. (A): RS of control, AMT-treated, and endogenous RBCs monitored throughout the 5 weeks post-transfusion. The bars represent mean with SD. The correlation between RS is R2=0.7628, p<0.0001. (B-D): Correlations between deformabilities of RBC subpopulations in recipient mice. (B): Correlation between AMT treated and control RBC RS Clearance of AMT-rigidified and control RBCs from recipient mice R2 =0.9304, p<0.0001. (C): Correlation between control and endogenous RBC RS is R2 =0.7628, p<0.0001. (D): Correlation between AMT-treated and endogenous RBC RS is R2 =0.7847, p<0.0001. 81 the RS of AMT-treated RBCs correlated best with control transfused RBCs (r2=0.9304). In contrast, endogenous RBCs were correlated to a lesser degree with both control (r2=0.7628) and AMT-treated RBCs (r2=0.7847). Together, these data indicate that AMT-treatment prior to transfusion does not have a significant effect on the deformability of transfused RBCs that remain in circulation over the course of weeks after transfusion. 6.3.5 Changes in RBC deformability correspond with cell loss in circulation. To estimate the number of labelled cells in circulation, we determined the frequency of control and AMT-treated in RBC samples at 24-hours post-transfusion as well as over the subsequent five weeks. Consistent with the change in RBC deformability at 24 hours post-transfusion (Fig. 6.3), we noted a significant decrease in the number of transfused AMT-treated rigid RBCs that could be recovered by sampling circulating blood (Fig. 6.5A, p<0.0001). This is consistent with our proposed model that the loss of apparent RBC rigidity in the RBC population is a consequence of selective removal of the most rigid cells. Over the subsequent 5 weeks of RBC sampling, we Figure 6.5. Percent remaining of transfused RBCs in circulation in recipients. (A): Comparison of percent remaining transfused control (red) versus AMT-treated (green) RBCs after first 24 hours post-transfusion. The error bars represent means with SD. A significant number of AMT-treated RBCs is cleared in first 24 hours post-transfusion, P<0.0001. (B): Clearance rate of AMT-treated and control RBCs from recipient mice throughout the 42-day post- transfusion monitoring period. 82 observed removal of transfused RBCs corresponding to both control and AMT-treated conditions (Fig. 6.5B), which is consistent with the anticipated senescence of these cells over time. Despite the significant loss of AMT-treated RBCs in the first 24 hours (Fig. 6.5A, p<0.0001), we did not observe further differences in the rate of RBC removal in both conditions or their circulatory half life (Fig. 6.5B). Consequently, we conclude that rigid RBCs that exist in donor blood due to either donor variation or storage lesion are eliminated from circulation within the first 24 hours, resulting in a lower abundance of RBCs that have similar cell deformability and longevity in circulation to healthy and deformable cells. This implies that detection of rigid RBCs in donor blood units can provide an indication of the proportion of functional cells immediately available post-transfusion. 6.4 Discussion The importance of RBC deformability in circulation and circulation longevity has long been known15,17–20,28,29. RBCs undergo a number of biochemical and morphologic changes, collectively known as storage lesion, as they are stored in blood banks10,11,15,30. These lesions include reversible changes, such as changes in ATP, 2,3-DPG, and ion levels, and irreversible damage, such as reactive oxygen species formation, protein denaturation, and membrane composition changes8,12,13,31. In addition, donor-specific differences, such as ethnicity, sex, and age, as well as donor health status, enzyme polymorphisms and protein variants, all can affect post-transfusion clearance time of donor RBCs10,15,28,32. So far, no single cellular marker has been established as a reliable and repeatable biomarker for RBC clearance. Most importantly, all of the above changes will contribute to loss of deformability of RBCs7,16–21. In this work, we employed a mouse transfusion model to examine the role of RBC deformability changes and its direct impact on circulation clearance time. 83 Mouse transfusion studies have been established and used in transfusion studies before226–229. The research so far suggests that similar to human RBCs, murine RBCs undergo a number of biochemical and metabolic changes during storage, some of which are irreversible, which then affect the post-transfusion circulation time in murine recipients17,211. The difference in storage quality has also shown to be donor strain-specific226,228. Post-transfusion circulation timespan of murine RBCs has been demonstrated previously226. Similar to human RBCs, murine transfused RBCs are gradually cleared from circulation and are below detection limits by the end of their circulation lifespan of 55 days173,216,218,219. Despite mouse RBCs being different from human RBCs, mouse models still offer a valuable tool for studying post-transfusion clearance of cells. Previous studies have not investigated how changes in RBC deformability impact post-transfusion clearance rate in mouse models. Our study bridges this gap by demonstrating that RBCs with reduced deformability are preferentially and rapidly cleared from the mouse circulation. Notably, the deformability profile of the remaining RBCs remained consistent with endogenous RBCs and controls throughout the 5-week sampling period, suggesting a routine and efficient clearance mechanism for less deformable RBCs in mice. Clearance of transfused RBCs from donor circulation is dictated by a combination of both donor and recipient factors228,230. Consequently, we took into account and sought to minimize the potential variability between donors, as well as between donor and recipient mice. To mitigate variability that might come from variability in strain, age and sex of the mice, we used BL6 mice of the same strain for donors and recipients, focusing on female mice aged 3-4 months42,53,60. Further consistency was ensured by using littermates as donors and recipients. To minimize variations in clearance rates between control and AMT-treated rigidified donor RBCs, we transfused the same mouse with both sample types at a 1:1 ratio. This approach led to consistent 84 RBC clearance patterns observed in all mice during the first 24 hours post-transfusion and over the subsequent 5-week period. Together, these findings offer a mouse transfusion model as a suitable model for studying the effects of deformability on RBC clearance post-transfusion. Utilizing the microfluidic ratchet device, we successfully characterized and monitored deformability in both transfused and native RBC subpopulations within recipients. These results pave the way for further research into the pathophysiological effects of transfusing rigid versus deformable RBCs. Additionally, it's crucial to extend these findings to human models to understand their applicability in human RBC clearance post-transfusion. The potential to predict donor RBC circulation rates in recipients based on deformability profiles could significantly enhance patient care, allowing for more targeted unit-to-donor matching, particularly for vulnerable patient groups. 85 Chapter 7: Conclusion This dissertation investigated the potential of using RBC deformability as a surrogate marker for circulatory clearance time. We initially focused on validating the use of our microfluidic ratchet device for measuring RBC deformability. We sought to determine if the deformability profiles of individual donors were significantly different and measurable using this device. Our findings revealed significant variability in the aging curve of RBC deformability across donors, while consistency was observed for individual donors over multiple donations. Building upon the validation of our device, we extended our research to explore the changes in RBC deformability in standard blood bag units which are used for RBC storage. The aim was to observe any relationship between RBC deformability changes throughout the cold storage in these units. Our findings demonstrated that storage in blood bags typically prevented RBC deformability loss within the standard 42-day storage period. However, beyond the standard storage period, between 42 and 56 days, deformability loss varied significantly among donors. We observed greater loss of RBC deformability in male donors than female donors. Furthermore, our found a strong correlation between deformability trends in blood unit segments and blood bag data. As a result, we concluded that RBC deformability loss could be used to screen for donors who can provide stable RBCs. Our finding provides a non-destructive method to sample RBC for future clinical studies. Our next aim was to examine how gamma irradiation affects RBC deformability. Gamma irradiation of RBC is used to reduce immune activation in sensitive patients. Our research revealed that irradiation of RBC units had an immediate and significant impact on deformability, making the cells more rigid. Importantly, the observed loss of deformability from irradiation did not 86 increase with cold storage. These results show the potential cost of irradiation in the functional properties of RBC. Finally, we studied how RBC deformability affect circulation time in transfusion recipients using a mouse transfusion model. Our aim was to determine the correlation between RBC deformability and post-transfusion clearance rate. We found that rigid RBC are rapidly and preferentially cleared within 24 hours after transfusion, while RBCs remaining in circulation exhibited a similar deformability profile to endogenous RBCs, highlighting the role of RBC deformability assessment in transfusion medicine. The results obtained from our research contribute to our understanding of how biophysical properties of RBCs affect circulation outcomes in transfusion recipients. These findings demonstrate the potential to use RBC deformability as a biomarker for predicting the post-transfusion circulation time of RBC units. This capability could ultimately enable the identification of long- and short-circulating RBC units, which could be selectively given to chronic and acute transfusion recipients, respectively. This separation of RBC unit pool has the potential to improve outcomes for chronic transfusion recipients by decreasing transfusion number and frequency, as well as increase the overall blood supply without negatively impacting acute recipients. To help enable the potential implementation of deformability assessments by blood banking services like Canadian Blood Services, we plan to conduct a clinical study to investigate the relationship between RBC deformability of transfused RBCs and their circulatory clearance time in chronic transfusion recipients. Collaborations with hematologists, blood bankers, and transfusion medicine specialists will be essential to design and execute these studies. We will investigate whether a more deformable RBC units correspond to better transfusion outcomes. Additionally to the clinical study planned, longitudinal studies tracking the long-term outcomes of 87 transfusion recipients who receive RBC units based on deformability assessment will be beneficial. These studies could assess not only the clinical efficacy but also the cost-effectiveness and quality-of-life improvements associated with personalized transfusion strategies in chronic transfusion patients. The future work will move patient care one step closer to precision transfusion medicine, where RBC deformability assessment could be used to match specific RBC units to recipients who would derive the most benefit. 88 References 1. Akash Gupta. Blood components. CBS Professional Education https://professionaleducation.blood.ca/en/transfusion/clinical-guide/blood-components (2023). 2. Hadjesfandiari, N., Khorshidfar, M. & Devine, D. V. Current Understanding of the Relationship between Blood Donor Variability and Blood Component Quality. IJMS 22, 3943 (2021). 3. Acker, J. P., Marks, D. C. & Sheffield, W. P. Quality Assessment of Established and Emerging Blood Components for Transfusion. Journal of Blood Transfusion 2016, 1–28 (2016). 4. Secomb, T. W. Mechanics of blood flow in the microcirculation. Symp Soc Exp Biol 49, 305–321 (1995). 5. Pries, A. R., Secomb, T. W. & Gaehtgens, P. Biophysical aspects of blood flow in the microvasculature. Cardiovascular Research 32, 654–667 (1996). 6. Peter Klinken, S. Red blood cells. The International Journal of Biochemistry & Cell Biology 34, 1513–1518 (2002). 7. Kruse, A., Uehlinger, D. E., Gotch, F., Kotanko, P. & Levin, N. W. Red Blood Cell Lifespan, Erythropoiesis and Hemoglobin Control. in Contributions to Nephrology (eds. Ronco, C. & Cruz, D. N.) vol. 161 247–254 (KARGER, Basel, 2008). 8. Lasch, J., Küllertz, G. & Opalka, J. R. Separation of Erythrocytes into Age-Related Fractions by Density or Size? Counterflow Centrifugation. cclm 38, 629–632 (2000). 9. D’Alessandro, A., Liumbruno, G. M., Grazzini, G. & Zolla, L. Red blood cell storage: the story so far. Blood Transfusion (2010) doi:10.2450/2009.0122-09. 89 10. Huisjes, R. et al. Squeezing for Life – Properties of Red Blood Cell Deformability. Front. Physiol. 9, 656 (2018). 11. Card, R. T., Mohandas, N. & Mollison, P. L. Relationship of post‐transfusion viability to deformability of stored red cells. Br J Haematol 53, 237–240 (1983). 12. Tomaiuolo, G. Biomechanical properties of red blood cells in health and disease towards microfluidics. Biomicrofluidics 8, 051501 (2014). 13. Blasi, B., D’Alessandro, A., Ramundo, N. & Zolla, L. Red blood cell storage and cell morphology. Transfusion Medicine 22, 90–96 (2012). 14. Yoshida, T., Prudent, M. & D’Alessandro, A. Red blood cell storage lesion: causes and potential clinical consequences. Blood Transfusion 27–52 (2019) doi:10.2450/2019.0217-18. 15. Sosale, N. G. et al. Cell rigidity and shape override CD47’s “self”-signaling in phagocytosis by hyperactivating myosin-II. Blood 125, 542–552 (2015). 16. Kumar, P., Sarkar, S. & Narang, A. Acute intravascular haemolysis following exchange transfusion with G-6-PD deficient blood. Eur J Pediatr 153, 98–99 (1994). 17. Karafin, M. S. & Francis, R. O. Impact of G6PD status on red cell storage and transfusion outcomes. Blood Transfusion 289–295 (2019) doi:10.2450/2019.0092-19. 18. Salomao, M. et al. Protein 4.1R-dependent multiprotein complex: New insights into the structural organization of the red blood cell membrane. Proc. Natl. Acad. Sci. U.S.A. 105, 8026–8031 (2008). 19. Perrotta, S., Gallagher, P. G. & Mohandas, N. Hereditary spherocytosis. The Lancet 372, 1411–1426 (2008). 90 20. Scott, M. D., Rouyer‐Fessard, P., Ba, M. S., Lubin, B. H. & Beuzard, Y. α‐ and β‐haemoglobin chain induced changes in normal erythrocyte deformability: comparison to β thalassaemia intermedia and Hb H disease. Br J Haematol 80, 519–526 (1992). 21. Scott, M. D. et al. Effect of excess alpha-hemoglobin chains on cellular and membrane oxidation in model beta-thalassemic erythrocytes. J. Clin. Invest. 91, 1706–1712 (1993). 22. Gallagher, P. G. Disorders of red cell volume regulation: Current Opinion in Hematology 20, 201–207 (2013). 23. Van Wijk, R. & Van Solinge, W. W. The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis. Blood 106, 4034–4042 (2005). 24. Tiffert, T., Bookchin, R. M. & Lew, V. L. Calcium Homeostasis in Normal and Abnormal Human Red Cells. in Red Cell Membrane Transport in Health and Disease (eds. Bernhardt, I. & Ellory, J. C.) 373–405 (Springer Berlin Heidelberg, Berlin, Heidelberg, 2003). doi:10.1007/978-3-662-05181-8_15. 25. Melo, D., Rocha, S., Coimbra, S. & Santos Silva, A. Interplay between Erythrocyte Peroxidases and Membrane. in Erythrocyte (ed. Tombak, A.) (IntechOpen, 2019). doi:10.5772/intechopen.83590. 26. Bosch, F. H. et al. Determinants of red blood cell deformability in relation to cell age. European Journal of Haematology 52, 35–41 (2009). 27. Kor, D. J., Van Buskirk, C. M. & Gajic, O. Red Blood Cell Storage Lesion. Bosn J of Basic Med Sci 9, S21–S27 (2009). 28. Neri, S., Swinkels, D. W., Matlung, H. L. & Van Bruggen, R. Novel concepts in red blood cell clearance. Current Opinion in Hematology 28, 438–444 (2021). 91 29. Kaestner, L. & Bogdanova, A. Regulation of red cell life-span, erythropoiesis, senescence, and clearance. Front. Physiol. 5, (2014). 30. Lutz, H. U. & Bogdanova, A. Mechanisms tagging senescent red blood cells for clearance in healthy humans. Front. Physiol. 4, (2013). 31. De Back, D. Z., Kostova, E. B., Van Kraaij, M., Van Den Berg, T. K. & Van Bruggen, R. Of macrophages and red blood cells; a complex love story. Front. Physiol. 5, (2014). 32. Ning, S., Heddle, N. M. & Acker, J. P. Exploring donor and product factors and their impact on red cell post-transfusion outcomes. Transfusion Medicine Reviews 32, 28–35 (2018). 33. Napolitano, L. M. et al. Clinical practice guideline: Red blood cell transfusion in adult trauma and critical care*: Critical Care Medicine 37, 3124–3157 (2009). 34. Chassé, M. et al. Association of Blood Donor Age and Sex With Recipient Survival After Red Blood Cell Transfusion. JAMA Intern Med 176, 1307 (2016). 35. Shi, P. A. Transfusion Management in Patients with Hemoglobinopathies. in Transfusion Medicine and Hemostasis 327–336 (Elsevier, 2013). doi:10.1016/B978-0-12-397164-7.00050-1. 36. Platzbecker, U., Hofbauer, L. C., Ehninger, G. & Hölig, K. The clinical, quality of life, and economic consequences of chronic anemia and transfusion support in patients with myelodysplastic syndromes. Leukemia Research 36, 525–536 (2012). 37. Liu, C. & Grossman, B. J. Red blood cell transfusion for hematologic disorders. Hematology 2015, 454–461 (2015). 38. Compernolle, V. et al. Red blood cell specifications for patients with hemoglobinopathies: a systematic review and guideline. Transfusion 58, 1555–1566 (2018). 92 39. Lagerquist, O. et al. The cost of transfusing a unit of red blood cells: a costing model for Canadian hospital use. VOXS 12, 375–380 (2017). 40. Klein, H. G., Spahn, D. R. & Carson, J. L. Red blood cell transfusion in clinical practice. The Lancet 370, 415–426 (2007). 41. Drews Steven, Khandelwal Aditi, Goldman Mindy, & Devine Dana. Donor selection, donor testing and pathogen reduction. CBS Professional Education https://professionaleducation.blood.ca/en/transfusion/clinical-guide/donor-selection-donor-testing-and-pathogen-reduction (2021). 42. Jordan, A. et al. Assessing the influence of component processing and donor characteristics on quality of red cell concentrates using quality control data. Vox Sanguinis 111, 8–15 (2016). 43. Kopolovic, I. et al. A systematic review of transfusion-associated graft-versus-host disease. Blood 126, 406–414 (2015). 44. Shaz, B. H. & Hillyer, C. D. Irradiation of Blood Products. in Transfusion Medicine and Hemostasis 209–214 (Elsevier, 2009). doi:10.1016/B978-0-12-374432-6.00036-1. 45. Zantek, N. D. et al. Recommendations on Selection and Processing of RBC Components for Pediatric Patients From the Pediatric Critical Care Transfusion and Anemia Expertise Initiative. Pediatric Critical Care Medicine 19, S163–S169 (2018). 46. Foukaneli, T. et al. Guidelines on the use of irradiated blood components. Br. J. Haematol. 191, 704–724 (2020). 47. Oksana, P.-G. & Solh, Z. Irradiated, washed and CMV seronegative blood components. Professional Education https://professionaleducation.blood.ca/en/transfusion/clinical-guide/irradiated-washed-and-cmv-seronegative-blood-components (2021). 93 48. Ohto, H., Yasuda, H., Noguchi, M. & Abe, R. Risk of transfusion‐associated graft‐versus‐host disease as a result of directed donations from relatives. Transfusion 32, 691–693 (1992). 49. Hess, J. R. Measures of stored red blood cell quality. Vox Sang 107, 1–9 (2014). 50. Almizraq, R., Tchir, J. D. R., Holovati, J. L. & Acker, J. P. Storage of red blood cells affects membrane composition, microvesiculation, and in vitro quality: RMPs and RBC quality measures. Transfusion n/a-n/a (2013) doi:10.1111/trf.12080. 51. Chen, D., Serrano, K. & Devine, D. V. Introducing the red cell storage lesion. VOXS 11, 26–33 (2016). 52. Zimring, J. C. Established and theoretical factors to consider in assessing the red cell storage lesion. Blood 125, 2185–2190 (2015). 53. Kanias, T. et al. Ethnicity, sex, and age are determinants of red blood cell storage and stress hemolysis: results of the REDS-III RBC-Omics study. Blood Advances 1, 1132–1141 (2017). 54. Roubinian, N. H. et al. Association of donor age, body mass index, hemoglobin, and smoking status with in‐hospital mortality and length of stay among red blood cell–transfused recipients. Transfusion 59, 3362–3370 (2019). 55. Zeller, M. P. et al. Sex‐mismatched red blood cell transfusions and mortality: A systematic review and meta‐analysis. Vox Sanguinis 114, 505–516 (2019). 56. Tzounakas, V. L. et al. Glucose 6-phosphate dehydrogenase deficient subjects may be better “storers” than donors of red blood cells. Free Radical Biology and Medicine 96, 152–165 (2016). 57. Vayá, A. et al. Rheological red blood cell behaviour in minor α-thalassaemia carriers. Clinical Hemorheology and Microcirculation 48, 241–246 (2011). 94 58. Vayá, A. et al. Rheological behaviour of red blood cells in beta and deltabeta thalassemia trait. Clin Hemorheol Microcirc 28, 71–78 (2003). 59. DeVenuto, F. & Wilson, S. Distribution of progesterone and its effect on human blood during storage. Transfusion 16, 107–112 (1976). 60. Sparrow, R. L., Payne, K. A. & Adams, G. G. Higher donor body mass index is associated with increased hemolysis of red blood cells at 42‐days of storage: A retrospective analysis of routine quality control data. Transfusion 61, 449–463 (2021). 61. Brinkmann, C. & Brixius, K. Peroxiredoxins and sports: new insights on the antioxidative defense. J Physiol Sci 63, 1–5 (2013). 62. de Groot, R. et al. Lifestyle behaviours are not associated with haemolysis: results from Donor InSight. Blood Transfusion 86–95 (2019) doi:10.2450/2019.0151-19. 63. Patel, R. M. & Josephson, C. D. Storage Age of Red Blood Cells for Transfusion of Premature Infants. JAMA 309, 544 (2013). 64. Fergusson, D. A. et al. Effect of Fresh Red Blood Cell Transfusions on Clinical Outcomes in Premature, Very Low-Birth-Weight Infants: The ARIPI Randomized Trial. JAMA 308, 1443 (2012). 65. Lacroix, J. et al. Age of Transfused Blood in Critically Ill Adults. N Engl J Med 372, 1410–1418 (2015). 66. Steiner, M. E. et al. Effects of Red-Cell Storage Duration on Patients Undergoing Cardiac Surgery. N Engl J Med 372, 1419–1429 (2015). 67. Lin, Y., Saw, C.-L., Hannach, B. & Goldman, M. Transfusion-related acute lung injury prevention measures and their impact at Canadian Blood Services: TRALI AT CANADIAN BLOOD SERVICES. Transfusion 52, 567–574 (2012). 95 68. Matthews, K., Lamoureux, E. S., Myrand-Lapierre, M.-E., Duffy, S. P. & Ma, H. Technologies for measuring red blood cell deformability. Lab Chip 22, 1254–1274 (2022). 69. Kuypers, F. A., Scott, M. D., Schott, M. A., Lubin, B. & Chiu, D. T. Use of ektacytometry to determine red cell susceptibility to oxidative stress. J Lab Clin Med 116, 535–545 (1990). 70. Wen, Z. et al. Biophysical meanings of orientation and deformation of RBCs in shear flow field of low viscosity with new Ektacytometry. Sci. China Ser. C.-Life Sci. 41, 195–202 (1998). 71. Baskurt, O. K. et al. Comparison of three commercially available ektacytometers with different shearing geometries. Biorheology 46, 251–264 (2009). 72. Dondorp, A. M. et al. The role of reduced red cell deformability in the pathogenesis of severe falciparum malaria and its restoration by blood transfusion. Transactions of the Royal Society of Tropical Medicine and Hygiene 96, 282–286 (2002). 73. Stuart, J. Erythrocyte rheology. Journal of Clinical Pathology 38, 965–977 (1985). 74. Reid, H. L., Barnes, A. J., Lock, P. J., Dormandy, J. A. & Dormandy, T. L. A simple method for measuring erythrocyte deformability. Journal of Clinical Pathology 29, 855–858 (1976). 75. Berezina, T. L. et al. Influence of Storage on Red Blood Cell Rheological Properties. Journal of Surgical Research 102, 6–12 (2002). 76. Sosa, J. M., Nielsen, N. D., Vignes, S. M., Chen, T. G. & Shevkoplyas, S. S. The relationship between red blood cell deformability metrics and perfusion of an artificial microvascular network. Clin Hemorheol Microcirc 57, 275–289 (2014). 77. Shevkoplyas, S. S., Yoshida, T., Gifford, S. C. & Bitensky, M. W. Direct measurement of the impact of impaired erythrocyte deformability on microvascular network perfusion in a microfluidic device. Lab Chip 6, 914 (2006). 96 78. Nash, G. B., O’Brien, E., Gordon-Smith, E. C. & Dormandy, J. A. Abnormalities in the mechanical properties of red blood cells caused by Plasmodium falciparum. Blood 74, 855–861 (1989). 79. Hebbel, R., Leung, A. & Mohandas, N. Oxidation-induced changes in microrheologic properties of the red blood cell membrane. Blood 76, 1015–1020 (1990). 80. Evans, E., Mohandas, N. & Leung, A. Static and dynamic rigidities of normal and sickle erythrocytes. Major influence of cell hemoglobin concentration. J. Clin. Invest. 73, 477–488 (1984). 81. Mills, J. P., Qie, L., Dao, M., Lim, C. T. & Suresh, S. Nonlinear elastic and viscoelastic deformation of the human red blood cell with optical tweezers. Mech Chem Biosyst 1, 169–180 (2004). 82. Dao, M., Lim, C. T. & Suresh, S. Mechanics of the human red blood cell deformed by optical tweezers. Journal of the Mechanics and Physics of Solids 51, 2259–2280 (2003). 83. Blood Cell - An Overview of Studies in Hematology. (InTech, 2012). doi:10.5772/2979. 84. Dulińska, I. et al. Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy. Journal of Biochemical and Biophysical Methods 66, 1–11 (2006). 85. Lekka, M. et al. Erythrocyte stiffness probed using atomic force microscope. Biorheology 42, 307–317 (2005). 86. Buys, A. V. et al. Changes in red blood cell membrane structure in type 2 diabetes: a scanning electron and atomic force microscopy study. Cardiovasc Diabetol 12, 25 (2013). 87. Abay, A. et al. Glutaraldehyde – A Subtle Tool in the Investigation of Healthy and Pathologic Red Blood Cells. Front. Physiol. 10, 514 (2019). 97 88. Herricks, T., Antia, M. & Rathod, P. K. Deformability limits of Plasmodium falciparum-infected red blood cells. Cell Microbiol 11, 1340–1353 (2009). 89. Gifford, S. C. et al. Parallel Microchannel-Based Measurements of Individual Erythrocyte Areas and Volumes. Biophysical Journal 84, 623–633 (2003). 90. Faustino, V. et al. A Microfluidic Deformability Assessment of Pathological Red Blood Cells Flowing in a Hyperbolic Converging Microchannel. Micromachines (Basel) 10, 645 (2019). 91. Forsyth, A. M., Wan, J., Ristenpart, W. D. & Stone, H. A. The dynamic behavior of chemically “stiffened” red blood cells in microchannel flows. Microvascular Research 80, 37–43 (2010). 92. Zheng, Y., Nguyen, J., Wang, C. & Sun, Y. Electrical measurement of red blood cell deformability on a microfluidic device. Lab Chip 13, 3275 (2013). 93. Abkarian, M., Faivre, M. & Stone, H. A. High-speed microfluidic differential manometer for cellular-scale hydrodynamics. Proc. Natl. Acad. Sci. U.S.A. 103, 538–542 (2006). 94. Guo, Q., Reiling, S. J., Rohrbach, P. & Ma, H. Microfluidic biomechanical assay for red blood cells parasitized by Plasmodium falciparum. Lab Chip 12, 1143 (2012). 95. Guo, Q., Park, S. & Ma, H. Microfluidic micropipette aspiration for measuring the deformability of single cells. Lab Chip 12, 2687 (2012). 96. Kwan, J. M., Guo, Q., Kyluik‐Price, D. L., Ma, H. & Scott, M. D. Microfluidic analysis of cellular deformability of normal and oxidatively damaged red blood cells. American J Hematol 88, 682–689 (2013). 97. Guo, Q., McFaul, S. M. & Ma, H. Deterministic microfluidic ratchet based on the deformation of individual cells. Physical Review E 83, 051910 (2011). 98 98. McFaul, S. M., Lin, B. K. & Ma, H. Cell separation based on size and deformability using microfluidic funnel ratchets. Lab Chip 12, 2369–2376 (2012). 99. Park, E. S. et al. Continuous Flow Deformability-Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets. Small 12, 1909–1919 (2016). 100. Guo, Q. et al. Deformability based sorting of red blood cells improves diagnostic sensitivity for malaria caused by Plasmodium falciparum. Lab Chip 16, 645–654 (2016). 101. Islamzada, E. et al. Deformability based sorting of stored red blood cells reveals donor-dependent aging curves. Lab Chip 20, 226–235 (2020). 102. Islamzada, E. et al. Degradation of red blood cell deformability during cold storage in blood bags. eJHaem 3, 63–71 (2022). 103. Islamzada, E. et al. Blood unit segments accurately represent the biophysical properties of red blood cells in blood bags but not hemolysis. Transfusion 62, 448–456 (2022). 104. Guo, Q., McFaul, S. M. & Ma, H. Deterministic microfluidic ratchet based on the deformation of individual cells. Phys. Rev. E 83, 051910 (2011). 105. McFaul, S. M., Lin, B. K. & Ma, H. Cell separation based on size and deformability using microfluidic funnel ratchets. Lab Chip 12, 2369 (2012). 106. Guo, Q., Duffy, S. P., Matthews, K., Islamzada, E. & Ma, H. Deformability based Cell Sorting using Microfluidic Ratchets Enabling Phenotypic Separation of Leukocytes Directly from Whole Blood. Sci Rep 7, 6627 (2017). 107. Desai, S. P., Freeman, D. M. & Voldman, J. Plastic masters—rigid templates for soft lithography. Lab Chip 9, 1631 (2009). 108. Han, V., Serrano, K. & Devine, D. V. A comparative study of common techniques used to measure haemolysis in stored red cell concentrates. Vox Sanguinis 98, 116–123 (2010). 99 109. Takei, T., Amin, N. A., Schmid, G., Dhingra-Kumar, N. & Rugg, D. Progress in global blood safety for HIV. J. Acquir. Immune Defic. Syndr. 52 Suppl 2, S127-131 (2009). 110. Hess, J. R. Conventional blood banking and blood component storage regulation: opportunities for improvement. Blood Transfus 8 Suppl 3, s9-15 (2010). 111. Garraud, O. & Tissot, J.-D. Blood and Blood Components: From Similarities to Differences. Front Med (Lausanne) 5, 84 (2018). 112. Franco, R. S. Measurement of red cell lifespan and aging. Transfus Med Hemother 39, 302–307 (2012). 113. Cohen, R. M. et al. Red cell life span heterogeneity in hematologically normal people is sufficient to alter HbA1c. Blood 112, 4284–4291 (2008). 114. Carson, J. L. Red Blood Cell Transfusion: A Clinical Practice Guideline From the AABB*. Annals of Internal Medicine 157, 49 (2012). 115. Tzounakas, V. L., Kriebardis, A. G., Papassideri, I. S. & Antonelou, M. H. Donor-variation effect on red blood cell storage lesion: A close relationship emerges. PROTEOMICS - Clinical Applications 10, 791–804 (2016). 116. Tzounakas, V. L. et al. Donor variation effect on red blood cell storage lesion: a multivariable, yet consistent, story. Transfusion 56, 1274–1286 (2016). 117. Wagner, S. J. Transfusion-transmitted bacterial infection: risks, sources and interventions. Vox Sang. 86, 157–163 (2004). 118. Hendrickson, J. E. & Hillyer, C. D. Noninfectious serious hazards of transfusion. Anesth. Analg. 108, 759–769 (2009). 119. Bux, J. Transfusion-related acute lung injury (TRALI): a serious adverse event of blood transfusion. Vox Sanguinis 89, 1–10 (2005). 100 120. Ng, M. S. Y., Ng, A. S. Y., Chan, J., Tung, J.-P. & Fraser, J. F. Effects of packed red blood cell storage duration on post-transfusion clinical outcomes: a meta-analysis and systematic review. Intensive Care Med 41, 2087–2097 (2015). 121. D’Alessandro, A. et al. An update on red blood cell storage lesions, as gleaned through biochemistry and omics technologies. Transfusion 55, 205–219 (2015). 122. Tzounakas, V. L. et al. Glucose 6-phosphate dehydrogenase deficient subjects may be better ‘storers’ than donors of red blood cells. Free Radic. Biol. Med. 96, 152–165 (2016). 123. Mebius, R. E. & Kraal, G. Structure and function of the spleen. Nat. Rev. Immunol. 5, 606–616 (2005). 124. Burger, P., Hilarius-Stokman, P., De Korte, D., Van Den Berg, T. & Van Bruggen, R. CD47 functions as a molecular switch for erythrocyte phagocytosis. Blood 119, 5512–5521 (2012). 125. Matthews, K. et al. Microfluidic deformability analysis of the red cell storage lesion. Journal of Biomechanics 48, 4065–4072 (2015). 126. Tinmouth, A., Fergusson, D., Yee, I. C., Hébert, P. C., & ABLE Investigators and the Canadian Critical Care Trials Group. Clinical consequences of red cell storage in the critically ill. Transfusion 46, 2014–2027 (2006). 127. Pavenski, K., Saidenberg, E., Lavoie, M., Tokessy, M. & Branch, D. Red Blood Cell Storage Lesions and Related Transfusion Issues: A Canadian Blood Services Research and Development Symposium. Transfusion Medicine Reviews 26, 68–84 (2011). 128. Zallen, G. et al. Age of transfused blood is an independent risk factor for postinjury multiple organ failure. Am J Surg 178, 570–572 (1999). 101 129. Bennett-Guerrero, E. et al. Evolution of adverse changes in stored RBCs. Proc. Natl. Acad. Sci. U.S.A. 104, 17063–17068 (2007). 130. Li, Y., Xiong, Y., Wang, R., Tang, F. & Wang, X. Blood banking-induced alteration of red blood cell oxygen release ability. Blood Transfus 14, 238–244 (2016). 131. Rael, L. T. et al. The effect of storage on the accumulation of oxidative biomarkers in donated packed red blood cells. J Trauma 66, 76–81 (2009). 132. Racek, J., Herynková, R., Holecek, V., Jerábek, Z. & Sláma, V. Influence of antioxidants on the quality of stored blood. Vox Sang. 72, 16–19 (1997). 133. Wither, M. et al. Hemoglobin oxidation at functional amino acid residues during routine storage of red blood cells. Transfusion 56, 421–426 (2016). 134. Bosman, G. J. C. G. M. et al. The proteome of red cell membranes and vesicles during storage in blood bank conditions. Transfusion 48, 827–835 (2008). 135. Jy, W. et al. Microparticles in Stored RBC as Potential Mediators of Transfusion Complications. Transfusion 51, 886–893 (2011). 136. Park, E. S. et al. Continuous Flow Deformability‐Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets. Small 12, 1909–1919 (2016). 137. Cluitmans, J., Hardeman, M., Dinkla, S., Brock, R. & Bosman, G. Red blood cell deformability during storage: towards functional proteomics and metabolomics in the Blood Bank. Blood Transfus 10 Suppl 2, s12-8 (2012). 138. Safeukui, I. et al. Sensing of red blood cells with decreased membrane deformability by the human spleen. Blood Adv 2, 2581–2587 (2018). 139. Koch, C. G. et al. Real Age: Red Blood Cell Aging During Storage. Ann. Thorac. Surg. 107, 973–980 (2019). 102 140. Bosch, F. H. et al. Determinants of Red-Blood-Cell Deformability in Relation to Cell Age. Eur J Haematol 52, 35–41 (1994). 141. Bessis, M., Mohandas, N. & Feo, C. Automated ektacytometry: a new method of measuring red cell deformability and red cell indices. Blood cells 6, 315–27 (1980). 142. Clark, M. R., Mohandas, N. & Shohet, S. B. Osmotic gradient ektacytometry: comprehensive characterization of red cell volume and surface maintenance. Blood 61, 899–910 (1983). 143. Baskurt, O. K. et al. Comparison of three commercially available ektacytometers with different shearing geometries. Biorheology 46, 251–64 (2009). 144. Streekstra, G. J., Dobbe, J. G. G. & Hoekstra, a G. Quantification of the fraction poorly deformable red blood cells using ektacytometry. Optics express 18, 14173–82 (2010). 145. Jani, V. P. et al. Blood Quality Diagnostic Device Detects Storage Differences Between Donors. IEEE Trans Biomed Circuits Syst 11, 1400–1405 (2017). 146. Tarasev, M. et al. Similar donors-similar blood? Transfusion 54, 933–941 (2014). 147. Sosa, J. M., Nielsen, N. D., Vignes, S. M., Chen, T. G. & Shevkoplyas, S. S. The relationship between red blood cell deformability metrics and perfusion of an artificial microvascular network. Clin. Hemorheol. Microcirc. (2013) doi:10.3233/CH-131719. 148. Hou, H. W. et al. Deformability based cell margination--a simple microfluidic design for malaria-infected erythrocyte separation. Lab Chip 10, 2605–2613 (2010). 149. Henry, E. et al. Sorting cells by their dynamical properties. Sci Rep 6, 34375 (2016). 150. Jani, V. P. et al. Blood Quality Diagnostic Device Detects Storage Differences Between Donors. IEEE Trans. Biomed. Circuits Syst. 11, 1400–1405 (2017). 151. Tarasev, M. et al. Similar donors—similar blood? Transfusion 54, 933–941 (2014). 103 152. Dern, R. J., Gwinn, R. P. & Wiorkowski, J. J. Studies on the preservation of human blood. I. Variability in erythrocyte storage characteristics among healthy donors. J Lab Clin Med 67, 955–965 (1966). 153. Dumont, L. J., AuBuchon, J. P., & Biomedical Excellence for Safer Transfusion (BEST) Collaborative. Evaluation of proposed FDA criteria for the evaluation of radiolabeled red cell recovery trials. Transfusion 48, 1053–1060 (2008). 154. McAteer, M. J. et al. Multi-institutional randomized control study of haemolysis in stored red cell units prepared manually or by an automated system: Potential association between whole blood donor and haemolysis. Vox Sanguinis 99, 34–43 (2010). 155. Dumont, L. J. & AuBuchon, J. P. Evaluation of proposed FDA criteria for the evaluation of radiolabeled red cell recovery trials. Transfusion 48, 1053–1060 (2008). 156. Bratosin, D., Estaquier, J., Ameisen, J. c. & Montreuil, J. Molecular and Cellular Mechanisms of Erythrocyte Programmed Cell Death: Impact on blood Transfusion. Vox Sanguinis 83, 307 (2002). 157. Bennett-Guerrero, E. et al. Evolution of adverse changes in stored RBCs. PNAS 104, 17063–17068 (2007). 158. D’Alessandro, A., Liumbruno, G., Grazzini, G. & Zolla, L. Red blood cell storage: the story so far. Blood Transfus 8, 82–88 (2010). 159. Tinmouth, A., Fergusson, D., Yee, I. C. & Hébert, P. C. Clinical consequences of red cell storage in the critically ill. Transfusion 46, 2014–2027 (2006). 160. Yoshida, T., Prudent, M. & D’Alessandro, A. Red blood cell storage lesion: causes and potential clinical consequences. Blood Transfus 17, 27–52 (2019). 104 161. Högman, C. F. & Meryman, H. T. Red blood cells intended for transfusion: quality criteria revisited. Transfusion 46, 137–142 (2006). 162. Pavenski, K., Saidenberg, E., Lavoie, M., Tokessy, M. & Branch, D. R. Red Blood Cell Storage Lesions and Related Transfusion Issues: A Canadian Blood Services Research and Development Symposium. Transfusion Medicine Reviews 26, 68–84 (2012). 163. Remy, K. E. & Spinella, P. C. Red blood cell storage age - what we know from clinical trials. Expert Rev Hematol 9, 1011–1013 (2016). 164. Berezina, T. L. et al. Influence of Storage on Red Blood Cell Rheological Properties. Journal of Surgical Research 102, 6–12 (2002). 165. Huruta, R. R. et al. Mechanical Properties of Stored Red Blood Cells Using Optical Tweezers. Blood 92, 2975–2977 (1998). 166. Nagaprasad, V. & Singh, M. Sequential analysis of the influence of blood storage on aggregation, deformability and shape parameters of erythrocytes. Clinical Hemorheology and Microcirculation 18, 273–284 (1998). 167. Ng, M. S. Y. et al. Transfusion of packed red blood cells at the end of shelf life is associated with increased risk of mortality - a pooled patient data analysis of 16 observational trials. Haematologica 103, 1542–1548 (2018). 168. Goel, R. et al. Red blood cells stored 35 days or more are associated with adverse outcomes in high-risk patients. Transfusion 56, 1690–1698 (2016). 169. Bratosin, D. et al. Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review. Biochimie 80, 173–195 (1998). 170. Chin-Yee I, Statchuk L, Milkovich S. Transfusion of red blood cells under shock conditions in the rat microvasculature. Blood 2713A (2004). 105 171. Kurach, J., Hansen, A., Turner, T., Jenkins, C. & Acker, J. Segments from red blood cell units should not be used for quality testing. Transfusion n/a-n/a (2013) doi:10.1111/trf.12303. 172. Islamzada, E. et al. Deformability based sorting of stored red blood cells reveals donor-dependent aging curves. Lab Chip 20, 226–235 (2020). 173. Gifford, S. C., Derganc, J., Shevkoplyas, S. S., Yoshida, T. & Bitensky, M. W. A detailed study of time‐dependent changes in human red blood cells: from reticulocyte maturation to erythrocyte senescence. Br J Haematol 135, 395–404 (2006). 174. Henkelman, S. et al. Is red blood cell rheology preserved during routine blood bank storage? Transfusion 50, 941–948 (2010). 175. List of normal lab values | Medical Council of Canada. https://mcc.ca/examinations-assessments/resources-to-help-with-exam-prep/normal-lab-values/ (2023). 176. Roubinian, N. H. et al. Effect of donor, component, and recipient characteristics on hemoglobin increments following red blood cell transfusion. Blood 134, 1003–1013 (2019). 177. Dhabangi, A. et al. Effect of Transfusion of Red Blood Cells With Longer vs Shorter Storage Duration on Elevated Blood Lactate Levels in Children With Severe Anemia: The TOTAL Randomized Clinical Trial. JAMA 314, 2514 (2015). 178. Heddle, N. M. et al. Effect of Short-Term vs. Long-Term Blood Storage on Mortality after Transfusion. N Engl J Med 375, 1937–1945 (2016). 179. Matthews, K. et al. Microfluidic deformability analysis of the red cell storage lesion. Journal of Biomechanics 48, 4065–4072 (2015). 180. Huruta, R. R. et al. Mechanical properties of stored red blood cells using optical tweezers. Blood 92, 2975–2977 (1998). 106 181. Barshtein, G. et al. Deformability of transfused red blood cells is a potent determinant of transfusion‐induced change in recipient’s blood flow. Microcirculation 23, 479–486 (2016). 182. Kurach, J. D. R., Hansen, A. L., Turner, T. R., Jenkins, C. & Acker, J. P. Segments from red blood cell units should not be used for quality testing. Transfusion 54, 451–455 (2014). 183. Kim‐Shapiro, D. B., Lee, J. & Gladwin, M. T. Storage lesion: role of red blood cell breakdown. Transfusion 51, 844–851 (2011). 184. Hébert, P. C. et al. Clinical Outcomes Following Institution of the Canadian Universal Leukoreduction Program for Red Blood Cell Transfusions. JAMA 289, 1941 (2003). 185. Serrano, K. et al. The effect of timing of gamma-irradiation on hemolysis and potassium release in leukoreduced red cell concentrates stored in SAGM. Vox Sang 106, 379–381 (2014). 186. Cicha, I. et al. Gamma-Ray-Irradiated Red Blood Cells Stored in Mannitol-Adenine-Phosphate Medium: Rheological Evaluation and Susceptibility to Oxidative Stress. Vox Sang 79, 75–82 (2000). 187. Rodriguez, J. V. & Tormey, C. A. Can transfusion-associated graft-versus-host disease (TA-GvHD) be prevented with leukoreduction alone? Transfusion and Apheresis Science 61, 103402 (2022). 188. Gifford, S. C., Derganc, J., Shevkoplyas, S. S., Yoshida, T. & Bitensky, M. W. A detailed study of time-dependent changes in human red blood cells: from reticulocyte maturation to erythrocyte senescence. Brit J Haematol 135, 395–404 (2006). 189. Bennett-Guerrero, E. et al. Evolution of adverse changes in stored RBCs. Proc. Natl. Acad. Sci. U.S.A. 104, 17063–17068 (2007). 107 190. D’Alessandro, A., Liumbruno, G., Grazzini, G. & Zolla, L. Red blood cell storage: the story so far. Blood Transfus 8, 82–88 (2010). 191. Barshtein, G., Pajic-Lijakovic, I. & Gural, A. Deformability of Stored Red Blood Cells. Front. Physiol. 12, 722896 (2021). 192. Weed, R. I. Importance of Erythrocyte Deformability. Am J Med 49, 147- (1970). 193. Card, R. T., Mohandas, N. & Mollison, P. L. Relationship of post-transfusion viability to deformability of stored red cells. British Journal of Haematology 53, 237–240 (1983). 194. Zbikowska, H. M. & Antosik, A. Irradiation dose-dependent oxidative changes in red blood cells for transfusion. Int J Radiat Biol 88, 654–660 (2012). 195. Katharia, R., Chaudhary, R. & Agarwal, P. Prestorage gamma irradiation induces oxidative injury to red cells. Transfus Apher Sci 48, 39–43 (2013). 196. Anand, A., Dzik, W., Imam, A. & Sadrzadeh, S. Radiation-induced red cell damage: role of reactive oxygen species. Transfusion 37, 160–165 (1997). 197. Singh, A. & Singh, H. Time-scale and nature of radiation-biological damage: approaches to radiation protection and post-irradiation therapy. Prog Biophys Mol Biol 39, 69–107 (1982). 198. Harm, S. K., Raval, J. S., Cramer, J., Waters, J. H. & Yazer, M. H. Haemolysis and sublethal injury of RBCs after routine blood bank manipulations. Transfus Med 22, 181–185 (2012). 199. Zimmermann, R. et al. Influence of late irradiation on the in vitro RBC storage variables of leucoreduced RBCs in SAGM additive solution. Vox Sang 100, 279–284 (2011). 108 200. Serrano, K. et al. The effect of timing of gamma-irradiation on hemolysis and potassium release in leukoreduced red cell concentrates stored in SAGM. Vox Sang 106, 379–381 (2014). 201. El Kenz, H., Corazza, F., Van Der Linden, P., Chabab, S. & Vandenvelde, C. Potassium content of irradiated packed red blood cells in different storage media: is there a need for additive solution-dependent recommendations for infant transfusion? Transfus Apher Sci 49, 249–253 (2013). 202. Puchala, M., Szweda-Lewandowska, Z. & Kiefer, J. The Influence of Radiation Quality on Radiation-induced Hemolysis and Hemoglobin Oxidation of Human Erythrocytes. JRR 45, 275–279 (2004). 203. Moreira, O. C. et al. Effects of γ-irradiation on the membrane ATPases of human erythrocytes from transfusional blood concentrates. Ann Hematol 87, 113–119 (2008). 204. Moroff, G. & Luban, N. L. C. The influence of gamma irradiation on red cell and platelet properties. Transfusion Science 15, 141–148 (1994). 205. Islamzada, E. et al. Degradation of red blood cell deformability during cold storage in blood bags. eJHaem 3, 63–71 (2022). 206. Zbikowska, H. M. & Antosik, A. Irradiation dose-dependent oxidative changes in red blood cells for transfusion. International Journal of Radiation Biology 88, 654–660 (2012). 207. Moroff, G. et al. Viability and in vitro properties of AS-1 red cells after gamma irradiation. Transfusion 39, 128–134 (1999). 208. Carson, J. L. Red Blood Cell Transfusion: A Clinical Practice Guideline From the AABB*. Ann Intern Med 157, 49 (2012). 109 209. Wither, M. et al. Hemoglobin oxidation at functional amino acid residues during routine storage of red blood cells. Transfusion 56, 421–426 (2016). 210. Racek, J., Herynková, R., Holeček, V., Jeřábek, Z. & Sláma, V. Influence of Antioxidants on the Quality of Stored Blood. Vox Sanguinis 72, 16–19 (1997). 211. Rael, L. T. et al. The Effect of Storage on the Accumulation of Oxidative Biomarkers in Donated Packed Red Blood Cells. Journal of Trauma: Injury, Infection & Critical Care 66, 76–81 (2009). 212. Huisjes, R. et al. Squeezing for Life – Properties of Red Blood Cell Deformability. Front. Physiol. 9, 656 (2018). 213. Högman, C. F., Verdier, C.-H., Ericson, å., Hedlund, K. & Sandhagen, B. Studies on the Mechanism of Human Red Cell Loss of Viability during Storage at +4°C in vitro. Vox Sanguinis 48, 257–268 (1985). 214. Barshtein, G. et al. Deformability of transfused red blood cells is a potent effector of transfusion-induced hemoglobin increment: A study with β-thalassemia major patients: BARSHTEIN et al. Am J Hematol 92, E559–E560 (2017). 215. Nagaprasad, V. & Singh, M. Sequential analysis of the influence of blood storage on aggregation, deformability and shape parameters of erythrocytes. Clin Hemorheol Microcirc 18, 273–284 (1998). 216. Arese, P., Turrini, F. & Schwarzer, E. Band 3/complement-mediated recognition and removal of normally senescent and pathological human erythrocytes. Cell Physiol Biochem 16, 133–146 (2005). 217. Franco, R. S. et al. Changes in the properties of normal human red blood cells during in vivo aging. Am J Hematol 88, 44–51 (2013). 110 218. Antonelou, M. H., Kriebardis, A. G. & Papassideri, I. S. Aging and death signalling in mature red cells: from basic science to transfusion practice. Blood Transfus 8 Suppl 3, s39-47 (2010). 219. Darr, D. & Fridovich, I. Irreversible inactivation of catalase by 3-amino-1,2,4-triazole. Biochemical Pharmacology 35, 3642 (1986). 220. Deng, X. et al. Aminotriazole attenuated carbon tetrachloride-induced oxidative liver injury in mice. Food and Chemical Toxicology 50, 3073–3078 (2012). 221. Scott, M. D., Wagner, T. C. & Chiu, D. T.-Y. Decreased catalase activity is the underlying mechanism of oxidant susceptibility in glucose-6-phosphate dehydrogenase-deficient erythrocytes. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1181, 163–168 (1993). 222. Kwan, J., Guo, Q., Kyluik-Price, D., Ma, H. & Scott, M. Microfluidic analysis of cellular deformability of normal and oxidatively damaged red blood cells. Am. J. Hematol. 88, 682–689 (2013). 223. Tjälve, H. Accumulation of Labelled Aminotriazole in Some Transplanted Tumours in Mice. Br J Cancer 30, 136–141 (1974). 224. Wood, R. L. & Legg, P. G. PEROXIDASE ACTIVITY IN RAT LIVER MICROBODIES AFTER AMINO-TRIAZOLE INHIBITION. Journal of Cell Biology 45, 576–585 (1970). 225. Khayat, A. & Dencker, L. Whole body and liver distribution of inhaled mercury vapor in the mouse: Influence of ethanol and aminotriazole pretreatment. J. Appl. Toxicol. 3, 66–74 (1983). 111 226. Gilson, C. R. et al. A novel mouse model of red blood cell storage and posttransfusion in vivo survival. Transfusion 49, 1546–1553 (2009). 227. Moller, G. SURVIVAL OF H-2 INCOMPATIBLE MOUSE ERYTHROCYTES IN UNTREATED AND ISOIMMUNE RECIPIENTS. Immunology 8, 360–374 (1965). 228. Zimring, J. C. et al. Strain-specific red blood cell storage, metabolism, and eicosanoid generation in a mouse model: Metabolomics of RBC Donor Variability in Mice. Transfusion 54, 137–148 (2014). 229. Hod, E. A. et al. Use of mouse models to study the mechanisms and consequences of RBC clearance. Vox Sanguinis 99, 99–111 (2010). 230. Ryder, A. B., Zimring, J. C. & Hendrickson, J. E. Factors Influencing RBC Alloimmunization: Lessons Learned from Murine Models. Transfus Med Hemother 41, 406–419 (2014). 112 Appendix: Supplementary Data for Chapter 6 A 1. Transfused RBC clearance rate and rigidity scores through 5 weeks post-transfusion. (A): Percent of transfused RBCs remaining in recipient’s circulation throughout the 5-week post-transfusion sampling period. Red bars: % control RBCs remaining, green bars: % AMT-rigidified RBCs remaining. (B): The rigidity scores of transfused control, AMT-rigidified, and endogenous RBCs throughout the 5-week post-transfusion sampling period. 0 1 2 3 4 5050100Recipient 10 1 2 3 4 5050100Recipient 40 1 2 3 4 5012345Recipient 10 1 2 3 4 5012345Recipient 40 1 2 3 4 5050100Recipient 20 1 2 3 4 5050100Recipient 50 1 2 3 4 5012345Recipient 2*0 1 2 3 4 5012345Recipient 50 1 2 3 4 5050100Recipient 30 1 2 3 4 5050100Recipient 60 1 2 3 4 5012345Recipient 30 1 2 3 4 5012345Recipient 60 1 2 3 4 5050100Recipient 70 1 2 3 4 5012345Recipient 7Weeks post-transfusionWeeks post-transfusion% Remaining RBCsRigidity Score AB* * * ** * ** * * ** *Ctrl AMT Endogenous Data missing ⋇*113 A 2. Murine transfusion study data analysis. A sample cell counts for percent of transfused RBCs remaining in circulation. (A): The RBC sample from each microfluidic ratchet device outlet is transferred to a 96-well imaging plate. A set area is chosen for analysis. (B): for estimating the total number of RBCs, five sample points of 101 µm each are chosen at the edges and center of the analysis area, and the total number of RBCs is estimated from that. (C): The number of fluorescent red (control) and green (AMT-rigidified) RBCs is manually counted in the same area. From these counts, the percent remaining of control and AMT-treated RBCs is estimated at each timepoint and deformability fraction.
Thesis/Dissertation
eng
Pathology and Laboratory Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Ma, Hongshen
Scott, Mark Douglas
Deformability based cell sorting enabling quality control of donated red blood cells
Text