Dissertations / Theses on the topic 'Red blood cell (RBC)'

Red blood cells (RBC), the oxygen-carriers within blood, eject their nuclei and other organelles to optimise cellular mechanics for gas exchange in capillary networks. Lack of organelles, however, strictly limits circulatory longevity of these cells, due to the inability to repair damaged cellular components. Given the turnover of RBC, the cell population within blood is inherently heterogenous, comprising RBC across the whole spectrum of in vivo age. Moreover, surrender of translational capacity restricts cellular signalling within RBC to modifications of existing proteins and/or flux of ions through membrane-embedded channels, rather than alterations in protein expression. The traversal of the cardiovascular system for the purpose of gas exchange exposes RBC to varying mechanical forces. Exposure to mechanical force physically deforms the RBC membrane, which, upon cessation of force exposure, readopts its native bi-concave disc chape. Novel observations support that these mechanical forces also activate biochemical pathways that may acutely and transiently alter RBC mechanics. The molecular machinery facilitating these mechanotransduction processes in RBC, however, is largely undescribed. The aim of the present body of work was thus to elucidate i. mechanotransductive pathways in mature, enucleated RBC; ii. the contribution of mechanically-activated signalling to the regulation of RBC mechanics; and iii. the impact of sub-populations of RBC with abnormal mechanical properties on blood fluid behaviour. The salient findings of the present dissertation support the presence of a relevant post-translational signalling network in circulating, enucleated RBC, some of which is sensitive to activation by mechanical forces. The cation channel Piezo1 appears to be a central mechanism of ‘force sensing’ in these cells. That is, opening of Piezo1 in response to mechanical force facilitates influx of calciumions, which regulate RBC mechanics via diverse mechanisms, including acute shifts in cell volume, selective removal of susceptible cells within a given RBC population, and initiation of nitric oxide production. Collectively, the herein presented results enhance the current understanding of fundamental RBC physiology by elucidating hitherto unrecognised signalling pathways. Given the demonstrated relevance of these processes to the regulation of RBC mechanical properties, which determine blood fluid properties and effective gas exchange, components of mechanically-activated signalling in these cells may provide novel therapeutic targets. Moreover, adverse complications arising in scenarios where blood is exposed to mechanical forces far exceeding those investigated here, for example during transit of mechanical circulatory support devices or dialysis machines, may be linked to overactivation of mechanically-sensitive signalling.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sci & Soc Wrk
Griffith Health
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Motivation: Red blood cells (RBCs) are the most common cells in the blood due to their high concentration. The RBC has a deformable membrane enclosing a jelly-like fluid known as the cytosol. For many years, the dynamics of RBCs has attracted growing interest both numerically and experimentally in various fields of research on biological systems. Owing to their high deformability, RBCs exhibit complex dynamic behaviours in micro-vessels where Reynolds numbers (Re) are less than unity (Re < 1). First, a healthy RBC at a low shear rate or a high viscosity contrast (λ - defined as the ratio of viscosities between RBC cytosol and external fluid), may tumble, i.e., the whole RBC rotates continuously in the original shape like a rigid body. Second, at a high shear rate or a low viscosity contrast (λ), the RBC may exhibit a tank-treading motion, i.e., its membrane rotates around the cytosol which maintains a fixed angle with respect to the flow direction. Finally, a healthy RBC migrates in the lateral direction towards the micro-vessel axis while moving in the longitudinal direction (downstream) of a micro-vessel. Under physiological conditions, the RBC experiences a varying range of shear stresses (typically in the range of 1-10 Pa) in the circulatory system without exhibiting any physical signs of mechanical damage. Upon exposure to high shear stresses, such as those present within mechanical circulatory support, RBCs exhibit irreversible functional impairment called sub-haemolytic/sub-lethal damage. Sub-haemolytically damaged RBCs exhibit impaired mechanical properties that substantially alter bulk flow behaviour when compared with healthy RBCs. However, there has been little attention directed toward characterizing sub-haemolytic damage in literature. For better understanding, it is necessary to have a reliable model to predict the dynamics of sub-haemolytically damaged RBCs in micro-vessels in comparison with healthy RBCs. Methods: Highly-efficient numerical approaches have been developed to investigate blood flow, with particular emphasis on the motion and deformation of RBCs under shear flow. Among these methods, the integration of the lattice Boltzmann method (LBM) and immersed boundary method (IBM) has received considerable attention. In this dissertation, a 2D in-house generated algorithm based on the LBM-IBM was utilised for the numerical simulations. Moreover, a spring-based model was applied to simulate the elastic behaviour of the RBC membrane. Finally, a microfluidic experimental system including flow control, image capture, and data acquisition was established to validate the numerical results with the experimental results. Goal: The main focus of this dissertation was to establish a 2D LBM-IBM coupled with a spring-based model to simulate the trajectory of both healthy RBC and damaged RBC in Poiseuille flow in low Reynolds numbers (Re < 1), in which the numerical results are compared with the experimental ones to allow for model validation. The second aim of this study was to numerically simulate the tumbling and tank-treading-like motion of a single RBC (healthy and damaged) in a micro-channel. Finally, the third aim was to numerically simulate the effect of the viscosity contrast (λ) on the trajectory of an RBC in a micro-channel. λ is one of the important factors that can severely affect RBC dynamics and cell deformation in a shear flow. Because of computational complexity, little effort has been made to numerically model the effect of λ on RBC dynamics in flow in the literature, for this reason, most of the current simulation studies assume for simplicity the viscosity contrast of unity. Results: Overall, the numerical results indicated a reasonable agreement with the observed experimental results. However, the numerical simulation predicts a larger migration (1.81 μm for the healthy RBC and 0.96 μm for the damaged RBC) compared to the experimental tests (1.20 μm for the healthy RBC and 0.41 μm for the damaged RBC). Moreover, the experimental results showed that at a certain distance from the entrance of the micro-channel, the RBCs have a rolling motion like a wheel but without lateral migration. Due to the deformability of the RBCs, this motion is unstable so that later on, the RBCs migrate laterally toward the centreline of the micro-channel. The results also showed that the distance at which rolling motion happens is greater for the damaged RBCs (~ 150 μm) compared to the healthy RBCs (~ 25 μm) because the damaged cells deform less. The numerical results confirm this result. It can be seen from the numerical results that the healthy RBC experiences the tank-treading motion compared to the damaged RBC that exhibits the tumbling motion. Furthermore, the numerical results indicated a significant impact on the RBC trajectory when λ = 5 compared to λ = 1. The higher viscosity contrast of 5 has less lift (5.06 μm) in comparison with the lower viscosity contrast of 1 (6.56 μm). In addition, for a fixed viscosity contrast λ of 10, as the rigidity of the RBC increases, its final lateral and longitudinal displacements decrease.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Eng & Built Env
Science, Environment, Engineering and Technology
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Field flow fractionation of particles in rotating coiled column has been investigated in recent year. In contrast to the classical mode of field flow fractionation in narrow channels, the use of rotating coiled columns offers the possibility of large sample loading. In this thesis, the potential for new cell separation methods based on the use of flow fractionation in fluctuating g-fields generated in rotating coil columns is examined. The effects of operational conditions (flow rate and rotational speed – Chapter 3 and Chapter 5); cell properties (cell flexibility – Chapter 4); and column shapes (different inner diameters and coil geometries – Chapter 6) on the flow behaviour of a model system of red blood cells (RBCs) from different species, which differ markedly in size, shape & density, flowing in a single phase of buffered saline have been characterised. Operational Conditions: For a particular rotational speed, there was a minimum flow rate which caused all the cells to be retained in the column and a maximum flow rate at which all cells were eluted. Both the minimum and maximum flow rate were increased when a higher rotational speed was applied. Differences in the behaviour of sheep & hen RBCs have been used to develop a separation method using a continuously increasing flow gradient. This separation could be speeded up by using a step flow gradient. The effects of cell load and rotational direction on the behaviour of RBCs in the column was also studied in this thesis. Cell Properties: The minimum flow rate was found to correlate with cell diameter/cell volume of the RBCs as expected for a sedimentation related process and was partially described by a theoretic equation developed for particles by Fedotov and colleagues (Fedotov et al. 2005). However cell dependent departures from this equation were found which appear to indicate that cell specific surface properties may also be involved for cells (Chapter 3). By contrast the maximum flow rate showed no correlation with cell diameter/cell volume. An effect of cell deformability on the flow separation behaviour of the cells has been demonstrated. Chemical fixation of sheep RBCs with glutaraldehyde rendered the normally deformable RBCs rigid and non-deformable and resulted in the fixed sheep RBCs eluting significantly earlier than unfixed sheep RBCs. This difference was great enough that a mixture of deformable (unfixed) and non-deformable (fixed) sheep RBCs could be separated. Fixed cells tended to show cell aggregation, which could be reduced by the addition of surfactant. Column Geometry: An effect of column shapes on the flow separation behaviour of cells has been demonstrated showing that the optimisation of column design is an important feature of this mode of cell separation. For columns with the same cross sectional area, a “horizontal” rectangular column provided better separation than a circular column and a “vertical” rectangular column gave the least efficient separation. A possible explanation for this behaviour is suggested the thinner sedimentation layer and less secondary flow. Differences in the behaviour of various species of RBCs in the “horizontal” rectangular column have been used to study the efficiency of separation of a mixture of sheep and hen RBCs, and a mixture of rabbit and hen RBCs. This work shows similarities and differences with other reports on cell/particle separations in rotating coiled columns in single phases and also in aqueous two phases systems (ATPS) and these are discussed. Fedotov P.S., Kronrod V.A. & Kasatonova O.N. (2005). Simulation of the motion of solid particles in the carries liquid flow in a rotating coiled column. J. Anal. Chem., 60, 4, 310-316.

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O objetivo do presente trabalho, foi o de correlacionar os resultados obtidos em Teste convencional in vivo, de Draize de Irritação ocular, em coelhos, que preconiza o estudo quantitativo das lesões induzidas por produtos cosmétidos na conjuntiva, córnea e íris, com os resultados do Teste RBC, in vitro, que preconiza a análise quantitativa da hemólise e desnaturação protéica induzidas pelos mesmos produtos, em eritrócitos de mamíferos, com a finalidade não só de validar este último como um Teste preliminar capaz de selecionar produtos altamente irritantes, excluindo-os do Teste de Draize, bem como avaliar o Teste in vitro, como uma alternativa válida para uma eventual substituição do Teste in vivo. Para isto, realizamos estudo paralelo in vivo/in vitro de 23 substâncias-teste (19 podutos cosméticos e 4 tensoativos) procurando relacionar as lesões das 3 estruturas oculares, quantificadas por escores, com os 3 parâmetros in vitro, referentes à indução, em eritrócitos de carneiro, (i) de hemólise (H50) e (ii) de desnaturação protéica (ID), cuja razão H 50/ID, reflete o Potencial de Irritação(PI) das substâncias-teste. Em termos de tensoativos, obtivemos expressivas correlações entre as médias dos escores máximos (MEM) das lesões das três estruturas oculares, com os efeitos induzidos nos 3 parâmetros in vitro, com Coefs. Correlação de Pearson (CCP) de 0,900 a 0,988. Resultados análogos ocorreram com os produtos cosméticos em relação à conjuntiva e córnea (CCP: 0,682 a 0,788) porém, em menor escala, em relação à íris. (CCP: 0,513 a 0,519), Portanto, o Ensaio RBC pode ser usado como screening para avaliar o PI de produtos cosméticos que contenham tensioativos pois revelou-se capaz de predizer, com um elevado nível de precisão (96 por cento) o valor desse PI. Além disso mostrou elevados graus de sensibilidade e especificidade, com índices da ordem de 94 e 100 por cento respectivamente.
The objective of the present study was to correlate the results obtained in vivo with the standard Draize test of ocular irritation, which permits a quantitative study of the lesions induced by cosmetic products in the conjunctiva, cornea and iris, with the in vitro results of the sheep red blood cell (RBC) test, which permits a quantitative analysis of the hemolysis and protein denaturation induced by the same products in mammalian erythrocytes, in order not only to validate the latter as a preliminary test capable of selecting highly irritating products, excluding them from the Draize test, but also to evaluate the in vitro test as a valid alternative for an eventual replacement of the in vivo test. To this end, we performed a parallel in vivo/in vitro study of 23 test substances (19 cosmetic products and 4 tensoactive agents) in order to relate the lesions of the 3 ocular structures, quantified by scores, to the 3 in vitro parameters concerning the induction of (i) hemolysis (H50) and (ii) protein denaturation (ID) in sheep RBC whose H 50/ID ratio reflects the irritation potential (IP) of the test substances. With respect to the tensoactive agents, we obtained significant correlations between mean maximum scores for the lesions of the three ocular structures and the effects induced on the 3 in vitro parameters, with Pearson correlation coefficients (PCC) of 0.900 to 0.988. Similar results were obtained for the cosmetic products with respect to the conjunctiva and the cornea (PCC: 0.682 to 0.788) although to a lesser extent compared to the iris (PCC: 0.513 to 0.519), Thus, the RBC assay can be used as a screening method to assess the IP of cosmetic products containing tensoactive agents since it proved to be able to predict the IP value with a high level of accuracy (96%). In addition, the assay showed high sensitivity and specificity rates of 94 and 100%, respectively.

Os eicosanoides, produtos do metabolismo do ácido araquidônico, apresentam papel importante na homeostasia e na patogênese de diversas doenças humanas. A biossíntese desses compostos pode ser estimulada por agentes farmacológicos como ionóforos e inibidores da Ca2+-ATPase, e também por agonistas naturais como o formil-metionil-leucil-fenialanina (fMLP). Considerando os interesses em avaliar e comparar o perfil de mediadores lipídicos, como os leucotrienos (LTs), as prostaglandinas (PGs), os ácidos epoxieicosatrienoicos (EETs), os ácidos dihidroxitetraenoicos (DiHETEs) e os ácidos hidroxieicosatetraenoicos (HETEs), na saúde e na doença, o objetivo deste trabalho foi padronizar um método analítico para determinar do perfil de eicosanoides em plasma humano após estimulação do sangue total, e assim observar diferenças entre indivíduos saudáveis e doentes. Dessa forma, um método por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (HPLC-MS/MS) foi validado para quantificação de 22 eicosanoides em plasma de indivíduos saudáveis. A análise por HPLCMS/ MS foi realizada em modo negativo pelo modo de varredura por monitoramento de reações múltiplas (MRM). A linearidade do método apresentou coeficiente de correlação (r) maior que 0,98 para todos os eicosanoides analisados. A precisão e exatidão intra e inter-ensaios tiveram desvio padrão e erro relativo menores que 15%, exceto para o limite inferior de quantificação cujos valores foram menores que 20%. Para estimulação das células do sangue total, quatro estímulos (fMLP, ionomicina, A23187 e tapsigargina) foram utilizados. A análise estatística mostrou que o A23187 e a tapsigargina foram os estímulos mais potentes na indução da produção de eicosanoides. Em seguida, comparamos o perfil de eicosanoides em amostras de plasma de indivíduos saudáveis com pacientes com anemia falciforme (AF), em tratamento com hidroxiureia (HU) ou transfusão sanguínea crônica. Os resultados demonstraram que o método é preciso para determinação de diferenças entre os pacientes e indivíduos saudáveis quanto à produção dos mediadores lipídicos 5-HETE, 12-HETE, LTB4, LTE4, TXB2 e PGE2. Portanto, nosso método analítico é sensível, específico e reprodutível para identificar e quantificar diferenças no perfil de eicosanoides em amostras de sangue estimuladas in vitro, e poderá contribuir para o estabelecimento do perfil de mediadores lipídicos em diferentes doenças inflamatórias e infecciosas.
Eicosanoids, products from arachidonic acid metabolism, play an important role in the homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca2+ ionophores and Ca2+-ATPase inhibitors, as well as natural agonists such as fMet-leu-Phe (fMLP) can stimulate eicosanoid biosynthesis. Considering the interests in evaluate and compare the profile of lipid mediators, as leukotriens (LTs), prostaglandins (PGs), epoxyeicosatrienoic acids (EETs), dihydroxytetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acids (HETEs), in healthy and disease, the aim of this work was to standardize a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation, and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient > 0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of < 15%, except for the lower limit of quantification, where these values were < 20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were used. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli to induce the production of eicosanoids. We next compared the eicosanoid profiles of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients compared to healthy subjects for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method is sensitive, specific and reproducible for indentify and quantify changes in eicosanoid profiles in whole blood stimulated in vitro, which can contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

Red blood cells (RBCs) are the most abundant cells in human blood, representing 40 to 45% of the blood volume (hematocrit). These cells have the particular ability to deform and bridge together to form aggregates under very low shear rates. The theory and mechanics behind aggregation are, however, not yet completely understood. The purpose of this work is to provide a novel method to analyze, understand and mimic blood behaviour in microcirculation. The main objective is to develop a methodology to quantify and characterize RBC aggregates and hence enhance the current understanding of the non-Newtonian behaviour of blood at the microscale. For this purpose, suspensions of porcine blood and human blood are tested in vitro in a Poly-di-methylsiloxane (PDMS) microchannel to characterize RBC aggregates within these two types of blood. These microchannels are fabricated using standard photolithography methods. Experiments are performed using a micro Particle Image Velocimetry ( PIV) system for shear rate measurements coupled with a high speed camera for the flow visualization. Corresponding numerical simulations are conducted using a research Computational Fluid Dynamic (CFD) solver, Nek5000, based on the spectral element method solution to the incompressible non-Newtonian Navier-Stokes equations. RBC aggregate sizes are quantified in controlled and measurable shear rate environments for 5, 10 and 15% hematocrit. Aggregate sizes are determined using image processing techniques. Velocity fields of the blood flow are measured experimentally and compared to numerical simulations using simple non-Newtonian models (Power law and Carreau models). This work establishes for the first time a relationship between RBC aggregate sizes and corresponding shear rates in a microfluidic environment as well as one between RBC aggregate sizes and apparent blood viscosity at body temperature in a microfluidic controlled environment. The results of the investigation can be used to help develop new numerical models for non-Newtonian blood flow, provide a better understanding of the mechanics of RBC aggregation and help determine aggregate behaviour in clinical settings such as for degenerative diseases like diabetes and heart disease.

The mechanics and elasticity of red blood cells (RBCs) determine the capability to deform of these cells when passing through the thinnest capillaries, where the delivery of oxygen takes place. The understanding of the elastic properties of RBCs is fundamental for improving our knowledge about microcirculation and it also has important biomedical applications, such as control of blood storage, or cell manipulation for pathology diagnosis. In this Thesis, we study the elasticity of RBCs under different conditions, understanding their mechanical response to different type of perturbations. In a first Part, we study the shape morphologies observed in the disco-echinocyte transition, when the cell is subjected to an imbalance in the membrane asymmetry, for instance after ATP depletion when lipids flip from the inner to the outer leaflet. Affected cells deform, adopting crenated morphologies known as echinocytes. We develop a theoretical study which allows us to identify and quantify the relevant aspects that trigger the shape transition. The lipid bilayer tries to expand its outer leaflet in order to accommodate the excess area, whereas the cytoskeleton opposes resistance to this type of deformations, preserving more compact shapes. The subtle interplay between both membrane structures determines the equilibrium morphology of the cell. The cytoskeleton is fundamental to ensure the stability of the healthy shape, the discocyte, against changes in the membrane composition. However, it is not severely stressed under weak deformations in which low curvatures are involved. Our results show that the energetic scale of these shape transitions is of hundreds of kbT, demonstrating the large stability of these shapes. Based on the knowledge gained from the theoretical study we also analyze a series of experiments in which echinocytes are mechanically perturbed by a AFM tip, inducing shape transitions towards the healthy discocyte in a controlled manner. In the second Part, we derive a phase-field method for membrane modeling. Phase-field methods have been extensively used for the study of interface phenomena, though with few applications to membranes. We present a new model which accounts for the membrane elasticity, and couples the membrane dynamics with an external fluid, whose hydrodynamics is dictated by the Navier-Stokes equation. We derive the expression of the stress tensor which allows us to recover the stress profile of the membrane. We also obtain the membrane equilibrium equations, proving that in the macroscopic limit our phase-field model recovers the correct expressions given by the elastic theory of membranes. In the third Part we make use of this phase-field model to study the behaviour of RBCs in flow in narrow channels, of width similar to that of the cell. We consider pressure-driven flows as they relevant for both in vivo and in vitro circulation. We carry out simulations by means of a lattice-Bolztmann method. Our study highlights the crucial role of the RBC shape, softness and deformability to explain its complex behaviour and rheological properties. RBCs flowing at low concentratrions, when they do not interact with other cells and the dynamics is governed by the interaction with the cell, are shown to migrate lateral towards the wall, avoiding the axial position. The RBC assumes an asymmetric shape and orients with the flow, reducing the viscosity of the fluid which presents a shear-thinning behaviour. The lateral position can be controlled by tuning the channel geometry and flow velocity, and it is also dependent on the shape of the cell, as sherical cells as shown to occupy and axial position. The control of these factors is important for the manipulation of different cell species, such as RBCs and leukocytes, in microfluidic devices. Finally, we study the behaviour of RBC suspensions at intermediate concentrations, when hydrodynamic interactions between RBCs govern the dynamics. The focusing to lateral positions induced by the walls is inhibited and cells are shown to order along the channel section, occupying the core of the channel. RBCs adopt and horizontal inclination, forming a relatively ordered structure of parallel rows. The rheology of the suspension is also affected, as the interactions between cells attenuate the orientation with the flux and higher flow velocities are required to induce the shear-thinning decay of the viscosity. The results presented in this Thesis highlight the delicate dependence of the cell mechanics in the balance of the cell membrane composition and elastic properties. They also demonstrate that the elastic behaviour of the cell, determined by its membrane, is also crucial for the rheological behaviour of blood, and any process of membrane damage or stiffening can substantially alter the correct blood functioning.
El estudio del comportamiento mecánico de los glóbulos rojos es fundamental para entender aspectos relevantes acerca de la elasticidad de membranas y reología de la sangre, incluyendo importantes aplicaciones biomédicas. En esta tesis se aborda la respuesta elástica de estas células bajo diferentes tipos de deformaciones morfológicas. Por un lado, se estudia el efecto de la microestructura de la membrana en las formas de equilibrio de los glóbulos, identificando la función del citoesqueleto celular cuando la asimetría en la bicapa lipídica es alterada (por ejemplo, reduciendo los niveles de ATP). Nuestros resultados muestran que la bicapa tiende a expandirse formando estructuras puntiagudas, mientras que el citoesqueleto se opone a estas deformaciones y mantiene formas más compactas cercanas al discocito. El citoesqueleto aparece como un elemento fundamental para estabilizar la célula en su conformación de equilibrio. En la segunda parte de la tesis, se deriva un modelo de interfase difusa para membranas. Para ello obtenemos el perfil de esfuerzos que muestra cómo el modelo captura correctamente las propiedades elásticas de las membranas. También se obtienen las ecuaciones macroscópicas que definen el comportamiento de equilibrio y dinámico del modelo, y que convergen correctamente a los resultados clásicos de la teoría general de membranas. Finalmente, en la tercera parte realizamos simulaciones haciendo uso de este modelo de interfase difusa para estudiar el comportamiento de glóbulos rojos fluyendo en canales confinados. El estudio refleja la compleja respuesta de las células, en las que la elasticidad y deformabilidad forman un papel clave. Los glóbulos a bajas concentraciones evitan la posición central del canal y se desplazan hacia un lateral, adquiriendo morfologías asimétricas y orientándose con el flujo. Esto permite que la viscosidad del fluído disminuya. En cambio, a mayores concentraciones, cuando varias células fluyen juntas, la interacciones hidrodinámicas inhiben este comportamiento, y las células fluyen alineadas con una orientación horizontal, organizadas en filas tanto en los laterales como en el centro del canal. La interacción y apantallamiento entre las células hace que el decaimiento en la viscosidad requiera de velocidades considerablemente mayores.

Determining the fragility of the red blood cell (RBC) is important for the diagnosis of and evaluation for treatment of several RBC diseases. In part RBC production is controlled through the hormone erythropoietin secreted by the kidney. In a previous study from this laboratory, it was found that RBC were more stable in uremic male rats compared to controls. In this experiment, uremia was induced in four groups of female rats through a two stage nephrectomy. The nephrectomy involved the removal of two-thirds of the left kidney, followed by the removal of the entire right kidney one week later. The animals were divided into three groups; NX-(5/6 nephrectomy), SH-(sham surgery), and PF-(sham surgery, but were fed the same food weight as the NX animals). The samples obtained in Trial I and Trial II were divided into two categories; initial and final. The initial samples were collected 14 days after the sham and five- sixth nephrectomy surgeries. The final sample were collected at the time of sacrifice. The samples obtained in Trial 11 consisted only of initial samples, taken fourteen days after the five-sixth nephrectomy and sham surgeries were completed. The samples in Trial IV were final samples, obtained at the time of sacrifice. Decreasing hypotonic %NaCl solutions were used to determine the hemolysis of RBC from the rats. RBC hemolysis was determined spectrophotometrically by monitoring hemoglobin absorbance at 540nm in the supernatant fluid. Analytic precision experiments using multiple assays of the same blood sample for 50% RBC hemolysis showed a coefficient of variation of only 1.1%. Analysis of the %NaCl at 50% RBC hemolysis did not differ significantly between the three groups of animals suggesting that although the NX animals were uremic, the RBC did not differ in stability to hypoosmotic shock. Future direction for this type of research will be extended to human studies where kidney failure patients (dialysis patients) can have both the age of the RBC and their fragility determined under therapy. The erythrocyte hemolysis peroxide test (HPT) was also performed on rat blood samples from Trial IV and on five human blood samples, in order to determine hemolysis in the RBC. A 2% H2O2 solution was used to determine RBC stability and %Hemolysis was calculated by dividing the value for hemolysis due to H2O2 by the 100% hemolysis value and multiplying by 100. Analysis of the %Hemolysis by HPT for each animal sample also showed no significant difference between the three subgroups of animals. Future direction for this type of research will be extended to include all four trials of animals as well as human studies involving patients with kidney failure (dialysis patients).

Les transfusions de globules rouges représentent le traitement principal de l’anémie. La décision de transfuser représente un vrai dilemme clinique. L’anémie et les transfusions de globules rouges sont toutes les deux associées à des risques et à un moins bon devenir des patients, alors que le bénéfice des transfusions sanguines reste difficile à démontrer. C’est pour cela que la décision de transfuser ne doit pas être pris à la légère et qu’elle doit tenir compte de la balance antre les risques des transfusions de globules rouges et les risques de l’anémie. L’anémie, définie comme un taux d’hémoglobine sous la moyenne pour l’âge, est fréquente chez les enfants en péri-opératoire de chirurgie cardiaque. Les conséquences de l’anémie sont une diminution du transport en oxygène vers les cellules. Le taux d’hémoglobine sous lequel la demande tissulaire en oxygène est compromise n’est pas connue et dépend de l’état de santé du patient et de ses comorbidités. Les causes peropératoires de l’anémie sont surtout le saignement et l’hémodilution. Une diminution de la production d’érythropoïétine endogène, une dérégulation du métabolisme du fer, une production défectueuse de la moelle et la répétition des prélèvements sanguins contribuent à l’anémie postopératoire. L’anémie est associée à des évènements indésirables et un moins bon devenir, mais cette association semble en grande partie expliquée par la pathologie sous-jacente, elle-même associée à l’anémie. Les transfusions en globules rouges sont fréquentes en chirurgie cardiaque pédiatrique. Le rapport bénéfice-risque des transfusions sanguines reste difficile à évaluer. Alors que les études rapportant des bénéfices clairs des transfusions sanguines restent rares, plusieurs travaux observent une association entre les transfusions en globules rouges et une augmentation de la morbidité et mortalité. En outre, les transfusions sanguines demeurent une ressource rare et chère.

Le but de ce travail est de contribuer à une meilleure utilisation des transfusions sanguines chez les patients de chirurgie cardiaque pédiatrique. Dans la première partie du travail, nous avons étudié les déterminants des transfusions en globules rouges et du saignement, qui représentent une des causes principales de transfusion sanguine chez ces patients. Une meilleure identification et une prise en charge adéquate des facteurs qui mènent aux transfusions sanguines devraient diminuer le nombre de transfusions inappropriées. Dans la deuxième partie de ce travail, nous nous sommes penchés sur l’association entre les transfusions sanguines et le mauvais pronostic des patients en étudiant deux approches :l’âge des globules rouges transfusés et l’indication transfusionnelle. Une meilleure compréhension des facteurs associés à un moins bon pronostic devrait permettre de mieux définir les patients qui bénéficieraient réellement de transfusions en globules rouges.

En ce qui concerne les déterminants des transfusions sanguines, nous avons démontré que l’anémie préopératoire était significativement associée aux transfusions sanguines péri-opératoires. Les enfants qui saignent reçoivent beaucoup de produits sanguins. Nous avons déterminé les patients à risque de saignement afin de les reconnaître et les soumettre à des tests de coagulation rapides pour orienter le type de produits sanguins à transfuser en fonction des anomalies de coagulation mises en évidence. Puisque l’anticoagulation par héparine est systématique chez les patients opérés sous circulation corporelle, nous avons étudié si notre protocole de neutralisation de l’héparine avec de la protamine était adéquat. En effet, la persistance d’héparine circulante ainsi qu’un surdosage en protamine sont associés à des saignements postopératoires. Un ratio protamine-héparine de 1:2 semble permettre une neutralisation adéquate de l’héparine chez la majorité des patients sans les exposer à un surdosage en protamine. Finalement, nous avons démontré qu’une stratégie transfusionnelle restrictive en postopératoire permettait de diminuer l’exposition aux transfusions sanguines sans augmenter la morbidité et mortalité de ces enfants. Cela signifie qu’on pourrait éviter des transfusions en globules rouges en prenant en charge l’anémie préopératoire, en développant un algorithme de prise en charge précoce du saignement peropératoire et en diminuant le seuil transfusionnel postopératoire.

La deuxième partie de ce travail avait pour but de préciser l’association qu’il existe entre les transfusions en globules rouges et la morbidité et mortalité postopératoire. L’âge du sang n’a pas l’air d’être un facteur influençant le pronostic des enfants opérés de chirurgie cardiaque. Par contre, ce travail a permis de montrer que c’est probablement l’indication transfusionnelle ou la raison qui mène à la transfusion, plutôt que la transfusion en elle-même qui est associée à un moins bon pronostic. L’association entre les transfusions sanguines et un moins bon pronostic est probablement surestimée par la présence de facteurs confondants comme l’indication transfusionnelle. Les transfusions en globules rouges seraient plutôt un marqueur de risque qu’un facteur de risque de mauvais pronostic.

En conclusion, ce travail contribue au développement de stratégies transfusionnelles plus rationnelles en chirurgie cardiaque pédiatrique. Reposant sur une approche multidisciplinaire, elles assurent une prise en charge structurée et orientée permettant de diminuer l’exposition des enfants aux produits sanguins, avec pour objectif une amélioration du pronostic et une réduction des coûts de prise en charge de ceux-ci.
Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

Continuous multidisciplinary advancements in medicine, science and engineering have led to the rise of biomedical microfluidic devices for clinical diagnoses, laboratory research for modeling and screening of drugs or disease states, and implantable organs such as artificial kidneys. Blood is often the biological fluid of choice for these purposes. However, unique hemodynamic properties observed only in microscale channels complicate experimental repeatability and reliability.

For vessels with 10-300μm diameters, red blood cell properties such as deformability have a significant impact on hemorheology, and the blood can no longer be considered as a homogeneous fluid. The flowing blood segregates into a red blood cell rich core bounded by a cell-free layer composed almost entirely of plasma. Viscous forces dominate flow behavior, and shear rates at the wall are much higher than in arteries and veins. The overall viscosity becomes dependent on vessel diameter. These unique characteristics are interesting from a biophysics perspective, but the value of biomedical microfluidic technologies makes research in this regime even more critical. Accordingly, this work focuses on experimental comparison of the microfluidic flow properties of red blood cells with varying physical characteristics. Blood viscosity in microscale tubes was investigated experimentally for 6 blood types (goat, sheep, pig, llama, chicken and turkey) at a range of hematocrits (0-50%). The selected blood types represented a small sample of the wide-ranging red blood cell characteristics found in mammals, birds, reptiles, amphibians and fish. These red blood cells vary in size over an order of magnitude, represent shapes ranging from biconcave to ellipsoidal, and include both nucleated cell types found in birds, amphibians and reptiles and denucleated mammalian cells. Pressure drop experiments at physiologically relevant flow rates were carried out for rigid tubing diameters ranging from 73μm - 161μm. The resulting viscosities were normalized relative to the measurements made of the homologous plasma for each species. The viscosity of blood in this regime is much different than in larger vessels (>500μm) or in small capillaries (<10μm), but existing studies in this size range focus only on human blood.

The results analyzed in the context of four primary variables: hematocrit, red blood cell size, red blood cell shape, and red blood cell deformability. The aggregation of the porcine blood complicated the data collection process, resulting in only a few usable data points.

Examination of the role of hematocrit yielded results which aligned well with existing hemorheology research: viscosity increases with hematocrit. After applying an existing fitting equation the collected data, three primary trends were observed. First, the chicken blood had the highest viscosity at every hematocrit regardless of tubing diameter. Secondly, the viscosities of the goat and sheep blood were very similar at all hematocrits, and ultimately had the lowest viscosity of all samples at the highest measured hematocrit values. Finally, the turkey and llama blood generally had the lowest viscosity at low hematocrit, with a deflection point around 30% hematocrit where viscosity began to increase much more sharply.

The role of cell size was considered in the context of both mean cell volume and major cell axis relative to vessel diameter, to account for the elongated shape of the llama, chicken and turkey red blood cells. The results indicated that cell major axis is better correlated with viscosity than cell volume, suggesting the potential importance of cell shape. The red blood cells were then characterized as either oblate or prolate to further investigate the importance of shape. The results further supported the idea that overall blood viscosity in small vessels depends on both cell size and shape. However, as with the hematocrit analysis, the chicken blood was an outlier. The chicken red blood cells are quite similar to turkey red blood cells in both size and shape, yet the chicken blood was consistently far more viscous than turkey blood. A comparison with theoretical rigid particles suggested that the chicken red blood cells may be the least deformable of the sampled blood types.

Two additional experiments were performed to assess the potential importance of deformability. Additional pressure drop measurements with chemically-hardened red blood cells demonstrated that the measurement system is quite sensitive to changes in cell deformability. Flow visualization in a microfluidic contraction indicated that the high viscosity of the chicken blood relative to the turkey blood could be attributed to differences in deformability.

Blood viscosity is influenced by multiple cell characteristics, including size, shape and deformability. The role of these parameters is worthy of further investigation alongside ongoing research in the rheology of human blood. The impact of red blood cell deformability on viscosity in small vessels is particularly interesting. The described experimental apparatus is easily replicable and highly customizable, and may serve as a helpful tool to analyze blood parameters in biomedical microfluidic device research and development. The collected data sets are available to interested researchers, and can currently be obtained by direct request. Ultimately, an online database will be made available via the Liepmann lab website.

Margination is the migration of particles in a channel towards the outer walls of the channel. In blood microcirculation, studying the margination of microparticles is important to understand platelet migration and the kinetics of drug delivery. Many new topics in drug delivery research examine the slow release of drugs through micro particles, such as micelles. The margination of such drug carriers is related to tissue absorption and, consequently, to the efficiency of drug delivery. We hypothesized that the intensity of red blood cell (RBC) aggregation will change the level of margination in a cylindrical channel. RBC aggregation is the reversible process of RBCs clumping together over time, under low fluid shear rate. A higher level of aggregation means that this clumping occurs more quickly. The goal of this thesis is to design an experiment that measures the level margination of microparticles and the effect that RBC aggregation has on margination, in a controlled in vitro environment. Fluorescent microparticles were added to human blood preparations. The aggregation properties of the blood preparation were modulated by the addition of a macromolecule (Dextran 500). The blood preparations were injected into PDMS microfluidic devices that were modified to have circular channels in order to better mimic the geometry of physiological microcirculation. We designed a circular microchannel that worked to capture the marginating microparticles and it was found that the level of margination of the microparticles increased with an increase in aggregation of the RBCs. This increase in margination was especially sensitive to aggregation levels in the range of physiological aggregation levels of whole blood, suggesting that aggregation plays an important role in margination in vivo.

Theoretical studies have demonstrated a pronounced effect of red blood cell (RBC) spacing on tissue oxygen supply. In spite of this, realistic values for RBC spacing and related capillary hematocrit (Hct) are not known in the heart. One of the possible reasons is the lack of proper methodology. Thus, the goal of this research study was twofold: (i) to develop a method to rapidly freeze rat heart in situ, following which RBCs and capillary walls could be simultaneously visualized, and (ii) to apply this methodology in rat hearts to establish whether differences exist in capillary Hct and RBC spacing in two distinct locations within the capillary bed, in the subendo- and midmyocardium, during diastole or systole. The results of this study suggest that different geometrical conditions (e.g. RBC spacing and capillary Hct) exist at the two distinct locations of the capillary bed studied. Presumably, the oxygen supply conditions in distal portions of the capillary bed are improved by these geometrical adjustments, preventing hypoxic or anoxic conditions in the tissue, which would be detrimental to cardiac function. (Abstract shortened by UMI.)

Traumatic haemorrhage is a leading preventable cause of critical mortality in mass casualty events (MCEs). Treatment requires the rapid provision of high volumes of packed red blood cells (PRBC) to meet the surge in casualty demand these events generate. The increasing frequency of MCEs coupled with the threat of more violent mechanisms risks overwhelming hospital based transfusion systems. The overall objective of this research was to improve understanding of blood use in MCEs using a mathematical modelling approach. A computerised discrete event simulation model was designed, developed and validated using civilian and military transfusion databases, a review of historical MCEs and discussion with experts involved in all aspects of in-hospital MCE PRBC provision. The model was experimented with across increasing casualty loads to optimise event outcomes under varied conditions of: stock availability, laboratory processing procedures and individual PRBC supply. The model indicated even in events of limited size the standard on-shelf PRBC stock level was insufficient to adequately meet demand amongst bleeding casualties. Restocking during an event allowed for equivocal treatment results if performed early following an event and this would be most effective if activated by central suppliers. Modifications to transfusion laboratory processing procedures were found to be of limited benefit in improving outcomes due to the principally automated nature of the techniques they employ. Conversely, the use of restricting excessive individual provision of both overall PRBC and emergency type O PRBC to individual casualties did show potential for managing scenarios where only a finite supply of stock existed or an accurate estimation of expected casualties was available.

This thesis developed a numerical model for simulating red blood cell shape and deformability. The model was applied to understand how changes in membrane mechanical properties impact upon the cells' exhibited shape and ability to deform. This was done in the context of two loading scenarios, indentation and stretching. The project highlights some important factors to consider when developing experimental methods that quantify red blood cell deformability, of interest in transfusion medicine and for the diagnosis of certain diseases.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 136-144).
Red blood cells (RBCs) play a crucial role in delivering oxygen to the body tissues. During the 120 days of their lifespan, average RBCs would circulate for approximately 500,000 times and undergo repeated deformations in small blood capillaries and splenic cords. Increased RBC clearance in the spleen is considered as one of the direct consequences of reduced RBC deformability. On the other hand, deformability is also indirectly impacting on RBC functionality through its complex connections with underlying molecular mechanisms. With the aid of microfabrication and microfluidic, we are able to perform high-throughput single cell deformability measurement. Overall, we established RBC deformability as an important biomarker for several blood related real world problems such as malaria and blood storage lesion. The ultimate goal is through our quantitative assessment of population-wide single cell deformability, we could aid in the decision-making of various clinical scenarios including drug screening and blood transfusion. Malaria is the most deadly parasitic disease affecting hundred millions of people worldwide. Infected RBCs are found to be less deformable and therefore more susceptible to splenic RBC clearance. In this thesis, we identified several clinically used anti-malarial drugs that are capable of altering RBC deformability and ultimately modifying RBC retention in spleen. We also employed a rodent malarial model, confirming the important connection of RBC deformability with splenic RBC retention and consequently malarial anemia in vivo. Blood storage lesion is another important application of our work. Taking the advantage of device high-throughput, we profiled hundreds of single RBC deformability from a large population and identified subpopulations that are less deformable. These subpopulations also exhibited higher osmotic fragility and were therefore predicted to pose higher transfusion risk according clinical standard. Furthermore, a deformability based sorting device was also developed to filter the less deformable blood subpopulations, improving overall blood quality.
by Sha Huang.
Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2010.
Cataloged PDF version of thesis.
Includes bibliographical references (p. 118-126).
Decreased deformability of human red blood cells (RBCs) is both a cause of disease and biomarker for disease (1). To traverse blood capillaries, the biconcave disk-shaped RBC must deform dramatically, since the diameter of the unconstrained RBC is larger than that of the capillaries. If the RBC becomes immobilized in a capillary, hypoxia and tissue injury may result, potentially leading to death. Changes in RBC deformability may be attributable to genetics (e.g. sickle cell anemia (2) and spherocytosis (3)), drug exposure (e.g. pentoxifylline (4)), and disease (e.g. diabetes (5) and malaria (6)). Within the past 15 years, microfabrication techniques have enabled the creation of pores comparable in size and shape to the smallest human capillaries (7) and slits in the spleen (8). We use this microfabrication ability to create devices that analyze and separate RBCs of different deformability. The first device we create is an automated 'deformability cytometer' that measures dynamic mechanical responses of 103~104 individual cells in a cell population. Fluorescence measurements of each cell are simultaneously acquired, resulting in a population-based correlation between biochemical properties (e.g. cell surface markers) and dynamic mechanical deformability. This device is especially applicable to heterogeneous cell populations, and we demonstrate its ability to mechanically characterize a small number of ring-stage malaria-infected RBCs in a large population of healthy RBCs. Next we present a device whose design is based on the architecture of the human spleen. This device is able to continuously separate more deformable from less deformable RBCs. We demonstrate the ability of this device to separate schizont-stage malaria-infected RBCs from healthy RBCs. Together, these devices enable the analysis and separation of single-RBCs based on deformability.
by Hansen Chang Bow.
Ph.D.

Erythrocytes are one of the cellular models frequently used to decipher the physiology of membrane transport. However, if pumps, antiporters and cotransporters are now well defined, the molecular identity and the physiological role of the conductive pathways present in the membrane of red blood cell (RBC) is still elusive albeit the growing evidences of their role in physiological and pathological conditions. The present work, using the patch clamp technique and biophysical studies, shows that: 1) The changes in osmotic fragility observed after Gardos channel dependent dehydration is mainly not due to rehydration of the cell via membrane transporters but rather due to change in membrane properties elicited by the Ca2+ loading. It reflects a specific calcium-induced lytic vulnerability of the membrane leading to rupture before the cells attain their maximal spherical volumes. 2) The Gardos channel can be transiently activated when seal formation induces membrane deformation. This phenomenon can result only from activation of a permeability pathway with a finite Ca2+ conductance. This transient activity generates secondary transient anionic channel activity that has been studied further. 3) The diversity of anionic channel activities recorded in normal human RBCs, as well as in P. Falciparum-infected RBCs, corresponds to different kinetic modalities of a unique type of maxi anion channel with multiple conductance levels, gating properties and pharmacology. Finally, the present work contributes to the understanding of the role of ion channels in RBCs and opens questions on the contribution of ion channels in the rheological properties of RBCs. Keywords: erythrocyte, ionic channels, osmotic fragility, patch clamp technique
Les érythrocytes sont un des modèles pour l’étude des voies de transport membranaire. Si les pompes, les antiports et les cotransports sont bien définis, l’identité moléculaire et le rôle physiologique des voies de conductances du globule rouge restent méconnus. Le présent travail réalisé à l’aide des techniques de patch-clamp montre que : 1) Les changements de fragilité osmotique observés lors d’une déhydratation des cellules après stimulation du canal Gardos, ne sont pas dus à une réhydration des cellules via l’activation de transporteurs, mais à des changements de propriétés de la membrane liées à l’augmentation du [Ca2+]i. Elle reflète une vulnérabilité spécifique de la membrane au Ca2+, induisant la rupture de la membrane avant que la cellule n’ait atteint son volume critique. 2) Le canal Gardos peut-être activé transitoirement lors d’épisode de déformation de la membrane. Ce phénomène ne peut être que le résultat de l’activation d’une voie de perméabilité au Ca2+ ayant une conductance déterminée. 3) L’activité transitoire du canal Gardos active secondairement une voie de conductance anionique. La diversité des canaux anioniques précédemment décrits dans la membrane des érythrocytes humains correspond à l’activité d’un unique canal anionique de type maxi-chlorure ayant des modalités d’activation et de fonctionnement (état d’ouverture multiple, cinétique, pharmacologie…) différentes en fonction des conditions expérimentales. Ce travail contribue à une meilleure compréhension des canaux ioniques présents dans la membrane du globule rouge et permet d’envisager la participation de ces canaux dans la régulation des propriétés rhéologiques des globules rouges. Mots clés: érythrocyte, canaux ioniques, fragilité osmotique, patch clamp

Microvascular transport is strongly influenced by the nonuniform partitioning of red blood cells at diverging microvessel bifurcations, where blood flows from one mother vessel into two daughter vessels. In such bifurcations, the volume fractions of red blood cells in the blood entering each daughter vessel typically differ significantly from the volume fraction in the mother vessel. This phenomenon is caused, to a first approximation, by nonuniform distribution of red blood cells in the cross-section of the mother vessel and the tendency of red blood cells to follow background fluid streamlines. In smaller vessels, however, red blood cell trajectories can deviate significantly from fluid streamlines. In this dissertation, the mechanical reasons for these deviations and their contributions to nonuniform partitioning are analyzed.A two-dimensional model is used to simulate the motion and deformation of flexible particles as they travel alone through a diverging microvessel bifurcation. Deviations of particle trajectories from background fluid streamlines result from migration towards the mother vessel centerline upstream of the bifurcation and flow perturbations caused by cell obstruction in the bifurcation region. Cell migration, which arises because of flexibility, causes more nonuniform partitioning while cell obstruction causes more uniform partitioning. Bifurcations with differently sized daughter vessels experience, on average, a higher red blood cell flux into the smaller branch. Partitioning is unaffected by daughter branching angles.The motion of two interacting cells is also considered. In diverging bifurcations several types of interactions were found, in which the presence of a nearby cell causes a cell to enter a different branch than it would have otherwise. The net effect of these interactions is to cause more uniform partitioning. In wall-bounded linear shear flow, a two-dimensional swapping interaction was identified, in which two cells on different background fluid streamlines approach each other, slowly move onto their partner's streamline, and then move away from each other.The simulations produced by this two-dimensional model provide insight into the effects of red blood cell deformability, bifurcation geometry and volume fraction of red blood cells on red blood cell partitioning and on the resultant distribution and transport of materials in the microvasculature.

Sickle cell anemia – the most common form of sickle cell disease (SCD) – is a debilitating condition that presents with health complications ranging from severe pain due to vaso-occlusion and acute chest syndrome, to a reduced ability to sufficiently transport oxygen through the body due to accelerated hemolysis of red blood cells. The underlying cause of SCD is a genetic mutation in which valine is replaced by glutamic acid at the sixth position of the β-globin chain, leading to the production of an abnormal sickle hemoglobin (hemoglobin S). This abnormal protein within the red blood cells have an increased propensity to polymerize into long fibers when deoxygenated, decreasing red-cell deformability while increasing the rate of cell death by damaging the cell membrane. These sickled red blood cells can then clump together to occlude blood vessels causing pain and organ damage. The impact of SCD has led to the evaluation of different treatment modalities in attempt to cure or ameliorate the manifestations of the disease. This thesis is intended to review the underlying disease process, elucidate the treatment modalities that have been previously proposed, and highlight the effect of hydroxyurea as the primary approved treatment of sickle cell anemia due to its ability to reverse the polymerization of already sickled cells, and increase the levels of fetal hemoglobin (HbF). The increase of HbF levels is a major goal of treatment that can achieved by increasing the expression of gamma-globin genes, therefore we can hypothesize that upregulating the activity of enzyme Demethylase will reverse the methylation of the CpG islands and reverse the binding of Histone deacetylase allowing the transcription of already silenced genes to resume.

This thesis develops a mesoscopic technique based on the numerical concepts of Lattice Boltzmann Method (LBM) to simulate blood flow in microvessels. The central theme of the thesis is to simulate the two- dimensional elastic deformation of Red Blood Cells (RBCs), and stream- ing under various types of loadings in the systemic microvessels. Two types of elastic deformation of RBCs that arise from the RBC-plasma and RBC- RBC interaction have been studied by using the most common Two-Dimensional LBM abbreviated as the D2Q9-LBM model. Since the model cannot handle the deformation of the cell independently, the Immersed Boundary Method (IBM) was incorporated in the D2Q9- LBM to simulate the neo-Hookean deformable RBCs due to the interactions of RBCs with plasma, where the deformation, angular shift and motion of RBCs in plasma have been tracked without involving in the measure of their size and volume change. In addition, further local RBCs deformation enhanced by RBC- RBC interaction has been investigated by utilizing the Morse potential model in the combined LBM-IBM model. Prior to using the LBM on the RBCs deformation, we have performed a successful benchmark on Poiseuille flow between a pair of parallel plates by comparing the D2Q9- LBM model with known analytical solution. After a full agreement between both methods was achieved, the D2Q9- LBM code has been applied to more realistic flow problems, such as flow through constricting, expanding and branching channels, and flow past miscellaneous obstacles as the real blood flow experiences. The combination scheme of the LBM- IBM is based on solving the flow dynamics by the D2Q9- LBM on fixed and regular Eulerian grid of lattices. On the other hand, the hydrodynamic features of neo- Hookean deformable RBCs are transported through IBM on moving and non- regular Lagrangian grid of points that follow the RBCs evolution in the blood. Three different sizes of the deformable RBCs on the basis of the lattice unit have been studied. The deformation of the RBCs due to cell-plasma interaction has been investigated under two varying mechanicalloadings namely; the stretching and bending modulus while the deformation due to cell-cell interaction has been investigated under three different values of the interaction strength potential.

Red blood cell (RBC) deformability plays an important role in the pathogenesis of Plasmodium falciparum malaria, and therefore could potentially enable simple, rapid, and reagent-free biophysical assays. A key challenge, however, is that pathological cells often only represent a small fraction of the sample, which requires testing a large number of individual cells to enable their detection. Additionally, it is often desirable to perform multiple assays simultaneously, which require technologies capable of parallelized analysis. Traditional technologies for analyzing RBC deformability are limited by their experimental difficulty, extensive instrumentation requirements, as well as their lack of throughput and parallelizability. Here, a new microfluidic mechanism called trans-dispersion is developed to address these issues, enabling a high-throughput and parallelized analysis of RBC deformability. The trans-dispersion mechanism transports single RBCs through a series of constrictions in a microfluidic channel, where their transit speed is a function of their deformability. This process is analogous to gel-electrophoresis, where the migration speed of molecules depends on their length. To ensure a sensitive and consistent measurement, the geometry of the constriction is sized such that the transiting cell forms a temporary seal with each constriction while supporting microchannels ensure consistent forces are applied to each deformation channel. After undergoing repeated deformations, the final position of each RBC, indicating its deformability, is determined using simple bright-field microscopy and automated image processing, and thereby resulting in a repeatable, high-throughput and parallelized process. The performance of this mechanism was evaluated by detecting changes in RBC deformability resulting from chemical degradation, malaria parasitism and exposure to anti-malarial drugs. This device can distinguish variation in RBC deformability following chemical degradation using small concentrations (0.0005%) of glutaraldehyde (GTA). P. falciparum-infected RBCs (iRBCs) show distinct deformability curves compared to the uninfected controls. The linear correlation between the parasitemia and the percentage of non-transiting cells could potentially be used to infer the parasitemia of clinical specimen. Furthermore, this device was able to simultaneously assess the efficacy of several antimalarial compounds; showing that rigidification of P. falciparum-iRBCs can potentially be used to evaluate antimalarial drug efficacy, as well as serve as a functional screen for new antimalarials.
Applied Science, Faculty of
Mechanical Engineering, Department of
Graduate

The theoretical modeling and computational simulation of human red blood cells is of interest to researchers for both academic and practical reasons. The red blood cell is one of the simplest in the body, yet its complex behaviors are not fully understood. The ability to perform accurate simulations of the cell will assist efforts to treat disorders of the cell. In this thesis, a computational model of a human red blood cell that combines preexisting mechanical and metabolic models is proposed. The mechanical model is a coarse-grained molecular dynamics model, while the metabolic model considers the set of chemical reactions as a system of first-order ordinary differential equations. The models are coupled via the connectivity of the cytoskeleton with a novel method. A simulation environment is developed in MATLAB® to evaluate the combined model. The combined model and the simulation environment are described in detail and illustrated in this thesis.

NK cells are known to respond in vitro to P. falciparum-infected red blood cells (iRBCs), although responses are highly heterogeneous between donors. Although their role during malaria infection is not fully understood, they may play a role in cytokine production during early infection, and furthermore may interact with and kill iRBCs. The work described in this thesis examines the role of NK cell receptors in determining the functional outcomes of NK cell activation by iRBCs, focusing on NK cell responses to exogenous cytokines and the phenotypic and functional profiles of NK cells from malaria naive (LSHTM) and malaria exposed (Ugandan) subjects. In a model of early activation of NK cells by accessory cell-derived cytokines, I have shown a key role for IL-18 in mediating NK cell responses during both primary and secondary immune responses as IL-18 synergises with cytokines from the common gamma chain family. NK cells from LSHTM donors showed low background expression of IFN- and CD25, but responded to iRBCs by secretion of IFN-, which was potentiated by exogenous IL-15. By contrast, NK cells from Ugandan donors showed higher background CD25 expression and signs of in vivo/ex vivo preactivation and enhanced responsiveness to IL-15, but did not make any appreciable response to iRBCs. Potential explanations for these findings are explored and discussed. KIR genotype and KIR expression also varied between LSHTM and Ugandan donors. Specifically, expansions of KIR2DL1+ CD57+ NKG2C+ NK cell populations (possibly driven by human cytomegalovirus (HCMV) infection) were observed in the Ugandan donors. Conversely, percentages of KIR2DL3+ and 2DS4+ NK cells were higher among LSHTM donors, indicating that HLA genotype or allelic KIR polymorphisms may influence KIR expression. Finally, the formation of NK-iRBC conjugates, which may be a precursor to NK cellmediated killing of iRBCs, was observed in cells from nearly all donors, but did not correlate with other functional responses. Analysis of KIR expression and NK cell functional responses indicated that donors expressing inhibitory KIR2DL5 had reduced numbers of conjugates. Further experiments indicated that KIR2DL5 might be specifically upregulated after incubation with iRBCs, and that individuals carrying a normally non-expressed KIR2DL5 gene may be able to express this gene under certain circumstances. This tentatively suggests a role for KIR2DL5 during NK cell responses to malaria infection, and suggests a possible function for a common but frequently non-expressed gene. In summary, my work suggests that NK cell responses are strongly influenced by cytokine receptor and KIR expression, which in turn depend on NK cell maturation status. KIR expression patterns may in part explain differential NK responses to iRBCs between LSHTM and Ugandan donors. I also propose a possible role for KIR2DL5 in malaria infection, and a reason for the low expression of this gene in African populations.

In some species, including humans, red blood cells (RBCs) under low shear stress tend to clump together and form into regular stacks called rouleaux. These stacks are not static, and constantly move and break apart. This phenomenon is referred to as red blood cell aggregation and disaggregation. When modelled as a single liquid, blood behaves as a non-Newtonian fluid. Its viscosity varies, mainly due to the aggregation of RBCs. The aim of this research is to develop a mesoscale computational model for the simulation of RBCs in plasma. This model considers RBC interaction and aggregation to predict blood-flow characteristics such as viscosity, rouleaux size and velocity distribution. In this work, the population-balance modelling (PBM) approach is utilized to model the RBC aggregation process. The PBM approach is a known method that is used for modelling agglomeration and breakage in two-phase flow fluid mechanics to find aggregate size. The PBM model is coupled to the incompressible Navier-Stokes equations for the plasma. Both models are numerically solved simultaneously. The population-balance equation has been used previously in a more restricted form, the Smoluchowski equation, to model blood viscosity, but it has never been fully coupled with the Navier-Stokes equation directly for the numerical modelling of blood flow. This approach results in a comprehensive model which aims to predict RBC aggregate size and their velocities for different flow configurations, as well as their effects on the apparent macro-scale viscosity. The PBM approach does not treat the microscopic physics of aggregation directly but rather uses experimental correlations for aggregation and disaggregation rates to account for the effects of aggregation on the bulk. To find the aggregation rate, a series of experiments on RBC sedimentation due to gravity is designed. In these tests, aggregated RBCs (rouleaux) tend to settle faster than single RBCs and, due to low shear stresses, disaggregation is very low and can be neglected. A high-speed camera is used to acquire video-microscopic pictures of the process. The size of the aggregates and their velocities are extracted using image processing techniques. For image processing, a general Matlab program is developed which can analyze all the images and report the velocity and size distribution of rouleaux. An experimental correlation for disaggregation rate is found using results from a previous steady-state Couette flow experiment. Aggregation and disaggregation rates from these experiments are used to complete the PBM model. Pressure-driven channel flow experiments are then used for the final validation of the model. Comparisons of the apparent viscosity of whole blood in previous experiments show reasonable agreement with the developed model. This model fills a gap between micro-scale and macro-scale treatments and should be more accurate than traditional macro-scale models while being cheaper than direct treatment of RBCs at the micro-scale.

Le globule rouge est une cellule simple avec l’une des fonctions les plus importantes de l’organisme : participer aux échanges gazeux et fournir l’oxygène aux tissus. C'est un disque biconcave hautement élastique grâce à un réseau de protéines du cytosquelette et de protéines membranaires spécifiques. La fonction, ainsi que la structure des globules rouges sont altérées dans plusieurs pathologies humaines telles que les hémoglobinopathies et les anomalies de membrane. La drépanocytose est une maladie héréditaire génétique caractérisée par une hémoglobine anormale qui polymérise en conditions hypoxiques provoquant la déformation des globules rouges circulants. La drépanocytose se caractérise également par une anémie hémolytique chronique et des crises vaso-occlusives douloureuses dues à l'obstruction des capillaires.Dans le but de mieux comprendre les mécanismes responsables des manifestations cliniques, nous avons étudié les propriétés mécaniques et adhésives des globules rouges de patients atteints de drépanocytose en évaluant 1) l'impact de contraintes mécaniques répétées sur la survie des globules rouges à l'aide d'un appareil microfluidique qui mime la microcirculation et 2) le rôle du stress oxydant dans l'activation des protéines d'adhérence érythroïde.Nous avons conçu une puce microfluidique et montré que le stress mécanique est un paramètre critique sous-jacent à l'hémolyse intravasculaire dans la drépanocytose et que des taux intracellulaires élevés d'hémoglobine fœtale préviennent cette lyse. Outre ces résultats, nous avons constaté que le traitement à l'hydroxyurée protège les globules rouges de la lyse lors d'un stress mécanique, même en l'absence d'expression de l'hémoglobine fœtale. D'autre part, nous avons étudié la structure et la fonction de la protéine d'adhérence érythroïde Lu/BCAM dans des conditions oxydantes en utilisant des approches biochimiques et d'imagerie. Nous avons observé que le stress oxydant active la fonction adhésive de Lu/BCAM par des modifications post-traductionnelles qui modifient sa distribution membranaire. Nous décrivons un nouveau mécanisme qui affecte les interactions en cis de Lu/BCAM à la surface cellulaire et qui pourraient expliquer l'adhérence anormale des globules rouges à la laminine en l'absence d'événements de phosphorylation.En conclusion, nous avons développé un modèle microfluidique reproduisant les dimensions physiologiques des microvaisseaux humains, permettant d’évaluer les caractéristiques cellulaires jusque-là inexplorées dans la drépanocytose. Nous montrons que le stress mécanique répété est en partie responsable de l'hémolyse chez les patients atteints d'anémie falciforme, ce qui pourrait contribuer aux niveaux élevés de stress oxydant en raison de l'hème libre dans la circulation. Nos travaux démontrent l'importance de la dimension mécanique dans l’obstruction des capillaires et la contribution critique du stress oxydant dans l'adhérence anormale des globules rouges dans cette maladie. Améliorer la déformabilité des globules rouges et cibler le stress oxydant pour inhiber l'adhérence des globules rouges serait une stratégie prometteuse pour cibler les principales caractéristiques de cette pathologie et alléger le fardeau de la maladie
The red blood cell is a simple cell with one of the most important functions in the organism, that is fulfilling the gas exchange function and delivering oxygen to the tissues. It is a highly elastic biconcave disk thanks to a network of specific skeletal and membrane proteins. The function and structure of the red cell are altered in several human pathologies like hemoglobinopathies and membrane disorders. Sickle cell disease is a genetic hereditary disorder characterized by abnormal hemoglobin that polymerizes under hypoxic conditions leading to the sickling and alteration of circulating red cells. The hallmarks of sickle cell disease are hemolytic anemia and painful vaso-occlusive crises due to the obstruction of fine capillaries.With the aim of better understanding the mechanisms behind these clinical manifestations we investigated the mechanical and adhesive properties of red blood cells from patients with sickle cell disease by assessing 1) the impact of repeated mechanical stress on red cell survival using a microfluidic device that mimic human microcirculation, and 2) the role of oxidative stress in the activation of erythroid adhesion proteins.We designed a microfluidic device that allowed us to show that mechanical stress is a critical parameter underlying intravascular hemolysis in sickle cell disease and that high intracellular levels of fetal hemoglobin protect against lysis. Furthermore, we found that treatment with hydroxyurea protects red blood cells from lysis upon mechanical stress even in the absence of fetal hemoglobin expression. On the other hand, we investigated the structure and function of the erythroid adhesion protein Lu/BCAM under oxidative conditions using biochemical and imaging approaches. We observed that oxidative stress activates the adhesive function of Lu/BCAM through post-translational modifications that alter its membrane distribution. We describe a novel mechanism that affects Lu/BCAM cis-interactions at the cell surface that might account for the abnormal adhesion of sickle red cells to laminin in the absence of phosphorylation events.In conclusion, we developed a microfluidic device replicating the physiological dimensions of human microvessels that allows assessing previously unexplored cellular characteristics in sickle cell disease. We show that repeated mechanical stress is partly responsible for hemolysis in patients with sickle cell disease, which might contribute to the high levels of oxidative stress because of free heme in the circulation. Our work demonstrates the importance of the mechanical dimension in the blockade of small capillaries and the critical contribution of oxidative stress in the abnormal adhesion of red cells in this disease. Improving red cell deformability and targeting oxidative stress to inhibit red cell adhesion would be promising strategies to target the main hallmarks of this pathology and alleviate the disease burden

This project used mechanical testing and imaging techniques to understand how red blood cells age in an in vitro environment, and identified markers of red blood cell product quality. The damage caused to the cells by cold storage could lead to a reduced transfusion efficiency and potential improvements to current storage protocols were proposed as a result of this research.

The ability to measure the oxygen saturation, oximetry, of retinal blood both non-invasively and in-vivo has been a goal of eye research for years. Retinal oximetry can in principle be achieved from the measurement of the reflectance spectrum of the ocular fundus. Oximetry calculations are however complicated by the scattering of red blood cells, the different pathways of light through blood and the ocular tissues that light interacts with before exiting the eye. The goal of this thesis was to investigate the influence of red blood cell scattering for different light paths relevant to retinal oximetry. Results of in-vitro whole blood experiments found calculated oxygen saturation differences between blood samples measured under different retinal light paths, and these differences did not depend on the absorbance path length. We also showed that the calculated oxygen saturation value determined by a multiple linear regression Beer-Lambert absorbance model depended on the wavelength range chosen for analysis. The wavelength dependency on the calculated oxygen saturation value is due in part to the correlation that exists between the oxyhaemoglobin and deoxyhaemoglobin extinction coefficient spectra and to errors in the assumptions built into the Beer-Lambert absorbance model. A wavelength region with low correlation between the oxyhaemoglobin and deoxyhaemoglobin extinction coefficients was found that is hypothesized to be a good range to calculate oxygen saturation using a multiple linear regression approach.

Aptamers are small DNA ligands that have been manually selected to strongly and specifically bind a target of interest. These molecules may prove superior to modern antibodies in a number of ways including price and reproducibility. One of the major advantages of using aptamers as opposed to antibodies is the relative speed of development. This, coupled with the repetitive nature of aptamer selection, means that the entire process is a possible target for automation. In the following experiments, a ssDNA aptamer is developed against the human red blood cell protein glycophorin A, partially through the novel use of a robotized benchtop. The process also utilizes an adapted protocol for emulsion PCR to further increase the efficiency of the selection process. After 11 rounds of selection, the DNA pools were sequenced leading to the generation of 14 potential aptamers. These aptamers were tested with the isolated protein and with human red blood cells resulting in several of the aptamers being deemed potential binders. Further work with these identified sequences could result in aptamers that can be reliably used to tag and delicately separate red blood cells from other cells of interest within blood, such as stem cells. The novel approaches to selection used in this work may also lead to quicker and more efficient generation of future aptamers.

The thesis reports on the application of advanced microanalytical techniques to answer a fundamental open question on the homeostasis of Plasmodium falciparum infected red blood cells, namely how infected cells retain their integrity for the duration of the parasite asexual reproduction cycle. The volume and shape changes of infected cells were measured and characterized at femtolitre resolution throughout the intraerythrocytic cycle using confocal microscopy. Fluorescence lifetime imaging and electron probe X-ray microanalysis were applied for the quantification of intracellular haemoglobin and electrolyte concentrations. The cytomechanical properties of uninfected and infected red cells were studied using a novel optical stretcher device, which enabled individual cells to be trapped and manipulated optomechanically in microfluidic channels. Combined, these methods offered a unique insight into the homeostatic and rheological behaviour of malaria-infected red cells. The results were analysed by comparison with predictions from a detailed physiological model of the homeostasis and volume regulation of infected cells, providing broad support to the view that excess haemoglobin consumptions by the parasite was necessary for the integrity of infected cells (the colloidosmotic hypothesis). The dissertation is introduced with an overview of malaria, red blood cells homeostasis and the changes induced by Plasmodium falciparum infection. In the following, this description is extended to an in-depth theoretical analysis of the infected red blood cell homeostasis, from which the need to characterise certain parameters arises. The subsequent chapters address sequentially the assessment of the haemoglobin and electrolyte concentration, cell shape and volume changes and ultimately alterations in cell elasticity. The experimental part is complemented with a comparison of the resulting data to the predictions from the theoretical analysis and an outlook on future work.

In this thesis we investigate the interaction between drugs and toxins with membranes using red blood cells (RBCs) as morphoelastic probes. Using fluctuation spectroscopy, we were able to probe the RBC mechanical response to a simulated diabetic environment and to investigate the effect of metformin, one of the most widely used medicines to treat diabetes. Healthy RBCs were incubated in high levels of glucose or glucose and metformin and their mechanical properties were tested upon the exposure to oxidation with hydrogen peroxide (H2O2). My results show that the response to oxidation and glycation is different for different donors, with some donors more susceptible to oxidation than others. I have also found that glycated cells are more susceptible to oxidation with H2O2 than control and metformin treated RBCs. Metformin treated RBCs show a response to oxidation similar to control cells which suggests that metformin may have some antihyperglycaemic and antioxidant effects which could preserve the RBCs membrane elasticity within the normal limits, counteracting the adverse effects of oxidative stress. The interaction between the RBC membrane and two of the Clostridium perfingens toxins, α-toxin and NetB, is next studied in this thesis. Using fluctuation and absorbance spectroscopy, changes in the RBCs morphology caused by the toxins can be monitored allowing us to describe the course of the toxin membrane interaction. I conclude that the two toxins studied in this thesis have two different mechanisms of action. Both toxins produce a decrease in the cell radius but through two different mechanisms. NetB causes a decrease in the cell radius by forming large pores in the red cell membrane allowing for quick lysis and the exchange of material across the membrane. Whereas α-toxin causes a decrease in the cell radius by hydrolysing specific lipids in the cell membrane without necessarily causing the formation of membrane pores. These differences in the interactions between the two toxins and the red cell membrane have distinct fingerprints in the evolution of the cell shape and membrane thermal fluctuation dynamics. Fluctuation and absorbance spectroscopy were also used to investigate the effect of nitroglycerine (GTN) on the RBC morphology and mechanical properties. This study was prompted by a recent report in the literature that related decreases in the viscosity of whole blood to changes in the membrane surface charge. My results show that changes in the electrophoretic mobility of GTN-treated RBCs strongly depend on the incubation time. Cells incubated in GTN for 5 minutes decreased their mobility by about 20% whereas cells incubated for 20 minutes increased their mobility by about the same amount. Further investigations on the RBC morphology showed that GTN causes changes in the RBC shape. The matching times scales between those experiments and the electrophoretic experiments made me conclude that RBCs shape may play a role in the electrophoretic mobility of the RBCs treated with GTN. The main results obtained in this thesis demonstrate the viability of the idea of using RBCs as morphoelastic probes, which can provide detailed information about the interaction of solutes of interest and the plasma membrane. At the end of this thesis I propose use of RBCs in such a capacity to monitor the progression of disease by comparing the cell elastic state to clinical markers of disease.

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Bachelors
Arts and Sciences
Humanities

Background: The Community Intravenous Therapy (CIVT) service gives patients the benefit of receiving IV therapies (including blood transfusion) in their own home, avoiding hospital admission. It is important to ensure this service can be offered to as many patients as possible. If red cell transfusion could be safely performed over a shorter duration (accelerated transfusion), this could theoretically increase the capacity of the service without additional resource. Red cell transfusions are usually administered over a minimum of 90 minutes to a maximum of four hours per unit. It was proposed that one unit could be given in 60 minutes (up to a maximum of 2 units per transfusion episode) to medically selected patients who do not have heart failure or other risk factors for circulatory overload. Methods: Physiological tolerability and safety of accelerated transfusion was evaluated by clinically assessing patients for symptoms and signs of transfusion-associated circulatory overload (TACO) after standard and accelerated rate transfusions. The impact on service capacity and staff resource was evaluated by auditing home transfusion workload data to determine the number of patients who were eligible for accelerated rate transfusion and the potential impact this had on treatment delivery time. Patient and practitioner experiences of accelerated transfusion were evaluated by conducting thematic analysis on semi-structured interviews to assess the acceptability and desirability of service change. Results: When accelerated red cell transfusion was performed on medically selected patients who had been screened for risk factors for circulatory overload, accelerated transfusion appeared to be safe. None of the patients in the study (n=25) developed transfusion associated circulatory overload across 269 accelerated transfusions performed. The mean arterial pressure appeared to statistically significantly increase up to 24 hours after blood transfusion regardless of whether it was infused at a standard or accelerated rate, with the group mean remaining within the normal range (standard rate transfusion: p = 0.0441; accelerated rate average across three transfusions: p = 0.009). There was no statistically significant difference between pre and post-transfusion mean arterial pressure measurements when standard and accelerated rate transfusions were compared (average across three accelerated rate transfusions: p = 0.473), showing that accelerated transfusion itself did not cause an increase in mean arterial pressure above that of standard rate transfusion. A significant proportion of haematology patients (57%, 26/46) were medically eligible for accelerated transfusion, and 49% of total transfusion episodes (224/459) were performed as such. Performing accelerated transfusion on eligible patients could potentially save 105 nursing hours, allowing an additional 35 three hour visits or 26 four hour visits per year. Accelerated transfusion was well received by patients. Positive themes from the data included less time receiving healthcare allowing freedom and time to do other things, improvements in comfort and altruism from knowledge that other patients and the service was benefitting. CIVT practitioners were highly motivated and positive about accelerated transfusion. Themes included satisfaction in seeing positive benefits in quality of life and social aspects of patient's lives; improved continuity of care, better work scheduling; increased service capacity, job satisfaction; better working conditions and professional autonomy in clinical decision-making. Conclusion: Accelerated red cell transfusion appears to be safe in medically selected patients. It can potentially increase service capacity through efficient use of staff resource whilst maintaining a safe and high quality service. Understanding of patient and practitioner experience suggested that changing the service to offer accelerated transfusion would be both acceptable and desirable.

De récents travaux ont montré que les voies métaboliques sont fortement impliquées dans la régulation de l’engagement et la différentiation des cellules souches hématopoïétiques (CSHs) vers les différentes lignées hématopoïétiques. Les nucléosides sont des métabolites et précurseurs indispensables pour la biosynthèse des nucléotides, et dont la disponibilité intracellulaire dépend de leur transport à travers la membrane plasmique. Cependant, le rôle des transporteurs de nucléosides et de nucléotides dans l’engagement érythroïde des CSHs reste mal défini. Au cours de ma thèse, l’analyse fonctionnelle des cellules sanguines de patients déficitaires de ENT1 ou ABCC4 a démontré pour la première fois que le transport des nucléosides (ENT1) et nucléotides (ABCC4) est essentiel pour maintenir (i) le métabolisme des nucléotides cycliques et désoxynucléotide, (ii) une morphologie et une déformabilité normales des globules rouges, (iii) la phosphorylation des protéines de la membrane érythrocytaire ainsi que l’organisation du cytosquelette, (iv) la physiologie plaquettaire et (v) l’érythropoïèse ex vivo. De plus, nos expériences mécanistiques ont démontré que ENT1 joue un rôle crucial dans l’engagement des CSHs vers la lignée érythroïde en contrôlant le métabolisme des nucléotides cycliques et l’activation des facteurs de transcriptions érythroïdes. Ce travail a également montré qu’une mutation compensatoire dans le gène ABCC4, retrouvée chez deux patients ENT1null, diminuait les altérations érythroïdes chez ces patients, ce qui confirme le rôle de ces deux transporteurs dans la régulation intracellulaire de l’AMP cyclique. De plus, ce résultat a été confirmé par un modèle souris Ent1-/- puisque le traitement de ces souris avec un inhibiteur de ABCC4 augmente l’engagement érythroïde des CSHs, l’érythropoïèse et l’hématocrite. En outre, une étude protéomique et génomique sur des sujets PEL-négatif a montré qu’une large délétion dans le gène ABCC4 est responsable de ce groupe sanguin. L’inactivation du gène ABCC4 dans une lignée cellulaire par CRISPR-Cas9 a confirmé que l’antigène de groupe sanguin PEL est portée par la protéine ABCC4. Contrairement à ENT1, l’absence totale de ABCC4 est associée à une altération de la fonction plaquettaire sans anomalie de la lignée érythroïde et dont le mécanisme de compensation n’implique pas ENT1. En conclusion, mon travail de thèse dévoile un nouveau mécanisme moléculaire régulant l’érythropoïèse, et démontre le rôle important du métabolisme des nucléotides dans l’engagement des CSHs et dans la biologie érythroïde. Nos résultats ouvrent de nouvelles perspectives pour le développement de stratégies thérapeutiques pour le traitement de l’anémie basées sur la disponibilité intracellulaire des nucléosides et des nucléotides
Recently, metabolic pathways have emerged as critical components in the regulation of hematopoietic stem cell (HSC) renewal, as well as in lineage commitment and differentiation. Nucleosides are major metabolite precursors for nucleotide biosynthesis and their availability in HSCs is dependent on their transport through specific membrane transporters. However, the role of nucleoside and nucleotide transporters in the differentiation of HSCs to the erythroid lineage remains undefined. In the present work, we demonstrate for the first time that nucleoside (ENT1) and nucleotide (ABCC4) transport is essential for the (i) metabolism of cyclic nucleotides and deoxynucleotides (ii) erythrocyte morphology and deformability, (iii) erythrocyte membrane protein phosphorylation and skeleton organization, (iv) platelet function, and (v) ex vivo erythropoiesis. Interestingly, functional and mechanistic experiments showed that the equilibrative nucleoside transporter 1 (ENT1) controls the nucleotide metabolism and the activation of erythroid transcription factors. This finding explains the role of ENT1 in maintaining the optimal erythroid commitment and differentiation of HSCs. On the other hand, although the downregulation of the ABC nucleotide transporter ABCC4 attenuates the erythroid disorders in ENT1null patients, its total loss results in abnormal platelet function without erythroid disorders. Importantly, we demonstrate that a large deletion in ABCC4 gene is associated with the PEL-negative null blood phenotype. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Targeting ENT1 and ABCC4 transporters either by knockout or pharmacological inhibition in mice lead to a marked change in blood cells counts. Specifically, the genetic deletion of Ent1 in mice results in a decreased RBC production and macrocytosis. While mice treated with ABCC4 inhibitor increased the erythroid commitment of HSCs, and enhanced erythropoiesis as demonstrated by the increase of circulating erythrocytes and reticulocytes. Overall, our findings reveal a new molecular mechanism regulating erythropoiesis and highlight the important role of nucleotide metabolism in the lineage commitment of HSCs and erythroid biology. Our findings open new avenues for the development of novel therapeutic strategies for the treatment of anemia

Objectives: The new red blood cell (RBC) parameters from automated haematology analyzers, such as reticulocyte haemoglobin content and percentage of hypochromic red cells or equivalent, are useful in the laboratory assessment of iron deficiency anaemia (IDA). However, the clinical utility of these parameters are confounded by thalassaemia trait (TT) especially in geographical areas of high thalassaemia prevalence. We aimed to evaluate the new formula derived from some red cell parameters on the Beckman Coulter DxH800 in distinguishing between IDA and TT in Hong Kong population. Methods: A total of 246 normal subjects, 102 patients with IDA and 115 subjects with TT were accrued for the study. Concurrent chronic disorder and ovelapped IDA and TT cases were excluded. The new red cell parameters studied were red blood cell size factor (RSF), low haemoglobin density (LHD%), microcytic anaemia factor (MAF), standard deviation of conductivity of the non-reticulocyte population (SD-C-NRET) and unghosted cell (UGC). Comparison between groups was performed by student t-test and the diagnostic performance of the parameters was determined by receiver operating characteristic (ROC) curve analysis. Results: Both LHD% and RSF were significantly higher in IDA than TT, whereas MAF was significantly lower. Although the biological basis was uncertain, SD-C-NRET was significantly lower in IDA than TT and showed the best diagnostic performance as a single parameter. A formula ((RBC+HB)*(HCT+SD-C-NRET))/(RDW-SD ) was devised to distinguish between IDA and TT. With a cutoff value of 23, the area under curve (AUC) was 0.995 (95% CI of 0.99 – 1.00), the sensitivity was 97% and the specificity was 99.1%. This formula was superior to other discriminant functions in the literature. The UGC number was not significantly different between alpha- and beta-TT. Conclusions: The new RBC parameters on Beckman Coulter DxH800 provided useful information in distinguishing between IDA and TT, which is important for clinical decision making and streamlining further laboratory testing. This index can be used to screen patients who are likely to be IDA or TT for further hematological studies to confirm diagnosis.
published_or_final_version
Pathology
Master
Master of Medical Sciences

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 95-103).
Malaria kills over 500,000 people annually, the majority of whom are children under five years old in sub-Saharan Africa. This disease is caused by several parasite species, of which Plasmodium falciparum is associated with the highest mortality. The clinical manifestations of malaria are associated with the phase of infection where parasites develop within red blood cells (RBCs). Infected RBCs can adhere to the host microvasculature, triggering inflammatory responses in affected organs that contribute to the pathophysiology of life threatening cerebral malaria and pregnancy-associated malaria. The expression of specific Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) variants on the RBC surface is associated with severe disease, such as VAR2CSA-mediated placental sequestration during pregnancy-associated malaria. While parasite proteins expressed on the surface of infected RBCs are linked to disease pathogenesis, this surface proteome is poorly characterized. Identifying parasite-derived antigens on the infected RBC surface could facilitate diagnosis, monitoring, and prevention of sequestration. To interrogate the infected RBC surface proteome, we require a panel of affinity reagents that robustly distinguish the parasite-derived proteins from the elaborate RBC surface milieu. Nucleic acid aptamers are widely used in biological applications for their high specificity and affinity to targets and are highly suitable for malaria applications. Efficiently generating aptamers against complex targets-such as whole cells-remains a challenge. Here we develop a novel strategy (I-SELEX) that utilizes inertial focusing in spiral microfluidic channels to stringently partition cells from unbound oligonucleotides. We use I-SELEX to efficiently discover high affinity aptamers that selectively recognize distinct epitopes present on target cells. Using first an engineered RBC model displaying a non-native antigen and, second, live malaria parasite-infected RBCs as targets, we establish suitability of this strategy for de novo aptamer selections. We demonstrate recovery of a diverse set of aptamers that recognize distinct epitopes on parasite-infected RBCs with nanomolar affinity, including an aptamer against the protein responsible for placental sequestration, VAR2CSA. These findings validate I-SELEX as a broadly applicable aptamer discovery platform that enables identification of new reagents for mapping the parasite-infected RBC surface proteome at higher molecular resolution to potentially contribute to malaria diagnostics, therapeutics and vaccine efforts.
by Christina M. Birch.
Ph. D.