Red blood cells (RBCs) are vital for oxygen delivery to tissues and constitute the vast majority of all cells in blood. After leaving the red bone marrow as mature cells, RBCs have a lifespan of approximately 120 days before they are removed from the circulation by macrophages, mainly in the spleen and liver. RBC transfusion is a common therapy in modern healthcare. Major surgery, numerous cancer treatments and other, often lifesaving, interventions would be unthinkable without available blood supply. For this reason, hospitals store donated RBCs in blood banks. The metabolic and structural changes that occur during prolonged storage of RBCs (the storage lesion) have been studied in detail in vitro and include oxidative stress, a reduction in glycolysis, increased membrane rigidity and shedding of microparticles from the RBC membrane. Stored RBCs share several features of senescent RBCs, but also with RBCs undergoing an apoptotic-like process called eryptosis. A consequence of the storage lesion is the fact that as much as 25% of stored RBCs could be rapidly removed from the circulation within 24 hours after transfusion. The mechanisms behind this rapid macrophage-mediated recognition and removal of stored RBCs, and its immunological consequences, remain largely unknown. Therefore, the aims of this thesis were to investigate if cryopreserved human RBCs induced an inflammatory response following autologous transfusion into healthy volunteers, and to further understand the mechanisms behind macrophage recognition of stored RBCs in vitro and in vivo. Autologous transfusion of two units of cryopreserved RBCs into healthy human recipients was found to be associated with an increased extravascular RBC elimination already at 2 hours after transfusion. However, there were no signs of an increased production of any of the investigated pro-inflammatory cytokines, indicating that an increase in the destruction of RBCs per se did not induce an inflammatory response. Eryptosis is a form of induced RBC death associated with an increased cytoplasmic Ca2+ uptake. We found that a subset of human RBCs increased their Ca2+ permeability during prolonged storage at +4°C. Using a murine model, to further understand how RBCs with an increased Ca2+ permeability were eliminated by phagocytic cells in the spleen, it was found that such RBCs were taken up by marginal zone macrophages and dendritic cells (DCs) in a manner distinct from that of naturally senescent RBCs. The DC population particularly efficient in this process expressed CD207 and are known for their ability to promote immunological tolerance. Eryptotic cell uptake was not regulated by the phagocytosis-inhibitory protein CD47 on the RBCs. To investigate how RBCs damaged during liquid storage are recognized and taken up by macrophages, a model to store and transfuse murine RBCs was developed. This storage model generated murine RBCs with several characteristics similar to that of stored human RBCs (i.e. loss of ATP, formation of RBC microparticles and rapid clearance of up to 35% of the RBCs during the first 24 h after transfusion). In vitro phagocytosis of human as well as murine stored RBCs was serum dependent and could be inhibited by blocking class A scavenger receptors using fucoidan or dextran sulphate. In conclusion, the findings of this thesis contribute to further understanding how changes inflicted to RBCs during storage direct the fate of these cells in their interaction with cells of the immune system after transfusion. The observation of an increased Ca2+ permeability of stored RBCs, and the possible recognition of such cells by tolerance-promoting DCs, in combination with the findings that class A scavenger receptors and serum factors may mediate recognition of stored RBCs, may result in novel new directions of research within the field of transfusion medicine.

Studies were conducted to investigate the effects of in ovo injection of selenium (Se) either as seleno-methionine (Se-Met) or sodium selenite (Na2SeO3) into the yolk of incubating eggs on tissue Se concentration, embryo livability, lipid peroxidation, immune response and growth performance. When white-shelled eggs were injected with 0.1ml of solutions providing 0, 2.5, 5, 10 or 20 μg Se/egg, no detrimental effects on embryo viability at 20 days of incubation were noted. The effects on tissue Se concentrations suggested that Se-Met and Na2SeO3 were metabolized differently by the chick embryo. In a subsequent study using injection doses up to 60 μg/egg, a greater linear response in tissue Se was obtained with Se-Met, compared with Na2SeO3 (P < 0.01). Minimal changes in heart and breast muscle Se concentrations were noted above the 40 μg dose when Na2SeO3 was used (P > 0.05). In a study with broiler eggs, injection doses of 0, 2.5, 5, 10, 20 and 40 μg Se/egg were used. Se-Met or Na2SeO3 at doses up to 40 μg Se/egg had little effect on embryo viability. Injecting Se-Met resulted in greater tissue Se accumulation than Na2SeO3 at 20 days of incubation. In another study with broiler eggs using injection doses up to 40 μg Se/egg, Se-Met injection resulted in higher hatchability, reduced lipid peroxidation in the lung and heart muscle of the embryos after 20 days incubation and higher Se concentrations in heart and breast muscle of hatched chicks through 7 days and in lung through 21 days of growth. In a feeding trial with broiler breeder hens, adding 0.3 mg/kg of Se as Se yeast or Na2SeO3 to the diet improved tissue Se status at hatching of progeny chicks. Taken together, these results indicate that injection of Se into the yolk of incubating eggs may be useful for enhancing Se status during embryonic and early post-hatch development. Therefore, the improvement in Se status using this method in conjunction with dietary Se supplementation of breeder hens would be much greater than with only using dietary supplementation.

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.

Segundo a legislação brasileira e normas internacionais, os concentrados de hemácias (CH) devem ser mantidos sob controle rigoroso de temperatura, armazenados de 2 a 6°C e transportados de 1 a 10°C, por até 24 horas. Entretanto, não existem evidências científicas de que estes valores sejam relevantes para manutenção de qualidade, segurança e viabilidade dos CH. O objetivo deste trabalho foi monitorar parâmetros laboratoriais in vitro em dois tipos de CH (CPDA-1, preparado pela metodologia do plasma rico em plaquetas e em SAGM preparado pelo buffy-coat), no decorrer de condições normais de armazenamento e em temperaturas de transporte inadequadas. O trabalho foi executado em três etapas. Na primeira etapa, foram estabelecidos os valores de referência para os dois tipos de CH armazenados em condições normais, através de testes laboratoriais semanais para determinação de hematócrito, hemoglobina total, hemoglobina plasmática, grau de hemólise, glicose, lactato, desidrogenase lática e potássio. Na segunda etapa, amostras de CH foram submetidas a temperaturas entre -2°C e +1°C, ou entre 11 e 15°C, por um período de 6 horas ou 24 horas, no 14º dia de armazenamento. Na terceira etapa os CH foram expostos a condições de estresse extremo, submetendo-os a temperaturas entre -2°C e +1°C ou entre 22°C e 25°C, por período de 48 horas. Os resultados dos testes laboratoriais das segunda e terceira etapas foram comparados com controles e aos valores de referência da primeira etapa. Nas unidades de CH em CPDA-1 foram encontrados valores significativamente mais elevados de potássio, hemoglobina livre, grau de hemólise, lactato e LDH do que nos CH em SAGM, enquanto que a concentração de glicose foi muito mais baixa em CH-CPDA1. Na 2ª etapa, exposições por até 24 horas em temperaturas de até - 2°C ou até 15°C não causaram diferenças significativas nas dosagens de hemoglobina livre, grau de hemólise, glicose, lactato, LDH e potássio quando comparados aos controles. Amostras de CH-CPDA1, quando avaliadas imediatamente após período de 48 horas de exposição, apresentaram resultados de glicose superiores aos controles quando submetidas a -2°C e inferiores ao controle quando a 25°C. Exposições por até 48 horas em temperaturas de até -2°C ou até 25°C não causaram diferenças significativas na hemoglobina livre, grau de hemólise, lactato, LDH e potássio quando comparados aos controles, nos dois tipos de CH. Nesse trabalho foram elaborados perfis padrão de parâmetros laboratoriais para dois tipos de CH. Estes perfis padrão constituirão uma ferramenta de grande utilidade para controle de qualidade e monitoramento das lesões de armazenamento em bolsas de CH produzidas na Fundação HEMOMINAS. Nossos resultados demonstraram que bolsas dos dois tipos de CH submetidas a temperaturas de - 2°C ou 14°C por períodos inferiores a 24 horas não apresentam alterações significativas nos parâmetros laboratoriais avaliados in vitro e permitiram algumas elaboramos recomendações práticas e importantes para serviços de hemoterapia. Entretanto, ainda não existem ainda evidências suficientes na literatura para modificação das normas de temperatura e tempo de transporte atualmente preconizadas pela legislação nacional e internacional. / The Brazilian legislation and international guidelines require that red blood cell concentrates (RBC) are kept under a strict temperature control, stored between 2 and 6°C and transported between 1 and 10°C for up to 24 hours. Nevertheless, there is no scientific evidence that these values are relevant to maintain RBC quality, safety and viability. This study was performed in order to monitor in vitro laboratorial variables of two different RBC types (CPDA-1, prepared using the platelet-rich plasma methodology, or SAGM, prepared using the buffy-coat) stored under normal conditions or under inadequate temperatures during transportation. The study had three phases. In the first phase, weekly laboratorial tests were performed to establish the reference values of hematocrit, total hemoglobin, plasma hemoglobin, rate of hemolysis, glucose, lactate, lactic acid dehydrogenase (LDH) and potassium, for each of the RBC types stored under normal conditions. In the second phase, RBC samples were submitted to temperatures between -2°C and +1°C, or between 11 and 15°C, for 6 hours or 24 hours, on the 14th storage day. Laboratorial test results were compared to a control group (2-6°C) and to the reference values established in the 1st phase. In the third phase, RBCs were exposed to extreme stress, i.e., temperatures between -2°C and +1°C or between 22°C and 25°C, for 48 hours, and the laboratory test results were compared to a control group. CPDA1-RBC had higher levels of potassium, free hemoglobin, rate of hemolysis, lactate and LDH compared to SAGM-RBC, whereas glucose was significantly lower in CPDA1. In the second phase, exposure for up to 24 hours in temperatures until -2°C or 15°C had no effect on free hemoglobin, rate of hemolysis, glucose, lactate, LDH and potassium when compared to control. CPDA1 samples right after the 48-h exposure had higher glucose levels than controls when kept at -2°C and lower than control if exposed to 25°C. Exposures up to -2°C or 25°C for up to 48 hours had no effect on free hemoglobin, rate of hemolysis, lactate, LDH and potassium when compared to control groups, both for CPDA1-RBC and SAGM-RBC. In this study, it was established standards for laboratory analyses for two different RBC types. Such standards will comprise valuable and useful tools for the quality control and monitoring of storage lesions of RBC units produced by Fundação HEMOMINAS. Our results demonstrate that units from both RBC types submitted to -2°C or 14°C for up to 24 hours had no significant changes in in vitro laboratory variables and allow some practical and important recommendations for hemotherapy services. Nevertheless, there are not enough evidences in the literature to support changes in the current guidelines for transportation recommended by national and international legislation.

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.

In USA more than 12.5% of all infants are born preterm. Approximately 75% of all perinatal deaths occur among these preterm infants. Preterm infants are frequently very low in birth weight (VLBW) and receive multiple red blood cell (RBC) transfusions. These transfusions pose increased risk of infections and other complications. Since erythropoietin (EPO) stimulates RBC production, EPO treatment of VLBW infants has received attention as a modality for reducing transfusions in this group. The overall hypothesis of this work is that treatment optimization of EPO of anemia in preterm infants requires a comprehensive knowledge of the behavior of RBC and the pharmacokinetic/pharmacodynamics (PK/PD) relationship between EPO and erythropoiesis. Under that overall hypothesis, the specific aims were: 1) To describe erythropoiesis dynamics in preterm infants, 2) To determine and explain the variability in the response to EPO in preterm infants, 3) To evaluate newborn sheep as an experimental model for erythropoiesis in preterm infants, 4) To test the hypothesis that RBC lifespan is shortened under acute hypoxic stress conditions, 5) To test the hypothesis that EPO receptor (EPOR) pool size increases under hypoxic stress conditions and the change in EPOR pool size can be predicted using EPO clearance measurements, 6) To describe the effect of EPOR pool size changes on erythropoiesis kinetics. A model that describes erythropoiesis dynamics in preterm infants as a function of the plasma EPO concentration is presented in Chapter 2. In Chapter 3, several covariates are tested for their ability to identify infants with good EPO responsiveness. The lamb is also tested as an animal model for the erythropoiesis in preterm infants (Chapter 4). In Chapters 5-7, the effect of hypoxic stress conditions on RBC survival was explored defining the relation between the efficacy of EPO and survival of RBC produced as a result of EPO administration. RBC lifespan measurement methods are reviewed in Chapter 5. In Chapter 6, a new methodology for the measurement of RBC lifespan under stress conditions is developed. This new methodology is applied in Chapter 7 to explore the effect of hypoxic stress conditions on the survival of RBC. The study presented in Chapter 8 is undertaken to investigate changes in both EPOR pool size and EPO clearance under hypoxic conditions. An erythropoiesis model that accounts for change in the EPOR pool size under stress conditions is presented in Chapter 9. Analysis of erythropoiesis dynamics in preterm infants demonstrated that a three fold increase in the amount of RBC produced by preterm infants is possible by EPO administration. This emphasizes the potential of using EPO for the management of anemia in preterm infants. Covariate screening identified gestational age as a potential marker for the responsiveness to EPO treatment. PD analysis results in lambs demonstrated similarities between lambs and preterm infants in different erythropoietic characteristics such as sensitivity to EPO in producing RBC, Hb production rate before birth and blood volume. Survival analysis demonstrated that RBC lifespan is not shortened under acute hypoxic conditions Analysis of EPOR mRNA level demonstrated an up regulation of EPOR level under stress conditions accompanied by a parallel increase in EPO clearance. EPOR up regulation under stress conditions level was incorporated in a PD model presented in Chapter 9. The developed model provides a framework for optimizing EPO dosing. Accordingly, an optimal dosing strategy should in general maximize the interaction between EPO and EPOR. Specifically, EPO should be administered when the number of EPOR are close to maximally up-regulated.

<p>Platelets are the smallest cells in the blood. They are formed in the bone-marrow and are important for the blood coagulation. Platelet tranfusions are given to patients propyhlactically before an operation but also in therapeutical purpose in connection with bleeding. It’s importent that the quality controls of the platelet concentrates are reliable.</p><p>ADVIA®60 (Bayer HealthCare) is a fully automated cell counter which uses impedance principle to count platelets in blood samples. The purpose of the study was to evaluate this new instrument for use in the blood bank of Akademiska Sjukhuset in Uppsala. The instrument was bought to be used for quality control of platelet concentrates. 30 samples from platelet concentrates, from both apheresis and from buffy coats, were analyzed 10 times each on ADVIA®60 and the coefficient of variation (CV) was calculated for each sample. CV variated from 0,8 % to 2,9 % which is good considering that according to Bayer HealthCare the CV should be < 5 % for thrombocytes on ADVIA®60. The instrument was newly calibrated when the study was performed. Platelet count can also be performed by immunological or optical principles.</p>

La microcirculation est rapidement altérée dans le sepsis et la persistance de ces altérations est associée à un mauvais pronostic. La microcirculation est composée de vaisseaux invisibles à l’œil (< 100 µm), de l’endothélium, du glycocalyx, des cellules musculaires lisses et des éléments sanguins dont les GR. De nombreuses études animales et humaines ont rapporté des altérations rhéologiques des GR dans le sepsis. Ces modifications comprennent une diminution de la déformabilité, une augmentation de l’agrégation et de l’adhérence globulaire. De plus, l’altération de la déformabilité peut induire des altérations du flux microcirculatoire dans des modèles expérimentaux animaux. Ces mêmes altérations rhéologiques sont rapportées dans le diabète. Dans cette pathologie, les GR présentent une diminution du contenu membranaire en AS, comme dans les processus de sénescence. La déformabilité des GR dépend des caractéristiques cellulaires incluant surtout les propriétés de la membrane, la géométrie cellulaire et dans une moindre mesure la viscosité cellulaire. Malgré la connaissance des altérations de la rhéologie dans le sepsis, peu de travaux, au contraire du diabète, s’interessent aux modifications de la membrane. Nous avons étudié, par analogie aux altérations globulaires rapportées dans le diabète, la membrane des GR de patients admis en soins intensifs pour un sepsis, et comparé à des GR de patients non septiques et de volontaires sains. Le contenu membranaire en AS était significativement diminué chez les patients septiques par rapport aux patients non-septiques et aux volontaires sains. De plus, les GR des patients septiques, analysés par une technique de cytométrie en flux indépendante de la température de l’échantillon, étaient rapidement plus sphériques (dans les 24 heures du sepsis) et incapables de modifier leurs formes en hypoosmolalité. Cette technique de cytométrie a par ailleurs aussi été utilisée pour l’analyse de GR de patients diabétiques et en insuffisance rénale terminale. La diminution du contenu en AS est aussi rapidement observée sur la transferrine, suggérant une augmentation de la concentration et/ou de l’activité de la neuraminidase, enzyme clivant l’AS. Dans un modèle de choc septique induit chez l’ovin, nous avons confirmé la rapidité de ce phénomène. En effet, la concentration en AS libre augmente dès la 15ième heure après induction du sepsis. In-vitro, nous avons pu reproduire les modifications de forme des GR observés chez les patients septiques par incubation de GR de volontaire avec de la neuraminidase, et ce en 10 heures, quelles que soient les concentrations utilisées. Ces modifications de forme et de membrane s’accompagnent d’une augmentation significative du contenu en lactate, suggérant une stimulation de la glycolyse érythrocytaire et en 2,3-DPG, facilitant la libération de l’O2 de l’Hb vers les tissus. Toutes ces modifications touchant la membrane des GR des patients de soins intensifs, surtout septiques, peuvent être responsables des altérations de rhéologie que nous avons observé grâce au LORCA sur une large population admis aux soins intensifs. Une meilleure compréhension des mécanismes conduisant aux altérations rhéologiques des GR dans le sepsis, et ses effets potentiellement déletères sur la microcirculation, sont nécessaires avant d’envisager les GR comme cible thérapeutique.

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.

Controlling fates of stem and progenitor cells is one of the central goals of regenerative medicine. However, conventional cell enumeration methods are unable to distinguish between the effects of cell death, proliferation, and differentiation through molecular interventions on the output of specific cell types. We have devised a strategy to simultaneously obtain proliferation and death rates in cultures of highly purified erythroid progenitors. The approach is based on combining cell-surface marker analysis, cell-division tracking and 7-amino-actinomycin-D staining to monitor cell death. A compartment model of cell proliferation was developed to evaluate cell generation-specific length of cell-division, rates of entry into division, and cell death, from the experimental cell-division tracking data obtained following stimulation with erythropoietin (EPO) and Stem cell factor (SCF). The results indicated that EPO and SCF, either as single factor or in combination, differentially affect the rates of differentiation, length of cell-division and rates of death.