Title of Diploma Thesis: Production and use of transfusion products in the District Hospital Trutnov. Aim of the work: The aim was to evaluate the production of transfusion products (TP) in the period 2014-2019 at the Transfusion and Hematology Department (THO) in the District Hospital Trutnov and their use for hemotherapy by individual medical disciplines. Futhermore, to compare the production of TP within the Czech Republic together with selected European countries. The incidence of post-transfusion reactions and other adverse events during the reporting period is also included. Material and methods: The methodological part describes the standard procedures of pre- transfusion examination using the column agglutination method from the company Grifols, S.A., laboratory examination of donors and transfusion products manufactured at the Transfusion Department Trutnov. Conclusion: Despite all the recommendations and efforts to reduce the use of transfusion products, this condition has not been proven both in our department, within the Czech Republic and in Europe. This work recommends deleucotization of transfusion products for the national and European part, unification of procedures for transfusion in a given disease, mandatory introduction of NAT testing to minimize the risk of transmission...

The manual differential count of leukocytes is a common analysis in the hematological laboratory. It is used for morphological assessment of the blood cells and can get valuable information according to diagnosis of hematological diseases. The microscopy assessment is dependent of a good staining result of the blood smear in order to get the best conditions to differentiate the different cell types and detect morphological or pathological findings.The aim of this study was to determine if it was possible to change the staining method without any compromises to the quality of the staining results. For this study 25 whole blood samples were collected, and blood smears was made and stained with four different staining protocols, including the one currently used in the routine practice at laboratoriemedicin Sundsvall. The samples were examined by three biomedical scientists and the staining quality of the cells was graded on a four-point scale. The statistical results with Friedmans and Wilcoxons signed-rank test showed differences between the methods on the nuclear and cytoplasm of lymphocytes and the nuclear of monocytes and neutrophils. The recommended staining protocol from the manufacturer was the method that had highest frequency of statistically significant differences compared to the other methods for those cell types. The differences were in favour for the other methods, and the current method showed the best performance. In conclusion it’s not recommended to continue the study with the manufacturer´s staining protocol, but its valuable to continue compare the best performed staining protocols.

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. <p>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. <p>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. <p>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.<p>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. <p> 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.<p>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. <p>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.<p>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.<p><p><p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: With the recent advances in the field of molecular biology, there is an increased need to integrate data on the various constituents of the cell in kinetic models that can predict and describe cellular behavior. When working towards a description of the entire cell using such kinetic models, the question arises: How do we compare different models for a given biological network? This is the central question addressed in my thesis and I developed and applied mathematical and computational methods for comparing dozens of existing models of erythrocyte and yeast glycolysis. To compare the steady-state behavior in models of erythrocyte glycolysis, I focussed on the function of the pathway, which is to supply the cell with Gibbs-free energy (γ- phosphate of ATP). I used supply-demand analysis in the framework of metabolic control analysis to make this comparison, which revealed that the ATP concentrations were homeostatically buffered at varying supply rates. I also applied this approach to compare steady-state behavior in models of yeast glycolysis, finding that they were not necessarily optimized for homeostatic maintenance of the ATP concentration and that in models for this organism the rate of ATP production is often determined by the supply reactions of glycolysis. In addition, I tested whether a kinetic model can describe novel behavior if it is adjusted to conditions different from those for which the model was originally constructed. More specifically, using a model of steady-state yeast glycolysis, I showed that small adjustments to the original enzyme concentrations are enough to obtain an oscillating model, which shows a remarkable resemblance to the experimentally observed oscillations. Importantly, some of these enzyme concentrations changes are known to occur during the pre-treatment of the cells which is necessary to obtain oscillatory behavior. To the best of my knowledge, the resulting model is the first detailed kinetic model that describes the experimentally observed strong synchronization of glycolytic oscillations in yeast populations. To analyze the dynamic behavior of yeast glycolytic models and to compare different models in terms of dynamics, I introduced a framework used in physics and engineering to create a vector based, two dimensional graphical representation of the oscillating metabolites and reactions of glycolysis. Not only was it possible to make a concise comparison of the set of models, but with the method I could also quantify the contribution of the interactions in the network to the transduction of the oscillations. Furthermore I could distinguish between different mechanisms of oscillation for each of the models, and demonstrated how the framework can be used to create such representations for experimental data sets. / AFRIKAANSE OPSOMMING: Met die onlangse vooruitgang in die veld van molekulere biologie, is daar ?n toenemende behoefte om data rakende die verskeie komponente van die sel in kinetiese modelle te integreer, om sodanig selgedrag te voorspel en te beskryf. As daar gepoog word om ’n beskrywing van die sel as geheel te verkry d.m.v. sulke kinetiese modelle, onstaan die vraag: Hoe vergelyk ons verskillende modelle van ’n gegewe biologiese netwerk? Dit is die sentrale vraag wat my tesis aanspreek en ek het wiskundige en numeriese metodes ontwikkel en toegepas om talle bestaande modelle van gis- en eritrosietglikolise te vergelyk. Om die bestendige-toestand gedrag in modelle van eritrosietglikolise te vergelyk, het ek gefokus op die funksie van die padweg, naamlik om die sel met Gibbs-vrye energie (γ-fosfaat van ATP) te voorsien. Ek het vraag-aanbod analiese in die raamwerk van metaboliese kontrole analiese gebruik om hierdie vergelyking te maak, wat getoon het dat die ATP konsentrasies homeostaties gebuffer was by verskillende aanbod tempos. Ek het ook hierdie aanpak gebruik om die bestendige-toestand gedrag in modelle van gisglikolise te vergelyk, en het bevind dat hulle nie noodwendig geoptimiseer is om ?n homeostatiese balans in die ATP konsentrasie te handhaaf nie, en dat in modelle vir hierdie organisme, die tempo van ATP produksie dikwels bepaal word deur die aanbod reaksies van glikoliese. Ek het verder ook bepaal of so ?n kinetiese model nuwe soorte gedrag kan beskryf, as dit aangepas word aan omstandighede wat verskil van dié waarvoor die model oorspronklik gekonstrueer was. Meer spesifiek, deur ?n model van bestendige-toestand gisglikolise te gebruik, kon ek wys dat klein veranderinge aan die oorspronkline ensiem konsentrasies genoeg was om ?n ossilerende model te verkry, wat opmerklik ooreenstem met die eksperimenteel waargenome ossilasies. Let ook daarop dat sommige van hierdie ensiem konsentrasie veranderinge plaasvind tydens die voorafbehandeling van die selle, wat essensieel is om die ossilasies waar te neem. Tot die beste van my kennis is die model wat ek met hierdie prosedures verkry het, die eerste gedetaileerde kinetiese model wat die eksperimenteel waargenome sterk sinkronisasie in ossilerende gis populasies voorspel. Om gis glikolitiese modelle te vergelyk in terme van hul dinamiese gedrag, het ek ?n raamwerk wat in fisika en ingeneurswese gebruik word, ingespan om ?n vektor-gebasseerde, twee dimensionele grafiese voorstelling van die ossilerende metaboliete en reaksies te maak. Hierdie raamwerk het dit nie net moontlik gemaak om ?n kompakte vergelyking van ?n stel modelle te maak nie, maar ek kon ook die bydrae van interaksies in die netwerk tot transduksie van die ossilasies kwantifiseer. Ek kon verder onderskeid tref tussen die verskillende ossilasiemeganismes vir elk van die modelle, en het ook gedemonstreer hoe die raamwerk gebruik kan word om sulke voorstellings vir eksperimentele datastelle te skep.

Objetivos: Transfusão de sangue é fundamental para a sobrevida de pacientes selecionados, porém é associada a complicações. A literatura é controversa em relação aos efeitos pulmonares, hemodinâmicos e inflamatórios da transfusão de glóbulos vermelhos (GV). Este estudo teve dois objetivos principais: 1- validar um modelo de transfusão homologa de GV estocados em suínos com hipovolemia aguda por hemorragia controlada; 2- avaliar os efeitos agudos da transfusão de GV nas trocas gasosas, mecânica respiratória, hemodinâmica e na resposta inflamatória pulmonar e sistêmica. Métodos: Este estudo foi dividido em duas etapas: 1.Coleta, processamento e estocagem por 14 dias de GV provenientes de um suino Agroceres®, avaliado antes (in vitro) e após (in vivo - marcação com cromato de sódio radioativo) à sua transfusão em suínos sadios , um autólogo e quatro homologos (n=cinco); 2. Outro grupo de suínos foi submetido à hemorragia aguda controlada (25% de sua volemia) e então dividido em dois grupos: grupo transfusão (n= oito) recebeu duas unidades de GV e solução de ringer lactato (RL) para restabelecer a volemia; grupo controle (n=oito) que recebeu somente RL. Ambos os grupos foram seguidos até 6horas após o final da ressuscitação volêmica. Dados hemodinâmicos e respiratórios foram coletados a cada hora após o inicio do estudo. Mediadores inflamatórios e expressão de RNAmensageiro(RNAm) foram medidos no plasma e no tecido pulmonar. Resultados: Houve recuperação de 97,5%±19% dos GV marcados com cromato de sódio radioativo 24 horas após a transfusão. Houve aumento significativo da saturação venosa mista, conteúdo arterial de oxigênio e dos níveis de hemoglobina e hematócrito no grupo transfundido comparado ao controle. Os parâmetros medidos para a avaliação da microcirculação e as trocas gasosas foram similares em ambos os grupos. Observou-se um aumento significativo na energia gasta na histerese pulmonar no grupo controle quando comparado ao grupo transfundido (p=0,002), bem como uma tendência á diminuição da energia inspiratória no grupo transfusão. As concentrações plasmáticas das diversas citocinas avaliadas antes e após a transfusão de GV mostraram-se abaixo dos limites de detecção do teste ELISA na maioria dos animais estudados em ambos os grupos; não houve diferença significativa nas concentrações de nitrato no plasma e no tecido pulmonar. Observou-se uma diferença discreta, porem estatisticamente significativa, entre os grupos na quantificação do RNAm da oxido nítrico sintetase induzida (iNOS) e da IL-21 no tecido pulmonar (aumento de 50% na iNOS e decréscimo de 50% na IL-21 no grupo transfundido comparado com o controle). Conclusão: Demonstramos a viabilidade \"in vitro\" e \"in vivo\" de GV suínos, estocados por até 14 dias. A transfusão de GV homólogos não causou alterações significativas na hemodinâmica, função pulmonar e resposta inflamatória nas primeiras 6 horas após a transfusão / Objectives: Blood transfusion is critical to the survival of selected patients, but may be associated with complications. Previous data related to pulmonary, hemodynamic and inflammatory effects of red blood cells (RBC) are still controversial. This study has two main objectives: 1- Validate a homologous stored red blood cell transfusion model in swine with acute hypovolemia by controlled bleeding; 2- Assess the acute effects of RBC transfusion on gas exchange, respiratory mechanics and hemodynamics, pulmonary and systemic inflammatory response. Methods: This study was divided into two phases: 1. Collection, processing and storing RBC from Agroceres® swines for 14 days and evaluation before (in vitro) and after (in vivo - labelling with radioactive sodium chromate) transfusion in one autologous and four homologous healthy swines (n = five); 2. Controlled acute hemorrhage (25% of blood volume) of sixteen pigs and then allocation in two groups: transfusion group (n = eight) received two units of RBC and Lactaded Ringer\'s solution (RL) to restore blood volume; control group (n = eight) that received only LR. Both groups were followed up to 6 hours after the end of resuscitation. Hemodynamic and respiratory data were collected hourly after the start of the study. Inflammatory mediators and messenger RNA(mRNA) expression were measured in plasma and lung tissue. Results: The 24-hour recovery of RBC labeled with radioactive sodium chromate was 97.5% ± 19%. We found significant increase of mixed venous oxygen saturation, oxygen arterial content, and hemoglobin and hematocrit levels in the transfused group compared to control. There were no significant differences between the two groups in microcirculation and gas exchange. There was a significant increase in the energy spent in lung hysteresis in the control group compared to the transfused group (p=0,002), as well as a tendency to decrease inspiratory energy in the transfusion group. The concentrations of the various cytokines evaluated before and after RBC transfusions were below the ELISA detection limit in most animals studied; there were no significative difference in nitrate concentrations in plasma and lung tissue. There was a statistically significant difference between groups in the quantification of mRNA induced nitric oxide synthase (iNOS) and IL-21 in lung tissue (increase in iNOS 50% and 50% decrease in IL-21 in the transfused group as compared to control). Conclusion: We demonstrated in this study the viability of 14-days stored swines RBC \"in vitro\" and \"in vivo\". Homologous stored red blood cell transfusion in swine did not cause significant hemodynamic changes in the pulmonary function and inflammatory response within the first six hours after transfusion

Made available in DSpace on 2017-07-21T20:42:30Z (GMT). No. of bitstreams: 1 Conclusao.pdf: 46180 bytes, checksum: 47dccc8736224bc604a0ca46bd517b13 (MD5) Previous issue date: 2008-04-25 / Hydroxyapatite (HAP) samples were prepared by the chemical precipitation method and were added niobium phosphate (NbOPO4) through wet grinding, up to 1%, 5% and 10% in mass of NbOPO4. The samples were thermally treated under 1100°C and 1300°C. Analysis through X-ray powder diffractometry using the Rietveld method identified the phases in each sample. In vitro tests were conducted with HAP powders activated and non activated with NbOPO4. Analysis by scanning electron microscopy (SEM) allowed the observation of erythrocyte cell’s morphology after incubation of the samples with HAP and spectrometry on the visible region was used to measure the hemolytic index (H.I.) in these cells as well as the functionality of the intra cellular hemoglobin after exposition to the samples. In the samples thermally treated under 1300°C, calcium phosphate phases (a-TCP and b-TCP) were observed and for the thermal treatment up to 1100°C, non stequiometric hydroxyapatite, tricalcium phosphate (b-TCP), CaNb2O6 and Ca2Nb2O7 phases were detected. Increasing the NbOPO4 percentage produced enhancement in the quantities of tricalcium phosphate phases as well as in the unit cell volume of these samples. That result was observed at both temperatures of thermal treatment. There were no observable isolated peaks referring to the phases with Nb in the samples thermally treated under 1300°C; at 1100°C, the increase in the cell volume of the phase b-TCP was followed by um decrease of the cell volume of the phase Ca2Nb2O7. These effects suggest that the deformation observed in the cell parameters of the mentioned phases were caused by the entrance of the Nb ion in to the unit cell of these phases, under substitutional or interstitial conditions. The photomicrographs revealed changes of the erythrocytes morphology of cells incubated with the HAP samples. The results show that these samples do not have a significant hemolytic index (H.I.<2), getting slightly hemolytic to the percentage of 10% of NbOPO4 (10>H.I.>20) in a 0.8% hematocrit. There were no alteration in the functionality of the intra cellular hemoglobin (O2 transport), according to the reoxigenation tests under a controlled atmosphere, for hematocrit 0.8% and 40%. / Amostras de hidroxiapatita (HAP) foram obtidas pelo método da precipitação química e receberam adição de oxifosfato de nióbio (NbOPO4) por moagem úmida. As amostras aditivadas com 1%, 5% e 10% em massa de NbOPO4 foram tratadas termicamente a 1100°C e 1300°C. Análises de difratometria de raios X do pó utilizando o método de Rietveld identificaram as fases presentes em cada amostra. Foram conduzidos testes in vitro dos pós de HAP não aditivada e aditivada com NbOPO4. Análises por microscopia eletrônica de varredura (MEV) permitiram observar a morfologia eritrocitária pós-incubação com as amostras de HAP e a espectrometria na região do visível permitiu medir o índice hemolítico (I.H.) dessas amostras e a funcionalidade da hemoglobina intracelular face à exposição à droga. Com o tratamento térmico a 1300°C foram observadas fases de fosfato tricálcico (a- TCP e b-TCP) e com o tratamento a 1100°C fases de hidroxiapatita não estequiométrica, fosfatos de cálcio (b-TCP), CaNb2O6 e Ca2Nb2O7. Uma quantidade maior de NbOPO4 produziu aumento da quantidade das fases de fosfato tricálcico bem como no volume da cela unitária destas amostras. Esse resultado foi observado para ambas as temperaturas de tratamento térmico. Não foram observados picos isolados referentes a fases com Nb nas amostras tratadas termicamente a 1300°C; já a 1100°C o aumento do volume de cela da fase b-TCP foi acompanhado por decréscimo do volume de cela da fase Ca2Nb2O7. Esses efeitos sugerem que a deformação observada nos parâmetros da cela unitária das fases citadas foi causada pela entrada do íon Nb na cela unitária destas fases, em condição substitucional ou intersticial. As fotomicrografias mostraram alteração da morfologia eritrocitária para as células incubadas com as amostras de HAP. Os resultados mostraram que essas amostras não têm índice hemolítico significativo (I.H.<2), chegando a moderadamente hemolítico para a porcentagem de 10% de NbOPO4 (10>I.H.>20) em hematócrito de 0,8%. Não houve alteração na funcionalidade da hemoglobina intracelular quanto ao transporte de O2, conforme verificado nos testes de reoxigenação em atmosfera controlada para hematócritos de 0,8% e 40%.

Fresh isolated erythrocyte ghost membranes and erythrocyte suspensions were incubated with an organic hydroperoxide, tert-butyl hydroperoxide, to establish the in vitro oxidative stress models. Circulating erythrocytes from normal individuals were fractionated into subpopulations of different ages by ultracentrifugation and used as an in vivo model. In these models, membrane iPF2alpha-III content accumulation was proportional to oxidative stress and correlated with decreased membrane fluidity. In circulating erythrocytes, membrane iPF2alpha-III increased with age and inversely correlated with membrane fluidity only in the core region. / Oxidative stress is involved in the pathophysiology of a wide variety of human diseases. Isoprostanes, a family of prostaglandin derivatives, are mainly derived from free radical peroxidation of specific polyunsaturated fatty acids (PUFA). Measurement of F2-isoprostanes (F2-iPs) or one specific biologically active isomer (iPF2alpha-III) is considered to be a reliable lipid peroxidation marker in human diseases. However, the association observed between increased plasma/urine F2-iPs and diseases does not necessarily reflect tissue oxidative damages. Circulating erythrocytes, a tissue with limited biosynthetic capacity and poor repair mechanism, would offer a number of advantages for assessment of in vivo oxidative damages. In this thesis, human erythrocyte membrane iPF2alpha-III content was investigated as a new marker for in vivo oxidative stress assessment. Membrane fluidity was used as an indirect marker of cellular function. / To use membrane iPF2alpha-III in a human disease with known oxidative stress burden, 49 Chinese patients on long-term haemodialysis and 31 healthy Chinese subjects were recruited. Both plasma and membrane iPF 2alpha-III showed that haemodialysis patients had increased oxidative stress. Only membrane iPF2alpha-III, but not the conventional used plasma iPF2alpha-III, correlated with membrane fluidity. Furthermore, the significant inverse correlation between membrane iPF 2alpha-III and the core region of membrane fluidity was observed for this group of patients too. Since membrane iPF2alpha-III was shown to provide a link between oxidative stress and erythrocyte function, it would be considered as a new marker of in vivo erythrocyte oxidative stress assessment. (Abstract shortened by UMI.) / Yu Xiongwen. / "July 2005." / Advisers: Wai Kei Christopher Lam; Chung Shun Ho. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3724. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 198-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Koblasa,Vladimír - Comparison of control measurements on various types of haematology analyzers used by ÚKBLD CHLTC at University Hospital in Prague First Faculty of Medicine, Charles University in Prague, Praha 2, Kateřinská 32 Head of the work: prof. MUDr. Jan Kvasnička, DrSc. Supervisor -consultant: Mgr. Ivana Malíková Blood cell count is essential testing method in hematology, where it is necessary to ensure properquality control. Aim of this study was to compare the results of measurement control materials with defined parameters and the same samples at different haematological analyzers to obtain evidence for the expression of measurement uncertainties. There are used more types of blood analyzers in ÚKBLD CHLTC at University Hospital in Prague, which operate on different principles. For comparsion were selected analyzers using the impedance working principle, where individual blood cell passes between two electrodes controlled by low voltage. Variation of this voltage is recorded and accurately defined for each type of blood cells. It was also chosen analyzer that works with optical detection. Analyzer illuminates the individual blood cell by light beam. A cell that enters into the path of light rays, reduce its optical density incident on the photocell. The change of the light density...

Objetivos: Transfusão de sangue é fundamental para a sobrevida de pacientes selecionados, porém é associada a complicações. A literatura é controversa em relação aos efeitos pulmonares, hemodinâmicos e inflamatórios da transfusão de glóbulos vermelhos (GV). Este estudo teve dois objetivos principais: 1- validar um modelo de transfusão homologa de GV estocados em suínos com hipovolemia aguda por hemorragia controlada; 2- avaliar os efeitos agudos da transfusão de GV nas trocas gasosas, mecânica respiratória, hemodinâmica e na resposta inflamatória pulmonar e sistêmica. Métodos: Este estudo foi dividido em duas etapas: 1.Coleta, processamento e estocagem por 14 dias de GV provenientes de um suino Agroceres®, avaliado antes (in vitro) e após (in vivo - marcação com cromato de sódio radioativo) à sua transfusão em suínos sadios , um autólogo e quatro homologos (n=cinco); 2. Outro grupo de suínos foi submetido à hemorragia aguda controlada (25% de sua volemia) e então dividido em dois grupos: grupo transfusão (n= oito) recebeu duas unidades de GV e solução de ringer lactato (RL) para restabelecer a volemia; grupo controle (n=oito) que recebeu somente RL. Ambos os grupos foram seguidos até 6horas após o final da ressuscitação volêmica. Dados hemodinâmicos e respiratórios foram coletados a cada hora após o inicio do estudo. Mediadores inflamatórios e expressão de RNAmensageiro(RNAm) foram medidos no plasma e no tecido pulmonar. Resultados: Houve recuperação de 97,5%±19% dos GV marcados com cromato de sódio radioativo 24 horas após a transfusão. Houve aumento significativo da saturação venosa mista, conteúdo arterial de oxigênio e dos níveis de hemoglobina e hematócrito no grupo transfundido comparado ao controle. Os parâmetros medidos para a avaliação da microcirculação e as trocas gasosas foram similares em ambos os grupos. Observou-se um aumento significativo na energia gasta na histerese pulmonar no grupo controle quando comparado ao grupo transfundido (p=0,002), bem como uma tendência á diminuição da energia inspiratória no grupo transfusão. As concentrações plasmáticas das diversas citocinas avaliadas antes e após a transfusão de GV mostraram-se abaixo dos limites de detecção do teste ELISA na maioria dos animais estudados em ambos os grupos; não houve diferença significativa nas concentrações de nitrato no plasma e no tecido pulmonar. Observou-se uma diferença discreta, porem estatisticamente significativa, entre os grupos na quantificação do RNAm da oxido nítrico sintetase induzida (iNOS) e da IL-21 no tecido pulmonar (aumento de 50% na iNOS e decréscimo de 50% na IL-21 no grupo transfundido comparado com o controle). Conclusão: Demonstramos a viabilidade \"in vitro\" e \"in vivo\" de GV suínos, estocados por até 14 dias. A transfusão de GV homólogos não causou alterações significativas na hemodinâmica, função pulmonar e resposta inflamatória nas primeiras 6 horas após a transfusão / Objectives: Blood transfusion is critical to the survival of selected patients, but may be associated with complications. Previous data related to pulmonary, hemodynamic and inflammatory effects of red blood cells (RBC) are still controversial. This study has two main objectives: 1- Validate a homologous stored red blood cell transfusion model in swine with acute hypovolemia by controlled bleeding; 2- Assess the acute effects of RBC transfusion on gas exchange, respiratory mechanics and hemodynamics, pulmonary and systemic inflammatory response. Methods: This study was divided into two phases: 1. Collection, processing and storing RBC from Agroceres® swines for 14 days and evaluation before (in vitro) and after (in vivo - labelling with radioactive sodium chromate) transfusion in one autologous and four homologous healthy swines (n = five); 2. Controlled acute hemorrhage (25% of blood volume) of sixteen pigs and then allocation in two groups: transfusion group (n = eight) received two units of RBC and Lactaded Ringer\'s solution (RL) to restore blood volume; control group (n = eight) that received only LR. Both groups were followed up to 6 hours after the end of resuscitation. Hemodynamic and respiratory data were collected hourly after the start of the study. Inflammatory mediators and messenger RNA(mRNA) expression were measured in plasma and lung tissue. Results: The 24-hour recovery of RBC labeled with radioactive sodium chromate was 97.5% ± 19%. We found significant increase of mixed venous oxygen saturation, oxygen arterial content, and hemoglobin and hematocrit levels in the transfused group compared to control. There were no significant differences between the two groups in microcirculation and gas exchange. There was a significant increase in the energy spent in lung hysteresis in the control group compared to the transfused group (p=0,002), as well as a tendency to decrease inspiratory energy in the transfusion group. The concentrations of the various cytokines evaluated before and after RBC transfusions were below the ELISA detection limit in most animals studied; there were no significative difference in nitrate concentrations in plasma and lung tissue. There was a statistically significant difference between groups in the quantification of mRNA induced nitric oxide synthase (iNOS) and IL-21 in lung tissue (increase in iNOS 50% and 50% decrease in IL-21 in the transfused group as compared to control). Conclusion: We demonstrated in this study the viability of 14-days stored swines RBC \"in vitro\" and \"in vivo\". Homologous stored red blood cell transfusion in swine did not cause significant hemodynamic changes in the pulmonary function and inflammatory response within the first six hours after transfusion

Cerebral malaria is characterised by an accumulation of infected erythrocytes in the microvasculature of the brain. Plasmodium falciparum infected erythrocytes have been shown to bind to a Human Brain Endothelial Cell line (HBEC-5i) in vitro. This provides a model for the investigation of interactions between P. falcuparum and human brain endothelium. Currently neither the parasite adhesion ligands on infected erythrocytes, nor the host endothelial cell receptors necessary for this interaction have been identified. In this work, the identity of the host receptor on brain endothelial cells was addressed by binding assays of selected and unselected parasites on a wide range of malaria-associated host molecules. The identity of the parasite ligand was investigated by microarray analysis of parasites after selection for cytoadherence to HBEC-5i. The hypothesis being tested was that the gene encoding the parasite cytoadherence ligand would show significant upregulation in selected compared to unselected paarasites. The P. falciparum laboratory strains 3D7, HB3 and IT/FCR3 were selected for binding to HBEC-5i using a panning assay. Compared to unselected parasites, HBEC-5i selected parasites showed a distinct phenotype with reduced platelet-mediated clumping. There was no significant increase in binding of parasites to any of the known endothelial cytoadherence receptors for P. falciparum after selection on HBEC-5i. Binding inhibition assays with various antibodies and soluble receptors did not greatly block the adhesion of parasites to HBEC-5i except for heparin. Altogether, the receptor(s) mediating the interation with HBEC-5i remains unknown. In order to carry out transcriptional analysis of selected and unselected paarasites form all three parasite strains, it was necessary to update the existing microarray chip which is based on the 3D7 genome. This is because each parasite train has a unique repertoire of variant surface antigens (VSAs) including var, rif and stevor genes. Therefore, to fully analysis HB3 and IT genomes. Unique oligonnucleotide probes were then designed for each new sequence and the 3D7-based microarray chip was updated. Transcriptional analysis was then carried out on selected and unselected parasites of all strains. Microarray data clearly indicated that the most highly upregulated genes after selection were group A or group A-like var genes (HB3var3, 3D7_PFDOO2Oc, ITvar7 and ITvar19), showing 11 to over 100 fold upregulation in selected parasites. The rif gene adjacent to the upregulated var gene was also highly expressed. To a lesser extent some exported proteins like RESA-1, PfEMP3 or PHIST family members also showed increased transcription in HBEC-selected parasites (2-3 fold upregulation). Reverse transcriptase-PCR confirmed the upregulation of group A var genes in selected parasites, suggessted that the group A PfEMP1 variants are major candidate ligands for parasite binding to HBEC-5i. These findings are consistent with previous work showing an association between Group A var genes and cerebral malaria.