Dans cette thèse, les propriétés rhéologiques du sang sont étudiées suivant deux approches differentes. Les propriétés de l'écoulement du plasma sont analysées selon trois modes différents : sous cisaillement, en extension et en constriction. Jusqu'à présent, le plasma était considéré comme un fluide newtonien, et le comportement complexe du sang était simplement attribué à la présence des globules rouges. Les expériences menées ont montré un comportement visco-élastique du plasma, que doit désomais être pris en compte dans les études futures. La deuxième axe traite des globules rouges. Leur assemblage en agrégats rectilignes est à l'origine du comportement rhéofluidifiant, mais les causes de la formation des agrégats restent encore vagues. L'énergie d'interaction entre deux cellules et la distribution des tailles des clusters dans des canaux microfluidiques ont été mesurées en présence de dextran et de fibrinogène. Comme les agrégats sont normalement cassés à des taux de cisaillement élevés, on a cru qu'ils ne jouaient pas de rôle dans l'écoulement du sang. Mais le fait que le nombre de clusters augmente à des concentrations physiologiques de fibrinogène, même pour des taux de cisaillement correspondant à ceux du système microvasculaire, il est clair que l'agrégation ne peut pas être négligée dans la description de l'écoulement du sang en le réseau capillaire. / In this work, the rheological properties of human blood are investigated by two different approaches. The flow properties of plasma, the liquid component of blood, is analyzed under three different conditions: shear flow, elongational flow and contraction flow. Up to now, the plasma was considered as a Newtonian fluid, while the non-Newtonian properties of blood were only attributed to the red blood cells. The performed experiments reveal a viscoelastic behavior of the plasma which has to be considered in future studies. In addition to the plasma, also diluted polymer solutions are analyzed in order to find a good model solution for plasma. The second part concerns the red blood cells. Their adhesion to linear aggregates is held responsible for the well-known shear thinning behavior of blood but the reason for the cluster formation is still not clear. The interaction energy between two red blood cells and the distribution of different sized clusters flowing through narrow channels are measured under the influence of the two macromolecules dextran and fibrinogen. As the aggregates are actually broken at high shear rates, the current understanding is that they would not play a role for the properties of blood flow. However, an increased amount of clusters at physiological fibrinogen concentrations can be shown, even at shear rates which are common in the microvascular system, which clarifies that the aggregation cannot be neglected in the description of blood flow through the capillary network.

L'athérosclérose est une maladie cardiovasculaire complexe affectant la paroi artérielle où le développement et la progression de la plaque sont fortement favorisés par une inflammation chronique. L'instabilité de la plaque carotidienne peut conduire à de potentiels évènements ischémiques majeurs tels que l'accident vasculaire cérébral (AVC) dont le caractère imprévisible rend la prévention primaire très compliquée. Ainsi, il n'existe pas à l'heure actuelle de biomarqueurs prédictifs efficaces de la rupture de plaque. Néanmoins, il est maintenant clairement établi que l'hémorragie intraplaque (IPH), la néovascularisation et l'accumulation excessive de macrophages sont les principaux facteurs d'instabilité de la plaque. Sur la base de travaux précédents, l'objectif de cette thèse était d'évaluer de manière indépendante les effets de l'activité physique (AP) et de la sédentarité, premièrement sur les paramètres histologiques d'instabilité de plaque, et deuxièmement, sur les facteurs de risque secondaires de l'athérosclérose, que sont l'inflammation, le stress oxydant et le profil hémorhéologique de patients asymptomatiques à risque d'AVC traités chirurgicalement. La 1ère étude a montré que l'AP régulière était associée à une prévalence d'IPH diminuée, et était l'unique facteur protecteur de l'IPH. Cette étude a également suggéré un effet bénéfique de l'AP sur le stress oxydant, ainsi que sur l'accumulation de macrophages. Dans une 2ème étude, nous avons caractérisé l'état fonctionnel de protéines potentiellement impliquées dans les dysfonctions du système immunitaire, et l'implication des cellules inflammatoires dans ces mécanismes. Nous avons alors identifié une cytokine pro-inflammatoire jouant un rôle déterminant dans les processus inflammatoires de déstabilisation de plaque. L'étude 3 nous a permis de caractériser l'effet du niveau d'AP sur la réponse monocytaire chez des patients avec plaque d'athérosclérose, et d'identifier une chimiokine qui pourrait avoir un rôle dans la modulation de la réponse monocytaire par l'AP. Enfin, la 4ème étude démontre l'altération de paramètres hémorhéologiques chez des patients atteints de maladie carotidienne sévère, et comment l'AP permet de limiter cette altération via la diminution de l'agrégation érythrocytaire. Ce travail de thèse apporte des informations quant à la pratique de l'AP dans la prévention primaire de l'athérosclérose. Des études complémentaires seront toutefois nécessaires afin de confirmer ces résultats, en proposant notamment une approche interventionnelle en activité physique / Atherosclerosis is a complex cardiovascular disease that affects the arterial wall where plaque development and progression are severely promoted by chronic inflammation. Carotid plaque destabilization could lead to potential major ischemic events such stroke which is still unpredictable, making primary prevention very complex. Thus, there is still currently no suitable predictive biomarker of plaque rupture. Nevertheless, it is now clearly established that intraplaque hemorrhage (IPH), neovascularization and excessive macrophage accumulation are the three main risk factors of plaque instability. Based on previous studies, the aim of this work was to evaluate independently the impact of physical activity (PA) and sedentary behavior, first on histological parameters of plaque instability, and secondly on secondary risk factors of atherosclerosis, such as inflammation, oxidative stress and hemorheological profile of asymptomatic patients at high-risk of stroke who underwent endarterectomy surgery. The first study shows that regular PA was associated to a decreased occurrence of IPH, and was the only protective factor for IPH. This study also suggested a beneficial effect of PA on macrophage accumulation as well as on oxidative stress. Then, in the 2nd study, we have characterized the functional state of proteins potentially implicated in immune system dysfunctions, and the implication of inflammatory cells in these mechanisms. We have identified a pro-inflammatory cytokine as a key driver of disrupting inflammatory process of plaque. In the same way, we have characterized in the 3rd study, the effect of PA on the monocytic response in atherosclerosis patients, and identified a chemokine associated that could explain the modulation of this monocytic response by PA. Finally, the 4th study demonstrates the hemorheological parameters alteration in carotid artery disease patients, and how PA could limit this alteration via red blood cell aggregation. This PhD thesis provided information regarding regular PA in primary prevention of atherosclerosis. However, additional studies are required to confirm these results, using in particular PA interventional approach

Le carcinome de Merkel est un cancer cutané de différenciation neuroendocrine rare, mais agressif, dont le facteur étiologique principal est le polyomavirus de Merkel (MCPyV). L’objectif de ce travail a été d’identifier des marqueurs virologiques et cellulaires pronostiques ou théranostiques à l’aide d’une cohorte historicoprospective de patients ayant un carcinome de Merkel. Les patients ayant des titres élevés d’anticorps dirigés contre la protéine de capside VP1 du MCPyV ont un pronostic favorable, tandis les anticorps dirigés contre les oncoprotéines virales reflètent l’évolution tumorale. Par ailleurs, il existe une hétérogénéité d’expression des récepteurs à la somatostatine dans les carcinomes de Merkel. Ce marqueur cellulaire peut constituer un outil théranostique lors de thérapies ciblées utilisant les analogues de la somatostatine. Enfin, nos travaux actuels portent sur l’évaluation de l’immunité cellulaire chez ces patients, avec une étude ayant montré la valeur pronostique du ratio neutrophiles/lymphocytes sanguin. / Merkel Cell Carcinoma is a rare and aggressive neuroendocrine skin cancer. The Merkel cell polymavirus has been identified as the main etiological agent of such cancer. We aimed to identify viral and cellular markers relevant as prognostic and theranostic tools in patients with Merkel Cell Carcinoma. Using serological immunoassays, we observed that patients with high levels of serum antibodies against the MCPyV major capsid protein at baseline had better outcomes, whereas antibodies directed against the MCPyV oncoproteins reflected the tumor burden and course of disease. We also demonstrated that Merkel Cell Carcinoma tumors displayed a heterogeneous expression of receptors to somatostatin, which could constitute a theranostic tool for the use of targeted therapies using somatostatin analogs. Finally, current studies focus on the assessment of cellular immunity in Merkel Cell Carcinoma patients. Our results indicate that the blood neutrophil-to-lymphocyte ratio is an independent marker of mortality in Merkel Cell Carcinoma patients.

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.

A leishmaniose visceral, causada no Brasil por protozoários da espécie Leishmania (L.) infantum, apresenta-se nos canídeos com manifestações similares à doença humana. Diversos trabalhos tem investigado a relação entre raças caninas e suscetibilidade/resistência à doença sendo as alterações lipídicas consideradas também nessa avaliação. Para avaliar o grau de comprometimento conforme a raça e as alternações lipídicas nos cães portadores de leishmaniose visceral, analisamos alterações hematológicas e bioquímicas frente à manifestação da doença em diferentes raças ou grupos raciais. Para tanto, incluímos 162 cães de área endêmica, sem histórico de outras patologias ou de vacinação contra leishmaniose, organizados em grupos de cães naturalmente infectados segundo a raça, quais sejam, Boxer, Labrador, Pit Bull, Sem Raça Definida (SRD) e Outras Raças, e grupo controle não infectados. Na avaliação das manifestações clínicas, dividimos os animais, dentro de cada grupo de cães infectados em assintomáticos (sem nenhum sinal clínico), oligossintomáticos (de 1 a 3 sinais clínicos) e polissintomáticos (acima de 3 sinais clínicos). As raças/grupos formadas pelo total de cães infectados (assintomáticos + oligossintomáticos + polissintomáticos) também foram avaliados segundo o escore de gravidade da infecção. Para analisar as possíveis alterações laboratoriais decorrentes da infecção de cães por Leishmania (L.) infantum foram realizados hemograma, leucograma, dosagens de albumina, globulinas, proteínas plasmáticas totais, creatinina, ureia, alanino aminotransferase (ALT), Fosfatase Alcalina (FA), colesterol total, lipoproteínas de alta densidade (high density lipoproteins, HDL), lipoproteínas de baixa densidade (low density lipoproteins, LDL), lipoproteínas de densidade muito baixa (very low density lipoproteins, VLDL) e triglicérides. Analisados em conjunto, observamos que os animais assintomáticos, independentemente de raça/grupo, apresentaram parâmetros bioquímicos e hematológicos similares aos valores descritos para o grupo controle não infectado. Não observamos diferença na gravidade das manifestações da doença entre as raças ou grupos raciais para o desenvolvimento da Leishmaniose Visceral Canina. As diversas raças e grupos raciais apresentaram alterações nos parâmetros pesquisados, porém os quadros de hipoalbuminemia, hiperglobulinemia e anemia foram identificados com maior frequência, sendo correlacionados à gravidade na raça Labrador e nos grupos SRD e Outras Raças. O quadro de anemia foi mais importante na raça Pit Bull. O grupo Outras Raças apresentou o maior número de alterações correlacionadas à gravidade. A raça Labrador apresentou concentração de HDL diminuída em relação às demais raças. As alterações lipídicas, principalmente em HDL, VLDL e triglicérides foram correlacionadas à gravidade no grupo Outras Raças. / Visceral leishmaniasis caused in Brazil by protozoa Leishmania (L.) infantum, presents in dogs similar manifestations as human disease. Some work has investigated the relationship between dog breeds and susceptibility/resistance to disease and lipid changes has been also considered in this evaluation. To assess the degree of commitment according breed and lipid changes in dogs with visceral leishmaniasis, we analyzed hematological and biochemical parameters and the manifestation of the disease in different breeds or groups. Therefore, we included 162 dogs from an endemic area of Brazil with no history of other diseases or vaccination against leishmaniasis, organized in groups of dogs naturally infected by breed, namely Boxer, Labrador, Pit Bull, Mogrel dogs, Other Breeds and uninfected control group. In the evaluation of the clinical manifestations, we divided the animals in infected asymptomatic (no clinical signs), oligosymptomatic (1-3 clinical signs) and polisymptomatic dogs (above 3 clinical signs). Breeds/groups formed by the total of infected dogs (asymptomatic+oligosymptomatic+polisymptomatic) were also assessed according to the severity of the infection. To analyze the possible laboratory changes resulting from infection of dogs by Leishmania (L.) infantum were performed red blood cell count, white blood cell count, evaluation of albumin, globulin, total plasma protein (TPP), creatinine, urea, alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol, high density lipoproteins (HDL), low density lipoproteins (LDL), very low density lipoprotein (VLDL) and triglycerides. Taken together, we observed that asymptomatic animals, regardless of breeds/group, showed biochemical and hematological parameters similar to values reported for the uninfected control group. There were no differences in severity of disease manifestations among breed or racial groups for the development of Canine Visceral Leishmaniasis. The different breeds and racial groups showed changes in the parameters studied, but hypoalbuminemia, hypergammaglobulinemia and anemia were identified more frequently being correlated to the severity in Labrador, mongrel and Other Breeds groups. The anemia was most important in Pit Bull. The group Other Breeds had the largest number of changes correlated to the severity. The Labrador showed decreased HDL levels compared to other breeds. Lipid changes, especially in HDL, VLDL and triglycerides were correlated to the severity in the Other Breeds group.

OBJETIVOS: O objetivo do estudo foi avaliar se a transfusão de hemácias no intraoperatório de cirurgia cardíaca com circulação extracorpórea está associada a complicações clínicas incluindo choque cardiogênico, arritmia, insuficiência renal aguda, isquemia miocárdica, choque séptico, necessidade de reintubação orotraqueal, acidente vascular cerebral ou mortalidade durante a internação hospitalar. DESENHO: Estudo clínico de coorte retrospectivo e unicêntrico com escore de propensão, realizado no Instituto do Coração (InCor) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. PACIENTES: Pacientes adultos submetidos à cirurgia cardíaca eletiva com circulação extracorpórea no período de janeiro de 2004 a dezembro de 2008. DESFECHO PRIMÁRIO: Complicações clínicas durante a internação hospitalar (choque cardiogênico, arritmia, insuficiência renal aguda, isquemia miocárdica, choque séptico, necessidade de reintubação orotraqueal, acidente vascular cerebral ou mortalidade hospitalar). DESFECHO SECUNDÁRIO: 1- Avaliar o efeito da transfusão de hemácias no intraoperatório no tempo livre de inotrópicos e vasopressores, tempo de ventilação mecânica e tempo de permanência na UTI e internação hospitalar. 2- Avaliar o efeito do número das unidades de hemácias transfundidas no intraoperatório na ocorrência de mortalidade hospitalar, choque cardiogênico, arritmia, isquemia miocárdica, choque séptico, acidente vascular cerebral e reintubação orotraqueal. 3- Avaliar o efeito da anemia à admissão e durante internação hospitalar na ocorrência de complicações pós-operatórias. INTERVENÇÃO: Não houve intervenção. RESULTADOS: Foram incluídos 2851 pacientes na análise final, dos quais 1471(51,6%) foram expostos a transfusão de hemácias e 1380 (48,4%) não receberam transfusão no intraoperatório. Os pacientes transfundidos apresentaram maior incidência das seguintes complicações: mortalidade (2,1% vs 0,4%, P < 0,001), insuficiência renal aguda (9,1% vs 3,9%, P<0,001), reintubação orotraqueal (3,8% vs 1,4%, P < 0,001) e choque séptico (2,2% vs 0,4%, P < 0,001). Os pacientes transfundidos também apresentaram maior tempo de internação hospitalar [16 dias (12-23) vs 13 dias (9-18), P < 0,001] e em unidade de terapia intensiva [3 dias (2-6) vs 2 dias (2-4), P < 0,001]. A concentração da hemoglobina menor que 9 g/dL ocorreu em 1847 pacientes (64,7%) durante a internação hospitalar e foi associada a maior risco de insuficiência renal aguda e de acidente vascular cerebral. O escore de propensão identificou 588 pacientes pareados em relação à exposição à transfusão, e essa análise demonstrou que a transfusão intraoperatória de hemácias não aumentou a ocorrência de complicações no período de internação hospitalar. Contudo a transfusão de 4 ou mais unidades de hemácias está associada a maior ocorrência de mortalidade hospitalar, choque cardiogênico e IRA, maior incidência de reintubação orotraqueal, choque séptico e AVC. Além de uma relação direta entre as unidades de hemácias transfundidas e a ocorrência de morte. CONCLUSÃO: Esse estudo observacional demonstrou que a anemia é frequentemente detectada no pós-operatório de cirurgia cardíaca, e está associada a maior incidência de complicações. Além disso, a transfusão de hemácias no intraoperatório não modifica a ocorrência das complicações pós-operatórias em pacientes submetidos a cirurgia cardíaca. No entanto a transfusão de 4 ou mais hemácias está associada a maior incidência de complicações clínicas, além de uma relação dose-dependente. Estratégias como detecção precoce de anemia e emprego de técnicas alternativas à transfusão no manejo devem ser estimuladas no ambiente perioperatório / OBJECTIVE: The objective of this study was to evaluate whether the transfusion of red blood cells in the intraoperative cardiac surgery with extracorporeal circulation is associated with complications after cardiac surgery. DESIGN: A retrospective cohort study with a propensity score analysis, performed at Instituto do Coração (InCor) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. PATIENTS: Adult patients undergoing elective cardiac surgery with cardiopulmonary bypass in the period of January to 2008 December. PRIMARY OUTCOME: Clinical complications during hospital stay (cardiogenic shock, arrhythmia, cardiogenic shock, acute kidney injury, myocardial ischemia, septic shock, tracheal reintubation, stroke or hospital mortality). SECONDARY OUTCOME: 1- Evaluate the effect of intraoperative red blood cell transfusion in inotropic and vasopressor free time, mechanical ventilation time, length of ICU stay and hospital stay. 2- Evaluate the effect of the number of units of transfused red blood cells intraoperatively on the occurrence of hospital mortality, cardiogenic shock, arrhythmia, myocardial ischemia, septic shock, stroke and orotracheal reintubation. 3- Evaluate the effect of anemia on admission and during hospitalization in the occurrence of postoperative complications. RESULTS: In the final analysis, 2851 patients were included. Of these patients, 1471(51.6%) were exposed to red blood cell transfusion (RBC) and 1380 (48.4%) were not exposed to RBC during intraoperative. Transfused patients had higher incidence of the following complications: mortality (2.1% vs. 0.4%, P < 0.001), acute kidney injury (9.1% vs. 3.9%, P < 0,001), tracheal reintubation (3.8% vs. 1.4%, P < 0.001) and septic shock (2.2% vs. 0.4%, P < 0.001). Transfused patients also had a longer length of hospital stay [16 days (12-23) vs. 13 days (9-18), P<0.001] and prolonged intensive care unit stay [3 days (2-6) vs. 2 days (2-4), P < 0.001]. Hemoglobin lower than 9 g/dL was found in 1847 patients (64.7%) during hospital stay and was associated to a higher risk of acute kidney injury and stroke. The propensity score identified 588 paired patients in relation to transfusion exposure, and this analysis demonstrated that intraoperative transfusion of red blood cells did not increase the occurrence of complications during hospitalization. However, transfusion of 4 or more units of red blood cells is associated with a higher occurrence of hospital mortality, cardiogenic shock and acute renal failure, a higher incidence of orotracheal reintubation, septic shock and stroke. In addition to a direct relationship between the units of transfused red blood cells and the occurrence of death. CONCLUSIONS: This observational study demonstrated that anemia is frequently detected in the postoperative period of cardiac surgery, and is associated with a higher incidence of complications. In addition, red blood cell transfusion in the intraoperative does not modify the occurrence of postoperative complications in patients undergoing cardiac surgery. However, transfusion of 4 or more erythrocytes is associated with a higher incidence of clinical complications, in addition to a dose-dependent relationship. Strategies such as early detection of anemia and use of alternative techniques to transfusion in management should be stimulated in the perioperative environment

Objetivos: O objetivo do estudo foi avaliar se uma estratégia restritiva de transfusão de hemácias era superior a uma estratégia liberal na redução de mortalidade em 28 dias de pacientes oncológicos críticos com choque séptico. Desenho: unicêntrico, randomizado, duplo cego e controlado. Local: Unidade de Terapia Intensiva do Instituto do Câncer do Estado de São Paulo da Faculdade de Medicina da Universidade de São Paulo. Pacientes: Pacientes adultos com neoplasia admitidos na Unidade de Terapia Intensiva (UTI) nas primeiras 6 horas do diagnóstico de choque séptico. Intervenção: Os pacientes foram randomizados para uma estratégia liberal (transfusão de hemácias se hemoglobina < 9 g/dL) ou para uma estratégia restritiva (transfusão de hemácias se hemoglobina < 7 g/dL) durante a permanência na Unidade de Terapia Intensiva. Desfecho primário: Mortalidade por todas as causas após 28 dias. Resultados: O estudo foi realizado no período de junho de 2012 a maio de 2014. Foram randomizados 300 pacientes para as estratégias de transfusão liberal (n = 149) ou restritiva (n = 151). A mortalidade após 28 dias da randomização ocorreu em 67 pacientes do grupo liberal (45%) e em 84 pacientes do grupo restritivo (56%) (RR 1,53; intervalo de confiança 95%, 0,97-2,52, P=0,07). A mortalidade 90 dias após a randomização foi de 59% no grupo liberal e 70% no grupo restritivo (RR 1,63; intervalo de confiança 95%, 1,01-2,63; P=0,044). Os pacientes do grupo liberal receberam mais unidades de transfusão de hemácias quando comparados aos pacientes do grupo restritivo (1 [0-3] unidade vs 0 [0-2] unidade, P < 0,001). Conclusões: O estudo confirmou que uma estratégia restritiva de transfusão de hemácias quando comparada a uma estratégia liberal, não reduziu a mortalidade em 28 dias de pacientes oncológicos com choque séptico / Objective: To assess whether a restrictive strategy of red blood cell (RBC) transfusion reduces 28-day mortality when compared to a liberal strategy in cancer patients with septic shock. Design: Single center, randomized, double-blind controlled trial. Setting: Teaching hospital. Patients: Adult cancer patients with septic shock in the first 6 hours of Intensive Care Unit (ICU) admission. Interventions: Patients were randomized to a liberal (hemoglobin threshold < 9 g/dL) or to a restrictive strategy (hemoglobin threshold < 7 g/dL) of red blood cell transfusion during ICU stay. Measurements and Main Results: The primary outcome was 28-day all-cause mortality after randomization. Between June 2012 and May 2014, 300 patients were randomized to the liberal transfusion strategy (n=149) or to the restrictive transfusion strategy (n=151) strategy. At 28 days after randomization, mortality rate in the liberal group was 45% (67 patients) compared with 56% (84 patients) in the restrictive group (hazard ratio, 1.53; 95% confidence interval, 0.97 to 2.52; P=0.07). At 90 days after randomization, mortality rate in the liberal group was 59% compared with 70% in the restrictive group (hazard ratio, 1.63; 95% confidence interval, 1.01 to 2.63; P=0.044). Patients in the liberal group received more RBC units than patients in the restrictive group (1 [0-3] unit vs. 0 [0-2] unit, P < 0.001). Conclusions: A restrictive strategy of RBC transfusion did not decrease 28-day mortality rate of patients admitted to ICU due to septic shock when compared to a liberal strategy

Introdução: A técnica de doação de duplos concentrados de hemácias por aférese está disponível nos hemocentros desde 1997, quando este procedimento foi aprovado pela Food and Drug Administration (FDA). A legislação brasileira vigente segue as normas americanas para seleção de potenciais doadores de hemocomponentes por aférese e orienta que cada serviço tenha seu próprio protocolo para realizar esses procedimentos. Objetivo: Analisar o comportamento das reservas de ferro de doadores de duplos concentrados de hemácias por aférese, selecionados de acordo com as normas brasileiras vigentes a fim de auxiliar na definição de critérios seguros para seleção desses doadores. Casuística e Métodos: Foram coletadas amostras de sangue para exames de hemograma, ferro sérico, ferritina e UIBC (capacidade latente de ligação de ferro) de 75 doadores de hemácias duplas por aférese nos momentos pré-doação e quatro, cinco, seis e sete meses após a doação. A análise dos dados foi realizada com o uso do Graph Pad Prism versão 5.0. Resultados: A dosagem de ferritina pré-doação foi significativamente maior que nos meses subsequentes, mesmo após sete meses da coleta (148 ng/mL x 84,7 ng/mL x 91,2 ng/mL x 93,9 ng/mL x 112 ng/mL, p<0,0001). Não foi observada diferença significativa dos valores de hematócrito, ferro sérico e UIBC ao longo do estudo. Interessantemente, os doadores apresentaram contagem de hemoglobina na pré-doação menor que a coletada no sétimo mês após a doação de duplos concentrados de hemácias por aférese (15,3 g/dL x 15,7 g/dL , p<0.05). Os valores do volume corpuscular médio (VCM) foram significativamente menores com quatro e cinco meses após doação, comparados com os valores basais (87,67 FL x 86,74 FL, p<0,0001 e 87,67 FL x 87,27 FL, p<0,0161) e significativamente maiores com sete meses após a doação (87,67 FL x 88,14 FL, p=0,01). Foi observado aumento dos níveis de Hemoglobina corpuscular média (HCM) nos sexto (29,45 PG x 29,84 PG, p=0,0044) e sétimo meses após a doação (29,45 PG x 30,22 PG, p<0,0001), em comparação com os valores basais. Verificou-se, também, que os estoques de ferro apresentaram queda significativa após uma doação de duplos concentrados de hemácias, obtidos por aférese e que até sete meses após a doação não houve a devida recuperação dos estoques de ferro ao nível basal dos doadores em estudo. Houve, ainda, aumento dos valores da hemoglobina e do HCM e VCM a partir do sexto mês após a doação de hemácias duplas por aférese. Conclusão: Concluiu-se que, em doadores do sexo masculino de duplos concentrados de hemácias por aférese, a dosagem de ferritina deve ser realizada para melhor controle e segurança dos mesmos, e que se os valores da dosagem da ferritina forem <30 ng/dl, o doador deve ser impedido de doar; e se >30 ng/mL, recomenda-se liberar para nova doação somente após seis meses da doação anterior. Assim, novos estudos serão necessários para se adequarem as normas vigentes à população de doadores brasileiros, visando maior segurança e menor impacto da doação nos estoques de ferro do doador. / Introduction: The technique of double red cell donation by apheresis has been available in blood establishments since 1997, when this procedure was approved by the American FDA. The Brazilian legislation follows the American standards for the selection of potential donors of blood components by apheresis and advises every establishment to have their own protocol to carry out these procedures. Aim: The aim of this study was to analyze the behavior of the iron reserves of donors of double red cells concentrate by apheresis, selected in accordance with current Brazilian standards, in order to help define safer criteria for the selection of these donors. Casuistic and Methods: Blood samples were collected from 75 donors of double red blood cells by apheresis for tests of blood count, serum iron, ferritin and UIBC at the moment before donation and at four, five, six and seven months after donation. Data analysis was done using Graph Pad Prism version 5.0. Results: Pre-donation ferritin was significantly higher than in the subsequent months, even after seven months of the collection (148 x 84.7 x 91.2 x 93.9 x 112 ng/mL, p<0.0001). Interestingly, donors showed hemoglobin count at pre-donation lower than the hemoglobin collected at the seventh month after the donation of double concentrates by apheresis (15.3 x 15.7 g/dL, p<0.05). No significant difference was observed in the values of hematocrit, serum iron, UIBC (iron binding capacity) four, five, six and seven months after the donation of double concentrates by apheresis when compared to basal levels. The MCV values were significantly lower four and five months after donation, compared to basal levels (87.67 x 86.74 FL, p<0.0001 and 87.67 x 87.27 FL, p<0.0161) and significantly higher after seven months after donation (87.67 x 88.14 FL, p=0.01). We observed an increase in MHC in the sixth month (29.45 x 29.84 PG, p=0.004 PG 4) and seventh month after donation compared to basal levels (29.45 x 30.22 PG, p<0.0001). This study observed that a donation of double blood red cell concentrates obtained by apheresis caused a significant drop in iron stores that persisted even after seven months after donation. Conclusion: We also verified an increase of hemoglobin, MCV and MHC values from the sixth month after donation, compared to basal levels. Probably, both the double red cell donation by apheresis and the stimulus of erythropoiesis might have influenced the slow recovery of donors\' iron stores in this study. Thus, novel studies are necessary to adjust the current standards to the population of Brazilian donors, aiming a greater safety and lower impact of donation in the donor\'s iron storage.

O uso de amostras \"Controle Interno\" (CI) para validação de técnicas e reagentes imunohematológicos é exigido por lei e indispensável para identificar erros nos processos laboratoriais. No Brasil, as legislações que regem a hemoterapia aprovam a produção dos reagentes CI pelos serviços de hemoterapia (SH), desde que o processo seja previamente validado e tenha critérios de aceitação bem definidos. O estudo analisou a estabilidade de anticorpos anti-eritrocitários irregulares, da classe IgG, presentes nos plasma fresco congelado (PFC) de doadores e nas amostras pré-transfusionais de pacientes com o objetivo de utilizar esta matéria prima para a produção de CI nos laboratórios de imuno-hematologia. Foram avaliados o título, o escore e a reatividade (testes de potência) de 12 amostras de PFC com anticorpos anti-D, -E e -K e de 25 amostras de pacientes com anticorpos anti-D, -E, -e, -c, -C e -K, ambas estocadas em freezer (-25°C) e em geladeira (2 a 8°C), durante seis e três meses respectivamente. Nas amostras de PFC os valores iniciais do título e reatividade foram mantidos durante o estudo, sendo que as quedas ocorridas não ultrapassaram uma titulação ou graduação em freezer e geladeira. Todavia, o tempo de armazenamento dos PFC antes do início do estudo impactou negativamente o escore das amostras estocadas em freezer (p=0,021) e geladeira (p=0,027). Nas amostras de pacientes o título sofreu um impacto negativo significante, sendo afetado tanto pelo tempo prévio de seu armazenamento antes do início do estudo quanto pelo tipo de estocagem durante a execução dos testes. Amostras com tempo de armazenamento prévio prolongado entre 2 e 8°C tiveram seus títulos significantemente impactados quando estocadas em freezer (p=0,014) e geladeira (p=0,039), indicando que sua permanência demorada em temperaturas mais altas pode acarretar a degradação dos anticorpos de forma mais acentuada. As alíquotas armazenadas em geladeira durante o estudo tiveram uma significante redução do título (p=0,016), quando comparadas com as amostras armazenadas em freezer, confirmando uma menor viabilidade dos anticorpos mantidos em temperaturas menos frias. Ao avaliarmos uma possível associação entre os valores iniciais do título, escore e reatividade e uma propensão à queda nos resultados dos testes de potência, verificamos que os anticorpos com esses valores inicialmente baixos não apresentaram menor estabilidade se comparados aos anticorpos com valores mais altos. Esta característica indica que anticorpos irregulares IgG com diferentes perfis iniciais de potência podem ser utilizados para a produção de CI desde que corretamente caracterizados, armazenados e validados. É pertinente ampliar o conhecimento acerca da estabilidade das diferentes especificidades de anticorpos que podem compor um painel de CI, além das avaliadas neste estudo. / The use of \"Internal Control\" (IC) samples for immunohematological techniques and reagents validation is required by law and necessary to identify laboratory processes errors. In Brazil the hemotherapy legislation approve the IC reagents production by hemotherapic services (HS), if previously validated and with well-defined acceptance criteria. The study examined the stability of irregular red blood cell antibodies (IgG class) present in fresh frozen plasma (FFP) of donor and pre-transfusion patients samples with the aim of using this raw material to produce IC in immunohematology laboratories. The title, score and reactivity (potency tests) of 12 FFP with anti-D, -E and -K and 25 patients samples with anti-D, -E, -e, - c, -C, and -K were evaluated, both stored in freezer (-25°C) and refrigerator (2 to 8°C), for six and three months respectively. In FFP samples the initial values of title and reactivity were maintained during the study and the falls didn\'t exceed a titration or graduation in freezer and refrigerator. However, the storage time of the FFP before the study beginning impacted negatively the score of samples stored in freezer (p=0.021) and refrigerator (p=0.027). In patient samples the title had a negative impact, being affected by the previous storage time before the study beginning and by the storage type during the execution of the tests (freezer and refrigerator). Samples with prolonged previous storage time between 2-8°C had their title significantly impacted when stored in freezer (p=0.014) and refrigerator (p=0.039), indicating that the delayed stay at highter temperatures may lead to degradation of antibodies more sharply. The samples stored in refrigerator during the study had a significant reduction in the title (p=0,016) when compared to the samples stored in freezer, confirming a lower viability of the antibodies kept in less cold temperatures. When was evaluated a possible association between initial title, score and reactivity values and a tendency to fall their potency tests results, it was found that antibodies with initially low values didn\'t show lower stability when compared to antibodies with initial higher values. This feature indicates that IgG irregular antibodies with different inicial potency profiles can be used for IC production provided they are properly characterized, stored and validated. It\'s pertinent to extend the knowledge about the stability of different antibody specificities that can compose a IC panel in addition to those evaluated in this study.

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. <p>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. <p>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. <p>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.<p>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