O Hemocentro São Paulo é responsável por manter um banco de dados com informações sobre cada doação ou tentativa de doação de sangue. No entanto, os dados desse banco de dados não possuem a qualidade requerida pelas ferramentas/técnicas de análise. Por essa razão, fica difícil utilizar tais dados para estabelecer relações sistemáticas entre as variáveis armazenadas. A principal contribuição desta tese é a descrição da proveniência para atributos selecionados usando critérios de classificação definidos por especialistas. Este trabalho mostra que é possível fazer investigações detalhadas usando a descrição dos dados sem a necessidade de alterar a estrutura do banco de dados. Durante o período de 1996 a 2006, 1.469.505 doadores foram responsáveis por mais de 2.8 milhões de doações. Após a descrição da proveniência, foram obtidos 252.301 doadores do sexo masculino e 133.056 doadores do sexo feminino e que atenderam aos critérios de inclusão usados nesta tese. Dos 385.357 doadores incluídos na análise, 21.954 (5,7%) tiveram suas doações adiadas devido a seus baixos níveis de hematócrito, 3.850 (1,5%) eram do sexo masculino e 18.104 (13,6%) do sexo feminino. Os resultados obtidos demonstram que, embora os intervalos de espera entre as doações de sangue sejam grandes entre os doadores do sexo feminino e masculino, as mulheres são recusadas mais cedo, por risco de desenvolver anemia, do que os homens. Aproximadamente 12,84% das mulheres e 1,21% dos homens desenvolveriam hematócrito baixo antes da sétima doação. Os dados sugerem que indivíduos com baixo nível de hematócrito devem esperar mais tempo antes de executarem a próxima doação. Portanto, é importante compreender se existe uma ligação entre a doação de sangue e a diminuição no nível de hematócrito, a fim de evitar resultados indesejáveis para os doadores de sangue. O modelo de proveniência apresentado nesta tese não foi definido de acordo com os modelos de proveniência genéricos já implementados. Esta tese apresenta um modelo de proveniência que foi capaz de acrescentar informações semânticas para adquirir conhecimento de um experimento in silico. Um dos principais objetivos foi desenvolver uma abordagem baseada em declarações, tentando responder a importantes questionamentos biológicos. O modelo descrito combina ricas informações em cada processo usando declarações, e se baseia no conhecimento de especialistas. Esta tese também utilizou estatística descritiva e Análise de Sobrevivência. Finalmente, com a validação do modelo em um domínio conhecido, é pretendido expandir esse método para outros sistemas de informação voltados para hemoterapia. / The São Paulo Blood Center is responsible to maintain a database with information on each donation. However, this database does not have the quality required by techniques of analysis. For this reason, it is difficult to use it directly to establish systematic relationships between the variables. The main contribution of this paper is a provenance description of attributes selected using classification criteria defined by specialists. We show that it is possible to make detailed investigations using the data description without the need to change the structure of the database. During 1996 2006, 1,469,505 donors were responsible for more than 2.8 million of donation. After the provenance description, we obtained 252,301 male and 133,056 female that met our inclusion criteria. Of the 385,357 donors included in the analysis, 21,954(5.7%) were deferred due to low hematocrit, 3,850(1.5%) were males and 18,104(13.6%) were females. Our results show that, although the intervals between donations for female and male donors are wider, women presented anemia earlier than men. Approximately 12,84% of the females and 1,21% of the males would develop low hematocrit before the 7th donation. Our data suggest that individuals with low hematocrit level should wait longer before the next donation. Therefore, it is important to understand if there is a connection between blood donation and decrease in hematocrit level in order to prevent undesirable outcomes to blood donors. The provenance model presented here was not defined according to the generic provenance models already implemented. This thesis presents a provenance model that is able to add semantic information to acquire knowledge of an in silico experiment. One of the main purposes is to develop an approach based on declarations in order to answer biological questions. The provenance model described in this paper combines rich information for each process using the declarations, each having expert knowledge as a basis. To evaluate this provenance model we use descriptive statistics and Survival Analysis. Finally, with the validation of the model in a known domain, we intent to apply and validate this provenance model to other hemotherapy information systems.

Um método cromatográfico foi desenvolvido para determinação dos antidepressivos mais prescritos no Brasil e seus produtos de biotransfomação (amitriptilina, imipramina, clomipramina, desmetilclomipramina, desipramina, nortriptilina, fluoxetina, norfluoxetina e sertralina) em sangue total por cromatografia em fase gasosa com detector de nitrogênio e fósforo. A extração em fase sólida (EFS) com o cartucho abselutTM NEXUS foi empregada de forma inovadora. O procedimento de extração consistiu na diluição de 0,5mL de sangue total em tampão (pH 9,5), aplicação da amostra no cartucho, remoção de interferentes usando tampão (pH 9,5) e eluição dos analitos com diclorometano/ isopropanol (17/3 v/v); a etidocaína foi adotada como padrão interno. Os limites de detecção (LD) e quantificação (LIQ) encontrados foram de 0,1mg/L a 0,4mg/L e de 0,4mg/L a 1,6mg/L, respectivamente. O método foi preciso, específico e linear na faixa de concentração estudada (do LIQ até 12mg/L). A recuperação média de todos os analitos foi 65,5%. O método foi aplicado em amostras de âmbito forense e de emergência clínica. / A gas chromatographic method was developed to determine antidepressants most prescribed in Brazil and their metabolites (amitriptyline, imipramine, clomipramine, desmethylclomipramine, desipramine, nortriptyline, fluoxetine, norfluoxetine, and sertralina) in whole blood, using solid phase extraction and gas chromatography with nitrogen-phosphorus detection. The solid-phase extraction (SPE) with abselutTM NEXUS was applied in an innovative manner. The extraction procedure consists of the dilution of 0.5mL of whole blood in buffer (pH 9.5), application of the sample in the cartridge, washing with buffer (pH 9.5) and elution of the analytes with dichloromethane/isopropanol (17/3, v/v). The limit of detection (LOD) and quantification (LLOQ) were from 0.1mg/L to 0.4mg/L and from 0.4mg/L to 1.6mg/L, respectively. Etidocaine was used as internal standard. The method was precise, specific and linear in the studied concentration (range from LLOQ to 12mg/L). The average recovery of all analytes was 65,5%. Forensic and clinical emergency samples were submitted to the validated method.

Three main projects are included in this dissertation. Though seemingly broad in scope, this research afforded a unique opportunity for comprehensive study of urban and rural environmental inhalation exposures to endotoxin (lipopolysaccharide LPS) and the associated immune response. In the LA PM10 Endotoxin Study, ambient concentration of LPS in PM (particulate matter) was quantified through analysis of air samples collected in Southern California. Endotoxin concentrations measured were lower than recognized thresholds for adverse health effects in occupational exposures, but in the same range as for indoor effects. This study provides the first extensive characterization of endotoxin concentration across a large metropolitan area in relation to PM10 and other pollutant monitoring, and supports the need for studies of the role of endotoxin in childhood asthma in urban settings. The Mouse Whole Blood Assay (WBA) Study replicated LPS-induced airway inflammation in a laboratory model. Presently, there is a need for less invasive options for evaluating pulmonary responses to occupational exposures. The whole blood assay (WBA), which measures cytokine production of leukocytes after ex vivo stimulation with LPS, may be one such option. This study used an endotoxin-tolerance model to demonstrate the efficacy of the WBA as a biomarker of inhalation exposure to swine concentrated animal feeding operation (CAFO) dust and showed the utility of the WBA for assessing susceptibility to organic dust-induced lung inflammation. Finally, The Human WBA Study applied the WBA outside the controlled environment of the laboratory. This study utilized pulmonary function testing (PFT), symptom questionnaires and the WBA to evaluate inflammatory responses following an inhalation exposure to purified LPS in CAFO workers and controls. Subjects were stratified into response groups for analysis of WBA results based on PFT response. All subjects demonstrated significant WBA LPS-stimulant dose-responses for all 3 cytokines measured. This study demonstrated that LPS-induced pulmonary and WBA responses are variable among individuals and offered insight into the use of the WBA in future studies. Information gained from these studies provides much insight into urban endotoxin concentrations, the use of the WBA as a biomarker of pulmonary exposure in the rural environment, and offers possibilities for further research.

<p>The aim of this study was to investigate the effects of Sutherlandia frutescens on the inflammatory response and T cell differentiation in vitro using cytokines as biomarkers. Whole blood cells containing various concentrations of Sutherlandia frutescens were stimulated in vitro with either Lipopolysaccharide (LPS) or Phytohaemagglutinin (PHA). Results show that Sutherlandia frutescens is not toxic at any of the concentrations tested. The addition of Sutherlandia frutescens at high concentrations to the stimulated whole blood cell cultures reflects a significant down regulation of Interleukin(IL) 6 and IL-10 compared to the control (P&lt / 0.05) hence suppressed the inflammatory and humoral immune response. Results obtained for Inteferon-gamma (IFN ) shows that Sutherlandia frutescens is donor specific as it reflects both up and down regulation in the release of IFN at the concentrations tested. The in vitro data generated by this study supports the use of Sutherlandia frutescens in the management of inflammatory conditions and allergies such as asthma. However the effects of Sutherlandia frutescens on cell mediated immunity was found to be donor specific. Further investigation of Sutherlandia frutescens on cellular immunity is advised.</p>

Although blood viscosity is an important component in determining vascular function, it is often assumed constant. Emerging evidence linking individual differences in viscosity to cardiovascular disease casts doubt on this assumption. The purpose of this study was to determine the contribution of whole blood viscosity to key measures of vascular function. To address this aim as comprehensively as possible, first, whole blood viscosity was compared with traditional risk factors for cardiovascular disease. Then flow-mediated dilation (FMD), carotid-femoral pulse wave velocity (cfPWV), and carotid artery compliance were calculated either with or without blood viscosity taken into account. Lastly, we tested whether the removal of blood viscosity could influence well-established associations between age and vascular function. Blood viscosity and vascular function were measured in 97 adults ranging in age from 18-63 years. No significant differences were observed between whole blood viscosity and traditional risk factors for cardiovascular disease. Whole blood viscosity was not significantly correlated with FMD, cfPWV, and carotid compliance. As expected, age was positively correlated with cfPWV (r=0.65, p<0.001) and negatively correlated with FMD (r=-0.21, p<0.05) and carotid compliance (r=-0.45, p<0.01). Even after controlling for viscosity, these relationships remained statistically significant (cfPWV r=0.65, p<0.001; FMD r=-0.24, p<0.05; carotid compliance r=-0.44, p<0.05). These results indicate that whole blood viscosity does not appear to significantly impact measures of vascular function and that the rationale for including whole blood viscosity in the calculation of vascular function remains weak. / text

<p>The aim of this study was to investigate the effects of Sutherlandia frutescens on the inflammatory response and T cell differentiation in vitro using cytokines as biomarkers. Whole blood cells containing various concentrations of Sutherlandia frutescens were stimulated in vitro with either Lipopolysaccharide (LPS) or Phytohaemagglutinin (PHA). Results show that Sutherlandia frutescens is not toxic at any of the concentrations tested. The addition of Sutherlandia frutescens at high concentrations to the stimulated whole blood cell cultures reflects a significant down regulation of Interleukin(IL) 6 and IL-10 compared to the control (P&lt / 0.05) hence suppressed the inflammatory and humoral immune response. Results obtained for Inteferon-gamma (IFN ) shows that Sutherlandia frutescens is donor specific as it reflects both up and down regulation in the release of IFN at the concentrations tested. The in vitro data generated by this study supports the use of Sutherlandia frutescens in the management of inflammatory conditions and allergies such as asthma. However the effects of Sutherlandia frutescens on cell mediated immunity was found to be donor specific. Further investigation of Sutherlandia frutescens on cellular immunity is advised.</p>

Clinical islet transplantation is today an established alternative treatment for a selected group of type 1 diabetes patients. The predominant technique for transplantation is infusion of islets in the liver via the portal vein. Obstacles to advancing islet transplantation include limited engraftment resulting from an immediate blood-mediated inflammatory reaction (IBMIR), a life-long need for immunosuppression and the shortage of organs available. In this thesis, innate and adaptive immunity were explored in allogeneic and xenogeneic settings, with the long-term goal of preventing islet graft destruction. Methods for studying immune responses to islets in blood and engrafted islets in liver tissue (intragraft gene expression) were developed and refined. The innate response to human islets and exocrine tissue in ABO-compatible blood was characterized up to 48 h using a novel whole-blood model. Physiological changes in the blood during incubations were explored and adjusted to allow prolonged experiments. Increased production of chemokines targeting CXCR1/2, CCR2 and CXCR3 was observed, accompanied by massive intra-islet neutrophil infiltration. Notably, endocrine and exocrine tissue triggered a similarly strong innate immune response. Two studies of adult porcine islet transplantation to non-human primates (NHPs) were performed. Expression of immune response genes induced in liver tissue of non-immunosuppressed NHPs (≤72 h) was evaluated after porcine islet transplantation. Up-regulation of CXCR3 mRNA, together with IP-10, Mig, MIP-1α, RANTES, MCP-1 and cytotoxic effector molecule transcripts, was associated with T-cell and macrophage infiltration at 48-72 h. Long-term survival (&gt;100 days) of adult porcine islets in a NHP model was later demonstrated using T-cell-based immunosuppression, including co-stimulatory blockade (anti-CD154 mAb). Graft failure was associated with increased levels of circulating, indirectly activated T cells, non-Gal pig-specific IgG and gene transcripts of inflammatory cytokines. Microarray analysis of the response to inflammatory cytokines in cultured porcine islets identified genes involved in cell death, immune responses and oxidative stress; this gene pattern coincided with physiological changes (decrease in insulin and ATP content). In summary, allogeneic whole-blood experiments and xenogeneic in vivo studies underscored the importance of preventing early inflammation and cell-recruitment to avoid islet graft loss in islet transplantation. Long-term survival of porcine islets in NHPs was shown to be feasible using T-cell-directed immunosuppression, including anti-CD154 mAb.

Fifty percent of solid cancers are treated with radiation therapy (RT). The dose used in RT is adjusted to the most sensitive individuals so that not more than 5% of the patients will have severe adverse healthy tissue effects. As a consequence, the majority of the patients will receive a suboptimal dose, as they would have tolerated a higher total dose and received a better tumor control. Thus, if RT could be individualized based on radiation sensitivity (RS), more patients would be cured and the most severe adverse reactions could be avoided. At present the mechanisms behind RS are not known. The long term aim of this thesis was to develop diagnostic tools to assess the individual RS of breast cancer patients and to better understand the mechanisms behind the RS and radiation effects after low dose exposures. The approach was based on the hypothesis that biomarkers of individual RS, in terms of acute adverse skin reactions after breast cancer RT, can be found in whole blood that has been stressed by low doses of ionizing radiation (IR).  To reach this goal two different approaches to identify biomarkers of RS have been investigated. A protocol for the analysis of differential protein expression in response to low dose in vitro irradiated whole blood was developed (paper I). This protocol was then used to investigate the proteomic profile of radiation sensitive and normo-sensitive patients, using isotope-coded protein labeled proteomics (ICPL). The results from the ICPL study (paper III) show that the two patient groups have different protein expression profiles both at the basal level and after IR. In paper II the potential biomarker 8-oxo-dG was investigated in serum after IR. The relative levels of IR induced 8-oxo-dG from radiation sensitive patients differ significantly from normo-sensitive patients. This indicates that the sensitive patients differ in their cellular response to IR and that 8-oxo-dG is a potential biomarker for RS. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Trombocytopeni behandlas primärt med trombocyttransfusion. Trombocytkoncentraten kan erhållas genom poolning av lättcellskikt framställda ur helblodsenheter från flera blodgivare. Helblodsenheterna kyls vanligen ner på en CompoCool®-platta för att snabbt komma ner till rumstemperatur och kan då prepareras redan efter 2 h. Detta brukar vara logistiskt fördelaktigt och gynnar erytrocyterna som framställs ur samma helblodsenheter. Det går även att låta helblodsenheterna kylas ner i rumstemperatur vilket å andra sidan sägs ge ett högre trombocytutbyte då studier visat att trombocyter är känsliga för kyla. Syftet med examensarbetet var att framställa och jämföra kvaliteten på trombocytkoncentrat där helblodsenheten hade kylts ner på CompoCool®-platta respektive kylts ner i rumstemperatur. Hypotesen var att trombocytutbytet skulle bli högre vid nedkylning av helblodsenheten i rumstemperatur än vid nedkylning på CompoCool®-platta. Framställningen av trombocytkoncentraten gjordes genom poolning av 5 st lättcellskikt och en påse trombocytsuspensionsmedium efterföljt av centrifugering och separation i en automatisk blodkomponents separator. Kvalitén utvärderades med avseende på trombocytkoncentration, leukocytkoncentration, swirling samt bakterieodling. Samtliga resultat för kvalitetskontrollerna låg inom de rekommenderade gränsvärdena. Det beräknade t-testet för trombocytkoncentrationen var högre än det kritiska t-värdet vilket innebar att det var en signifikant skillnad mellan de olika nedkylningsmetoderna. Genom användning av de erhållna resultaten kunde hypotesen bekräftas och slutsatsen dras att trombocytutbytet är signifikant högre då helblodsenheten kyls ner i rumstemperatur jämfört med CompoCool®-platta.

Warfarin är ett läkemedel som används för att förhindra att högriskpatienter såsom de med förmaksflimmer får tromboembolism. Denna verkan uppnås genom att hämma de K-vitaminberoende faktorerna VII, X och protrombin och på så sätt minska blodets förmåga att koagulera. Att hitta rätt dosering av läkemedlet för warfarinbehandlade patienter har visat sig vara svårt eftersom det kräver regelbunden provtagning och påverkas av mat- och levnadsvanor. Det vanligaste sättet att mäta protrombinkomplexhalten är med venös plasma men det är även möjligt att använda sig av kapillär plasma. Helblod kan användas för mekaniska metoder som inte använder sig av optisk detektion. Fördelen är att helblod inte kräver centrifugering. Studiens syfte var att undersöka om det fanns en signifikant skillnad (p≤0,05) mellan helblod och plasma som används i den nuvarande metoden för kapillära prover och om det finns en skillnad i stabiliteten av dessa prov. Dubbla prover togs från 30 warfarinbehandlade patienter och 5 icke warfarinbehandlade individer. Ett av proven centrifugerades och analyserades på plasma, det andra analyserades på helblod. Resultaten visade att det fanns en signifikant skillnad (p≤0,05) mellan metoderna. Bland-Altman diagrammet visade att 95 % av helblodsproverna inte var högre än 0,25 INR och lägre än 0,14 INR. Detta har en låg klinisk inverkan. 4 Proverna förvarades i rumstemperatur i upp till 24 timmar och analyserades sedan om. Ingen förändring över 10 % kunde observeras i hållbarheten. Studien visade att trots att det finns en signifikant skillnad är det möjligt att ersätta den nuvarande metoden med plasma och använda helblod istället. / Warfarin is a drug used to prevent high-risk patients such as those with atrial fibrillation from thromboembolisms. This effect is achieved by suppressing vitamin-K dependent factors VII, X and prothrombin and therefore decreasing the bloods ability to clot. Finding the right dosage of the drug for warfarin treated patients has proven difficult, as it demands regular blood draws to monitor their prothrombin complex level, which is affected by dietary and living habits. The most common way to measure prothrombin complex levels is by using venous plasma but it is also possible to use capillary plasma. Whole blood can be used for mechanical methods, which don’t use optical detection. The benefit is that whole blood doesn’t require centrifugation. The aim of this study was to investigate if there was a significant difference (p≤0,05) between using whole blood and plasma which is the existing method for capillary sample and also if there is any differences between the stability of these samples. Double samples from 30 warfarin treated patients and 5 non-treated persons were taken. One of the samples were centrifuged and analyzed on plasma and the other analyzed on whole blood. The results showed that there was a significant difference (p≤0,05) between the methods. Bland-Altman plot comparison showed that 95 % of the whole blood samples would not be higher than 0,25 INR and lower than 0,14 INR. This has low clinical impact. The samples were stored at room temperature for up to 24 hours and reanalyzed. No changes over 10 % in INR values were observed. This study showed that even though there is a significant difference, it is possible to replace the existing method which using plasma with the whole blood instead.