Diss. (sammanfattning) Linköping : Univ., 2003. / Härtill 6 uppsatser.

A Cannabis sativa L. é a droga de abuso de uso proscrito mais consumida no mundo. O consumo desta droga por condutores de veículo automotor está associado com o aumento do risco de acidentes de trânsito, com o aumento da gravidade e com o aumento das taxas de mortalidade. O objetivo deste trabalho foi propor métodos analíticos aplicáveis à rotina laboratorial forense para monitoramento do consumo de canábis por condutores. Para tanto, foi utilizada como metodologia a extração líquido-líquido seguida de análise por cromatografia em fase gasosa acoplada a detector de massas para pesquisa de canabinoides em matrizes biológicas como sangue total (ST) e fluido oral (FO). A análise de 11-nor-9-carbóxi-delta9-THC (THC-COOH) em amostras de ST por cromatografia gasosa envolve a derivatização desta molécula e, consequentemente, do THC extraído desta matriz (quando presente), uma vez que ambos estão presentes no mesmo extrato, constituindo mais uma etapa analítica e que requer maior controle das condições de reação. Por outro lado, a detecção de THC em FO pode ser realizada sem a realização desta etapa, o que constitui uma vantagem analítica. A determinação de THC-COOH e THC em ST não demonstrou repetibilidade, o que inviabilizou as análises qualitativas e quantitativas nesta matriz. A detecção de THC em FO se mostrou uma análise simples e passível de validação, porém com limite de detecção (200ng/mL) acima do recomendado pelas guias forenses internacionais (2 ng/mL). A metodologia analítica desenvolvida se mostrou compatível com aplicação na análise confirmatória em casos de intoxicação aguda pelo consumo de canábis, demonstrando a necessidade de utilização de técnicas de concentração de amostras como extração em fase sólida ou microextração em fase sólida, para obtenção de menores limites de detecção, podendo assim ser aplicado na rotina laboratorial para o monitoramento do uso frequente de canábis, e não apenas em casos de intoxicação aguda. Além disso, foi realizada uma abordagem sobre a interpretação da detecção de THC em diferentes matrizes biológicas. / Cannabis sativa L. is the illegal drug of abuse most consumed in the world. The consumption of this drug by motor vehicle drivers is associated with an increased risk of traffic accidents, increased severity and increased mortality rates. The objective of this work was to propose analytical methods applicable to forensic laboratories to verify the consumption of cannabis by drivers. Liquid-liquid extraction followed by gas chromatography (GC) coupled to mass spectrometry (MS) detector has been applied to cannabinoid analysis in biological samples such as whole blood (WB) and oral fluid (OF). The analysis of 11-nor-9-carboxy-delta9-THC (THC-COOH) in WB by GC required the derivatization of this molecule and also involved the derivatization of THC since both were extracted from the same sample. The derivatization constitutes another analytical step, which requires greater control of the reaction conditions. Fortunately, the detection of only THC in OF can be done without performing this step. The determination of THC-COOH and THC in WB did not demonstrate repeatability, which impaired the qualitative and quantitative analyzes in this matrix. The detection of THC in OF proved to be a simple analysis, that could be validated, but the limit of detection (200ng/mL) was higher than the recommended by the international forensic guides (2 ng/mL). The chromatographic method developed was compatible with the application of a confirmatory analysis in acute cannabis intoxication, demonstrating the need to use techniques of samples concentration such as SPE or SPME, in order to obtain lower limits of detection, thus being able to be applied in the laboratory routine for the monitoring of the frequent use of cannabis, and not only in cases of acute intoxication. In addition, it was made an approach of THC detection in different biological matrices.

A Cannabis sativa L. é a droga de abuso de uso proscrito mais consumida no mundo. O consumo desta droga por condutores de veículo automotor está associado com o aumento do risco de acidentes de trânsito, com o aumento da gravidade e com o aumento das taxas de mortalidade. O objetivo deste trabalho foi propor métodos analíticos aplicáveis à rotina laboratorial forense para monitoramento do consumo de canábis por condutores. Para tanto, foi utilizada como metodologia a extração líquido-líquido seguida de análise por cromatografia em fase gasosa acoplada a detector de massas para pesquisa de canabinoides em matrizes biológicas como sangue total (ST) e fluido oral (FO). A análise de 11-nor-9-carbóxi-delta9-THC (THC-COOH) em amostras de ST por cromatografia gasosa envolve a derivatização desta molécula e, consequentemente, do THC extraído desta matriz (quando presente), uma vez que ambos estão presentes no mesmo extrato, constituindo mais uma etapa analítica e que requer maior controle das condições de reação. Por outro lado, a detecção de THC em FO pode ser realizada sem a realização desta etapa, o que constitui uma vantagem analítica. A determinação de THC-COOH e THC em ST não demonstrou repetibilidade, o que inviabilizou as análises qualitativas e quantitativas nesta matriz. A detecção de THC em FO se mostrou uma análise simples e passível de validação, porém com limite de detecção (200ng/mL) acima do recomendado pelas guias forenses internacionais (2 ng/mL). A metodologia analítica desenvolvida se mostrou compatível com aplicação na análise confirmatória em casos de intoxicação aguda pelo consumo de canábis, demonstrando a necessidade de utilização de técnicas de concentração de amostras como extração em fase sólida ou microextração em fase sólida, para obtenção de menores limites de detecção, podendo assim ser aplicado na rotina laboratorial para o monitoramento do uso frequente de canábis, e não apenas em casos de intoxicação aguda. Além disso, foi realizada uma abordagem sobre a interpretação da detecção de THC em diferentes matrizes biológicas. / Cannabis sativa L. is the illegal drug of abuse most consumed in the world. The consumption of this drug by motor vehicle drivers is associated with an increased risk of traffic accidents, increased severity and increased mortality rates. The objective of this work was to propose analytical methods applicable to forensic laboratories to verify the consumption of cannabis by drivers. Liquid-liquid extraction followed by gas chromatography (GC) coupled to mass spectrometry (MS) detector has been applied to cannabinoid analysis in biological samples such as whole blood (WB) and oral fluid (OF). The analysis of 11-nor-9-carboxy-delta9-THC (THC-COOH) in WB by GC required the derivatization of this molecule and also involved the derivatization of THC since both were extracted from the same sample. The derivatization constitutes another analytical step, which requires greater control of the reaction conditions. Fortunately, the detection of only THC in OF can be done without performing this step. The determination of THC-COOH and THC in WB did not demonstrate repeatability, which impaired the qualitative and quantitative analyzes in this matrix. The detection of THC in OF proved to be a simple analysis, that could be validated, but the limit of detection (200ng/mL) was higher than the recommended by the international forensic guides (2 ng/mL). The chromatographic method developed was compatible with the application of a confirmatory analysis in acute cannabis intoxication, demonstrating the need to use techniques of samples concentration such as SPE or SPME, in order to obtain lower limits of detection, thus being able to be applied in the laboratory routine for the monitoring of the frequent use of cannabis, and not only in cases of acute intoxication. In addition, it was made an approach of THC detection in different biological matrices.

Pyrogens are substances that may induce fever in the human body. They can be parts of bacteria, virus or fungi and due to the reaction they may cause in the body, they are routinely looked for in the medical technology industries. A method called in vitro pyrogen test (IPT) has been developed to detect these pyrogens. It is based on the fever reaction in the human body and only requires blood in combination with a solution believed to contain pyrogens. If the result is positive, the production of cytokines is started. The cytokines of interest in the IPT method are those involved in the fever process and two of them are IL-1β and TNF-α, which are the cytokines used as markers of infection in this study. Since the production of cytokines is in proportion to the amount of pyrogens, the inflammation-inducing potential of the sample can be decided. Due to problems in standardizing the method, mainly because it handles with living blood cells, focus is still pointed at improving it. The aim of this study was to optimize parameters within the IPT method by analysing air samples taken in indoor surroundings believed to contain pyrogens. The different parameters included extraction of the filter from the air sampling, incubation of whole blood and sample extract and analysis of the incubation with ELISA (enzyme linked immunosorbent assay). More specific, some of the issues concerned extraction media, time and shaking intensity for the extraction, blood ratio for the whole blood incubation and cytokines suitable for the method. A possible approach for the IPT method, when analysing air samples containing pyrogens, was reached. / Pyrogener kallas ämnen som framkallar feber och de kan exempelvis bestå av hela eller delar av bakterier, virus eller svamp (fungi). En metod som kallas för in vitro pyrogen test (IPT) har utvecklats för att detektera dessa pyrogener. Metoden bygger på att en lösning som misstänks innehålla pyrogener får komma i kontakt med blod från en människa. Efter en inkubering på mellan 4-24 timmar har blodet reagerat på eventuella pyrogener och bildat cytokiner, där mängden cytokiner är proportionell mot mängden pyrogener. De intressanta cytokinerna i den här studien var IL-1β och TNF-α, som båda är involverade i feberprocessen. Det har varit svårigheter med att standardisera metoden, mycket beroende på att det är levande celler som hela metoden bygger på, så syftet med den här studien var att förbättra in vitro pyrogen test. Luftprover tagna i inomhusmiljöer som misstänks innehålla pyrogener har använts i försöken att optimera varje steg i processen. De olika stegen inkluderade extraktion av filter som använts vid luftprovtagningen, inkubering med helblod och provextrakt och analys av inkuberingen med ELISA (enzyme linked immunosorbent assay). Några av de parametrar som undersöktes gällde extraktionsmedium, skaktid och skakintensitet under extraktionen, blodförhållande under helblodsinkuberingen och lämpliga cytokiner för metoden. Studien resulterade i att en metodik, för att analysera luftprov innehållande pyrogener med in vitro pyrogen test, kunde tas fram.

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

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Candidate: Bc. Kateřina Nováková Supervisor: RNDr. Gabriela Červená, Ph.D. plk. MUDr. Miloš Bohoněk, Ph.D. Title of diploma thesis: Whole blood - New Blood Component Containing Platelets Background: The aim of this thesis was to evaluate in vitro quality parameters and haemostatic function of leucodepleted WB and their comparison with non-leucodepleted WB. The same way the quality parameters (RBC, PLT, HB and hemolysis) of erythrocyte blood component were measured and compared in day D14, D21, D35 and D42. Methods: WB collected from 30 healthy group A donors was divided into two groups - leucodepleted (LWB), using in-line platelet-sparing filters and collected to a blood bag system IMUFLEX® WB-SP (Terumo BCT, USA) and non-leucodepleted WB (NLWB), colletected to a blood bag system CompoFlex® Single System (Fresenius Kabi, Germany). Both groups were stored at 42řC for 14 days and following parameters were measured in days D0, D1, D3, D5, D10 and D14: WBC, RBC, PLT, HB, hemolysis, pH, TEG, FVIII, TT, PT, aPTT, aggregometry, concentration of PF4 and sCD40L (ELISA). Moreover, in days D0, D7 and D14 was measured the level of expression of platelet activation marker CD62P (P-selectin), CD42b, CD61 by...

Haemostasis is maintained by a dynamic balance between pro- and anti-thrombotic mediators. Its dysregulation can lead to bleeding or thrombosis, and is a major cause of morbidity and mortality. Thus, elucidation of the mechanisms involved in maintaining or disrupting this balance have important implications in health and disease. Investigative tools enable characterization of the haemostatic system, but are often associated with limitations. For instance, haemostasis in animal models is often investigated by assessing bleeding responses in one particular vessel or tissue without a complete understanding of how the results translate to the regulation of haemostasis in other vascular beds. As a second example, microparticles (MPs) are a heterogeneous population of submicron-sized vesicles that may be important in thrombosis. With the exception of a few subtypes, MPs cannot be reliably characterized using widely accessible techniques. Finally, the thrombin generation assay (TGA), which measures ex vivo activation and inhibition of thrombin, is a promising tool for clinical assessment of thrombosis and haemostasis. However, characterization of thrombin generation in the general population, and the development of point of care testing are in their infancies. As a result, the TGA remains largely a research tool. The works described in this thesis specifically seek to address these three limitations in thrombosis and haemostasis research. The first isolated murine arterial bleeding model is presented and its characterization with respect to bleeding in other vascular tissues is described. In addition, a solid-phase capture assay for evaluating procoagulant, P-selectin-binding MPs, which are postulated to be mediators of thrombosis, was developed in order to determine whether these MPs associate with risk of recurrent venous thromboembolism. Lastly, a 25 x 20 mm chip that performs four individual thrombin generation assays using ~10 µl of capillary blood was developed as a proof of concept for point of care thrombin generation testing. / Thesis / Doctor of Philosophy (PhD)

Actuellement, l'étude exploratoire de la fonction hémostatique en routine se fonde essentiellement sur les tests du bilan standard d'hémostase, c'est à dire la détermination du temps caractéristique de formation d'un gel de fibrine dans des conditions standardisées. Cependant, la dernière décennie a vu la naissance de nouveaux tests se focalisant sur les transformations mécaniques du sang lors de sa coagulation. Portés par les récentes avancées dans la connaissance des phénomènes biochimiques et biophysiques menant à ces transformations mécaniques, ces tests, basés sur une étude dynamique des propriétés viscoélastiques de la coagulation du sang total, sont aujourd'hui de plus en plus adoptés par les hématologues et sont au centre d'un nombre grandissant d'études cliniques. C'est dans ce contexte que, s'appuyant sur les récents développements des techniques ultrasonores haute-fréquence, un dispositif de monitoring ultrasonore haute-fréquence de la coagulation sanguine sur sang total a été développé au sein de notre équipe. Grâce à une analyse simultanée de plusieurs paramètres acoustiques, ce dispositif à montré ses capacités à suivre les transformations mécaniques du sang coagulant. Le travail de cette thèse a consisté à poursuivre le développement de ce dispositif en s'attachant notamment à discriminer le rôle respectif des différents phénomènes ayant lieu lors de la coagulation sur les cinétiques acoustiques mesurées. En analysant les effets de traitements anticoagulant et anti-agrégant plaquettaires sur notre monitoring ultrasonore dans le cadre d'une étude clinique pilote, un premier potentiel diagnostic du dispositif a été établi. Les résultats de cette étude ont ensuite mené à la mise en place de mesures spécifiques centrées sur deux phénomènes : la formation de la fibrine et la contraction plaquettaire. Une visualisation originale de la formation du réseau de fibrine a pu être mise en place et a mené à la détermination d'un nouveau paramètre capable de déterminer à la fois le temps de gélification et le temps de rétraction. La gélification du milieu s'est avéré être primordiale dans l'évolution de l'atténuation dans le milieu, tout comme la rétraction du caillot est essentiellement responsable de l'augmentation de la vitesse. / Today, routine blood coagulation tests rely principally on the measurement of the time for a blood sample to gel under standardized conditions. However, in the last decade, new tests focused on monitoring mechanical changes during blood coagulation have been developped. Thanks to a new understanding of the biochemical and biophysical phenomena leading to those mechanical changes, these tests, dynamically studying the viscoelastic properties of coagulating whole blood, tend to be more and more adopted by haematologists and are the focus of a tremendous amount of clinical studies. Within this context and due to the recent development of high-frequency ultrasound techniques, a high-frequency ultrasound apparatus allowing the monitoring of whole blood coagulaion has been developped by our team. Simultaneously analysing the kinetics of four acoustical parameters, it has shown its potential in monitoring the mechanical changes appearing in whole blood coagulation. In this PhD thesis, new developments of this technique have been carried out and allowed to discriminate the respective role of the different phenomena appearing during coagulation on our acoustical parameters. Analysing the effect of anticoagulant and antiplatelet therapy within a pilot clincal study, the diagnostic potential of our test has been established. Following the results of this study, specific measurements have been set up and have shown the importance of two phenomena : fibrin formation and platelet contraction. A new way to visualize the fibrin network formation has been devised and has led to the computation of a new parameter capable of defining gel time and retraction time. Gelation of the medium was shown to be linked to the changes in attenuation in the medium and retraction of the clot was found to be critical in the rise of longitudinal velocity.

O veneno de serpentes do gênero Bothrops (responsáveis por 80% dos envenenamentos no Brasil) é composto por metalo e serinoproteases, disintegrinas, fosfolipases, entre outros, e pode causar edema, hemorragia, necrose, e manifestações sistêmicas, como coagulação intravascular, choque, falência renal e hemorragia. O veneno da B. pirajai ativa o sistema complemento (C), sugerindo uma contribuição para o agravamento dos sintomas. Considerando a importância do C no processo inflamatório e o papel das metaloproteinases nos envenenamentos, verificou-se que o tratamento do sangue total humano (onde células e mediadores plasmáticos interagem entre si) com ou sem a compstatina (inibidor de C3 do C), e com a metaloproteinase de classe PI do veneno de B. pirajai, levou a diferenças bastante significativas na expressão dos marcadores analisados nos leucócitos, na geração de anafilatoxinas e TCC, e na quantificação de citocinas e quimiocinas no plasma, sugerindo que a inibição do C reduz o processo inflamatório, podendo ser uma terapia efetiva para envenenamentos botrópicos. / The snake venom of Bothrops genus (responsible for 80% of envenomation in Brazil) is composed by metallo and serine proteases, disintegrins, phospholipase, among others, and can cause edema, hemorrhage, necrosis, and systemic manifestations, such as intravascular coagulation, shock, renal failure and systemic hemorrhage. The venom of B. pirajai is able to activate the complement system (C), suggesting a contribution to the worsening of symptoms. Considering the importance of C in the inflammatory process and the role of metalloproteinases in envenomation, it was found that treatment of human whole blood (where cells and plasma mediators interact) with or without compstatin (C3 inhibitor), and with a class PI metalloproteinase from B. pirajai\'s venom led to highly significant differences in the expression of the markers analyzed in leukocytes, in generation of anaphylatoxins and TCC, and quantification of cytokines and chemokines in plasma, suggesting that inhibition of C reduces the inflammatory process and may be an effective therapy for bothropic envenomations.

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.