Дисертації з теми "Blood samples and stains"

The aim of the research reported herein was to identify and develop a dried blood spot (DBS) direct analysis technique that could support high sample throughput quantitative bioanalysis in a regulated drug development environment. An initial literature review, coupled with proof of concept testing of the most prominent direct analysis techniques coupled to mass spectrometers (MS), resulted in direct elution (direct extraction of DBS via a confined solvent, producing a liquid extract) being selected as the most suitable technique to develop for this application. Direct elution technology was then developed into fully automated techniques with sufficient functionality to enable compatibility with high sample throughput quantitative bioanalysis. Proof of concept robustness data demonstrated that direct elution, despite the lack of sample clean up, was a reliable technique which had no detrimental effects on detector or chromatographic performance compared to conventional wet plasma extraction and analysis. A proof of concept investigation also demonstrated that a method of improving internal standard (IS) performance by spraying IS solution onto DBS samples prior to extraction, allowed the analyte of interest and IS to be co-extracted, while retaining adequate analytical performance. The foregoing proof of concept data was then combined to produce a fully automated DBS direct elution instrument designed to introduce sample extracts into a LC-MS/MS system. This instrument incorporated a 500 DBS card capacity, an intelligent visual recognition system, a dynamic IS applicator module, and a highly effective wash system that virtually eliminates carryover. Ultimately, this work led to the production of a fully automated DBS direct elution system that is now commercially available. Subsequent research focused on optimising the system, and using this technology to address some of the issues that are currently inhibiting the development of DBS usage in drug development applications, namely haematocrit (HCT) based assay bias, and the decreased sensitivity on offer from DBS sampling. It was demonstrated that using the IS sprayer enabled the IS to integrate sufficiently with the DBS sample prior to extraction to nullify HCT based recovery bias. The direct elution mechanism was also optimised with a view to maximising assay sensitivity while retaining acceptable analytical and chromatographic (LC-MS/MS) performance. Generic optimised direct elution conditions were developed which demonstrated that increases in assay sensitivity of up to 30 fold (compared to conventional manual extraction methods) were possible using a set of representative small molecule compounds.

Bartonella bacilliformis is the etiologic agent of Carrion’s disease. This disease has two well established phases, the most relevant being the so called Oroya Fever, in which B. bacilliformis infect the erythrocytes resulting in severe anemia and transient immunosuppression, with a high lethality in the absence of adequate antibiotic treatment. The presence of B. bacilliformis was studied in 113 blood samples suspected of Carrion’s disease based on clinical criteria, despite the absence of a positive thin blood smear, by two different PCR techniques (using Bartonella-specific and universal 16S rRNA gene primers), and by bacterial culture. The specific 16S rRNA gene primers revealed the presence of 21 B. bacilliformis and 1 Bartonella elizabethae, while universal primers showed both the presence of 3 coinfections in which a concomitant pathogen was detected plus Bartonella, in addition to the presence of infections by other microorganisms such as Agrobacterium or Bacillus firmus. These data support the need to implement molecular tools to diagnose Carrion’s disease.

Background: A number of anticoagulants are available in clinical use to preserve blood samples in liquid form until a suitable time for laboratory testing. Rotational thromboelastography is usually performed on a blood sample that has been anticoagulated with sodium citrate and then recalcified immediately prior to testing. In our institution we have had shortages of citrated Vacutainer® sample tubes. The use of a single in vitro anticoagulant promises to cut costs, simplify laboratory processes as well as limit the amount of blood drawn from patients. This together with the known problems with using citrate as an anticoagulant for viscoelastic testing (VET) prompted us to investigate the suitability of EDTA as anticoagulant for VET. Method: Blood samples from 20 healthy volunteers were divided into citrated and EDTA Vacutainer® tubes. A ROTEM EXTEM® assay was performed on each sample in both groups following the manufacturer's guidelines. Clotting time (CT), clot formation time (CFT), alpha angle (α-angle) and maximum clot firmness (MCF) results were compared. Ionised calcium concentrations were measured on each sample before and after recalcification with CaCl2 to determine if there was a significant difference in post - recalcification ionised calcium concentrations between the groups. Results: The results from the two groups were treated by Bland-Altman analysis. Apart from MCF values there was significant bias between all parameters measured in the two groups. The limits of agreement for all parameters apart from MCF were unacceptable. Conclusion: We found that ROTEM EXTEM® results from EDTA samples were not comparable to or interchangeable with those from citrated samples. The difference in results is not due to differences in ionised calcium concentration levels in the samples post-recalcification as the ionised calcium concentrations in both groups post-recalcification were adequate for coagulation. EDTA samples did show superior consistency in all parameters and may be a suitable alternative for sample preservation for VET if reference ranges can be established.

An important aspect of forensic science is the detection and identification of biological fluids at a crime scene (Virkler and Lednev 2009). The determination of the type and origin of a biological sample can yield valuable information that supports a link between the criminal act and donor, which in turn may assist in the reconstruction and sequencing of a crime scene (An et al. 2012). A body and therefore any associated biological stains may not be located for a period of time, during which the decedent will begin to decompose. Blood and decomposition fluid stains have been reported to be visually similar (Comstock 2014) and therefore, it is important to determine the source of the stain. The presence of blood would suggest an injury has occurred before or shortly after death, whereas decomposition fluid is produced as a part of the naturally occurring decomposition process. Several approaches including visual examination, pH measurements, presumptive testing for blood, spectroscopic techniques, the analysis of volatile organic compounds, genomics, and proteomics may provide potential methods of biological stain identification and differentiation (Harbison and Fleming 2016; Stuart 2013; Virkler and Lednev 2009). However, there are associated limitations to these methods. This dissertation reviewed the effectiveness of these methods, which then informed the development of a proof--‐of--‐ concept study to assess if the technique of microfluidic proteomics by protein electrophoresis can identify potential differences between blood and decomposition fluid stains. When compared to conventional techniques, microfluidic devices offer many advantages including improved efficiency, a decrease in sample and reagent consumption, and automation (Li 2015). The potential results obtained from the proposed study design will assist in enhancing the knowledge base surrounding the differentiation of blood and decomposition fluid stains.

Blood stains are a relatively common occurrence at scenes of violent crime and are a source of much information including biological, circumstantial, positional and potentially, information on the timeline of the crime. Successfully aging blood on scene could aid in time of death estimation, time since incident or even verify alibies of potential suspects. Methods in use thus far for time since death currently hold high error rates due to fluctuations in environmental factors. Several different techniques for aging of blood stains have been analysed and compared for suitability as well as similar spectroscopic techniques. Some of these aging methods were found to be complimentary, as reflectance spectroscopy has a relatively low error rate for short term aging, followed by a gradual increase, making it unsuitable for long term aging. Whereas, RNA marker analysis which follows the general degradation pattern of select RNA, was able to show a more steady error rate making it a more viable method for long term aging but not well suited to short term. The major variable factors in determining the time since deposition of blood stains include; daily or person to person fluctuations in blood protein percentages, haematocrit values, drugs present and environmental considerations such as temperature, humidity and UV light exposure. In order for any method to be acceptable in court, the full extent to these factors needs determination and full error analysis implemented. Additionally, improvement in portability, efficiency and development of non-destructive methods should be prioritised.

Leishmaniasis is a disease affecting more than 12 million people worldwide. It is caused by the protozoan parasite Leishmania, which is transmitted to humans and dog hosts through bites of infected sand flies belonging to genus Phlebotomine. Several studies have shown Polymerase Chain Reaction (PCR) to be effective for the diagnosis of VL in clinical samples compared to the classical methods. The aims of this study were first to compare four different sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and furthermore to find a method that is sensitive, rapid, cost benefit, simple and easy to perform. Two preparation methods were compared for the isolation of leukocytes (with Ficoll and Tris –EDTA buffer) and two DNA isolation methods (with Proteinase K and QIAgen kit). From the methods that were compared, lysis of erythrocytes with TE and the QIAgen kit seems to be the most suitable to use.

Epigenome-wide association studies are used to link patterns in the epigenome to human phenotypes and disease. These studies continue to increase in num- ber, driven by improving technologies and decreasing costs. However, results from population-scale association studies are often difficult to interpret. One major chal- lenge to interpretation is separating biologically relevant epigenetic changes from changes to the underlying cell type composition. This thesis focuses on computa- tional methods for correcting cell type composition in epigenome-wide association studies measuring DNAm in blood. Specifically, we focus on a class of methods, called reference-based methods, that rely on measurements of DNAm from puri- fied constituent cell types. Currently, reference-based correction methods perform poorly on human cord blood. This is unusual because adult blood, a closely related tissue, is a case-study in successful computational correction. Several previous attempts at improving cord blood estimation were only partially successful. We demonstrate how reference-based estimation methods that rely on for cord blood can be improved. First, we validated that existing methods perform poorly on cord blood, especially in minor cell types. Then, we demonstrated how this low per- formance stems from missing cell type references, data normalization and violated assumptions in signature construction. Resolving these issues improved estimates in a validation set with experimentally generated ground truth. Finally, we com- pared our reference-based estimates against reference-free techniques, an alterna- tive class of computational correction methods. Going forward, this thesis provides a template for extending reference-based estimation to other heterogeneous tissues.
Science, Faculty of
Computer Science, Department of
Graduate

Reliable biomarkers of axonal damage are urgently needed in neurological diseases. Neurofilaments (Nf) are specific structural elements of neurons composed of at least three subunits: Nf light chain (NfL), Nf medium and Nf heavy chain (NfH). This PhD aimed to characterise NfL levels and their correlation with clinical features in patients with neurological diseases with a different rate of progression and following and under different treatment regimes. An important aim was also to develop a bioassay for NfL measurements in blood. Cerebrospinal fluid (CSF) NfL levels discriminated patients with a clinically isolated syndrome (CIS) (p=0.001) or multiple sclerosis (MS) (p=0.035) from healthy controls more efficiently, and was more sensitive to change after natalizumab therapy (p<0.0001) than CSF NfH (p=0.002). Further, CSF NfL levels decreased in fingolimodtreated MS patients (p=0.001), but not in those receiving placebo (p=0.433). Based on these findings, a sensitive method for the detection of NfL in serum was developed and validated. Patients with neurological diseases had higher serum NfL values than controls. In acute spinal cord injury (SCI), serum NfL levels correlated with injury severity and long-term motor outcome, and Minocycline treatment was associated with decreased NfL levels in complete SCI patients compared to placebo. Finally, I found that serum NfL levels were higher in CIS patients than in healthy controls but did not predict conversion to clinically definite MS (CDMS). Independent predictors of CDMS were instead oligoclonal bands, number of T2 lesions and age at CIS. Lower 25-OHvitamin D levels were associated with CDMS in univariate analysis, but this was attenuated in the multivariate model. In conclusion, NfL proved to be an analytically stable protein which is an important prerequisite for biomarkers. The role of NfL quantification as a surrogate measure of neuroaxonal damage is corroborated by my findings and further supports the usefulness of NfL as a putative biomarker of axonal damage in various neurological diseases.

Bakgrund: Sjukvårdsrelaterade infektioner komplicerar vården av miljontals patienter varje år och är mer frekventa i utvecklings länder. Hur sjuksköterskor arbetar med hygienrutiner har ett stort inflytande över spridningen av dessa sjukdomar. Att ta ett blodprov är en invasiv procedur och trots att det för många sjuksköterskor är rutin så kan det vara förenat med risker för patient och sjuksköterska. Syfte: Att observera och beskriva hur sjuksköterskor utför hygien rutiner vid blodprovstagning i högrisk områden. Metod: En empiriskt strukturerad observationsstudie med en kvantitativ ansats, genomförd på ett sjukhus i Mpongwe distriktet i Zambia. Resultatet har blivit analyserat med hjälp av manifest innehållsanalys. Resultat: Sjuksköterskornas arbete med hygienrutiner vid blodprovstagning följde ofta inte från sjukhuset uttalade riktlinjer. Majoriteten av de observerade sjuksköterskorna tvättade och desinfekterade inte händerna enligt angiven procedur. Skyddsutrustning användes ofta inte alls eller på ett felaktigt sätt. Omständigheterna för arbete uppmuntrade inte till utförande av hygienrutiner. Slutsats: Resultatet indikerar ett behov av mer finansiella medel såväl som mer utbildning och förespråkande för vikten av hygienrutiner vid blodprovstagning. Detta för an ökad förståelsen av värdet av dessa rutiner.
Background: Healthcare related infections complicates the care of millions of people world wide every year and is shown more frequent in developing countries. How nurses follow basic hygiene routines has a great impact of the spreading of such infections. Collecting blood is an invasive procedure and even though it is a routine procedure for most nurses it can still be related to a great risk of exposure for both patient and performer.Objective: To observe and describe how nurses follow hygiene routines when collecting blood samples in areas with high prevalence of infection diseases.Methods: Empirically structured observational study with a qualitative approach, carried out in a hospital in Mpongwe district, Zambia. The result has been analyzed through manifest content analysis. Result: Basic hygiene routines were often not followed when collecting blood specimen in the hospital who served as setting for this study. A majority of the nurses did not wash and disinfect hands in accordance with guidelines recognized by the hospital. Protective equipment was often not used, at all, or in a correct way even when available. The environment did not promote hygiene routines when collecting samples. Conclusion: The result indicate a need for more financial means as well as more persistent education and campaigning regarding the importance of preforming hygiene routines when collecting blood samples. This to promote a change in performance and attitudes among staff members regarding the importance of those routines.

Background The lack of an effective diagnostic tool for Carrion’s disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches. Methodology/Principal Findings We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. Methodology/Principal Findings We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. Conclusions/Significance From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion’s disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.

学位審査報告書には「分離剤入り採血管で貯蔵した血液検体を再遠心分離したために生じた偽性高カリウム血症」とあり
Kyoto University (京都大学)
0048
新制・課程博士
博士(医学)
甲第8860号
医博第2363号
新制||医||768(附属図書館)
UT51-2001-F190
京都大学大学院医学研究科内科系専攻
(主査)教授 一山 智, 教授 内山 卓, 教授 福井 次矢
学位規則第4条第1項該当

Molecular analysis of free circulating (fc)DNA has the potential to change the face of medicine, specifically in cancer diagnostics and in monitoring the efficacy of cancer treatments. In this study, a microfluidic device using AC electrokinetics is developed for rapid concentration and detection of fcDNA from blood. The device concentrates fcDNA using a combination of AC electrothermal flow and dielectrophoresis. The electrothermal fluid motion drives fcDNA towards the center of the electrode where dielectrophoretic trapping occurs. Once fcDNA is collected at the center, the concentration in the sample can be determined by fluorescent analysis using an intercalating dye binding to the double-stranded DNA. Effects of operating parameters are investigated to optimize the device's design. The electrokinetic device isolates high molecular weight DNA and can distinguish from low molecular weight DNA. Quantitative detection of fcDNA in physiologically relevant concentrations is demonstrated toward rapid diagnostics of cancer and monitoring of treatment efficacy.

The demand for devices capable of efficiently identifying the presence of biomarkers in human body, useful to detect a wide variety of pathologies, is progressively increasing. This urge is generated in the scientific community due to the pressing request of reliable screening protocols, able to enhance in the next future the detection capability on pathologies while not in terminal or harmful state of development. The main goals of this approach lay in the enhancement of the medical prevention, and consequentially the reduction of the national health systems expenses to cure harmful pathologies once degenerated. Nanostructured chemoresistive semiconductor sensors, which can change their conductance depending on the chemical reactions between their surface and the gaseous analytes, showed in the past to be a suitable choice for medical research, especially for oncological purposes (there is plenty of literature discussing volatile organic compounds and tumor detection), and in this work they are proposed as sensing units for a final screening devices implementation. In this thesis, a prototype hosting an array of metal-oxide and non-metal-oxide sensors was tested, in order to detect the presence of airborne tumor markers exhaled from three different kind of samples: human blood from medical sampling, human tissues from surgeries, and cell cultures of different nature. In all the experiments, these chemicals were conveyed to sensors with an air-flow circuit, equipped with antibacterial filters to maintain the sterility of the system. Resulting signals were acquired, processed and plotted thanks custom-made software, realized in Labview®. Statistics on these signals were performed with single sensors approach and Principal Component Analysis. Tests were carried on cell cultures, in order to verify if, having only one type of cell involved in the exhalation of markers, it was possible to observe differences between primary cell lines while compared to the immortalized and tumor strains, and the possibility to discriminate also between different kind of the second macro-group of cells, having them been plated at the same time and still having different metabolisms. For what concerns blood samples, both male and female subjects, ranging between 21 to 91 years of age, have accepted to participate in this study, donating their blood samples. Donors were patients affected by colorectal and stomach cancers, at different stages of evolution, and/or having differently localized metastasis, confronted to a healthy control group sharing same gender and age spectrum. At the end of this part of the study, the prototype proved capable to distinguish between tumor-affected individuals and healthy samples. Obtained results have shown also correlation between the amplitude of the responses and the level of tumor growth and vascularization, as well as a decrease of the response to the healthy threshold for the post-screening of the patients after surgery or chemotherapy. Finally, for human tissues, tests on samples from the tumor-affected area and the healthy surroundings have been effected (during surgical removal, parts from the healthy tissue of the subject are inevitably removed together with the cancer itself; also, for some kind of tumors – like colorectal – the removal of healthy tissue upstream and downstream of the cancer is also voluntary from the surgeons, in order to avoid field effect from the original tumor, which may spread the disease on healthy tissue even after the removal of the original neoplasia). The donors were patients already undergoing surgery, due to the pathologies they were affected from. The donors age, from both sexes, spanned between 41 and 91 years old. The results on human samples have shown that the sensor responses, once processed, well discriminate healthy and tumor-affected subjects, with the specimens tested after two hours and a half from the extraction.
La richiesta di strumenti in grado di identificare efficientemente la presenza di biomarcatori nel corpo umano, funzionali al rilevamento una grande varietà di patologie, è in progressivo aumento. Questa necessità è generata nella comunità scientifica dalla pressante richiesta dello sviluppo di protocolli di screening affidabili, in grado di migliorare la capacità di identificazione di patologie ancora non in stadio terminale o comunque dannoso per l’organismo. Gli obiettivi principali di questo approccio risiedono in un miglioramento generale della prevenzione medicale, ed in una conseguente riduzione delle spese per la cura di patologie per i sistemi sanitari nazionali. Sensori nanostrutturati chemoresistivi a semiconduttori, in grado di variare la propria conduttanza a seconda delle reazioni chimiche tra la loro superficie e gli analiti gassosi, hanno dimostrato in passato di essere una scelta papabile per la ricerca oncologica (esiste grande riscontro in letteratura scientifica trattante i composti organici volatili per il rivelamento tumorale), e in questo lavoro sono proposti come unità sensibili per dispositivi per lo screening. In questa tesi, un prototipo ospitante un array di sensori ad ossidi, metallici e non, è stato testato per individuare la presenza di marker tumorali esalati da tre differenti tipologie di campioni: sangue umano, tessuti umani da operazioni chirurgiche, e colture cellulari di diversa natura. In tutti gli esperimenti, questi agenti chimici sono stati convogliati ai sensori tramite un circuito per il flusso d’aria, dotato di filtri antibatterici per mantenere la sterilità del sistema. Le risposte sono state poi acquisite, processate e rappresentate graficamente grazie a dei software realizzati in Labview®. Lo studio statistico di questi segnali è stato effettuato mediante approccio a singolo sensore e l’Analisi delle Componenti Principali. Test sono stati effettuati su colture cellulari osservando che, avendo un solo tipo di cellule coinvolte nell’esalazione dei marcatori, fosse possibile differenziare quelle provenienti da coltura primaria e quelle da linee immortalizzate o tumorali, nonché la possibilità di discriminare anche tra diverse tipologie di cellule, una volta piastrate nello stesso momento ma mantenenti diverso metabolismo. Per ciò che riguarda i test sui campioni sanguigni, sia soggetti maschili che femminili aventi età tra 21 e 91 anni si sono prestati allo studio. I donatori sono stati pazienti affetti da cancro a colon-retto e stomaco, a differenti stadi evolutivi, e/o aventi metastasi localizzate differentemente, posti a confronto con un gruppo di controllo avente medesimi generi ed età. Il prototipo è stato in grado di distinguere tra campioni provenienti da individui affetti da tumori o sani. I risultati ottenuti hanno inoltre mostrato una correlazione tra l’ampiezza delle risposte ed il livello di crescita e vascolarizzazione del tumore, così come una decrescita nel post-screening operatorio a pazienti dopo intervento o chemioterapia. Infine, per i tessuti umani, test sono stati effettuati su campioni provenienti dall’area affetta dal tumore e su quella sana circostante ad esso (durante la rimozione chirurgia, parti del tessuto sano sono inevitabilmente rimosse assieme al cancro stesso; inoltre per alcuni tipi di tumori, come quello al colon-retto, la rimozione di tessuto sano a valle e a monte del tumore è volontaria da parte del chirurgo, così da evitare il field effect, che può diffondere la patologia anche dopo la rimozione della neoplasia originale). I donatori sono stati pazienti già registrati per operazioni chirurgiche, per via di patologie da cui già erano affetti. L’età dei donatori, di sesso maschile e femminile, va da 41 a 91 anni. I risultati sui tessuti umani mostrano che i sensori distinguono tra soggetti sani e affetti da tumori, con i campioni testati dopo due ore e mezza dall’estrazione chirurgica.

Engineered control over one-dimensional (1D) mesoporous silica nanotubes (NTs) inside anodic alumina membranes (AAMs) has led to various methods for detecting, visualizing, adsorbing, filtering, and recovering ultra-trace concentrations of toxic metal ions, such as Hg2+ and Pb2+, in water and blood. These often “one-pot” screening methods offer advantages over conventional methods in that they do not need sophisticated instruments or laborious sample preparation. This mesoscopic membrane sensor for the nakedeye detection micro-object have large surface area-to-volume ratios and uniformly shaped pores in threedimensional nanoscale gyroidal structures and its active sites consist of heteroatoms arranged around uniformly shaped pores in three-dimensional (3D) nanoscale gyroidal mesostructures densely coated with the chelating ligand so it permits ultra-fast, specific, pH-dependent visualization and removal of toxic metals at low concentrations from aqueous media, including drinking water and a suspension of red blood cells by means of a colorimetric signal visible to the naked eye, as well as by means of UV–vis reflectance spectroscopy. Removal of target ions from biological fluids was assessed by means of flow cytometric analysis. Our results demonstrate the potential for our membrane sensors to be used for preventing the health risks associated with exposure to toxic metal ions in the environment and blood.

Revisión por pares
16th International Congress on Infectious Diseases (ICID), 2014. 2-5 de Abril 2014. Cape Town, South Africa
Background: Bartonella bacilliformis is the etiological agent of Carrion’s disease, a neglected illness with a febrile lethal stage and a warty benign phase, being the human the only known reservoir. The diagnostic by microscopy in endemic areas is several times erroneous. Furthermore, the culture of this bacterium is time-consuming, being the diagnostic by PCR the easiest way to perform a correct diagnostic. The objective of this study was to evaluate the detection limit of three PCR schemes, designed to detect B.bacilliformis, both in blood and filter papers to test their potential use for transferring samples from endemic areas to reference centers. Moreover, the specificity was also observed as well as the applicability of the technique with clinical samples from different stages of the disease. Methods & Materials: Fragments of 16SrRNA and fla genes were amplified as well as the variable-intergenic region (its). The detection limit was determined by bacterial quantification with flow cytometry and performing dilutions (106cfu/ml-10cfu/ml) both in blood and filter papers. DNA was extracted and PCRs were performed. Specificity was tested by processing other bacteraemia microorganisms. Clinical samples, 12 from febrile patients, 13 from warty and 71 from healthy asymptomatic individuals living in endemic area(Mandinga-Cajamarca) were also processed. Results: The 16SrRNA PCR scheme showed the lower detection limit (5 cfu from blood and filter paper) being the PCR scheme chosen to be tested in clinical samples. All febrile patients’ samples were positive, whereas in warty individuals only 3(23%) faint bands were obtained. No amplification was obtained in samples from healthy people. Fainter bands were always obtained when PCRs were made of filter papers. All PCRs were specific for B.bacilliformis. Conclusion: The 16SrRNA PCR seems to be the best technique to detect feverish patients. However, the applicability to identify asymptomatic carriers was undetermined. Filter paper may be an alternative for easy transportation of samples but is need to consider the decreasing sensitivity of the results. It is critical to develop rapid, sensitive and specific technique capable of being applied in endemic rural areas, to avoid misdiagnosis and facilitate the detection of asymptomatic carriers that will allow progress towards the eradication of this disease.

Il tumore al colon-retto è, ad oggi, un grave problema di salute pubblica, in quanto costituisce il 10% di tutti i tumori annualmente diagnosticati e dei decessi strettamente correlati a questo tipo di patologia in tutto il mondo. Per questo motivo, il riconoscimento e monitoraggio dei marcatori tumorali per mezzo di tecniche completamente non invasive e affidabili, rappresenta un obiettivo di ricerca fortemente condiviso dalla comunità scientifica mondiale. ll riconoscimento univoco di questi marcatori rappresenta quindi un aspetto di cruciale importanza nello screening tumorale, che permetterebbe il miglioramento dell’affidabilità dei protocolli di screening attualmente in uso e, l’eventuale individuazione della malattia in stato precoce, aumentando così i margini di intervento e le probabilità di guarigione. Questa tesi rappresenta un lavoro di durata triennale, atto all’individuazione di marker tumorali volatili (VOC) emanati dal metabolismo cellulare alterato dal cancro al colon-retto (CCR), per mezzo di sensori chemoresistivi nanostrutturati a film spesso. A questo fine, sono state analizzate diverse tipologie di campioni biologici come feci, sangue, tessuti bioptici e cellule immortalizzate, tramite un dispositivo innovativo brevettato (Italia n° 102015000057717), nominato SCENT B1, che ospita un array di quattro sensori del suddetto tipo, basati su materiali sensibili diversi (combinazioni di ossidi di stagno, titanio, tungsteno, niobio, vanadio e tantalio). La scelta dei sensori per l’applicazione in campo biomedicale è stata effettuata sulla base di test di laboratorio (presso il Laboratorio Sensori, Dipartimento di Fisica, Università di Ferrara) che hanno messo in luce la loro idoneità nell’individuare i composti organici volatili di interesse con il miglior compromesso fra sensibilità e specificità. I risultati ottenuti nel corso dello studio sono stati piuttosto incoraggianti, in quanto mettono in luce la capacità dei sensori scelti di distinguere le feci, i tessuti e il sangue prelevati da soggetti affetti da cancro al colon-retto da quelli prelevati da soggetti sani (giovani e senza fattori di rischio rilevanti), inseriti nello studio come controparti di controllo (paragrafi 4.1 – 4.6). I test svolti sulle cellule immortalizzate hanno messo in luce la sensibilità dei sensori in relazione alla tipologia di cellule analizzate, alla loro concentrazione iniziale di piastratura e, alle ore di incubazione a cui sono state sottoposte (24, 48,72 ore). E’ stato inoltre presentato il progetto di rinnovamento del dispositivo SCENT, partendo dalla riprogettazione dell’involucro (con conseguente riarrangiamento degli spazi interni), alla riprogettazione dell’apparato elettronico e software. Quest’ultimo è stato riadattato, sfruttando componenti elettroniche di ultima generazione che esibiscono rumori elettronici trascurabili, portando a eccellenti rapporti di segnale-rumore, e ad un basso surriscaldamento di tutto l’apparato. Infine, è stato avviato un nuovo studio al termine del 2020, con lo scopo di eseguire un protocollo di follow-up nei pazienti oncologici tramite quattro prelievi di sangue effettuati in quattro momenti diversi del percorso post-operatorio. Questo processo ha lo scopo di eseguire un monitoraggio dinamico del paziente in seguito all’intervento chirurgico, rivelando così un’eventuale insorgenza di recidive e/o complicazioni.
Colorectal cancer (CRC) is, nowadays, a severe problem of public health, counting the 10% of all the tumors annually diagnosed worldwide and of their strictly related deaths. On this basis, the non-invasive tumoral markers recognition and monitoring represent a strongly shared research topic in the whole world scientific community. The detection of these markers represents a crucial aspect in field of tumor prevention, allowing to improve the current screening protocols. The establishment of a reliable and accurate novel screening protocol might promote the early-stage tumor detection, enhancing the healing probability. This thesis reports a three-year work, aimed to reveal the volatile tumor-markers (VOCs) exhaled by CRC-affected cells (mainly produced by their membrane peroxidation and altered metabolism), by means of thick-film nanostructured chemoresistive metal-oxide sensors. With this aim, a wide range of diverse biological samples have been investigated, such as feces, blood, tumor tissues, and immortalized cells, by using the innovative patented devices: SCENT B1 (Italian patent n° 102015000057717) and SCENT A1 (Italian patent n° RM2014A000595, European patent n° 3210013), both hosting an array of different sensors based on different active material blends (mixtures of tin, titanium, tungsten, tantalum, niobium, vanadium oxides). The sensor choice for the bio-medical field applications has been performed after several laboratory tests (at Laboratorio Sensori, Department of Physics, University of Ferrara), highlighting the sensor suitability for VOC-detection with the best compromise between sensitivity and specificity parameters. The attained results have been rather encouraging, highlighting the capability of sensors of distinguishing among feces, blood, and tumor tissues collected from CRC-affected subjects and the healthy subject ones, taken as controls (young and without relevant risk factors), by comparing the sensor response patterns. The tests carried out on immortalized cells underlined the dependence of the sensor responses on the cells type, on their initial plating concentration and on the incubation period length (24-48-72 hours). Moreover, the renewed SCENT device has been described (Chapter 5). It starts from the enclosure re-design (with consequent internal spaces rearrangement) and follows with the electronic system and management software re-design and production. The electronic system (most renewed device part) has been readapted exploiting the last generation electronic components, that exhibit a very low electronic noise and an excellent signal-to-noise ratio. Their employment, besides improving the device stability and reliability, allays the internal device overheating. Finally, a new study has been started at the end of 2020, aimed to carry out a follow-up protocol for CRC patients. It consists in the analysis of four blood samples during the post-surgery period. This process should allow a dynamic monitoring of the patient health status and the detection of possible relapses.

Thesis (MScMed)--Stellenbosch University, 2008.
ENGLISH ABSTRACT: Introduction: Flow cytometry has progressively replaced many traditional laboratory tests due to its greater accuracy, sensitivity and rapidity in the routine clinical settings especially clinical trails. It is a powerful tool for the measuring of chemical (the fluorochrome we add) and physical (size and complexity) characteristics of individual cells. As these instruments became major diagnostic and prognostic tools, the need for more advanced quality control, standardized procedures and proficiency testing programs increased as these instrumentations and their methodology evolve. Minor instrument settings can affect the reliability, reproducibility and sensitivity of the cytometer and should be monitored and documented in order to ensure identical conditions of measurement on a daily basis. This can be accomplished by following an Internal Quality Assurance (IQA) and/ or External Quality Assurance (EQA) program. Currently there are no such programs available in South Africa and poorer Africa countries. HIV is a global concern and the laboratories and clinics in these places are in need of such IQA programs to ensure quality of their instrumentation and accurate patient results. Quality assurance programs such as CD Chex® and UK Nequas are available but due to bad sample transport, leave the receiving laboratories with nightmares. It would be best if there was a laboratory in South Africa that could provide the surrounding laboratories with stabilized whole blood samples that can be utilized as IQA. The transport of these samples can be more efficient due to shorter distance and thus the temperature variations limited. Aims and Objectives: The aim of Chapter one is to familiarize the reader with general terminology and concepts of immunology. Chapter two describes in detail the impact stabilized whole blood had on clinical immunology concerning Quality Control and Quality Assurance. The objective of this study is to stabilize whole blood with a shelf life of greater than 30 days to serve as reference control material for South African Immunophenotyping. It is further an objective to use these in-house stabilized control samples for poorer African countries as Internal Quality Assurance reference material. It is a still further objective to stimulate various lymphocyte subsets to express activation antigens and then stabilize these cells for more specialized immunological test and can serve as a QC for those required samples. Study design: In Chapter three, the method currently used to stabilize whole blood was modified. The stability of different concentrations of a first stabilizing agent (Chromium Chloride hexahydrate) was investigated. Incubation periods and concentrations of paraformaldehyde as second stabilizing agent were investigated. Blood samples from healthy individuals (n=10) were stabilized and monitored for the routine HIV phenotypic surface antigens over a period of 40 days. These samples (n=10) were compared on the Becton Dickinson Biosciences (BD) FACSCalibur™ versus BD FACSCount™ instrumentation. Blood samples (n=3) were stabilized and monitored to identify phenotypic cell surface molecules for as long as possible. They were quantified on both flow cytrometric instruments. In addition, these stabilized samples (n=3) were investigated as control blood for calibration purposes on the BD FACSCount™ instrument. In Chapter four, lymphocytes were isolated and activated with various stimuli to express sufficient activation antigens such as CD25, CD69, HLA-DR and CD40 Ligand on the T helper cell surfaces. These activated antigens were analyzed on the BD FACSCalibur™ and further stabilized to serve as possible IQA samples in future. Results: In Chapter three, the ten individual stabilized samples had non-significant P values (P > 0.05) for CD3, CD4 and CD8 percentages and absolute values comparing day 3 until day 40. Comparing the BD FACSCalibur™ versus BD FACSCount™, resulted in a R2 = 0.9848 for CD4 absolute values and a R2 = 0.9636 for CD8 absolute values. Stabilized blood samples (n=3) were monitored for routine HIV phenotypic markers until day 84. The cells populations were easily identifiable and could be quantified on both BD FACSCalibur™ and BD FACSCount™ instruments. In Chapter four; for the activation study purposes, activated T helper lymphocytes expressed approximately 25 to 35% CD40 Ligand cell surface molecules. The stimulant of choice was Ionomycin at a 4μM concentration. Cells were incubated for four hours at 37 degree Celsius in a 5% CO2 environment. For CD69 surface expression, 6 hour incubation was optimum. The stimulus of choice in this case was 4μM Ionomycin which induced 84.21% CD69 expression in the test samples. For CD25 expression; 6 hour incubation with PHA resulted in approximately 43% of CD25 expression. For HLA-DR surface expression; 6 hour incubation with PHA resulted in approximately 43.32% of HLA-DR expression. Activated lymphocytes expressing CD40 Ligand showed stability until day 23. Activated Lymphocytes expressing CD69, CD25 and HLA-DR were stabilized in the same manner and stability could be achieved until day 16. Conclusion: This thesis was related to the preparation of control samples (IQA) designed to simulate whole blood having defined properties in clinical laboratory situations. In future kits can be developed with a low, medium and high control sample for the various immunological phenotypic determinants. Another kit can be compiled where various activation markers can be identified, quantified with a “zero”, low and high control. These whole blood IQA kits and “activation IQA kits” can be implemented for training of newly qualified staff, competency testing of staff, method development, software testing, panel settings and instrument setting testing. Control samples ideally must have a number of properties in order to be effective. For instance stability during storage times, preferably lasting more than a few weeks, reproducibility and ease of handling. These will provide the information on day-to-day variation of the technique or equipment which will enhance accuracy and improve patient care.
AFRIKAANSE OPSOMMING: Inleiding: Vloeisitometrie tegnologie het verskeie tradisionele laboratorium toetse vervang as gevolg van beter akuraadheid, sensitiwiteit en vinniger beskikbaarheid van resultate in ‘n kliniese omgewing, veral kliniese proewe. Vloeisitometrie is ‘n kragtige tegniek om chemiese (fluorokroom byvoeging) en fisiese (sel grote en kompleksiteit) karakter eienskappe van individuele selle te meet. Met die toename in gebruik en gewildheid van hiedie instrumente, neem die behoefde toe vir gevorderde kwaliteit kontroles, gestandardiseerde prosedures, met profesionele toets programme tesame met metode ontwikkeling. Klein verstellings aan instrument parameters beinvloed die betroubaarheid, herhaalbaarheid en sensitiwiteit van ‘n sitometer en moet gemonitor (en dokumenteer) word om identiese kondisies van leesings op ‘n daaglikse basis te verseker. Dit kan bereik word deur in te skakel met ‘n interne kwaliteits versekerings program [IQA: “Internal Quality Control”] en/of ‘n eksterne kwaliteits versekerings program [EQA: “External Quality Control”] te volg. Op die oomblik is daar geen sulke kwaliteits versekerings programme in Suid Afrika en/of in die verarmende Afrika lande beskikbaar nie. MIV is ‘n wêreldwye bekommernis en laboratoriums en klinieke in hierdie gedeeltes van die land verlang ‘n dringende behoefdte vir sulke “IQA” programme om kwaliteit van instrumentasie en akkurate pasiënt resultate te verseker wat tot beter behandeling van pasiënte lei. Kwaliteit versekerings programme soos “CD Chex®” en “UK Nequas” is beskikbaar, maar baie probleme met verwysing na monster integriteit as gevolg van tydsame vervoer en aflewering kondisies word hiermee geassosieër. Die behoefte het ontstaan vir ‘n laboratorium in Suid Afrika wat direk die omliggende laboratoriums, hospitale en klinieke kan voorsien met gestabiliseerde blood monsters wat gebruik kan word as “IQA”. Die vervoer en aflewerings kondisies van hierdie monsters sal aansienlik verbeter as gevolg van die korter aflewerings afstand wat direk die beperkte temperatuur wisseling beinvloed. Doel van studie: Die doelwit van hoofstuk een is om vir die leser ‘n inleiding te gee tot terminologie en konsepte van immunologie en die immune sisteem. Hoofstuk twee beskyf die impak wat gestabiliseerde heelbloed het op die kliniese immunologie met betrekking tot kwaliteit beheer en kwaliteit versekering. Die doelwit van hierdie studie is om heelbloed te stabiliseer sodat die rakleeftyd meer as 30 dae is en sodoende as verwysings-materiaal kontroles vir Suid Afrikaanse immunofenotipering kan dien. Dit is ‘n verdere doelwit om hierdie tuis-gestabiliseerde kontrole monsters te gebruik as “IQA” verwysings materiaal in verarmende Afrika lande. Die doelwit van hoofstuk vier is om limfosiete te stimuleer om verskeie aktiverings merkers uit te druk op hul selmembrane en dan te stabiliseer en dié te gebruik as Kwaliteits Kontroles vir die meer gespesialiseerde immunologiese toetse. Studie ontwerp: Hoofstuk drie beskryf ‘n aangepaste en verbeterde metode van heel bloed stabiliseering. Stabiliteit word ondersoek in ‘n verskyndenheid konsentrasies van ‘n primêre stabiliseerings agent (chromium chloried heksahidraat) en inkubasie periodes met paraformaldehied as tweede stabiliseerings agent word deeglik gedokumenteer. Bloedmonsters van gesonde indiwidië (n=10) was gestabiliseer en gemonitor vir roetine MIV membraanoppervlak antigene oor ‘n periode van 40 dae. Hierdie monsters (n=10) was gelees en geanaliseer op ‘n BD FACSCalibur™ en vergelyk met ‘n BD FACSCount™ vloeisitometer instrument. Drie gestabiliseerde heelbloed monsters (n=3) was gemonitor vir ‘n periode vir so lank moontlik die fenotipiese selmembraan molekules identifiseerbaar was en die kwantiteit bepaalbaar was. Hierdie drie monsters was gemeet op beide instrumente. As ‘n addisionele doelwit, was hierdie drie gestabiliseerde monsters ondersoek om as moontlike kalibrasie materiaal (verteenwoordig ‘n normale bloedmonster) te dien vir die BD FACSCount™ instrument in die oggende voor pasiënt monsters gelees kan word. In hoofstuk vier was limfosiete geϊsoleer en geaktiveer met ‘n verskyndenheid stimulante om optimale aktiveerings-antigene uit te druk op T helper selmembrane (byvoorbeeld CD25, CD69, HLA-DR en CD40 Ligand). Hierdie geaktiveerde monsters was geanaliseer op die BD FACSCalibur™ en daarna gestabiliseer. Na stabilisasie van die geaktiveerde limfosiet monsters was dit gemonitor oor ‘n tydperk so lank moontlik data plotte leesbaar en selpopulasies identifiseerbaar was. Hierdie monsters kan dien as ‘n moontlike “IQA” toets stel vir ‘n meer gespesialiseerde immunologiese aktiveerings kontrole doeleindes. Resultate: In hoofstuk drie; tien individiële gestabiliseerde heelbloed monsters het gedui op geen-beduidende P waardes (P > 0.05) vir CD3, CD4 en CD8 persentasies en absolute waardes; gemeet vanaf DAG 3 vergelykbaar tot-en-met DAG 40. Met korrelasie statistiek en vergelyking van die BD FACSCalibur™ met die FACSCount™ instrumente, is die volgende opgemerk; R2 = 0.9848 vir die CD4 absolute waardes en ‘n R2 = 0.9636 vir die CD8 absolute waardes. Drie gestabiliseerde monsters (n=3) was gemonitor vir MIV roetine fenotipeering tot en met DAG 84. Die selpopulasies was duidelik identifiseerbaar en die kwantitatief meetbaar op albei instrumente (BD FACSCalibur™ en BD FACSCount™). Hoofstuk vier: geaktiveerde T helper lymphosiete het 25 – 35% membraan CD40 Ligand uitgedruk op hul selmembrane. Die stimulant van keuse was ionomysien teen ‘n optimale konsentrasie van 4μM. Die optimale inkubasie tydperk was vier ure by 37°C in 5% CO2 kondisie. Ses uur inkubasie in 4μM ionomysien by 37°C in ‘n 5% CO2 omgewing was optimal vir die CD69 selmembraan uitdrukking en het 84.21% opgelewer. Vir CD25 selmembraan uitdrukking was die selle vir ses ure met phietoheamagglutinin (PHA) gestimuleer by 37°C in 5% CO2 kondisie en het 43% CD25 selmembraan uitdrukking opgelewer. HLA-DR selmembraan uitdrukking: selle was vir ses ure saam met PHA by 37°C in 5% CO2 kondisie inkubeer en het 43.32% opgelewer. CD40 Ligand aktivering/gestabiliseerde limfosiete het tot en met dag 23 stabiliteit getoon. Die ligand was duidelik identifiseerbaar en kwantifiseerbaar. Geaktiveerde lymphosiete wat CD69, CD25 en HLA-DR selmembraan merkers uitdruk het na die stabiliseerings proses stabiliteit getoon tot-en-met dag 16. Gevolgtrekking: Die doel van hierdie studie was om verwysingskontroles voor te berei sodat dit vars heelbloed naboots met uitkenbare eienskappe vir kliniese situasies. ‘n Toets kontrolestel met verwysings materiaal vir drie vlakke (byvoorbeeld ‘n lae, medium en hoë kontrole) absolute selwaardes en persentasies kan voorberei word vir roetine immunologiese fenotiperings merkers (CD3/CD4/CD8/CD45). Meer gespesialiseerde kontrolestelle vir meer spesifieke doeleindes kan opgemaak word wat ‘n verskydenheid van limfosiet aktiveringsmerkers bevat met byvoorbeeld ‘n “nul”, lae en hoë verwysings kontrole daarin. Hierdie heelbloed kan dien as “aktiveerde interne kwaliteits verwysings materiaal” en kan gebruik word om nuut aangestelde laboratorium werkers en nuut gekwalifiseerde studente op te lei. Hierdie verwysings materiaal / kontroles kan aangewend word vir bevoegdheids doeleindes (byvoorbeeld vir SANAS akkreditasie doeleindes), vir metode ontwikkeling, vir sagteware toetsing, vir paneel opstelling en instrument verstellings doeleindes. Die kontroles moet ‘n verskydenheid eienskappe bevat om effektief te wees. Byvoorbeeld, stabiliteit tydens storing, gewenslik meer as ‘n paar weke, herhaalbaar en maklik handteerbaar. Hierdie kontroles sal inligting voorsien op ‘n daaglikse basis tydens wisseling van tegnieke of instrumentasie wat akuraatheid beinvloed en op die ou-end direk pasiënt versorging bevoordeel.

This dissertation describes the elucidation and implementation of derivatisation in the quantification of biologically active vitamin D metabolites in limited volume serum and dry blood spot samples (DBS) using the liquid chromatography tandem-mass spectrometry (LC-MS/MS) analytical technique. This manuscript describes in detail the development and validation of an analytical methodology, highlighting the role derivatisation and mass spectrometry plays in the structural characterisation and quantification of vitamin D metabolites. The first chapter reviews comprehensively, the history of vitamin D biosynthesis discovery as an anti-rickets agent, the biochemistry of vitamin D, its metabolic pathway, functions in the different biological systems and the consequences of its deficiency in the body. The second chapter reviews the current methods and techniques utilised for the detection and characterization of vitamin D metabolites, with specific emphasis based on the contribution made by derivatisation and mass spectrometry. A brief introduction to derivatisation is provided, with specific focus on PTAD and Amplifex diene reagents (Cooksontype reagents) used in this study. The importance of sensitivity and selectivity of targeted analytes is described first in detail for underivatised analytes, followed by PTAD and Amplifex derivatised samples. Chapter 2 also describes the importance of vitamin D quantification using liquid chromatography, the strengths and limitations of LC-MS/MS when used in isolation and after derivatisation. Also discussed, is how combining these techniques can overcome inherent limitations in LCMS/MS and enhance analytical performance. In Chapter 3 the materials and methods used and the study design is laid out, describing a brief introduction of the routinely used clinical diagnostics assay enzyme-linked immunosorbent assay (ELISA) as a reference method and is compared to an LC-MS/MS assay, to ascertain discrepancies and agreement between both methodologies from the same volunteer samples. Chapters 3 and 4 describes the comprehensive development, optimisation and validation of the highly sensitive PTAD derivatives LC-MS/MS assay for the quantification of active vitamin D metabolites, as well as the development of method using Amplifex diene derivatisation. Also discussed, is sample preparation optimisation of DBS and Mitra micro-samples. A holistic approach was taken to the development of the methodologies to provide data from which the required analytical information can be obtained for method evaluation and statistical analysis. The validated PTAD derivatives method is applied to the quantification of vitamin D metabolites in limited volume (100 μL) clinical human serum samples from 30 volunteers compared to results obtained using the clinical diagnostics ELISA technique. In Chapter 4 data analysis is described and the results are further discussed and a conclusion made based on the findings from the study. This study envisaged that combination of limited sample volume and DBS, derivatisation and LCMS/ MS is a powerful tool in vitamin D metabolite analysis and provided evidence of a positive increase in sensitivity and selectivity between derivatised compared to underivatised samples. A 10-fold increase in signal-to-noise-ratio (S/N) was observed when comparing PTAD derivatised, and Amplifex diene derivatised versus underivatised samples. Chapter 5 presents suggested future directions and considerations in the areas of vitamin D metabolite derivatisation and DBS sampling technique analysis using LC-MS/MS research based on the results presented in this dissertation.
Dissertation (MSc)--University of Pretoria, 2017.
Pharmacology
MSc
Unrestricted

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.

Using existing software and six novel scripts, we developed a pipeline for variant calling using exome re-sequencing data of germline blood deoxyribonucleic acid (DNA) samples. We observed >99% (6,723/6,739) concordance between calls from our pipeline and previous genotyping. We identified >93% of single nucleotide variants (SNVs) and >94% of insertion/deletions called by a commercial partner using the same sequencing reads. Using a subset of genes, we showed that around half of predicted pathogenic variants could be validated in vitro. Together, these data showed that our pipeline was reliable for variant calling. Next generation sequencing (NGS) of DNA from formalin-fixed, paraffin-embedded (FFPE) tumours is technically challenging. We sought to determine the sensitivity of NGS to detect known somatic hotspot mutations (n=25) in 19 FFPE advanced colorectal cancers and to optimise it for the identification of novel somatic variants. Analysis using Illumina’s MiSeq software identified 100% of somatic mutations with 93% specificity, whereas the Genome Analysis Tool Kit’s (GATK) HaplotypeCaller identified 80-92% of somatic mutations with 100% specificity. We investigated the background mutator profile and found that normal FFPE DNA had 3-fold more SNVs than matched blood DNA, with an excess of FFPE-associated C:G>T:A substitutions (27.1 vs. 5.3%). Uracil DNA glycosylase treatment reduced this excess. Only ~5% of variants were consistently called in replicate runs and were likely to be genuine somatic variants. We detected potential candidates for genetic biomarkers of cetuximab-resistance in colorectal cancers that were previously shown to be wild type for KRAS, NRAS, BRAF and PIK3CA. We found that 7/21 (33%) of the CRCs analysed harboured mutations at either codon 12 or 61, whileother CRCs carried truncating KRAS mutations. NRAS also carried a codon 12 mutation in 1 CRC, and PIK3CA possessed mutations at codons 542 and 545. PTEN was found to harbour 5 truncating mutations at codons 71, 140, 155, 178 and 189, potentially leading to loss of function.

South Africa has one of the highest prevalences of drug misuse and abuse in Africa. Salt River Mortuary (Cape Town, South Africa), along with other national Forensic Pathology Service providers, receives many cases of suspected drug-related deaths. In some cases, the traditional autopsy – when viewed together with the decedent's history – is not able to indicate whether a drug-related death is accidental or suicidal in relation to altered drug metabolism. Literature has shown that this can be investigated by sequencing gene(s) encoding the implicated metabolising enzyme(s) in a postmortem genetic analysis. However, as such an analysis would normally be performed following the obtainment of postmortem toxicological results, it is imperative to investigate whether blood samples retrieved back from a toxicology laboratory would be sufficient for the said genetic analysis, despite the handling involved in the process of toxicological investigation. To this end, blood samples from 30 deceased individuals in which drug use/abuse may have contributed to death, were collected into two red-top tubes (plain), two grey-top tubes (containing sodium fluoride and potassium oxalate) and one EDTAcontaining purple-top tube (control). DNA was immediately extracted from one of each colour tube, while the duplicate red-top and grey-top tubes first underwent a process of toxicological analyses, and then underwent DNA extraction. The concentration, degradation, purity, contamination, and quality of DNA were assessed using real-time PCR, spectrophotometry, forensic DNA profiling, and Sanger sequencing. In contrast to the grey-top tubes, the results showed that the red-top tubes were most suitable for the aforementioned genetic analysis. Overall, the study not only demonstrated that postmortem genetic analysis using samples retrieved from a toxicology laboratory is possible in the local context, but also provided guidelines around the pre-analytical phase of the analysis. These results illustrate the opportunity to investigate these toxicogenetic avenues further, particularly in future expansion of services currently provided at Salt River Mortuary, which may provide families more information about circumstances of their relative’s death.

The feasibility of Fourier transform infrared (FTIR)-imaging spectroscopy as a tool to discriminate samples from patients suffering from benign prostatic hyperplasia (BPH) and prostate cancer (CaP) samples in blood serum was investigated. Prostate cancer is known to be an age related disease, with the risk of developing the disease dramatically increasing in men past forty years old. Currently the PSA blood test is notoriously unreliable and is non specific for CaP thus leading to overtreatment of the disease. It is important therefore to develop diagnostic method that is non-invasive, reliable, and specific for CaP.In order to achieve the objective of establishing a robust protocol, which could be applied to a clinical study, obtaining optimal sample preparation for the FTIR analysis of serum smears, had to be achieved. A protocol was developed to prepare the serum samples prior to their FTIR analysis. First, the samples were centrifuged with ultrafiltration devices of different sizes to obtain several fractions which were then smeared to obtain thin films of serum. The spectra from the larger (>100 kDa components) and medium (containing the 10–100 kDa components) fractions were utilised for both a pilot and a clinical study, while the spectra from the smaller fractions (containing the 3–10 and <3 kDa components) were affected by fringing and could therefore not be used. A major novelty of this project involved the application of FTIR-imaging to the analysis of serum smears. The use of the Focal Plane Array detector system enabled the collection of a spectral image containing 16,384 spectra, on which a Quality Testing and pre-processing techniques were applied to select the “good spectra” and reject the spectra that failed the Quality Test. Several types of substrates were assessed to determine the most appropriate for the analysis of the smears and it was established that the spectra obtained from the serum smeared on CaF2 windows gave the most reproducible results. 5 BPH and 5 CaP samples were analysed for the pilot study following the developed protocol. While no clear separation was observed in the Principal Component Analysis (PCA) plots between the BPH and the cancerous samples, a trend emerged throughout the results, with the CaP samples clustering together and the BPH samples scattered around them. A larger clinical study was conducted with 60 BPH samples and 60 CaP samples. PCA was applied on the “good spectra” and while the over 100 kDa fraction did not show a clear separation between the two types of samples, the 10–100 kDa fraction showed a distinct classification between the BPH and CaP samples. An artificial neural network was then applied to create a model using patients from the database used for the PCA analysis to determine whether the discrimination between the two types of samples could be increased or highlight different classification trends. For the >100 kDa fraction, the sensitivity value was calculated to be 97.8% and the specificity value was calculated to be 44.3% while the sensitivity and the specificity value for the 10 to 100 kDa fraction were calculated to be 78.9% and 60% respectively. A complementary study using mass spectrometry was carried out on healthy and diseased samples to identify the components contained within the different fractions and determine whether they could be correlated with the components identified from the spectral features of the FTIR data. While no quantitative information was obtained from this study, the components found in the different fractions were identified, confirming the results of the FTIR studies.

Recent studies have shown increasing levels of unidentified fluorinated compounds, with unknown toxic effects to living organisms. These unidentified fluorinated compounds might be those novel replacements for PFOS and PFOA and some other related chemicals. These fluorinated substances have proven to be a challenge to identify and measure in blood samples and the number has been approximated to over 4700 compounds. Analysing the extractable organic fluorine (EOF) in samples and performing a mass balance analysis on measurable per- and polyfluoroalkyl substances (PFAS) to total EOF can provide an estimate on the levels of unidentified fluorinated compounds.Following the contamination of drinking water in Ronneby, a third of the population in the affected area were exposed to elevated levels of PFASs. PFHxS, PFOS and PFOA were measured in serum samples. The present study measured 11 target PFAS as well as EOF in whole blood samples from Ronneby (with known exposure) together with samples from three other cities in Sweden to preform a mass balance analysis in order to estimate the levels of unidentified fluorinated compounds. Results showed measurable concentrations of 11 PFAS in all cities included in this study, and mass balance analysis of EOF indicated the presence of unidentified fluorinated compounds in all samples, ranging from 47 % to 89 % among the samples from Ronneby and 11 % to 42 % among samples from Stockholm, Örebro and Malmö. The composition of 11 PFAS in human blood samples might indicate the source of exposure.

The enzymatic method for ethanol measurement can detect very low concentration of ethanol at samples, consequently it can’t be applied for samples with high concentration and implies as very sensitive method at limited range of detection.The alcohol dehydrogenase method is based on oxidation of alcohol in the presence of ADH as enzyme and NAD+ as coenzyme and formation of acetaldehyde and NADH that can be monitored by spectrophotometric measurement at 334,340 or 365 nm wavelengths.Ethanol +NAD+ ADH↔ Acetaldehyde +NADH+H+For optimum conditions of measurements all the parameters that affect the enzymatic reaction including temperature, pH, trapping agent for product and proper mixing need to be optimized.In order to calculate the unknown concentration of ethanol in a sample based on this method,it is crucial to find right mathematical model to calculate the unknown concentrations of ethanol in the sample using a mathematical equation that generalizes relationships among the reactants in the reaction including the reaction products. In most enzymatic reactions many parameters are involved meaning that the reaction seldom follows simple linear relation between concentration and signal. Four-parameter logistic model is well suited for modeling sigmoid relationships frequently found in biology.The aim of this project is determination of ethanol at microdialysis samples and the fundamental reason for developing the present measurement method was to study changes in blood flow in living tissues using wash out of the very dissolvable ethanol as a flow marker using the Microdialysis technique.Result from this measurement technique for microdialysis samples shows that ethanol can be detected at range of 0,5-16mmol/L and whole detected concentration for different samples during one microdialysis test follows the inverse relation of blood flow changes in tissue.Also the reported result from Urea test as general method for studying blood flow changes and ethanol test for microdialysis sample has been compared and leads to this conclusion that ethanol techniques is as reliable tool for studying blood flow changes.
Uppsatsnivå: D

Este proyecto tiene como objetivo la implementación de algoritmos para la detección de defectos en las telas poliéster. Como sabemos, desde sus inicios la industria ha utilizado avances tecnológicos no sólo para optimizar los procesos de fabricación sino también para mejorar la calidad de los productos. Ahora, si bien, no es posible evitar las fallas que alteran la calidad de las telas poliéster, sí es posible su detección mediante una inspección visual dentro del proceso de fabricación. En el presente estudio se realizó algoritmos de procesamiento de imágenes mediante el uso de librerías del software LabView para la detección de defectos en las telas poliéster, basándonos en muestras de telas con manchas comunes (MC), manchas de aceite (MA) y puntadas erróneas (PE), las cuales nos permitieron realizar varias pruebas experimentales, utilizando un módulo de pruebas a pequeña escala el cual fue fabricado según el tamaño de las muestras de tela, con la utilización de la técnica de iluminación lateral doble, y basándonos en el análisis del histograma de la imagen original de las muestras de telas, se lograron obtener parámetros numéricos que permitieron la detección de manchas comunes, manchas de aceite y puntadas erróneas, basado en el histograma de cada imagen, el cual muestra la cantidad de píxeles (tamaño de imagen) y la intensidad que se encuentra comprendido en un rango de 0-255 (siendo 0 el valor mínimo y 255 el valor máximo), se logró parametrizar numéricamente cada rango de valores de detección para el caso de MC un rango de valores de intensidad de cada pixel, obteniendo como resultado un intervalo de detección para MC de 0-195 y para el caso de las MA obteniendo como resultado un intervalo de detección de 167-194, que ayudaron en la realización del algoritmo para cada tipo de defecto, que validaron lo planteado en un inicio en la presente investigación. This project takes the implementation of algorithms to detect faults in fabrics polyester. Now, though, it is not possible to avoid the faults that alter the quality of fabrics polyester, the detection is possible by means of a visual inspection of the product inside the manufacturing process. This study carried out algorithms of image processing through the use of libraries from LabView software for detection of defects in fabrics polyester, based on samples of fabrics with Common Stains (MC), Oil Stains (MA) and Erroneous Stitches (PE), which allowed us to carry out several experimental tests, using a module of small scale tests which was manufactured according to the size of the fabric samples , using the technique of double side lighting, and based on histogram analysis, we have managed to obtain parameters that allowed the detection of Common Stains, Oil Stains and Erroneous Stitches. In the case of common stains, was parameterized numerically every range of detection values for the case of MC a range of values of each pixel intensity, resulting in a detection interval for 0-195 MC and the case of the MA resulting in a 167-194 detection interval.

Drug usage, both of a recreational or pharmaceutical nature, is common, however the abuse of such substances is an international problem. In the Western cape, South Africa, the burden of drug-related fatalities is high compared to the rest of the country. The provincial Forensic Pathology Service may encounter cases where drug-related fatalities are unclear whether death was accidental or suicidal, or drug toxicity is inconsistent with the medical/social history. This may be due to genetic alterations with drug metabolism and it has been suggested that genetic analyses may be the next step in these cases. However, toxicology results from the National Forensic Chemistry Laboratory in the Western Cape may be delayed by months to years, meaning that upon interpretation of toxicology results, there is no chance to obtain another blood sample from the deceased individual for genetic analysis. It was therefore important to determine the suitability of blood samples collected and handled in toxicology environments for subsequent genetic tests. Previously, blood samples from 30 post-mortem cases were collected into two red-top (no additives), two grey-top (sodium fluoride/potassium oxalate) and one purple-top (EDTA) tubes. Samples from one red-top and one grey-top tube underwent toxicological analysis, followed by DNA analysis, while the remaining tubes (controls) underwent DNA analysis immediately. All samples were then stored for approximately one year, prior to this study. The DNA analysis was repeated on all blood samples (n = 150) and results were assessed in terms of storage time and tube type. DNA was not significantly degraded in any of the samples; however, DNA from red-top tubes had significantly lower concentrations compared to that from grey-top tubes (p < 0.001), regardless of whether the sample had undergone toxicological analysis. The very low yields of DNA from red-top tubes posed substantial challenges for PCR-based analysis, resulting in poor quality Sanger sequencing results. Some DNA from grey-top tubes, passed the quality assessments and hence further work is required to provide an informed decision on which tube type is better suited for genetic analyses.

Objectives: Aim of the present doctoral dissertation is independent confirmation in family-based population samples, using strict appropriate statistical approach, of the associations, if any, between blood pressure (BP) and related metabolic phenotypes, analysed as continuous traits, and variations in candidate genes arising from experimental animal models [Spontaneously hypertensive rat-clone A-Hypertension-associated (SAH) gene], and from physiological cascades of the adrenergic system [alfa2B- (ADRAB2) and beta1- (ADRB1) adrenergic receptors]. Methods and Results: The SAH gene variants were evaluated in the frame of the European Project On Genes in Hypertension. In details, 2603 relatives from 560 families and 31 unrelated subjects (mean age 38.8?15.7 years; 52.1% women) were randomly recruited from six European populations. Systolic/diastolic BP, body mass index, triceps skinfold, waist-to-hip ratio, serum total and HDL cholesterol, serum triglycerides and blood glucose were measured. All subjects were genotyped for the G-1606A and -962 del/ins polymorphisms and the allele frequencies were 11.8% and 29.5% for -1606A and -962del, respectively. Lewontin’s D’ was 0.97 (p<0.0001). Haplotype frequencies were 58.8% for -1606G plus -962ins, 29.5% for -1606G plus -962del, and 11.7% for -1606A plus -962ins. Both before and after adjustment for covariates, none of the phenotype-genotype associations approached statistical significance. Family-based analyses did not reveal any population stratification (P?0.67) as a possible explanation of those negative results. The association studies between ADRB1 Arg389Gly and ADRA2B I/D polymorphisms of the beta1- and alfa2B-adrenergic receptors with BP and metabolic phenotypes, were conducted in a subsample of the EPOGH cohort. 1802 relatives from 175 families and 79 unrelated subjects (mean age 45.5?15.7 years; 51.1% women) were randomly recruited from a Caucasian population living in Northern Belgium. Systolic/diastolic BP, body mass index, waist-to-hip ratio, serum total and HDL cholesterol were measured. All subjects were genotyped for the ADRA2B I/D and ADRB1 Arg389Gly polymorphisms. The ADRA2B genotypes (II 45.7%, ID 41.7%, and DD 12.5%; P=0.05) and the ADRB1 genotypes (ArgArg 56.2%, ArgGly 36.9%, and GlyGly 6.9%; P=0.66) did not deviate from Hardy–Weinberg proportions. ADRB1 ArgArg homozygotes, compared with Gly allele carriers, had higher diastolic BP (79.4 vs 78.4 mmHg; P=0.012), and higher serum HDL cholesterol (1.33 vs 1.29 mmol/l; P=0.020). None of the other cardiovascular or metabolic phenotypes in relation to the two polymorphisms reached significance. The family-based analyses did not reveal population stratification (P?0.23). Conclusions: The present study gives evidence in favour of association of diastolic BP and HDL cholesterol with the ADRB1 Arg389Gly polymorphism in the absence of population stratification. However, the evidences supporting association of hypertension or hypertension-related phenotypes with the SAH gene remain equivocal in human studies.

Bioanalytical chemistry is a challenging field, often involving complex samples, such as blood, plasma, serum or urine. In many applications, sample cleanup is the most demanding and time-consuming step. In the work underlying this thesis a novel dynamic miniature extractor, known as a hollow-fibre liquid-phase microextractor (HF-LPME), was designed, evaluated and studied closely when used to clean plasma samples. Aqueous-organic-aqueous liquid extraction, in which the organic liquid is immobilised in a porous polypropylene membrane, was the principle upon which the extractor was based, and this is discussed in all the papers associated with this thesis. This type of extraction is known as supported-liquid membrane extraction (SLM). The aim of this work was the development of a dynamic system for SLM. It was essential that the system could handle small sample volumes and had the potential for hyphenations and on-line connections to, for instance, LC/electrospray-MS. The design of a miniaturised HF-LPME device is presented in Paper I. The extraction method was developed for some weakly acidic pesticides and these were also used for evaluation. In the work described in Paper II, the method was optimised on the basis of an experimental design using spiked human plasma samples. Paper III presents a detailed study of the mass-transfer over the liquid membrane. The diffusion through the membrane pores was illustrated by a computer-simulation. Not surprisingly, the more lipophilic, the greater the retention of the compounds, as a result of dispersive forces. The main focus of the work described in Paper IV was to make the HF/LPME system more versatile and user-friendly; therefore, the extractor was automated by hyphenation to a SIA system.
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript.

Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs. In this project a new kit, TaqManÒ Sample-to-SNP KitÔ for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method. The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure. The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.

Tenofovir (TFV) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors, often used in preexposure prophylaxis (PrEP) trials: where antiretroviral drugs are administered to high-risk, HIV-negative individuals to prevent HIV infection. Both drugs are safe when taken either daily or intermittently, which is ideal for PrEP regimens where adherence may not be high. The minimum number of doses estimated to confer high PrEP efficacy for a TFV/FTC regimen is four or more doses per week, resulting in a 95% lower risk of HIV acquisition. However, this is highly dependent on various host factors, of which adherence plays the largest role. The aim of the project was to develop a novel sensitive, specific, and robust direct method for the measurement of adherence, utilising tenofovir-diphosphate (TFV-DP) in dry blood spots (DBS) through LC-MS/MS analysis, to replace the current costly and laborious indirect method currently used to elucidate adherence of patients. This indirect method faces challenges, due to the polar nature of TFV and its metabolites, leading to separation and retention issues. The existing method applied a technique which separated the parent drug from the metabolite and then back-converted all metabolites to the parent drug before analysing the samples on LC-MS/MS. The developed alternative method aimed to reduce the time taken for each assay and the associated cost of consumables. TFV-DP is a highly polar compound and traditional reverse-phase chromatography has poor retention and separation capabilities when used to retain polar compounds, therefore alternative strategies were implemented. In this developed direct method, an anion exchange column was used along with a pH gradient, with the aim of improving separation and chromatography of TFV, TFV-DP, and tenofovir-monophosphate (TFV-MP). The method was optimised and validated using current U.S. Food and Drug Administration (FDA) and European Medical Agency (EMA) guidelines. The use of the anion exchange column resulted in a marked increase in retention time and allowed baseline separation of TFV, TFV-DP, and TFV-MP. Determination of TFV-DP from DBS was performed using three 3 mm DBS punches per sample, which underwent an extraction procedure followed by high-performance liquid chromatography with tandem mass spectrometry detection on an AB Sciex Qtrap 5500 mass spectrometer. The transitions of the protonated precursor ions were monitored at m/z 448.0 and 452.9 to the product ions m/z 350.0 and 354.9 for TFV-DP and the deuterated TFVDP internal standard, respectively. The method was validated over a range of 50–6400 fmol/punch for TFV-DP. The developed direct method had a lower limit of quantification (LLOQ) of 50 fmol/punch, which was higher than that of the indirect method; therefore, it had less sensitivity. The reduced sensitivity was acceptable, since the methods were meant for the measurement of adherence. The direct method had an ULOQ of 6400 fmol/punch, which was similar to that of the indirect method. The direct method also required significantly less on-bench sample processing and, therefore, was less time consuming and costly. To determine the suitability and accuracy of the direct method in comparison to the indirect method a comparative analysis was completed by analysing the same samples using both the indirect and direct method. The developed method met all the validation requirements and a strong correlation was observed between the results of the indirect and direct methods during the comparative analysis.

The genotoxicological effects of five intra-peritoneal administered oestrogens (17β- oestradiol, daidzein, diethylstilboestrol, genistein, and equol), were examined. Male hooded- Lister rats were used to examine to what extent DNA damage occurred. The alkaline Comet assay was the chosen method used to assess double-strand DNA breakage by examining the Olive tail moment and %age tail DNA. Tissues from the testis, bone marrow, liver and blood were analysed after an 8-day duration of exposure. Statistically significant increases in DNA damage were observed in the testis with daidzein and in the blood with diethylstilboestrol. In addition, a further study was carried out to examine the effects of bulk and nanotised forms of non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and ibuprofen, in the Comet and micronucleus assays, on whole blood taken from prostate cancer patients or volunteers. These were used because it is known that the sensitivity of DNA to genotoxins can be heightened in patients with cancer. Patients’ and volunteers’ blood was cultured with either the bulk or nano-forms for 44 hours at 37°C, 5% CO2. Data were obtained for the Comet assay as above and the number of binucleated cells scored for the micronucelus assay. The results show the nanotised forms of the NSAIDs decreased the levels of strand breakage and lowered the numbers of micronuclei generated compared with their bulk forms. There was no clear difference between the sensitivity of the healthy controls and the prostate cancer patients, with only one individual showing evidence of heightened sensitivity.

INTRODUÇÃO: As mucopolissacaridoses (MPS) são doenças de depósito lisossômico, caracterizadas pela deficiência de enzimas lisossômicas envolvidas na degradação dos glicosaminoglicanos (GAGs). O acúmulo anormal dessas macromoléculas no interior dos lisossomos provoca alterações estruturais e funcionais, de caráter multissistêmico e progressivo. Os GAGs acumulados também são excretados na urina, onde podem ser identificados através de diversos métodos bioquímicos. Estas doenças estão presentes em todos os grupos étnicos e a incidência conjunta das MPS, é estimada entre 1:10.000 a 1:25.000 nascidos vivos. (Baehner, 2005). A causa das MPS é a deficiência de uma enzima específica na rota de degradação dos GAGs. As MPS são classificadas segundo o tipo de substrato (GAGs) acumulado e a enzima específica deficiente. Na síndrome de Morquio A, ou mucopolissacaridose tipo IVA (MPS IVA), o substrato acumulado é o queratan sulfato e a enzima deficiente é a N-acetilgalactosamina-6-sulfatase. (GALNS). Os pacientes afetados por MPS IVA apresentam baixa estatura, disostose múltipla, opacidade de córnea, entre outros sinais e sintomas. O desenvolvimento psicomotor e mental é normal. O método de detecção inicial das MPS baseia-se na identificação dos GAGs, que são excretados em excesso na urina destes pacientes. A presença de queratan sulfato na eletroforese ou a detecção de níveis aumentados na dosagem quantitativa, direciona a investigação laboratorial para a MPS IV. O diagnóstico definitivo se estabelece através da medida da atividade enzimática em leucócitos ou fibroblastos, onde se constata a deficiência enzimática. OBJETIVOS: Este estudo teve como objetivo principal, tornar disponível um novo método, mais simples, rápido e acessível, para o diagnóstico bioquímico de mucopolissacaridose tipo IVA, utilizando amostras de sangue impregnadas em papel filtro (SIPF). MATERIAIS E MÉTODOS: Amostras de SIPF e leucócitos de 35 pacientes de ambos os sexos, com idade entre 3 e 47 anos, com diagnóstico previamente estabelecido de MPS IVA, pelo método convencional, em leucócitos e /ou fibroblastos, foram analisadas. Para o estabelecimento dos valores de referência, foram estudadas amostras de leucócitos e de SIPF de 54 indivíduos saudáveis (18-50 anos), de ambos os sexos. Após assinatura do termo de consentimento, amostras de sangue periférico de pacientes e controles, foram coletadas, para a obtenção de leucócitos e sangue impregnado em papel filtro.(SIPF). Os ensaios enzimáticos foram realizados nas amostras de leucócitos e SIPF, simultaneamente, para comparar os resultados. RESULTADOS: Os resultados obtidos nos ensaios enzimáticos de todos os pacientes apresentando MPS IVA, confirmaram a deficiência da atividade enzimática em ambos materiais (leucócitos e SIPF) com uma diferença estatisticamente significativa em relação ao grupo controle. (Mann-Witney U test, p< 0,001). Neste estudo, a medida de GALNS em amostras de SIPF permitiu a identificação dos pacientes com MPS IVA, com sensibilidade de 100 %. Os testes de estabilidade realizados nas amostras de SIPF indicaram que amostras coletadas para a medida da atividade de GALNS devem ser mantidas a 4ºC sempre que possível, sendo estáveis nesta temperatura por mais de 30 dias. CONCLUSÕES: Nas condições utilizadas, amostras de SIPF se mostraram adequadas para a identificação segura de pacientes com MPS tipo IVA. O método que utiliza amostras de SIPF é mais acessível e rápido, simplificando a etapa de coleta e transporte, podendo ser utilizado para detectar pacientes afetados, especialmente em áreas de difícil acesso para a coleta e transporte de amostras líquidas.
INTRODUCTION: Mucopolysaccharidosis (MPS) are lysosomal deposit diseases characterized by lysosomal enzymes deficiency involved in the degradation of glycosaminoglycans (GAGs). The abnormal accumulation of these macromolecules inside the lysosomes provokes structural and functional alterations multi-systemically and progressively. The accumulated GAGs are also excreted in the urine, where they may be identified through many different biochemical methods. These diseases occur among all ethnical groups and the combined incidence of MPS is estimated at 1:10.000 to 1:25.000 live births. (Baehner, 2005). The MPS’ cause is the deficiency of a specific enzyme in the GAGs degradation route. The MPS are classified according to a type of substrate accumulated (GAGs) and the deficiency of a specific enzyme. In Morquio syndrome A or Mucopolysaccharidosis type IVA (MPS IVA), the accumulated substrate is the keratan sulfate and the deficient enzyme is the N-acetylgalactosamine-6-sulfatase (GALNS). The patients affected by MPS IVA present short stature, dysostosis multiplex, corneal opacity, among others signs and symptoms. The cognitive and mental developments are normal. The MPS initial detection method is based on the identification of the GAGs which are excreted in the patients’ urine. The presence of the keratan sulphate in the electrophoresis or the detection of the increased levels in the quantitative dosage directs the laboratory investigation to MPS IV. The definitive diagnosis is established through measuring the enzymatic activity in leukocytes or fibroblasts, in which the enzymatic deficiency is proved. OBJECTIVE: This study’s main purpose is to offer an original, simpler, faster and more accessible method for biochemical diagnosis of Mucopolysaccharidosis type IVA using dried blood samples (DBS). MATERIALS AND METHODS: DBS and leukocytes from 35 patients from both sexes between 3 and 47 years of age with previously established diagnosis of MPS IVA through the conventional method in leukocytes and/or fibroblasts were analyzed. In order to establish reference values DBS and leukocytes samples from 54 healthy people (18-50 years of age) from both sexes were studied. After signing a paper consent form, peripheral blood samples from patients and controls were collected for obtaining leukocytes and dried blood samples (DBS). To validate the method, we made a simultaneous GALNS assay in leukocytes and DBS. RESULTS: The results obtained in the enzymatic assays from all patients presenting MPS IVA confirmed the deficiency of enzymatic activity in both materials (leukocytes and DBS) with a significant statistical difference in relation to the control group. (Mann-Witney U tes, p< 0,001). In this study, the quantity of GALNS in DBS allowed the identification of patients with MPS IVA with sensibility of 100%. The stability tests indicate that DBS samples collected for measuring the activity of GALNS must be kept at 4ºC whenever possible, being stable in this temperature for more than 30 days. CONCLUSION: In the used conditions, DBS were adequate for a safe identification of patients with MPS type IVA. The method which utilizes DBS is cheaper and faster, what simplifies the collection and transportation stage and can be used to detect affected patients especially in difficult access areas for the collection and transportation of liquid samples.

In heroin fatalities the diagnosis of the cause of death may be particularly difficult because of several reasons, such as the relationship between lethal dose and current individual tolerance, the complexity of heroin metabolism, the presence of systemic dysfunction, and the contemporary use of other drugs of abuse. Thus, a wide variability is present in post-mortem blood concentration of morphine (MOR), the main metabolite of heroin, which is usually the most important analytical result for the interpretation of the cause of death. Recently, increasing interest has grown towards the role of the metabolites morphine-3-β-D-glucuronide (M3G) and morphine-6-β-D-glucuronide (M6G) in mediating heroin effects. The aim of this PhD study has been the development of a LC/MS-MS method for the determination of MOR, M3G and M6G in autopsy blood samples. An ESI-QqQ Mass Spectrometer, operating in positive ionisation and MRM mode, was used. Chromatographic separation was achieved thanks to a Reversed-Phase method, using a C18 column and a gradient elution with a binary mobile phase. SPE technique was employed to extract the analytes from biological samples. After validation, the method was applied to twenty-five blood specimens collected from cases of suspected fatal heroin overdose. The concentrations and the molar ratios of MOR, M3G and M6G were investigated. The influence of some risk factors, such as the contemporary use of alcohol, methadone or cocaine, was also studied.

Mestrado em Biomedicina Molecular
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the presence of extracellular amyloid plaques (senile plaques) and intracellular neurofibrillary tangles formed by hyperphosphorylated TAU protein. TAU protein when hyperphosphorylated loses the ability to bind to microtubules and can be released into peripheral fluids. This process leads to neuronal degradation and neuronal death. Phosphorylation at threonine 231 has been shown to be specific for AD and to precede assembly of paired helical filaments in the human brain. In order to understand more about this residue we analysed SH-SY5Y cells undifferentiated and in differentiated cells induced by retinoic acid (RA). Treatment with RA increased expression of TAU phosphorylated at Thr231 (TAUpThr231) as determined by Western blot analysis. We further explored TAU phosphorylation by immunocytochemistry and noticed that in undifferentiated SH-SY5Y cells, TAUpThr231 was located mainly in the nucleus. In contrast, TAU and TAUpThr231 was redistributed to the neurites and in the soma of SH-SY5Y cells, which were induced to differentiate by retinoic acid (RA). In order to evaluate the potential of TAUpThr231 as a biomarker, we measured TAUpThr231 in CSF by a sandwich enzyme immunoassay and observed that the ratio of TAUpThr231/TAU levels discriminated significantly the AD group for the non-AD group. These findings indicate that TAUpThr231/t-TAU ratio levels may be a valuable marker for the clinical diagnosis of AD, irrespective of age and gender.
A doença de Alzheimer (AD) é uma doença neurodegenerativa progressiva caracterizada pela presença de placas de amilóide extracelulares (placas senis) e tranças neurofibrilhares intracelulares formadas pela proteína TAU hiperfosforilada. A proteína TAU quando hiperfosforilada perde a capacidade de se ligar a microtúbulos e pode ser libertada para fluidos periféricos. Este processo leva à degradação neuronal e à morte neuronal. A fosforilação na treonina 231 tem-se demonstrado ser específica para a AD e preceder a formação de filamentos helicoidais emparelhados no cérebro humano. Para melhor perceber a função deste resíduo e a contribuição para a localização da TAU, analisámos células SH-SY5Y indiferenciadas e células diferenciadas, pela adição de ácido retinoico (RA). O tratamento com RA aumentou a expressão de TAU fosforilada na Thr231 (TAUpThr231) conforme determinado por Western blot. Explorámos ainda a fosforilação da TAU por imunocitoquímica e percebemos que em células SH-SY5Y indiferenciadas, a phosphoTAU231 estava localizada principalmente no núcleo. Em contraste, TAU e phosphoTAU231 foram redistribuídas para as dendrites e citosol das células SH-SY5Y diferenciadas pelo ácido retinoico (RA). Para avaliar o potencial deste resíduo como biomarcador; medimos TAUpThr231 em CSF por meio de um imunoensaio enzimático em sanduíche e observámos que a proporção de níveis de TAUpThr231 / TAU discriminou de forma significativa o grupo AD do grupo não-AD. Essas descobertas podem indicar que os níveis da relação TAUpThr231 / t-TAU podem ser um marcador valioso para o diagnóstico clínico de AD, independentemente da idade e do género.

A técnica analítica mais utilizada para o monitoramento de exposição a metais tóxicos ou para avaliação de deficiência a elementos essenciais é a espectrometria de absorção atômica chama (FAAS) ou com forno de grafite (GF AAS). Entretanto, cada vez mais os laboratórios de pesquisa na área clínica estão mudando seus métodos de análise, baseados nestas técnicas, para a utilização da espectrometria de massas com plasma acoplado (ICP-MS). Isso vem acontecendo porque o ICP-MS permite a determinação de elementos químicos, em diversas matrizes, numa ampla faixa de concentração (ng L-1 a mg L-1), além de possibilitar alta rapidez de análise, capacidade multielementar e limites de detecção menores que outras técnicas analíticas. Uma das principais características do método a ser utilizado para a análise de elementos químicos em rotina por ICP-MS é que ele seja rápido, com o mínimo de manipulação da amostra. Neste sentido, métodos que propõem a análise direta de amostra são mais atrativos. Todavia, ainda é limitado o número de métodos que propõem análise direta de fluidos biológicos por ICP-MS. Neste sentido, o presente trabalho teve como objetivo o desenvolvimento de um método para análise direta de sangue por ICP-MS para determinação de Ag, As, Cd, Co, Mn, Ni, Pb e Se. Para isso, amostras de sangue (200 µL) foram misturadas com 500 µL de hidróxido de tetrametilamônio (TMAH) (10% v/v) e incubadas por 10 minutos. Posteriormente, a solução resultante foi diluída para 10 mL com uma solução contendo 0,05% m/v EDTA; 0,005% v/v Triton® X-100. Em seguida, as amostras foram analisadas diretamente por ICP-MS (ELAN DRC II). Ródio (Rh) foi usado como padrão interno e a calibração das amostras foi feita por meio de ajuste de matriz com sangue base ovino. Os limites de detecção (LD) do método foram de 0,008; 0,02; 0,004; 0,009; 0,003; 0,09; 0,04; 0,1 µg L-1 para Ag, As, Cd, Co, Mn, Ni, Pb and Se, respectivamente. A validação dos resultados foi realizada com análise de um material de referência de sangue do Programa de Proficiência do Institut National de Santé Publique du Quebec, no Canadá. Validação adicional foi obtida pela comparação dos resultados obtidos pela análise de 20 amostras de sangue pelo método proposto e pela técnica de GF AAS. O método foi também comparado a outros dois existentes na literatura e comumente utilizados em laboratórios dos Estados Unidos e Suécia, apresentando limites de detecção comparáveis ou melhores e melhores exatidão e precisão.
The most used analytical technique for monitoring the exposure to toxic metals or for the assessment of the deficiency of essentials elements is the atomic absorption spectrometry with flame (FAAS) or graphite furnace (GF AAS). However, more and more clinical laboratories are changing their methods of analysis, based on this technique, to methods using inductively coupled plasma-mass spectrometry (ICP-MS). It occurs because ICP-MS allows the determination of chemical elements in various types of samples, at concentrations in a wide linear range (ng L-1 to mg L-1), providing high-throughput analysis with multielemental capability with lower detection limits. However, for routine porpuses the method of choice must be fast with minimal sample manipulation.On the other hand, the number of methods proposing direct introduction of biological fluids to the ICP-MS are still limited. This work aimed the development of a method for the direct analysis of blood samples by ICP-MS for the determination of Ag, As, Cd, Co, Mn, Ni, Pb and Se. For this, blood samples (200 L) were mixed with 500 L of tetramethylammonium hydroxide (TMAH) (10% v/v) and left at room temperature during 10 minutes. Subsequently, the resulting solution was diluted to 10 mL with a solution containing 0.05% m/v EDTA + 0005% v / v Triton ® X-100. Thus the samples were analyzed directly by ICP-MS (ELAN DRC II). Rhodium (Rh) was used as internal standard with matrix matching calibration. The method detection limits were: 0.008, 0.02, 0.004, 0.009, 0.003, 0.09, 0.04, 0.1 µg L-1 for Ag, As, Cd, Co, Mn, Ni , Pb, and Se respectively. Method validation was acquired with the analysis of blood reference material provided by the Institut National de Santé Publique du Quebec, Canada. Furthermore, for additional validation 20 ordinary blood samples were analyzed by the proposed method and by GF AAS. The method was also compared with two existing methods in the literature and commonly used in laboratories in the United States and Sweden where comparable or better detection limits and better accuracy and precision were obtained.

This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper.

Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated.

Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil).

Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE.

Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.