Lo, Yin Wai Wyatt. / "December 2010." / Thesis (M.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 186-206). / Abstracts in English and Chinese. / ABSTRACT --- p.II / 摘要 --- p.VII / ACKNOWLEDGEMENTS --- p.X / PUBLICATIONS --- p.XI / TABLE OF CONTENTS --- p.XII / LIST OF TABLES --- p.XVIII / LIST OF FIGURES --- p.XXI / LIST OF ABBREVIATIONS --- p.XXIV / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATALTESTNG --- p.2 / Chapter 1.1. --- THE AIM --- p.2 / Chapter 1.2. --- INVASIVE PRENATAL DIAGNOSIS --- p.4 / Chapter 1.3. --- NONINVASIVE PRENATAL SCREENING --- p.6 / Chapter CHAPTER 2: --- NONINVASIVE PRENATAL DIAGNOSIS --- p.10 / Chapter 2.1. --- CIRCULATING FETAL CELLS IN PRENATAL DIAGNOSIS --- p.10 / Chapter 2.2. --- CIRCULATING FETAL NUCLEIC ACIDS IN PRENATAL DIAGNOSIS --- p.12 / Chapter 2.3.1. --- Biology of circulating fetal DNA . --- p.14 / Chapter 2.3.2. --- Clinical applications of circulating fetal DNA --- p.15 / Chapter 2.3.2.1. --- Qualitative analysis of fetal DNA in maternal plasma --- p.16 / Chapter 2.3.2.2. --- Quantitative analysis of fetal DNA in maternal plasma --- p.17 / Chapter 2.4. --- CIRCULATING FETAL RNA IN MATERNAL PLASMA --- p.20 / Chapter 2.4.1. --- Biology of circulating fetal RNA --- p.20 / Chapter 2.4.2. --- Clinical applications of circulating fetal RNA --- p.22 / Chapter 2.4.2.1. --- Quantitative analysis of fetal RNA in maternal plasma --- p.22 / Chapter CHAPTER 3: --- TECHNICAL CHALLENGES IN ANALYZING CIRCULATING FETAL NUCLEIC ACIDS --- p.26 / Chapter 3.1. --- INTRODUCTION --- p.26 / Chapter 3.2. --- "PREANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYSE"";" --- p.28 / Chapter 3.2.1. --- Low abundance of cell-free fetal nucleic acids in maternal plasma --- p.28 / Chapter 3.2.2. --- High level of maternal background in maternal plasma --- p.30 / Chapter 3.3. --- ANALYTICAL ISSUES IN MATERNAL PLASMA NUCLEIC ACID ANALYS --- p.IS / Chapter 3.3.1. --- Imprecise measurement of fetal nucleic acid quantity --- p.33 / Chapter 3.3.2. --- Coexistence of fetal nucleic acids and maternal nucleic acid background in maternal plasma --- p.36 / Chapter 3.4. --- AIMS OF THIS THESIS --- p.41 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.42 / Chapter CHAPTER 4: --- QUANTITATIVE AND QUALITATIVE ANALYSIS OF NUCLEIC ACIDS --- p.43 / Chapter 4.1. --- SAMPLE COLLECTION AND PROCESSING --- p.43 / Chapter 4.1.1. --- Preparation of plasma and blood cells --- p.43 / Chapter 4.1.2. --- Preparation of placental tissues --- p.44 / Chapter 4.2. --- NUCLEIC ACID EXTRACTION --- p.45 / Chapter 4.2.1. --- Extraction of total RNA --- p.45 / Chapter 4.2.1.1. --- Plasma samples --- p.45 / Chapter 4.2.1.2. --- Placental tissue samples --- p.46 / Chapter 4.2.2. --- Extraction of genomic DNA --- p.48 / Chapter 4.2.2.1. --- Plasma samples --- p.48 / Chapter 4.2.2.2. --- Blood cell samples --- p.48 / Chapter 4.3. --- CONVENTIONAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.50 / Chapter 4.3.1. --- Principles of real-time polymerase chain reaction --- p.50 / Chapter 4.3.2. --- Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) --- p.52 / Chapter 4.3.3. --- Quantitative polymerase chain reaction (qPCR) --- p.53 / Chapter 4.4. --- DIGITAL REAL-TIME PCR ANALYSIS OF NUCLEIC ACIDS --- p.55 / Chapter 4.4.1. --- Principles of digital PCR (dPCR) --- p.55 / Chapter 4.4.2. --- 384-reaction well dPCR v --- p.56 / Chapter 4.4.3. --- Microfluidics dPCR --- p.57 / Chapter 4.5. --- MATRIX-ASSISTED LASER DESORPTIONIONIZATION/TIME-OF-FLIGHT MASS SPECTROMETRY (MALDI-TOF MS) ANALYSIS OF NUCLEIC ACIDS --- p.59 / Chapter 4.5.1. --- Principles of MALDI-TOF MS --- p.59 / Chapter 4.5.2. --- DNA genotyping analysis by MassArray Homogenous MassExtend (hME) assay --- p.60 / Chapter 4.6. --- CLONING AND DNA SEQUENCING --- p.63 / Chapter SECTION III: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING RNA --- p.65 / Chapter CHAPTER 5: --- ENRICHMENT OF PLACENTA EXPRESSED MRNA MARKERS BY WHOLE TRANSCRIPTOME PREAMPLIFICATION --- p.66 / Chapter 5.1. --- INTRODUCTION --- p.66 / Chapter 5.2. --- MATERIALS AND METHODS --- p.69 / Chapter 5.2.1. --- Study design --- p.69 / Chapter 5.2.2. --- Subjects and sample collection --- p.71 / Chapter 5.2.3. --- RNA extraction and sample dilution --- p.71 / Chapter 5.2.4. --- Preamplification --- p.73 / Chapter 5.2.5. --- qPCR analysis --- p.74 / Chapter 5.3. --- RESULTS --- p.83 / Chapter 5.3.1. --- Comparison of mRNA expression profiles in placental tissues with and without preamplification --- p.83 / Chapter 5.3.1.1. --- Undiluted placental tissue RNA --- p.84 / Chapter 5.3.1.2. --- Diluted placental tissue RNA --- p.88 / Chapter 5.3.2. --- The effect of RNA input on the degree of amplification --- p.92 / Chapter 5.3.2.1. --- Correlation between RNA input and RNA output --- p.94 / Chapter 5.3.2.2. --- Correlation between RNA input and output/input ratio --- p.96 / Chapter 5.3.3. --- Preamplification of maternal plasma RNA --- p.98 / Chapter 5.3.3.1. --- Concentrations of placenta expressed mRNA in third trimester maternal plasma --- p.98 / Chapter 5.3.3.2. --- Concentrations of placenta expressed mRNA in first trimester maternal plasma --- p.100 / Chapter 5.4. --- DISCUSSION --- p.102 / Chapter SECTION IV: --- IMPROVEMENTS ON MATERNAL PLASMA ANALYSIS OF CIRCULATING DNA --- p.105 / Chapter CHAPTER 6: --- ACCURATE GENE DOSAGE ANALYSIS BY MULTIPLEX QPCR --- p.106 / Chapter 6.1. --- INTRODUCTION --- p.106 / Chapter 6.2. --- MATERIALS AND METHODS --- p.110 / Chapter 6.2.1. --- Study design --- p.110 / Chapter 6.2.2. --- Subjects and sample collection --- p.112 / Chapter 6.2.3. --- DNA extraction and sample dilution --- p.113 / Chapter 6.2.4. --- qPCR analysis --- p.113 / Chapter 6.2.4.1. --- Monoplex assays --- p.114 / Chapter 6.2.4.2. --- Multiplex assays --- p.114 / Chapter 6.2.5. --- Microfluidics dPCR assay --- p.122 / Chapter 6.2.6. --- Gene Dosage Comparison --- p.122 / Chapter 6.2.6.1. --- In adult male samples --- p.123 / Chapter 6.2.6.2. --- In maternal plasma samples --- p.123 / Chapter 6.3. --- RESULTS --- p.125 / Chapter 6.3.1. --- The influence of using the same and different sets of primers for amplifying different chromosomal loci --- p.125 / Chapter 6.3.2. --- Effects of using monoplex and multiplex real-time PCR formulations --- p.130 / Chapter 6.3.3. --- Effects of incorporating calibration curves for template quantification in conventional qPCR --- p.135 / Chapter 6.4. --- DISCUSSION --- p.140 / Chapter CHAPTER 7: --- DPCR DETECTION OF PATERNALLY INHERITED POINT MUTATIONS --- p.144 / Chapter 7.1. --- INTRODUCTION --- p.144 / Chapter 7.2. --- MATERIALS AND METHODS --- p.153 / Chapter 7.2.1. --- Study design --- p.153 / Chapter 7.2.2. --- Subjects and sample collection --- p.157 / Chapter 7.2.3. --- DNA extraction and sample preparation --- p.158 / Chapter 7.2.4. --- MassArray hME assays --- p.159 / Chapter 7.2.5. --- dPCR assay --- p.159 / Chapter 7.3. --- RESULTS --- p.161 / Chapter 7.3.1. --- Validation of the digital HbE assay --- p.161 / Chapter 7.3.2. --- Determination of the minimum fetal DNA amount required for digital PCR detection --- p.165 / Chapter 7.3.3. --- Detection of paternally inherited fetal HbE mutation in maternal plasma --- p.172 / Chapter 7.4. --- DISCUSSION --- p.175 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.180 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.181 / Chapter 8.1. --- IMPROVEMENTS ON QUANTITATIVE AND QUALITATIVE ANALYSIS OF FETAL NUCLEIC ACIDS IN MATERNAL PLASMA --- p.181 / Chapter 8.2. --- PERSPECTIVES FOR FUTURE WORK --- p.184 / REFERENCES --- p.186

A insuficiência cardíaca é uma síndrome clínica de alta morbimortalidade e por este motivo é crescente o interesse em se estudar novos biomarcadores da doença visando buscar caminhos para novas estratégias terapêuticas. Neste contexto, a análise do ar exalado pode ser promissora. Baseado nestes dados e na observação de que pacientes com insuficiência cardíaca grave exalam odor peculiar, ainda em estudo piloto, nós identificamos acetona no ar exalado de pacientes com insuficiência cardíaca. Assim, nosso estudo teve por objetivo primário avaliar o papel da acetona exalada como biomarcador do diagnóstico de insuficiência cardíaca e de insuficiência cardíaca descompensada. Como objetivo secundário, avaliar sua relação com a classe funcional segundo a classificação da New York Heart Assocation (NYHA) e sua correlação com o peptídeo natriurético do tipo B (BNP). Entre maio de 2009 e setembro de 2010, pacientes consecutivos com disfunção sistólica (grupo IC) admitidos na emergência (insuficiência cardíaca descompensada grupo ICDESCOMP) e pacientes estáveis nos últimos três meses encaminhados para o teste cardiopulmonar (insuficiência cardíaca compensada grupo ICCOMP) foram submetidos à coleta de ar exalado (extração em água) para posterior análise qualitativa por cromatografia gasosa acoplado à espectrometria de massas e quantificação por espectrofotometria de absorção, através da reação com salicilaldeído. Entre os 235 pacientes avaliados, 89 foram incluídos (59 com insuficiência cardíaca descompensada e 30 com insuficiência cardíaca compensada), 61% do sexo masculino e com mediana de idade de 52 anos. Vinte indivíduos saudáveis (grupo controle) pareados por idade participaram do estudo. O valor mediano (intervalo interquartil) de acetona exalada foi maior no grupo IC em relação ao controle [3,70 g/L (1,69-10,45 g/L) versus 0,39 g/L (0,30-0,79 g/L), p < 0,001]. O valor mediano de acetona em pacientes com insuficiência cardíaca descompensada foi maior do que no grupo com insuficiência cardíaca compensada [7,80 g/L (3,60-15,20 g/L) versus 1,22 g/L (0,682,19 g/L), p < 0,001]. A acurácia do método tanto para o diagnóstico de insuficiência cardíaca (acetona > 1,16 g/L; área sob a curva = 0,94) quanto para o diagnóstico de insuficiência cardíaca descompensada (acetona > 2,50 g/L; área sob a curva = 0,93) foi aproximadamente 85 %, semelhante à acurácia do BNP (BNP > 42 pg/mL; área sob a curva = 0,97 e BNP > 424 pg/mL; área sob a curva = 0,94, respectivamente). Houve correlação positiva entre acetona exalada e BNP (r = 0,772, p < 0,001). Observamos aumento progressivo nas concentrações de acetona exalada de acordo com a piora da classe funcional segundo NYHA (p < 0,001). Assim, podemos concluir que nosso estudo revelou a acetona exalada como um novo biomarcador do diagnóstico de insuficiência cardíaca e de insuficiência cardíaca descompensada, que está associado à maior gravidade da doença e que apresenta correlação positiva com BNP. Sua dosagem é um novo método de diagnóstico não invasivo que pode ser realizado à beira leito, cuja acurácia é semelhante à do BNP / Heart failure (HF) is a condition associated with high mortality and frequent hospital admissions. In this context, multiple biomarkers of heart failure severity have emerged recently. However, the usefulness of most of these biomarkers has not been fully established. Exhaled breath has been considered a suitable tool (biomarker) to evaluate different diseases. Based on the clinical observation that patients with acute decompensated heart failure (ADHF) exhale a distinct odor, in a pilot study we have identified acetone in exhaled breath of heart failure patients and this study aimed to evaluate the role of acetone as a new biomarker of heart failure and ADHF disease. As secondary aims, we intended to analyze the relation to New York Heart Association (NYHA) class and the correlation with B-Type Natriuretic Peptide (BNP). Patients with systolic dysfunction (HF group) admitted consecutively at the emergency room (ADHF group) and stable patients referred to the cardiopulmonary test (chronic HF CHF group) between May 2009 and September 2010 were submitted to exhaled breath collection (extraction into water). Acetone identification was done by gas chromatography-mass spectrometry (GC-MS) and its determination by absorption spectrophotometry after reaction with salicylaldehyde. Twenty healthy subjects matched for age were enrolled (control group). Among 235 patients with HF, 89 were included in the study (59 ADHF and 30 CHF), 61% male, with median age of 52 years. Median exhaled breath acetone value (interquartile range) was higher in the HF group when compared to control group [3.7 g/L (1.69-10.45 g/L) versus 0.39 g/L (0.30-0.79 g/L), p < 0.001] and also higher in ADHF when compared to CHF group [7.80 g/L (3.60-15.20 g/L) versus 1.22 g/L (0.682.19 g/L), p < 0,001]. The accuracy of the method to diagnose CHF (Acetone > 1.16 g/L; AUC = 0.94) and ADHF (Acetone > 2.5 g/L; AUC = 0.93) was similar to the accuracy of BNP (BNP > 42 pg/mL; AUC = 0.97 and BNP > 424 pg/mL; AUC = 0.94, respectively). There was a positive correlation between exhaled breath acetone and plasmatic BNP (r = 0.772, p < 0.001). Levels of exhaled breath acetone were different among the four different NYHA classes (p<0.001). In summary, we can conclude that our study showed exhaled breath acetone as a new biomarker of heart failure and ADHF. It is associated with heart failure severity and has a good correlation with BNP. This is a promising non-invasive diagnostic method of heart failure, whose accuracy is equivalent to BNP

INTRODUÇÃO: A apnéia obstrutiva do sono é uma doença altamente prevalente na população adulta e associada à morbidade significante. A polissonografia noturna é o método padrão ouro para o diagnóstico de apnéia obstrutiva do sono. Entretanto seu custo é elevado e a disponibilidade de leitos para polissonografia é muito inferior a demanda. Por esta razão, estratégias para otimizar o diagnóstico de apnéia obstrutiva do sono são urgentes e necessárias. O objetivo deste estudo foi o de comparar um exame diurno de polissonografia, de curta duração e através de sono induzido por benzodiazepínico com a polissonografia noturna para o diagnóstico de apnéia obstrutiva do sono. MÉTODOS: Foram estudamos 40 pacientes divididos em dois grupos baseados no resultado da polissonografia noturna (Índice de Apnéia e Hipopnéia = 15 eventos/hora). Os dezoito Indivíduos portadores de apnéia obstrutiva do sono (id= 46 + 9 anos) e os vinte e dois controles (id= 38 + 10 anos) foram submetidos a uma polissonografia diurna, de curta duração, com indução de sono através de infusão intravenosa lenta de midazolam. RESULTADOS: O sono induzido foi obtido em todos indivíduos. O tempo total de sono foi de 41,5 + 18,9 minutos. A maioria dos eventos respiratórios durante o sono induzido forma obstrutivos e similares aos observados durante a polissonografia noturna. Não houve diferença estatisticamente significativa entre o índice de apnéia e hipopnéia bem como com a saturação mínima de oxigênio obtido pela polissonografia noturna e com sono induzido nos grupos estudados (p>0,05). Reunindo os dois grupos, o índice de apnéia e hipopnéia e a menor saturação de oxigênio obtidos pelos dois métodos tiveram correlação significativa (r=0,67 e r=0,77, respectivamente). A sensibilidade e especificidade para o diagnóstico de apnéia obstrutiva do sono através do sono induzido foi 0,83 e 0,72 respectivamente. Nenhuma complicação foi observada durante o sono induzido. CONCLUSÃO: A polissonografia com sono induzido é um método rápido e seguro que pode ser uma alternativa a polissonografia noturna para o diagnóstico de apnéia obstrutiva do sono. / Polysomnography is the gold standard method for diagnosing obstructive sleep apnea. However, the gap between demand and capacity in performig polysomnography is a major healthcare problem. We sought to compare a short day-time induced sleep with full overnight standard PSG (full PSG) monitoring for the diagnosis of obstructive sleep apnea. We studied 40 patients classified into subjects with obstructive sleep apnea (n=18, age= 46.8 + 9.1yr) and controls (n=22, age= 38.5 + 10,7yr) groups, based on the results of a full polysomnography (apnea-hypopnea index >= 15 events/hour). All subjects underwent a short day-time polysomnography. Sleep was induced by slow intravenous drip infusion of midazolam and achived in all subjects. Total time of induced sleep was 41.5 ± 18.9 min. The majority of the respiratory events during induced sleep were obstructive and similar to that observed during full polysomnography. There was no difference between apnea-hypopnea index obtained by full and short polysomnography in obstructive sleep apnea and control groups (p>0,05). The same occured to lowest O2 saturation. Taken all together, apnea-hypopnea index and lowest O2 saturation during short polysomnography correlated well with full polysomnography (r=0,67 and r=0,77, respectively). Sensitivity and specificity for the diagnosis of obstructive sleep apnea by induced sleep was 0,83 and 0,72, respectively. No complications were observed. Induced sleep PSG by midazolan is a short and safe study that may represent an alternative for full polysomnography in the diagnosis of obstructive sleep apnea.

O desempenho de seis métodos de extração de DNA de oocistos de Cryptosporidium spp. em fezes foram avaliados pela nested PCR do gene SSU rRNA. Os métodos consistem na combinação com pequenas variações de duas técnicas para a liberação de esporozoítos (indução de excistamento/E ou choque térmico/C) e três técnicas de purificação de DNA (fenol-clorofórmio/F, GuSCN-sílica/S ou kit QIAmp DNA Stool Mini/K). Para a avaliação da sensibilidade analítica, os testes foram realizados a partir de diluições seriadas de oocistos purificados, na ausência e na presença de 100 &mu;l de fezes bovinas. A sensibilidade diagnóstica foi avaliada pela comparação com o método microscópico de centrífugo-flutuação em sacarose (padrão-ouro) em 15 amostras fecais de diferentes hospedeiros naturalmente infectados. Na ausência de fezes, foram avaliados apenas os métodos EF, ES, CF e CS, sendo que EF apresentou sensibilidade analítica superior, possibilitando a detecção de até 1 oocisto. Na presença de fezes, os métodos EF, EK e CK apresentaram desempenhos equivalentes, com sensibilidade de 104 oocistos. As maiores sensibilidades diagnósticas foram obtidas pelos métodos EK e CK, que possibilitaram a detecção de 13 (86,7%) das 15 amostras. Devido à alta sensibilidade analítica de EF em amostras purificadas, avaliou-se sua sensibilidade diagnóstica em amostras de oocistos purificados pelo método de centrífugo-flutuacão em sacarose, obtendo-se 100% de detecção. A presença de inibidores nas amostras fecais reduziu fortemente a sensibilidade das reações de PCR a partir de DNA extraído pelos métodos avaliados, sendo recomendável o emprego de técnicas de purificação de oocistos previamente à extração de DNA. Os resultados apontam para uma melhor eficiência dos métodos de indução de excistamento e kit QIAmp DNA Stool Mini. / The performance of six methods for DNA extraction from Cryptosporidium spp. oocysts in feces were evaluated by nested PCR of SSU rRNA gene. The methods are the combination with small variations of two techniques for the release of sporozoites (induction of excystation/E or thermal shock/C) and three techniques for DNA purification (phenol-chloroform/F, GuSCN-silica/S or QIAmp DNA Stool Mini kit/K). For the evaluation of analytical sensitivity, tests were made from serial dilutions of purified oocysts, in absence and in presence of 100 &mu;l of cattle feces. The diagnostic sensitivity was assessed by comparison with the microscopic method of centrifugalflotation in sucrose (gold standard) in 15 fecal samples from different naturally infected hosts. In the absence of feces, only methods EF, ES, FC, and CS were tested. EF had the highest analytical sensitivity, enabling the detection of up to 1 oocyst. In the presence of feces, methods EF, EK, and CK showed similar performance, with sensitivity of 104 oocysts. The highest sensitivities were obtained by methods EK and CK, which enabled the detection of 13 (86.7%) of 15 samples. Due to the high analytical sensitivity of EF in purified samples, its diagnostic sensitivity was evaluated in samples of purified oocysts by the method of centrifugal-flotation in sucrose, resulting in 100% detection. The presence of inhibitors in fecal samples greatly reduced the sensitivity of the PCR reactions from DNA extraction methods evaluated, and the use of techniques for purification of oocysts prior to DNA extraction is recommended. The results show a better performance of the methods of induction of excystation and QIAmp DNA Stool Mini kit.

Alterações no aspecto do disco óptico e da camada de fibras nervosas da retina (CFN) freqüentemente precedem o aparecimento de defeitos de campo visual no glaucoma, o que faz com que a avaliação destas estruturas seja essencial para o diagnóstico precoce e prevenção da perda visual nesta doença. A polarimetria de varredura a laser (GDx VCC), a oftalmoscopia confocal de varredura a laser (HRT II [Heidelberg Retina Tomograph]) e a tomografia de coerência óptica (Stratus OCT) são tecnologias que permitem a avaliação objetiva e quantitativa do disco óptico e da CFN. No presente estudo, estas tecnologias foram comparadas em sua habilidade para diferenciar pacientes glaucomatosos de indivíduos normais. Pacientes com glaucoma foram selecionados com base na presença de defeitos reprodutíveis de campo visual na perimetria acromática automatizada (glaucoma perimétrico), ou com base na evidência documentada de progressão do dano glaucomatoso ao disco óptico, sem presença de defeitos de campo visual (glaucoma pré-perimétrico). Indivíduos normais apresentaram campos visuais e exame clínico dentro da normalidade. Todos os indivíduos foram submetidos a exames com o GDx VCC, HRT II, Stratus OCT e campo visual dentro de um período de três meses. Diversas medidas foram utilizadas para avaliação da acurácia diagnóstica, incluindo áreas sob as curvas receiver operating characteristic (AROC), sensibilidades para especificidades fixas, e razões de probabilidade. Modelos estatísticos foram utilizados para avaliação da influência da severidade do glaucoma e tamanho do disco óptico na performance diagnóstica dos diferentes instrumentos. Um olho de cada indivíduo foi utilizado para análise. Dos 258 sujeitos inicialmente avaliados, 33 (13%) foram posteriormente excluídos por apresentarem imagens de baixa qualidade em pelo menos um dos aparelhos, restando 225 indivíduos (133 glaucomatosos e 92 normais) para análise. Na comparação entre os parâmetros de cada instrumento com maiores valores de AROC, o parâmetro do GDx VCC, Nerve Fiber Indicator (NFI; AROC = 0,91), e o parâmetro do Stratus OCT, Espessura Média (AROC = 0,90), apresentaram áreas sob as curvas ROC significativamente superiores à do parâmetro do HRT II, função discriminante de Bathija (AROC = 0,84). A severidade do defeito de campo visual exerceu influência significativa sob a acurácia diagnóstica dos três instrumentos, com melhora no poder diagnóstico em casos mais avançados da doença. Para o GDx VCC e Stratus OCT, o aumento no tamanho do disco óptico foi associado à diminuição na sensibilidade para detecção do glaucoma; enquanto que, para o HRT II, diminuição no tamanho do disco óptico foi associada à diminuição na sensibilidade. Razões de probabilidade para resultados anormais nas xxv classificações finais de cada instrumento foram associadas a grandes efeitos de mudança na probabilidade pós-teste em relação à probabilidade préteste, sugerindo que o encontro de um resultado anormal em qualquer um destes testes, durante a avaliação de um paciente com suspeita de glaucoma, tem impacto significativo em aumentar a probabilidade de que a doença esteja presente. Além disso, os resultados obtidos na avaliação de pacientes com glaucoma pré-perimétrico sugerem que todos os três instrumentos sejam capazes de detectar alterações estruturais precoces no glaucoma, antes do aparecimento de defeitos de campo visual na perimetria acromática / Changes in the structural appearance of the optic nerve head (ONH) and retinal nerve fiber layer (RNFL) have been reported to precede the development of visual field loss in glaucoma. Detection of ONH and RNFL damage is therefore crucial for early diagnosis of glaucoma and prevention of functional loss from the disease. Scanning laser polarimetry (GDx VCC), confocal scanning laser ophthalmoscopy (HRT II [Heidelberg Retina Tomograph]) and optical coherence tomography (Stratus OCT) are different technologies capable of providing objective and quantitative information related to these structures. The purpose of the present study was to compare, in a single population, the diagnostic abilities of these technologies in the discrimination of glaucomatous patients from healthy subjects. Glaucoma patients were selected based on the presence of repeatable visual field defects, as identified by standard automated perimetry (perimetric glaucoma), or documented evidence of progressive damage to the optic disc, in the absence of detectable visual field loss (preperimetric glaucoma). Normal subjects had normal visual fields and normal clinical examination. All subjects underwent imaging with the GDx VCC, HRT II and Stratus OCT within a 3-month period. Several measures were used for evaluation of diagnostic accuracy, including the area under the receiver operating characteristic curve (AROC), sensitivities at fixed specifities, and likelihood ratios. Statistical models were used to evaluate the influence of glaucoma severity and optic disc size on the diagnostic performance of the different instruments. One eye of each individual was randomly selected for statistical analysis. From an initial group of 258 eligible subjects, 33 (13%) had images of unacceptable quality, leaving 133 glaucoma patients and 92 healthy subjects for further analysis. In the comparison of the parameters with highest values of AROC from each instrument, the GDx VCC Nerve Fiber Indicator (AROC = 0.91) and the Stratus OCT Average Thickness (AROC = 0.90) perfomed significanlty better than the HRT II Bathija discriminant function (AROC = 0.84). For all instruments, the diagnostic accuracy increased with increasing severity of visual field defects. For the GDx VCC and Stratus OCT parameters, an increase in the size of the optic disc was related to a decrease in the sensitivity for glaucoma detection. An opposite effect was observed with the HRT II: a decrease in the size of the optic disc was related to a decrease in the sensitivity for glaucoma diagnosis. Abnormal results for each of the instruments were associated with strong positive likelihood ratios, indicating a large change from prestest to posttest probability of glaucoma. These results suggest that the finding of an abnormal result in any of these tests, when assessing a patient suspect of having glaucoma, would substantially raise the probability of disease. Results of the evaluation of patients with preperimetric glaucoma also suggest that all three instruments are able to detect early glaucomatous structural damage in the absence of visual field loss

INTRODUÇÃO: A determinação do genótipo RHD fetal no plasma materno é um teste de diagnóstico pré-natal não invasivo oferecido a gestantes RhD negativo que apresentam potencial de sensibilização e/ou Doença Hemolítica Perinatal. Atualmente, este exame é realizado de rotina em diversos países, mas não no Brasil. A Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) oferece atendimento terciário a gestantes RhD negativo, com monitorização dos títulos de anticorpos irregulares, administração da imunoglobulina anti-D e/ou terapêutica fetal, quando necessários. OBJETIVO: Avaliar a acurácia do teste semiautomatizado para determinação do genótipo RHD fetal no plasma materno. METODOLOGIA: Foram coletadas prospectivamente amostras de sangue de 220 gestantes RhD negativo, com idade gestacional entre 8-28 semanas. O plasma foi obtido em no máximo 2 horas após a coleta, e uma alíquota de 1 mL foi submetida à extração de ácidos nucléicos no equipamento automatizado MagNA Pure Compact (Roche), empregando o kit Large Volume. O DNA extraído foi submetido a PCR em tempo real (Step One Plus - Applied Biosystems), usando o protocolo do grupo SAFE, que tem como alvos os éxons 5 e 7 do gene RHD. RESULTADOS: Ocorreu exclusão de 35 amostras devido a problemas pré-analíticos, aborto ou desconhecimento do fenótipo do recém-nascido. Entre as 185 amostras analisadas, 130 (70,2%) foram genotipadas como RHD+ e 55 (29,8%) RHD-. Os resultados obtidos foram comparados com a fenotipagem do cordão umbilical, e houve concordância completa (100%). Sete amostras exibiram amplificação exclusiva para o éxon 7. Essas amostras foram submetidas aos protocolos em PCR convencional, e PCR em tempo real específico para o pseudogene RHD. Ambos os ensaios apresentaram os mesmos resultados: cinco positivos e dois negativos. Nesses mesmos 7 casos, após extração da camada de leucócitos materna, os protocolos foram repetidos, e o resultado confirmou que cinco mães eram RHD. As duas amostras com resultado negativo foram submetidas ao protocolo Multiplex, envolvendo os éxons 3-9 do gene RHD, com resultados negativos, confirmando que as mães são verdadeiramente RHD- portanto o sinal do éxon 7 é provindo dos fetos que são D variantes. CONCLUSÃO: O método para a determinação do RHD fetal no plasma materno descrito demonstrou ser rápido, de fácil execução, alta precisão e reprodutível, além de indicar possíveis variantes RHD em nossa população / BACKGROUND: Fetal RHD genotype determination in maternal plasma is a noninvasive prenatal diagnostic test performed in RhD negative pregnant women at risk of alloimmunization and/or Hemolytic Disease of Fetus and Newborn. Currently, this test is routinely performed in many countries but not in Brazil. The Department of Obstetrics at Hospital das Clínicas, São Paulo University Medical School provides tertiary antenatal care for RhD negative pregnant women including anti-D immunoglobulin administration, antibody levels monitoring and intrauterine treatment if necessary. AIMS: To validate the accuracy of a semi-automated test for fetal RHD genotype determination in maternal plasma. METHODS: Two-hundred and twenty blood samples were prospectively collected between 8 and 28 weeks of gestational age. Plasma processing was performed within 2 hours after blood collection, and nucleic acids were extracted from 1mL aliquots with an automated extraction platform (MagNA Pure Compact Roche) and the Large Volume kit. RHD gene exons 5 and 7 were amplified with real-time PCR (Step One Plus - Applied Biosystems) using the SAFE group protocol. RESULTS: Thirty-five samples were excluded due to pre-analytical problems, miscarriage and missing follow-up. In the remaining 185 samples, 130 (70.2%) were genotyped as RhD+ and 55 (29.8%) RhD-. Comparison with umbilical cord blood group phenotype showed 100% concordance. Seven samples showed amplification for exon 7 only. These were further investigated with conventional and real-time PCR with an specific protocol for RHD? pseudogene: 5 were positive and 2, negative. In these 7 cases, maternal buffy-coat DNA analysis also confirmed that 5 women were RHD?. In the remaining 2 cases, a multiplex protocol directed at RHD gene exons 3-9 confirmed that both mothers were truly RhD negative so exon 7 signal comes from the fetuses, further found to harbor D variants. CONCLUSION: The present study demonstrates that fetal RHD determination in maternal plasma is a fast, easy-to-perform and reproducible technique with high accuracy in our population. Moreover, it helps in the identification of possible RHD variants in our population

INTRODUÇÃO: A determinação do genótipo RHD fetal no plasma materno é um teste de diagnóstico pré-natal não invasivo oferecido a gestantes RhD negativo que apresentam potencial de sensibilização e/ou Doença Hemolítica Perinatal. Atualmente, este exame é realizado de rotina em diversos países, mas não no Brasil. A Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) oferece atendimento terciário a gestantes RhD negativo, com monitorização dos títulos de anticorpos irregulares, administração da imunoglobulina anti-D e/ou terapêutica fetal, quando necessários. OBJETIVO: Avaliar a acurácia do teste semiautomatizado para determinação do genótipo RHD fetal no plasma materno. METODOLOGIA: Foram coletadas prospectivamente amostras de sangue de 220 gestantes RhD negativo, com idade gestacional entre 8-28 semanas. O plasma foi obtido em no máximo 2 horas após a coleta, e uma alíquota de 1 mL foi submetida à extração de ácidos nucléicos no equipamento automatizado MagNA Pure Compact (Roche), empregando o kit Large Volume. O DNA extraído foi submetido a PCR em tempo real (Step One Plus - Applied Biosystems), usando o protocolo do grupo SAFE, que tem como alvos os éxons 5 e 7 do gene RHD. RESULTADOS: Ocorreu exclusão de 35 amostras devido a problemas pré-analíticos, aborto ou desconhecimento do fenótipo do recém-nascido. Entre as 185 amostras analisadas, 130 (70,2%) foram genotipadas como RHD+ e 55 (29,8%) RHD-. Os resultados obtidos foram comparados com a fenotipagem do cordão umbilical, e houve concordância completa (100%). Sete amostras exibiram amplificação exclusiva para o éxon 7. Essas amostras foram submetidas aos protocolos em PCR convencional, e PCR em tempo real específico para o pseudogene RHD. Ambos os ensaios apresentaram os mesmos resultados: cinco positivos e dois negativos. Nesses mesmos 7 casos, após extração da camada de leucócitos materna, os protocolos foram repetidos, e o resultado confirmou que cinco mães eram RHD. As duas amostras com resultado negativo foram submetidas ao protocolo Multiplex, envolvendo os éxons 3-9 do gene RHD, com resultados negativos, confirmando que as mães são verdadeiramente RHD- portanto o sinal do éxon 7 é provindo dos fetos que são D variantes. CONCLUSÃO: O método para a determinação do RHD fetal no plasma materno descrito demonstrou ser rápido, de fácil execução, alta precisão e reprodutível, além de indicar possíveis variantes RHD em nossa população / BACKGROUND: Fetal RHD genotype determination in maternal plasma is a noninvasive prenatal diagnostic test performed in RhD negative pregnant women at risk of alloimmunization and/or Hemolytic Disease of Fetus and Newborn. Currently, this test is routinely performed in many countries but not in Brazil. The Department of Obstetrics at Hospital das Clínicas, São Paulo University Medical School provides tertiary antenatal care for RhD negative pregnant women including anti-D immunoglobulin administration, antibody levels monitoring and intrauterine treatment if necessary. AIMS: To validate the accuracy of a semi-automated test for fetal RHD genotype determination in maternal plasma. METHODS: Two-hundred and twenty blood samples were prospectively collected between 8 and 28 weeks of gestational age. Plasma processing was performed within 2 hours after blood collection, and nucleic acids were extracted from 1mL aliquots with an automated extraction platform (MagNA Pure Compact Roche) and the Large Volume kit. RHD gene exons 5 and 7 were amplified with real-time PCR (Step One Plus - Applied Biosystems) using the SAFE group protocol. RESULTS: Thirty-five samples were excluded due to pre-analytical problems, miscarriage and missing follow-up. In the remaining 185 samples, 130 (70.2%) were genotyped as RhD+ and 55 (29.8%) RhD-. Comparison with umbilical cord blood group phenotype showed 100% concordance. Seven samples showed amplification for exon 7 only. These were further investigated with conventional and real-time PCR with an specific protocol for RHD? pseudogene: 5 were positive and 2, negative. In these 7 cases, maternal buffy-coat DNA analysis also confirmed that 5 women were RHD?. In the remaining 2 cases, a multiplex protocol directed at RHD gene exons 3-9 confirmed that both mothers were truly RhD negative so exon 7 signal comes from the fetuses, further found to harbor D variants. CONCLUSION: The present study demonstrates that fetal RHD determination in maternal plasma is a fast, easy-to-perform and reproducible technique with high accuracy in our population. Moreover, it helps in the identification of possible RHD variants in our population

A ecocardiografia com perfusão miocárdica em tempo-real (EPTR) é uma técnica desenvolvida recentemente que utiliza baixa energia ultra-sônica e permite a avaliação da perfusão miocárdica e a quantificação do fluxo miocárdico regional. Embora estudos tenham demonstrado a possibilidade da medida da reserva de fluxo miocárdico (RFM) por esta técnica, sua acurácia para detecção de doença arterial coronariana (DAC) e sua correlação com a reserva de fluxo coronariano (RFC) obtida pelo estudo das velocidades de fluxo nos vasos epicárdicos, não estão definidas. Os objetivos deste estudo foram comparar a exeqüibilidade e acurácia da RFM medidas pela EPTR e da RFC obtida pela ecodopplercardiografia transtorácica (ETT) para a detecção de lesão obstrutiva na artéria coronária descendente anterior (ADA), tendo como padrão de referência a angiografia coronária quantitativa (ACQ), e correlacionar os valores ecodopplercardiográficos das reservas de fluxo miocárdico e coronariano com o grau de estenose coronariana. Foram Avaliados prospectivamente 71 pacientes, dos quais 56 (20 homens, média etária de 59 ± 11 anos) foram considerados para análise da acurácia. Os pacientes foram submetidos ao estudo da perfusão miocárdica pela EPTR em repouso e durante infusão de adenosina 140 mg/kg/min, usando como agente de contraste ecocardiográfico microbolhas encapsuladas por albumina e glicose. A quantificação do platô de intensidade miocárdica (A) que reflete o volume sangüíneo miocárdico, a velocidade de repreenchimento do miocárdio pelas microbolhas (ß) e o fluxo miocárdico (A x ß) foi realizada utilizando-se um programa computacional específico (Q-Lab 3.0, Philips Medical Systems). As velocidades de fluxo na porção distal da ADA foram avaliadas pela ETT, e a RFC definida como a relação entre a velocidade diastólica máxima durante hiperemia e no estado basal. Os pacientes foram submetidos à ACQ dentro de 30 dias do estudo ecocardiográfico. Lesão coronariana significativa foi definida como presença de obstrução >50% do diâmetro luminal. No presente estudo, a medida da RFC pelo Doppler da ADA apresentou exeqüibilidade global de 83% , enquanto que a quantificação da RFM pela EPTR mostrou exeqüibilidade de 99%. Os pacientes com lesão angiograficamente significativa na ADA apresentaram valores de RFC (2,86 ± 0,71 versus 1,57 ± 0,38; p = 0,0001), RFM (2,43 ± 0,80 versus 1,24 ± 0,48; p = 0,0001) e reserva b (2,08 ± 0,82 versus 1,23 ± 0,46; p = 0,001) menores que pacientes sem lesão significativa. O valor de corte utilizado para diferenciar pacientes com e sem lesão na ADA foi 1,84 para a RFC obtida pelo Doppler da ADA, 1,74 para a RFM e 1,68 para a reserva b. A sensibilidade, especificidade e acurácia para detecção de lesão angiograficamente significativa na ADA foram de 96%, 93%, e 95% para a RFC obtida pelo Doppler da ADA, 88%, 90% e 89% para a RFM obtida pela xxii EPTR, e 88%, 84%, e 86% para a reserva b. A análise de regressão logística demonstrou que o estudo com Doppler da ADA foi o parâmetro que melhor diferenciou os pacientes com e sem lesão na ADA (Razão de chances de 1,78, intervalo de confiança de 95% de 1,28 a 2,47). Houve uma boa correlação entre a medida da reserva b (r = 0,89; p <0,05), RFM (r = 0,79; p <0,05), e RFC (r = 0,88; p < 0,05) e o grau de estenose obtido pela ACQ. Conclui-se que a avaliação da RFC pelo Doppler da ADA e da RFM pela EPTR quantitativa apresentaram alta exeqüibilidade e foram capazes de diferenciar de modo preciso os indivíduos com e sem lesão angiográfica significativa na ADA. No entanto, a acurácia diagnóstica pelo Doppler da ADA foi discretamente superior aos outros parâmetros analisados e apresentou menor exeqüibilidade. Ambas as reservas de fluxo miocárdico e coronariano correlacionaram-se de modo inverso com o grau de estenose coronariana / Real-time myocardial contrast echocardiography (RTMCE) is a recently developed technique that utilizes low-mechanical index imaging and allows for noninvasive evaluation of myocardial perfusion as well as for quantification of regional myocardial blood flow. Although previous studies have demonstrated that RTMCE permits determining myocardial blood flow reserve (MBFR), its diagnostic accuracy and correlation with the measurement of coronary flow reserve (CFR) by transthoracic Doppler echocardiography (TTDE) has not been fully demonstrated. The aims of this study were to compare the feasibility and diagnostic accuracy of MBFR obtained by RTMCE and CFR obtained by TTE for detecting angiographically significant obstruction in the left anterior descending coronary artery (LAD), and to determine the correlation between MBFR and CFR and the severity of stenosis determined by quantitative coronary angiography. We prospectively studied 71 patients, among them 56 patients (20 men, 59 ± 11 years) were considered for the determination of diagnostic accuracy. All patients underwent RTMCE at rest and during 140mcg/kg/min of adenosine infusion. Plateau acoustic intensity (A), myocardial replenishment velocity slope (B) and myocardial blood flow (A x B) were quantified using Q-Lab 3.0 (Philips Medical Systems). Coronary flow velocities were evaluated in the distal LAD using TTE and CFR was defined as the ratio between maximal diastolic velocity during hiperemia and baseline. LAD stenosis (obstruction >50% of luminal diameter) was determined by quantitative coronary angiography (QCA) performed within one month of RTMCE. The feasibility of CFR measurement by TTE was 83%, while the feasibility of MBFR measurement by RTMCE was 99%. CFR was significantly lower in patients with than in patients without angiographically significant LAD stenosis (2.86 ± 0.71 versus 1.57 ± 0.38; p = 0.0001), as was the MBFR (2.43 ± 0.80 versus 1.24 ± 0.48; p = 0.0001) and b reserve (2.08 ± 0.82 versus 1.23 ± 0.46; p = 0.001). Cutoff values for differentiating patients with and without LAD stenosis were 1.84 for CFR, 1.74 for MBFR, and 1.68 for b reserve. The sensitivity, specificity and accuracy for detecting LAD stenosis were 96%, 93%, and 95% for CFR obtained by TTE, 88%, 90%, and 89% for MBFR, and 88%, 84%, and 86% for b reserve. Multivariate logistic regression analysis revealed that CFR as measured by TTE was the best predictor of LAD (Odds ratio = 1.78, 95% confidence interval 1.28 to 2.47). There was a good correlation between b reserve (r = 0.89; p <0.05), MBFR (r = 0.79; p <0.05), and CFR (r = 0.88; p < 0.05) and the severity of coronary obstruction determined by QCA. In conclusion, CFR obtained by TTE and MBFR obtained by RTMCE were highly feasible and accurate for differentiating patients with and without angiographically significant LAD obstruction. CFR had a slightly higher diagnostic accuracy than other xxv evaluated parameters, despite lower feasibility. Both the CFR and MBFR were inversely correlated with the degree of luminal coronary obstruction determined by QCA

Chen, Zhang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (p. 109-114). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.III / ACKNOWLEDGEMENTS --- p.IV / PUBLICATIONS --- p.VI / CONTRIBUTORS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XIII / LIST OF FIGURES --- p.XIV / LIST OF ABBREVIATIONS --- p.XVI / Chapter SECTION I : --- BACKGROUND --- p.1 / Chapter CHAPTER 1: --- PRENATAL DIAGNOSIS OF FETAL TRISOMY BY NEXT GENERATION SEQUENCING TECHNOLOGY --- p.2 / Chapter 1.1 --- FETAL TRISOMY --- p.2 / Chapter 1.2 --- CONVENTIONAL PRENATAL DIAGNOSIS OF FETAL TRISOMIES --- p.3 / Chapter 1.3 --- CELL FREE FETAL D N A AND ITS APPLICATION IN PRENATAL DIAGNOSIS --- p.5 / Chapter 1.4 --- NEXT GENERATION SEQUENCING TECHNOLOGY --- p.5 / Chapter 1.5 --- SUBSTANTIAL BIAS IN THE NEXT GENERATION SEQUENCING PLATFORM --- p.9 / Chapter 1.6 --- PRENATAL DIAGNOSIS OF TRISOMY BY NEXT GENERATION SEQUENCING --- p.10 / Chapter 1.7 --- AIMS OF THIS THESIS --- p.11 / Chapter SECTION I I : --- MATERIALS AND METHODS --- p.13 / Chapter CHAPTER 2: --- METHODS FOR NONINVASIVE PRENATAL DIAGNOSIS OF FETAL TRISOMY MATERNAL PLASMA DNA SEQUENCING --- p.14 / Chapter 2.1 --- STUDY DESIGN AND PARTICIPANTS --- p.14 / Chapter 2.1.1 --- Ethics Statement --- p.14 / Chapter 2.1.2 --- "Study design, setting and participants" --- p.14 / Chapter 2.2 --- MATERNAL PLASMA D N A SEQUENCING --- p.17 / Chapter 2.3 --- SEQUENCING DATA ANALYSIS --- p.18 / Chapter SECTION I I I : --- TRISOMY 13 AND 18 DETECTION BY THE T21 BIOINFORMATICS ANALYSIS PIPELINE --- p.21 / Chapter CHAPTER 3: --- THE T21 BIOINFORMATICS ANALYSIS PIPELINE FOR TRISOMY 13 AND 18 DETECTION --- p.22 / Chapter 3.1 --- INTRODUCTION --- p.22 / Chapter 3.2 --- METHODS --- p.23 / Chapter 3.2.1 --- Bioinformatics analysis pipeline for trisomy 13 and 18 detection --- p.23 / Chapter 3.3 --- RESULTS --- p.23 / Chapter 3.3.1 --- Performance of the T21 bioinformatics analysis pipeline for trisomy 13 and 18 detection --- p.23 / Chapter 3.3.2 --- The precision of quantifying chrl 3 and chrl 8 --- p.27 / Chapter 3.4 --- DISCUSSION --- p.29 / Chapter SECTION IV : --- IMPROVING THE T21 BIOINFORMATICS ANALYSIS PIPELINE FOR TRISOMY 13 AND 18 DETECTION --- p.30 / Chapter CHAPTER 4: --- IMPROVING THE ALIGNMENT --- p.31 / Chapter 4.1 --- INTRODUCTION --- p.31 / Chapter 4.2 --- METHODS --- p.32 / Chapter 4.2.1 --- Allowing mismatches in the index sequences --- p.32 / Chapter 4.2.2 --- Calculating the mappability of the human reference genome --- p.33 / Chapter 4.2.3 --- Aligning reads to the non-repeat masked human reference genome --- p.34 / Chapter 4.2.4 --- Trisomy 13 and 18 detection --- p.34 / Chapter 4.3 --- RESULTS --- p.34 / Chapter 4.3.1 --- Increasing read numbers by allowing mismatches in the index sequences --- p.34 / Chapter 4.3.2 --- Increasing read numbers by using the non-masked reference genome for alignment . --- p.38 / Chapter 4.3.3 --- Allowing mismatches in the read alignment --- p.42 / Chapter 4.3.4 --- The performance of trisomy 13 and 18 detection after improving the alignment --- p.47 / Chapter 4.4 --- DISCUSSION --- p.50 / Chapter CHAPTER 5: --- REDUCING THE GC BIAS BY CORRECTION OF READ COUNTS --- p.53 / Chapter 5.1 --- INTRODUCTION --- p.53 / Chapter 5.2 --- METHODS --- p.54 / Chapter 5.2.1 --- Read alignment --- p.54 / Chapter 5.2.2 --- Calculating the correlation between GC content and read counts --- p.55 / Chapter 5.2.3 --- GC correction in read counts --- p.55 / Chapter 5.2.4 --- Trisomy 13 and 18 detection --- p.56 / Chapter 5.3 --- RESULTS --- p.56 / Chapter 5.3.1 --- GC bias in plasma DNA sequencing --- p.56 / Chapter 5.3.2 --- Correcting the GC bias in read counts by linear regression --- p.59 / Chapter 5.3.3 --- Correcting the GC bias in read counts by LOESS regression --- p.65 / Chapter 5.3.4 --- Bin size --- p.72 / Chapter 5.4 --- DISCUSSION --- p.75 / Chapter CHAPTER 6: --- REDUCING THE GC BIAS BY MODIFYING THE GENOMIC REPRESENTATION CALCULATION --- p.77 / Chapter 6.1 --- INTRODUCTION --- p.77 / Chapter 6.2 --- METHODS --- p.78 / Chapter 6.2.1 --- Modifying the genomic representation calculation --- p.78 / Chapter 6.2.2 --- Trisomy 13 and 18 detection --- p.78 / Chapter 6.2.3 --- Combining GC correction and modified genomic representation --- p.78 / Chapter 6.3 --- RESULTS --- p.79 / Chapter 6.3.1 --- Reducing the GC bias by modifying genomic representation calculation --- p.79 / Chapter 6.3.2 --- Combining GC correction and modified genomic representation --- p.86 / Chapter 6.4 --- DISCUSSION --- p.89 / Chapter CHAPTER 7: --- IMPROVING THE STATISTICS FOR TRISOMY 13 AND 18 DETECTION --- p.91 / Chapter 7.1 --- INTRODUCTION --- p.91 / Chapter 7.2 --- METHODS --- p.92 / Chapter 7.2.1 --- Comparing chrl 3 or chrl8 with other chromosomes within the sample --- p.92 / Chapter 7.2.2 --- Comparing chrl 3 or chrl 8 with the artificial chromosomes --- p.92 / Chapter 7.3 --- RESULTS --- p.93 / Chapter 7.3.1 --- Determining the trisomy 13 and 18 status by comparing chromosomes within the samples --- p.93 / Chapter 7.3.2 --- Determining the trisomy 13 and 18 status by comparing chrl3 or chrl 8 with artificial chromosomes --- p.97 / Chapter 7.4 --- DISCUSSION --- p.100 / Chapter SECTION V : --- CONCLUDING REMARKS --- p.102 / Chapter CHAPTER 8: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.103 / Chapter 8.1 --- THE PERFORMANCE OF THE T21 BIOINFORMATICS ANALYSIS PIPELINE DEVELOPED FOR TRISOMY 21 DETECTION IS SUBOPTIMAL FOR TRISOMY 13 AND 18 DETECTION --- p.103 / Chapter 8.2 --- THE ALIGNMENT COULD BE IMPROVED BY ALLOWING ONE MISMATCH IN THE INDEX AND USING THE NON-REPEAT MASKED HUMAN REFERENCE GENOME AS THE ALIGNMENT REFERENCE --- p.104 / Chapter 8.3 --- THE PRECISION OF QUANTIFYING CHR13 AND CHR18 COULD BE IMPROVED BY THE G C CORRECTION OR THE MODIFIED GENOMIC REPRESENTATION --- p.104 / Chapter 8.4 --- THE STATISTICS FOR TRISOMY 13 AND 18 DETECTION COULD BE IMPROVED BY COMPARING CHR13 OR CHR18 WITH ARTIFICIAL CHROMOSOMES WITHIN THE SAMPLE --- p.105 / Chapter 8.5 --- PROSPECTS FOR FUTURE WORK --- p.106 / REFERENCE --- p.109

As Oclusões Venosas da Retina são complicações do sistema vascular retiniano com grande potencial de impacto no sentido da visão. As oclusões Venosas são classificadas de acordo com a anatomia da região acometida e podem ser divididas em oclusão de ramo e da veia central da retina. O edema macular é a principal causa de baixa acuidade visual na Oclusão de Ramo da Veia Central da Retina (OVR). Sua detecção, localização e classificação são cruciais para a prevenção e tratamento da perda visual na OVR. A Biomicrospia Macular e a Angiofluoresceinografia são os métodos tradicionais de avaliação do edema macular na OVR, a Tomografia de Coerência Óptica (TCO) é uma tecnologia nova, que permite uma avaliação objetiva da morfologia macular. O objetivo primário do presente estudo foi avaliar, em uma mesma população, a acurácia dos métodos de diagnóstico do edema macular na OVR através da análise de três quesitos, aumento da espessura macular e da sua localização, presença de cistos intra-retinianos e a presença de descolamento seroso macular (DSM). Indivíduos com OVR foram selecionados com base nos achados fundoscópicos da doença e com tempo de história da doença entre três e vinte quatro meses. Todos os sujeitos foram submetidos a exames com o Stratus OCT (TCO), Angiofluoresceinografia digital (Angio) e o exame da biomicroscopia macular com lente de Volk de 78 D (BM) dentro do período de uma semana, além de exame oftalmológico completo. As medidas utilizadas para avaliação da acurácia diagnóstica foram o índice de concordância Kappa e a sensibilidade. Dos 32 indivíduos inicialmente avaliados, 8 foram posteriormente excluídos por não cumprirem os critérios de inclusão ou de exclusão, restando 24 indivíduos para análise. Na avaliação da espessura macular, a concordância entre BM e a TCO foi substancial e significante para a detecção do aumento da espessura macular com Kappa = 0,778 (p < 0,001) e a sensibilidade foi 95,5%. Na comparação entre a BM e a TCO para a detecção de cistos intra-retinianos, a concordância foi fraca com Kappa = 0,250 (p = 0,066) e a sensibilidade foi 57,9 %. A concordância entre a Angio e a TCO para os cistos foi pobre e não significante com Kappa = 0,124 (p = 0,237) e a sensibilidade foi 58,7 %. Houve uma correlação significante entre a presença de extravasamento na angiofluoresceinografia e a presença de cistos intra-retinianos na TCO (p=0,042). Na avaliação do DSM a concordância entre a BM e a TCO foi fraca com Kappa = 0,314 (p = 0,062) e a sensibilidade foi 60%, não houve concordância entre a Angio e a TCO para a detecção do DSM com Kappa = 0 e sensibilidade de 0%, e também não houve correlação significante entre a presença de descolamento seroso na TCO e a presença de extravasamento na angiofluoresceinografia (p=0,615). Os dados obtidos no presente estudo sugeriram que o Stratus OCT têm acurácia superior para a detecção dos cistos intra-retinianos e do descolamento seroso macular, se comparado à biomicroscopia macular e à angiofluoresceinografia / Retinal Vein Occlusions are complications of the retinal vascular system that can cause a great impact on vision. The Vein Occlusions are classified according to the anatomy of the affected region and can be categorized as: branch retinal vein occlusion and central retinal vein occlusion. Macular edema is the main cause for low visual acuity in Branch Retinal Vein Occlusion (BRVO). Its detection, location and classification are crucial for the prevention and treatment of vision loss in BRVO. Both Macular Biomicroscopy and Angiofluoresceinography are traditional methods for the evaluation of the macular edema in BRVO; the Optical Coherence Tomography (OCT) is a new technology that allows for an objective evaluation of macular morphology. The main objective of the present study was to evaluate, among the same population, the accuracy of the diagnostic methods of macular edema in BRVO through the analysis of three items: the increase of macular thickness and its location, the presence of intraretinal cysts, and the presence of serous macular detachment. Patients with BRVO were selected according to the fundoscopic findings of the disease and a disease history of three to twenty-four months. All of the individuals underwent exams with the Stratus OCT, digital Angiofluoresceinography (Angio) and a macular biomicroscopy with a 78D Volk lens (MB), within a period of one week, as well as a complete ophthalmologic exam. The parameters used in the evaluation of the diagnostic accuracy were sensitivity and the kappa coefficient. Of the 32 subjects who were initially evaluated, 8 were later excluded since they did not meet inclusion or exclusion criteria. The remaining 24 subjects were analyzed. In the evaluation of macular thickness, the concordance between the MB and the OCT was substantial and significant in the detection of the increase in macular thickness, with Kappa = 0.778 (p<0.001) and a sensitivity of 95.5%. When comparing the MB and the OCT in the detection of intraretinal cysts, the concordance was fair, with Kappa = 0.250 (p = 0.066) and a sensitivity of 57.9%. The concordance between the Angio and the OCT for cysts detection was poor and non significant, with Kappa = 0.124 (p = 0.237) and a sensitivity of 58.7%. There was a significant correlation between the presence of leakage in the angiofluoresceinography and the presence of intraretinal cysts in the OCT (p = 0.042). In the evaluation of the SMD, the concordance between the MB and the OCT was fair, with Kappa = 0.314 (p = 0.062) and a sensitivity of 60% an there was no concordance between the Angio and the OCT in the detection of SMD, with Kappa = 0 and sensitivity of 0%. There was also no significant correlation between the presence of serous detachment in the OCT and the presence of leakage in the angiofluoresceinography (p = 0.615). The results obtained in the present study suggest that Stratus OCT is more accurate to the detection of intraretinal cysts and serous macular detachment than macular biomicroscopy with a 78D Volk lens, and by angiofluoresceinography