Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology

Paralytic shellfish poisoning (PSP) toxin monitoring in shellfish is currently performed using the internationally accredited AOAC mouse bioassay. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. The feasibility of using a surface plasmon resonance optical biosensor to detect PSP toxins in shellfish tissue below regulatory levels was examined. Three different PSP toxin protein binders were investigated: a sodium channel receptor (SCR) preparation derived from rat brains, a monoclonal antibody (GT13-A) raised to gonyautoxin 2/3, and a rabbit polyclonal antibody (R895) raised to saxitoxin (STX). Inhibition assay formats were used throughout. Immobilization of STX to the biosensor chip surface was achieved via amino-coupling. Specific binding and inhibition of binding to this surface was achieved using all proteins tested. For STX calibration curves, 0 - 1000 ng/mL, IC50 values for each binder were as follows: SCR 8.11 ng/mL; GT13-A 5.77 ng/mL; and R895 1.56 ng/mL. Each binder demonstrated a different cross-reactivity profile against a range of STX analogues. R895 delivered a profile that was most likely to detect the widest range of PSP toxins at or below the internationally adopted regulatory limits.

Assessment and detection of peer-bullying through analysis of the group context

Resumen tomado de la publicaci??n

Environmental assessment and the built environment : a highway engineer's perspective

This paper is an engineer's appreciation of environmental assessment with particular reference to highway development. While scheme-related Environmental Assessment for individual development may identify particular potential impacts, and may avoid or minimise some of the problems, in many cases it may be too late to take such actions. Ideally, Environmental Assessment should commence at the Strategic Level to cover policies, plan and programmes, and the scheme-related Environmental Assessments for individual projects should supplement those in the framework of Strategic Level. The utimate target is to assess the policy for their contribution to effecting sustainable development. Whole Life Environmental Impacts should be considered. These are the full impact consideration from planning, design and choice of materials, construction, operation and finally decommission. Most of the Environmental Assessments have not included the Whole Life Environmental Impacts. There is only limited monitoring in the operation stage after the construction of the scheme is complete, therefore, subsequent Environmental Assessments cannot benefit from the feedback of the scheme. No development should cost the Earth, hence Environmental Assessments have to be carried out thoroughly to serve as one of the instruments to meet the need of sustainable development.

Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media

Aims: To make a preliminary assessment of the incidence of Salmonella in Egyptian dairy products, and to investigate the effectiveness of various protocols for the detection of the pathogen in these products. Methods and Results: Samples of milk and related dairy products were randomly collected from local markets and examined for the presence of Salmonella. While most samples were free of the organism, isolates of Salmonella enterica subsp. enterica serovar Typhimurium PT 8 could be recovered from 'matared' cream specimens. These isolates were susceptible to antibiotics usually used to challenge infections caused by Salmonella. A combination of buffered peptone water, Muller-Kauffman tetrathionate broth, and brilliant green phenol red agar gave the best results for the detection of the pathogen. Selenite-cystine broth and Hektoen enteric agar were ineffective as an enrichment and a plating medium, respectively, in the isolation of Salmonella. A modified identification strategy that reduces the burden of serological testing of presumptive isolates is proposed. Conclusions, Significance and Impact of the Study: 'Matared' cream could be a vehicle for transmitting Salmonella. Using the above combination of media, beside the suggested modified confirmatory procedure, should increase the effectiveness and ease of the detection of Salmonella in milk and dairy products.

Analytical Assessment of a Home Made Capillary Electrophoresis Equipment with Linear Charge Coupled Device for Visible Light Absorption Detection in the Determination of Food Dyes

The performance of modular home made capillary electrophoresis equipment with spectrophotometric detection, at a visible region by means of a miniaturized linear charge coupled device, was evaluated for the determination of four food dyes. This system presents a simple but efficient home made cell detection scheme. A computer program that converts the spectral data after each run into the electropherograms was developed to evaluate the analytical parameters. The dyes selected for analytical evaluation of the system were Brilliant Blue FCF, Fast Green FCF, Sunset Yellow FCF, and Amaranth. Separation was carried out in a 29cm length and 75 mu m I.D fused silica capillary, using 10mmolL-1 borate buffer at pH 9, with separation voltage of 7.5kV. The detection limits for the dyes were between 0.3 and 1.5mgL-1 and the method presented adequate linearity over the ranges studied, with correlation coefficients greater than 0.99. The method was applied for determination and quantification of these dyes in fruit juices and candies.

Detection of hormones in surface and drinking water in Brazil by LC-ESI-MS/MS and ecotoxicological assessment with Daphnia magna

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Reactive power planning under conditional-value-at-risk assessment using chance-constrained optimisation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Accuracy of positron emission tomography/computed tomography and clinical assessment in the detection of complete rectal tumor regression after neoadjuvant chemoradiation Long-Term Results of a Prospective Trial (National Clinical Trial 00254683)

BACKGROUND: Neoadjuvant chemoradiation (CRT) therapy may result in significant tumor regression in patients with rectal cancer. Patients who develop complete tumor regression have been managed by treatment strategies that are alternatives to standard total mesorectal excision. Therefore, assessment of tumor response with positron emission tomography/computed tomography (PET/CT) after neoadjuvant treatment may offer relevant information for the selection of patients to receive alternative treatment strategies. METHODS: Patients with clinical T2 (cT2) through cT4NxM0 rectal adenocarcinoma were included prospectively. Neoadjuvant therapy consisted of 54 grays of radiation and 5-fluorouracil-based chemotherapy. Baseline PET/CT studies were obtained before CRT followed by PET/CT studies at 6 weeks and 12 weeks after the completion of CRT. Clinical assessment was performed at 12 weeks after CRT completion. PET/CT results were compared with clinical and pathologic data. RESULTS: In total, 99 patients were included in the study. Twenty-three patients were complete responders (16 had a complete clinical response, and 7 had a complete pathologic response). The PET/CT response evaluation at 12 weeks indicated that 18 patients had a complete response, and 81 patients had an incomplete response. There were 5 false-negative and 10 false-positive PET/CT results. PET/CT for the detection of residual cancer had 93% sensitivity, 53% specificity, a 73% negative predictive value, an 87% positive predictive value, and 85% accuracy. Clinical assessment alone resulted in an accuracy of 91%. PET/CT information may have detected misdiagnoses made by clinical assessment alone, improving overall accuracy to 96%. CONCLUSIONS: Assessment of tumor response at 12 weeks after CRT completion with PET/CT imaging may provide a useful additional tool with good overall accuracy for the selection of patients who may avoid unnecessary radical resection after achieving a complete clinical response. Cancer 2012;35013511. (C) 2011 American Cancer Society.

From fall-risk assessment to fall detection: inertial sensors in the clinical routine and daily life

Falls are caused by complex interaction between multiple risk factors which may be modified by age, disease and environment. A variety of methods and tools for fall risk assessment have been proposed, but none of which is universally accepted. Existing tools are generally not capable of providing a quantitative predictive assessment of fall risk. The need for objective, cost-effective and clinically applicable methods would enable quantitative assessment of fall risk on a subject-specific basis. Tracking objectively falls risk could provide timely feedback about the effectiveness of administered interventions enabling intervention strategies to be modified or changed if found to be ineffective. Moreover, some of the fundamental factors leading to falls and what actually happens during a fall remain unclear. Objectively documented and measured falls are needed to improve knowledge of fall in order to develop more effective prevention strategies and prolong independent living. In the last decade, several research groups have developed sensor-based automatic or semi-automatic fall risk assessment tools using wearable inertial sensors. This approach may also serve to detect falls. At the moment, i) several fall-risk assessment studies based on inertial sensors, even if promising, lack of a biomechanical model-based approach which could provide accurate and more detailed measurements of interests (e.g., joint moments, forces) and ii) the number of published real-world fall data of older people in a real-world environment is minimal since most authors have used simulations with healthy volunteers as a surrogate for real-world falls. With these limitations in mind, this thesis aims i) to suggest a novel method for the kinematics and dynamics evaluation of functional motor tasks, often used in clinics for the fall-risk evaluation, through a body sensor network and a biomechanical approach and ii) to define the guidelines for a fall detection algorithm based on a real-world fall database availability.

Estimating greenhouse gas emissions of highway construction and rehabilitation to support pavement life cycle assessment

Highway infrastructure plays a significant role in society. The building and upkeep of America’s highways provide society the necessary means of transportation for goods and services needed to develop as a nation. However, as a result of economic and social development, vast amounts of greenhouse gas emissions (GHG) are emitted into the atmosphere contributing to global climate change. In recognizing this, future policies may mandate the monitoring of GHG emissions from public agencies and private industries in order to reduce the effects of global climate change. To effectively reduce these emissions, there must be methods that agencies can use to quantify the GHG emissions associated with constructing and maintaining the nation’s highway infrastructure. Current methods for assessing the impacts of highway infrastructure include methodologies that look at the economic impacts (costs) of constructing and maintaining highway infrastructure over its life cycle. This is known as Life Cycle Cost Analysis (LCCA). With the recognition of global climate change, transportation agencies and contractors are also investigating the environmental impacts that are associated with highway infrastructure construction and rehabilitation. A common tool in doing so is the use of Life Cycle Assessment (LCA). Traditionally, LCA is used to assess the environmental impacts of products or processes. LCA is an emerging concept in highway infrastructure assessment and is now being implemented and applied to transportation systems. This research focuses on life cycle GHG emissions associated with the construction and rehabilitation of highway infrastructure using a LCA approach. Life cycle phases of the highway section include; the material acquisition and extraction, construction and rehabilitation, and service phases. Departing from traditional approaches that tend to use LCA as a way to compare alternative pavement materials or designs based on estimated inventories, this research proposes a shift to a context sensitive process-based approach that uses actual observed construction and performance data to calculate greenhouse gas emissions associated with highway construction and rehabilitation. The goal is to support strategies that reduce long-term environmental impacts. Ultimately, this thesis outlines techniques that can be used to assess GHG emissions associated with construction and rehabilitation operations to support the overall pavement LCA.

Dish Detection and Segmentation for Dietary Assessment on Smartphones

Diet-related chronic diseases severely affect personal and global health. However, managing or treating these diseases currently requires long training and high personal involvement to succeed. Computer vision systems could assist with the assessment of diet by detecting and recognizing different foods and their portions in images. We propose novel methods for detecting a dish in an image and segmenting its contents with and without user interaction. All methods were evaluated on a database of over 1600 manually annotated images. The dish detection scored an average of 99% accuracy with a .2s/image run time, while the automatic and semi-automatic dish segmentation methods reached average accuracies of 88% and 91% respectively, with an average run time of .5s/image, outperforming competing solutions.

Evaluación de la contribución y el impacto de las tecnologías del habla en la detección automática del Síndrome de la Apnea Obstructiva del Sueño = Contributions and impact assessment of speech technologies on the automatic detection of severe Obstructive Sleep Apnea syndrome

La presente Tesis analiza las posibilidades que ofrecen en la actualidad las tecnologías del habla para la detección de patologías clínicas asociadas a la vía aérea superior. El estudio del habla que tradicionalmente cubre tanto la producción como el proceso de transformación del mensaje y las señales involucradas, desde el emisor hasta alcanzar al receptor, ofrece una vía de estudio alternativa para estas patologías. El hecho de que la señal emitida no solo contiene este mensaje, sino también información acerca del locutor, ha motivado el desarrollo de sistemas orientados a la identificación y verificación de la identidad de los locutores. Estos trabajos han recibido recientemente un nuevo impulso, orientándose tanto hacia la caracterización de rasgos que son comunes a varios locutores, como a las diferencias existentes entre grabaciones de un mismo locutor. Los primeros resultan especialmente relevantes para esta Tesis dado que estos rasgos podrían evidenciar la presencia de características relacionadas con una cierta condición común a varios locutores, independiente de su identidad. Tal es el caso que se enfrenta en esta Tesis, donde los rasgos identificados se relacionarían con una de la patología particular y directamente vinculada con el sistema de físico de conformación del habla. El caso del Síndrome de Apneas Hipopneas durante el Sueno (SAHS) resulta paradigmático. Se trata de una patología con una elevada prevalencia mundo, que aumenta con la edad. Los pacientes de esta patología experimentan episodios de cese involuntario de la respiración durante el sueño, que se prolongan durante varios segundos y que se reproducen a lo largo de la noche impidiendo el correcto descanso. En el caso de la apnea obstructiva, estos episodios se deben a la imposibilidad de mantener un camino abierto a través de la vía aérea, de forma que el flujo de aire se ve interrumpido. En la actualidad, el diagnostico de estos pacientes se realiza a través de un estudio polisomnográfico, que se centra en el análisis de los episodios de apnea durante el sueño, requiriendo que el paciente permanezca en el hospital durante una noche. La complejidad y el elevado coste de estos procedimientos, unidos a las crecientes listas de espera, han evidenciado la necesidad de contar con técnicas rápidas de detección, que si bien podrían no obtener tasas tan elevadas, permitirían reorganizar las listas de espera en función del grado de severidad de la patología en cada paciente. Entre otros, los sistemas de diagnostico por imagen, así como la caracterización antropométrica de los pacientes, han evidenciado la existencia de patrones anatómicos que tendrían influencia directa sobre el habla. Los trabajos dedicados al estudio del SAHS en lo relativo a como esta afecta al habla han sido escasos y algunos de ellos incluso contradictorios. Sin embargo, desde finales de la década de 1980 se conoce la existencia de patrones específicos relativos a la articulación, la fonación y la resonancia. Sin embargo, su descripción resultaba difícilmente aprovechable a través de un sistema de reconocimiento automático, pero apuntaba la existencia de un nexo entre voz y SAHS. En los últimos anos las técnicas de procesado automático han permitido el desarrollo de sistemas automáticos que ya son capaces de identificar diferencias significativas en el habla de los pacientes del SAHS, y que los distinguen de los locutores sanos. Por contra, poco se conoce acerca de la conexión entre estos nuevos resultados, los sé que habían obtenido en el pasado y la patogénesis del SAHS. Esta Tesis continua la labor desarrollada en este ámbito considerando específicamente: el estudio de la forma en que el SAHS afecta el habla de los pacientes, la mejora en las tasas de clasificación automática y la combinación de la información obtenida con los predictores utilizados por los especialistas clínicos en sus evaluaciones preliminares. Las dos primeras tareas plantean problemas simbióticos, pero diferentes. Mientras el estudio de la conexión entre el SAHS y el habla requiere de modelos acotados que puedan ser interpretados con facilidad, los sistemas de reconocimiento se sirven de un elevado número de dimensiones para la caracterización y posterior identificación de patrones. Así, la primera tarea debe permitirnos avanzar en la segunda, al igual que la incorporación de los predictores utilizados por los especialistas clínicos. La Tesis aborda el estudio tanto del habla continua como del habla sostenida, con el fin de aprovechar las sinergias y diferencias existentes entre ambas. En el análisis del habla continua se tomo como punto de partida un esquema que ya fue evaluado con anterioridad, y sobre el cual se ha tratado la evaluación y optimización de la representación del habla, así como la caracterización de los patrones específicos asociados al SAHS. Ello ha evidenciado la conexión entre el SAHS y los elementos fundamentales de la señal de voz: los formantes. Los resultados obtenidos demuestran que el éxito de estos sistemas se debe, fundamentalmente, a la capacidad de estas representaciones para describir dichas componentes, obviando las dimensiones ruidosas o con poca capacidad discriminativa. El esquema resultante ofrece una tasa de error por debajo del 18%, sirviéndose de clasificadores notablemente menos complejos que los descritos en el estado del arte y de una única grabación de voz de corta duración. En relación a la conexión entre el SAHS y los patrones observados, fue necesario considerar las diferencias inter- e intra-grupo, centrándonos en la articulación característica del locutor, sustituyendo los complejos modelos de clasificación por el estudio de los promedios espectrales. El resultado apunta con claridad hacia ciertas regiones del eje de frecuencias, sugiriendo la existencia de un estrechamiento sistemático en la sección del tracto en la región de la orofaringe, ya prevista en la patogénesis de este síndrome. En cuanto al habla sostenida, se han reproducido los estudios realizados sobre el habla continua en grabaciones de la vocal /a/ sostenida. Los resultados son cualitativamente análogos a los anteriores, si bien en este caso las tasas de clasificación resultan ser más bajas. Con el objetivo de identificar el sentido de este resultado se reprodujo el estudio de los promedios espectrales y de la variabilidad inter e intra-grupo. Ambos estudios mostraron importantes diferencias con los anteriores que podrían explicar estos resultados. Sin embargo, el habla sostenida ofrece otras oportunidades al establecer un entorno controlado para el estudio de la fonación, que también había sido identificada como una fuente de información para la detección del SAHS. De su estudio se pudo observar que, en el conjunto de datos disponibles, no existen variaciones que pudieran asociarse fácilmente con la fonación. Únicamente aquellas dimensiones que describen la distribución de energía a lo largo del eje de frecuencia evidenciaron diferencias significativas, apuntando, una vez más, en la dirección de las resonancias espectrales. Analizados los resultados anteriores, la Tesis afronta la fusión de ambas fuentes de información en un único sistema de clasificación. Con ello es posible mejorar las tasas de clasificación, bajo la hipótesis de que la información presente en el habla continua y el habla sostenida es fundamentalmente distinta. Esta tarea se realizo a través de un sencillo esquema de fusión que obtuvo un 88.6% de aciertos en clasificación (tasa de error del 11.4%), lo que representa una mejora significativa respecto al estado del arte. Finalmente, la combinación de este clasificador con los predictores utilizados por los especialistas clínicos ofreció una tasa del 91.3% (tasa de error de 8.7%), que se encuentra dentro del margen ofrecido por esquemas más costosos e intrusivos, y que a diferencia del propuesto, no pueden ser utilizados en la evaluación previa de los pacientes. Con todo, la Tesis ofrece una visión clara sobre la relación entre el SAHS y el habla, evidenciando el grado de madurez alcanzado por la tecnología del habla en la caracterización y detección del SAHS, poniendo de manifiesto que su uso para la evaluación de los pacientes ya sería posible, y dejando la puerta abierta a futuras investigaciones que continúen el trabajo aquí iniciado. ABSTRACT This Thesis explores the potential of speech technologies for the detection of clinical disorders connected to the upper airway. The study of speech traditionally covers both the production process and post processing of the signals involved, from the speaker up to the listener, offering an alternative path to study these pathologies. The fact that utterances embed not just the encoded message but also information about the speaker, has motivated the development of automatic systems oriented to the identification and verificaton the speaker’s identity. These have recently been boosted and reoriented either towards the characterization of traits that are common to several speakers, or to the differences between records of the same speaker collected under different conditions. The first are particularly relevant to this Thesis as these patterns could reveal the presence of features that are related to a common condition shared among different speakers, regardless of their identity. Such is the case faced in this Thesis, where the traits identified would relate to a particular pathology, directly connected to the speech production system. The Obstructive Sleep Apnea syndrome (OSA) is a paradigmatic case for analysis. It is a disorder with high prevalence among adults and affecting a larger number of them as they grow older. Patients suffering from this disorder experience episodes of involuntary cessation of breath during sleep that may last a few seconds and reproduce throughout the night, preventing proper rest. In the case of obstructive apnea, these episodes are related to the collapse of the pharynx, which interrupts the air flow. Currently, OSA diagnosis is done through a polysomnographic study, which focuses on the analysis of apnea episodes during sleep, requiring the patient to stay at the hospital for the whole night. The complexity and high cost of the procedures involved, combined with the waiting lists, have evidenced the need for screening techniques, which perhaps would not achieve outstanding performance rates but would allow clinicians to reorganize these lists ranking patients according to the severity of their condition. Among others, imaging diagnosis and anthropometric characterization of patients have evidenced the existence of anatomical patterns related to OSA that have direct influence on speech. Contributions devoted to the study of how this disorder affects scpeech are scarce and somehow contradictory. However, since the late 1980s the existence of specific patterns related to articulation, phonation and resonance is known. By that time these descriptions were virtually useless when coming to the development of an automatic system, but pointed out the existence of a link between speech and OSA. In recent years automatic processing techniques have evolved and are now able to identify significant differences in the speech of OSAS patients when compared to records from healthy subjects. Nevertheless, little is known about the connection between these new results with those published in the past and the pathogenesis of the OSA syndrome. This Thesis is aimed to progress beyond the previous research done in this area by addressing: the study of how OSA affects patients’ speech, the enhancement of automatic OSA classification based on speech analysis, and its integration with the information embedded in the predictors generally used by clinicians in preliminary patients’ examination. The first two tasks, though may appear symbiotic at first, are quite different. While studying the connection between speech and OSA requires simple narrow models that can be easily interpreted, classification requires larger models including a large number dimensions for the characterization and posterior identification of the observed patterns. Anyhow, it is clear that any progress made in the first task should allow us to improve our performance on the second one, and that the incorporation of the predictors used by clinicians shall contribute in this same direction. The Thesis considers both continuous and sustained speech analysis, to exploit the synergies and differences between them. On continuous speech analysis, a conventional speech processing scheme, designed and evaluated before this Thesis, was taken as a baseline. Over this initial system several alternative representations of the speech information were proposed, optimized and tested to select those more suitable for the characterization of OSA-specific patterns. Evidences were found on the existence of a connection between OSA and the fundamental constituents of the speech: the formants. Experimental results proved that the success of the proposed solution is well explained by the ability of speech representations to describe these specific OSA-related components, ignoring the noisy ones as well those presenting low discrimination capabilities. The resulting scheme obtained a 18% error rate, on a classification scheme significantly less complex than those described in the literature and operating on a single speech record. Regarding the connection between OSA and the observed patterns, it was necessary to consider inter-and intra-group differences for this analysis, and to focus on the articulation, replacing the complex classification models by the long-term average spectra. Results clearly point to certain regions on the frequency axis, suggesting the existence of a systematic narrowing in the vocal tract section at the oropharynx. This was already described in the pathogenesis of this syndrome. Regarding sustained speech, similar experiments as those conducted on continuous speech were reproduced on sustained phonations of vowel / a /. Results were qualitatively similar to the previous ones, though in this case perfomance rates were found to be noticeably lower. Trying to derive further knowledge from this result, experiments on the long-term average spectra and intraand inter-group variability ratios were also reproduced on sustained speech records. Results on both experiments showed significant differences from the previous ones obtained from continuous speech which could explain the differences observed on peformance. However, sustained speech also provided the opportunity to study phonation within the controlled framework it provides. This was also identified in the literature as a source of information for the detection of OSA. In this study it was found that, for the available dataset, no sistematic differences related to phonation could be found between the two groups of speakers. Only those dimensions which relate energy distribution along the frequency axis provided significant differences, pointing once again towards the direction of resonant components. Once classification schemes on both continuous and sustained speech were developed, the Thesis addressed their combination into a single classification system. Under the assumption that the information in continuous and sustained speech is fundamentally different, it should be possible to successfully merge the two of them. This was tested through a simple fusion scheme which obtained a 88.6% correct classification (11.4% error rate), which represents a significant improvement over the state of the art. Finally, the combination of this classifier with the variables used by clinicians obtained a 91.3% accuracy (8.7% error rate). This is within the range of alternative, but costly and intrusive schemes, which unlike the one proposed can not be used in the preliminary assessment of patients’ condition. In the end, this Thesis has shed new light on the underlying connection between OSA and speech, and evidenced the degree of maturity reached by speech technology on OSA characterization and detection, leaving the door open for future research which shall continue in the multiple directions that have been pointed out and left as future work.

Reduced carbon and energy footprint in highway operations: the Highway Energy Assessment (HERA) methodology

Global demand for mobility is increasing and the environmental impact of transport has become an important issue in transportation network planning and decision-making, as well as in the operational management phase. Suitable methods are required to assess emissions and fuel consumption reduction strategies that seek to improve energy efficiency and furthering decarbonization. This study describes the development and application of an improved modeling framework – the HERA (Highway EneRgy Assessment) methodology – that enables to assess the energy and carbon footprint of different highways and traffic flow scenarios and their comparison. HERA incorporates an average speed consumption model adjusted with a correction factor which takes into account the road gradient. It provides a more comprehensive method for estimating the footprint of particular highway segments under specific traffic conditions. It includes the application of the methodology to the Spanish highway network to validate it. Finally, a case study shows the benefits from using this methodology and how to integrate the objective of carbon footprint reductions into highway design, operation and scenario comparison.

Simultaneous assessment of loss of heterozygosity at multiple microsatellite loci using semi-automated fluorescence-based detection: subregional mapping of chromosome 4 in cervical carcinoma.

Detection of loss of heterozygosity (LOH) by comparison of normal and tumor genotypes using PCR-based microsatellite loci provides considerable advantages over traditional Southern blotting-based approaches. However, current methodologies are limited by several factors, including the numbers of loci that can be evaluated for LOH in a single experiment, the discrimination of true alleles versus "stutter bands," and the use of radionucleotides in detecting PCR products. Here we describe methods for high throughput simultaneous assessment of LOH at multiple loci in human tumors; these methods rely on the detection of amplified microsatellite loci by fluorescence-based DNA sequencing technology. Data generated by this approach are processed by several computer software programs that enable the automated linear quantitation and calculation of allelic ratios, allowing rapid ascertainment of LOH. As a test of this approach, genotypes at a series of loci on chromosome 4 were determined for 58 carcinomas of the uterine cervix. The results underscore the efficacy, sensitivity, and remarkable reproducibility of this approach to LOH detection and provide subchromosomal localization of two regions of chromosome 4 commonly altered in cervical tumors.