65 research outputs found

    A Protocol Generator Tool for Automatic In-Vitro HPV Robotic Analysis

    Get PDF
    Human Papilloma Virus (HPV) could develop precancerous lesions and invasive cancer, as it is the main cause of nearly all cases of cervical cancer. There are many strains of HPV and current vaccines can only protect against some of them. This makes the detection and genotyping of HPV a research area of utmost importance. Several biomedical systems can detect HPV in DNA samples; however, most of them do not have a procedure as fast, automatic or precise as it is actually needed in this field. This manuscript presents a novel XML-based hierarchical protocol architecture for biomedical robots to describe each protocol step and execute it sequentially, along with a robust and automatic robotic system for HPV DNA detection capable of processing from 1 to 24 samples simultaneously in a fast (from 45 to 162 min), efficient (100% markers effectiveness) and precise (able to detect 36 different HPV genotypes) way. It includes an efficient artificial vision process as the last step of the diagnostic.FIDETIA P055-12/E03Ministerio de Economía y Competitivida TEC2016-77785-

    Advanced microarray technologies for clinical diagnostics

    Get PDF
    DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains multiple copies of one oligonucleotide, called the capture probe, which has a sequence complementary to one single specific target molecule. A DNA microarray assay is based on the preferential binding between a DNA target molecule with its complementary probe present on the microarray surface. Provided appropriate conditions, these two single stranded DNA molecules with complementary DNA sequences form a double stranded molecule during a process called hybridization. When the target molecules are labeled with e.g. a fluorophore, the fluorescent microarray pattern achieved upon incubation of the sample with the microarray provides information on the presence of the target sequences in the sample. The key advantage of microarrays is their large degree of parallelism per sample due to the simultaneous reaction between multiple capture probes and target sequences. This thesis focuses on low density DNA arrays that are limited to a few hundred spots for diagnostic use, for the rapid detection of a constrained and specific set of target sequences. One major application of low density microarrays lies in the detection and identification of pathogens in the field of infectious diseases. So, high density arrays containing as high as millions of different spots that allows for the screening and detection of a much higher number of targets simultaneously, usually applied for studying gene expression studies, are beyond the scope of this thesis. The transfer of microarray-based assays from the research lab into the clinic has taken more time than anticipated and it faces a number of technical and regulatory challenges. These are especially related to quality control and data reproducibility caused by a lack of fundamental understanding of the biochemical processes involved. In this thesis, a number of these challenges are addressed and investigated, and potential routes for improvement are identified. This work focuses on the improvement of microarray technology in order to enable a faster implementation of microarray-based assays into clinical practice. Inkjet printing is a suitable technology for manufacturing low density microarrays. It is, however, an open-loop process and does therefore not provide any feedback on the quality of manufactured microarrays. E.g. missing spots are unacceptable since this could lead to potentially false negative results. Moreover, microarrays are generally being considered as high complexity products by the Clinical Laboratory Improvement Amendments (CLIA), or as high risk applications by the Food and Drug Administration (FDA). Therefore specific quality control measures on microarrays for clinical diagnostics are needed. In chapter 2, a closed-loop inkjet printing system equipped with an optical droplet detection system to investigate failure mechanisms of the printing process is described. It was found that of all microarrays analyzed, in 1.6 % jetting failed at some point in time. In 14 % of these cases, these failures could have been detected in advance by detection of changing droplet velocities during jetting. Real time analysis of droplet characteristics and closed-loop control can enable a higher yield of the microarray manufacturing process. The improved production process as explained in chapter 2 is the basis for the microarray experiments carried out for the results in experimental chapters 3-5. Many different surface chemistries exist that are applied to graft the DNA probes onto the microarray surface. A lack of understanding on the characteristics of DNA immobilization and hybridization as separate processes hampers the improvement in reproducibility of the use of microarrays. Immobilization and hybridization rates of oligonucleotides equipped with specific tails on amine-functionalized surfaces by using 254 nm UV-light are studied. This surface chemistry is attractive because of its ease-of-use, robustness and low cost. A method was developed that enables a systematic study of the immobilization and subsequent hybridization processes independently. It was found that immobilization efficiencies are greatly influenced both by the UV dose applied and by the composition, as well as the length of the specific tails attached to the oligonucleotides. Hybridization is mainly influenced by the length of the oligonucleotides, and much less by the composition of the tail as observed for immobilization. This means that within the UV-window as applied in this study, no significant UV-damage was found. The standard microarray procedure runs as follows: microarrays are incubated with the sample, followed by a washing step to remove non-specific binding, then dried and scanned. This process flow implies that only a single measurement (end) point is used to evaluate target and capture molecule interactions. Since these interactions depend on many factors, including the preceding steps and the composition of the mixture itself, a complete hybridization measurement would provide additional and more reliable data. This means, however, that measurements should be done in high background signals requiring the deployment of methods that enable background suppression. Chapter 4 describes a method that measures real time hybridization and melting curves based on the repeated flow of the solution containing the targets through a porous microarray substrate. During each hybridization cycle, background was suppressed by pumping the liquid through the substrate. This method, using the Human Papilloma Virus (HPV) assay as a model assay, was evaluated with clinical samples and benchmarked against a method commonly used in the clinic (Reverse Line Blot). Both methods show comparable results, while the flow-through method also enabled the identification of cross-hybridizations by the analysis of the binding and melting kinetics. This method can contribute to identification of false positive signals. The Polymerase Chain Reaction (PCR) is a method to amplify the concentration of specific DNA target sequences and is often performed prior to a microarray hybridization. Sample preparation variations including this amplification step contribute to a lower reproducibility of microarrays. In the real time array PCR concept the steps of real time amplification (qPCR) and detection are integrated, thereby combining the advantages of both methods: the high multiplexing capabilities of microarray based assays and the quantitative characteristics of real time PCR. In this procedure the hybridization of the formed amplicons on the microarray surface is monitored, during each annealing step of the amplification process. Consequently an amplification curve on the microarray surface is obtained from which quantitative information on the target input concentration can be derived for a much higher number of targets compared to qPCR. After the amplification, an additional hybridization and melting curve step can be performed to further assess the specificity. The detection of low signals within high background levels in relatively short times is the greatest technical challenge of this concept. This was achieved by the use of a confocal fluorescence scanner that significantly reduced background levels. A proof of concept study was carried out with a prototype instrument that was designed and built from standard components known from the optical storage technology. In order to minimize costs of goods, standard products (reagents and disposables) were also used for the biochemical processes of amplification and hybridization as much as possible. Amplification and detection were performed in a single and closed chamber, minimizing risks on cross-contamination and reducing manual steps, making this method especially suited for diagnostic testing. As low as 10 copies of a target sequence could be detected using this method. Chapter 6 contains a mathematical analysis of the real time array PCR process which was carried out to provide insights in the biochemical processes taking place. Models of qPCR and Langmuir adsorption were combined into a new model describing the different steps in the real time array protocol. Reasonably good agreement between the model and the experiments was found. Using this result for the experimental array-based assay, a number of measures have been identified and tested to increase the overall amplification efficiency which resulted in a similar performance compared to bulk qPCR

    Marshall Space Flight Center Research and Technology Report 2019

    Get PDF
    Today, our calling to explore is greater than ever before, and here at Marshall Space Flight Centerwe make human deep space exploration possible. A key goal for Artemis is demonstrating and perfecting capabilities on the Moon for technologies needed for humans to get to Mars. This years report features 10 of the Agencys 16 Technology Areas, and I am proud of Marshalls role in creating solutions for so many of these daunting technical challenges. Many of these projects will lead to sustainable in-space architecture for human space exploration that will allow us to travel to the Moon, on to Mars, and beyond. Others are developing new scientific instruments capable of providing an unprecedented glimpse into our universe. NASA has led the charge in space exploration for more than six decades, and through the Artemis program we will help build on our work in low Earth orbit and pave the way to the Moon and Mars. At Marshall, we leverage the skills and interest of the international community to conduct scientific research, develop and demonstrate technology, and train international crews to operate further from Earth for longer periods of time than ever before first at the lunar surface, then on to our next giant leap, human exploration of Mars. While each project in this report seeks to advance new technology and challenge conventions, it is important to recognize the diversity of activities and people supporting our mission. This report not only showcases the Centers capabilities and our partnerships, it also highlights the progress our people have achieved in the past year. These scientists, researchers and innovators are why Marshall and NASA will continue to be a leader in innovation, exploration, and discovery for years to come

    Modern Approaches To Quality Control

    Get PDF
    Rapid advance have been made in the last decade in the quality control procedures and techniques, most of the existing books try to cover specific techniques with all of their details. The aim of this book is to demonstrate quality control processes in a variety of areas, ranging from pharmaceutical and medical fields to construction engineering and data quality. A wide range of techniques and procedures have been covered

    A longitudinal study of the experiences and psychological well-being of Indian surrogates

    Get PDF
    Study question: What is the psychological well-being of Indian surrogates during and after the surrogacy pregnancy? Summary answer: Surrogates were similar to a matched group of expectant mothers on anxiety and stress. However, they scored higher on depression during and after pregnancy. What is known already: The recent ban on trans-national commercial surrogacy in India has led to urgent policy discussions regarding surrogacy. Whilst previous studies have reported the motivations and experiences of Indian surrogates no studies have systematically examined the psychological well-being of Indian surrogates, especially from a longitudinal perspective. Previous research has shown that Indian surrogates are motivated by financial payment and may face criticism from their family and community due to negative social stigma attached to surrogacy. Indian surrogates often recruited by agencies and mainly live together in a “surrogacy house.” Study design, size, duration: A longitudinal study was conducted comparing surrogates to a matched group of expectant mothers over two time points: (a) during pregnancy (Phase1: 50 surrogates, 70 expectant mothers) and (b) 4–6 months after delivery (Phase 2: 45 surrogates, 49 expectant mothers). The Surrogates were recruited from a fertility clinic in Mumbai and the matched comparison group was recruited from four public hospitals in Mumbai and Delhi. Data collection was completed over 2 years. Participants/materials, setting, methods: Surrogates and expectant mothers were aged between 23 and 36 years. All participants were from a low socio-economic background and had left school before 12–13 years of age. In-depth faceto-face semi-structured interviews and a psychological questionnaire assessing anxiety, stress and depression were administered in Hindi to both groups. Interviews took place in a private setting. Audio recordings of surrogate interviews were later translated and transcribed into English. Main results and the role of chance: Stress and anxiety levels did not significantly differ between the two groups for both phases of the study. For depression, surrogates were found to be significantly more depressed than expectant mothers at phase 1 (p = 0.012) and phase 2 (p = 0.017). Within the surrogacy group, stress and depression did not change during and after pregnancy. However, a non-significant trend was found showing that anxiety decreased after delivery (p = 0.086). No participants reported being coerced into surrogacy, however nearly all kept it a secret from their wider family and community and hence did not face criticism. Surrogates lived at the surrogate house for different durations. During pregnancy, 66% (N = 33/50) reported their experiences of the surrogate house as positive, 24% (N = 12/50) as negative and 10% (N = 5/50) as neutral. After delivery, most surrogates (66%, N = 30/45) reported their experiences of surrogacy to be positive, with the remainder viewing it as neutral (28%) or negative (4%). In addition, most (66%, N = 30/45) reported that they had felt “socially supported and loved” during the surrogacy arrangement by friends in the surrogate hostel, clinic staff or family. Most surrogates did not meet the intending parents (49%, N = 22/45) or the resultant child (75%, N = 34/45). Limitations, reasons for caution: Since the surrogates were recruited from only one clinic, the findings may not be representative of all Indian surrogates. Some were lost to follow-up which may have produced sampling bias. Wider implications of the findings: This is the first study to examine the psychological well-being of surrogates in India. This research is of relevance to current policy discussions in India regarding legislation on surrogacy. Moreover, the findings are of relevance to clinicians, counselors and other professionals involved in surrogacy. Trial registration number: N/A

    CT-PET guided target delineation in head and neck cancer and implications for improved outcome

    Get PDF
    Aim: Fifty percent of patients with squamous cell carcinoma of the Head and Neck develop loco-regional recurrence after treatment. Factors leading to this failure are most likely altered intra-tumoural glucose metabolism and increased hypoxia. Tissue glucose utilisation and the degree of hypoxia can be visualised by CTPET imaging with 18FDG and hypoxic radio-nuclides. This thesis has investigated 18FDG CT-PET guided target volume delineation methods and attempted to validate 64Cu-ATSM as a hypoxic radio-nuclide in patients with squamous cell carcinoma of the Head and Neck. Materials and Methods: Eight patients with locally advanced disease underwent 18FDG CT-PET imaging before and during curative radiotherapy or chemo-radiotherapy. Fixed (SUV cut off and percentage threshold of the SUVmax) and adaptive thresholds were investigated. The functional volumes automatically delineated by these methods and SUVmax were compared at each point, and between thresholds. Four patients with locally advanced disease, two to seven days prior to surgery, underwent 3D dynamic CT-PET imaging immediately after injection of 64Cu- ATSM. Two patients were also imaged 18 hours after injection, and two underwent a dynamic contrast-enhanced CT to evaluate intra-tumoural perfusion. All patients received pimonidazole before surgery. The pimonidazole, GLUT1, CAIX, and HIF1a immuno-histochemical hypoxic fractions were defined. Staining was correlated with the retention pattern of 64Cu-ATSM at 3 time points. Hypoxic target volumes were delineated according to tumour to muscle, blood and background ratios. Results: 18FDG primary and lymph node target volumes significantly reduced with radiation dose by the SUV cut off method and correlated with the reduction in the SUVmax within the volume. Volume reduction was also found between thresholds by the same delineation method. The volumes delineated by the other methods were not significantly reduced (except the lymph node functional volume when defined by the adaptive threshold). 64Cu-ATSM correlated with hypoxic immuno-histochemical staining but not with blood flow. Tumour ratios increased with time after injection, which influenced the delineated hypoxic target volume. Conclusion: Dose-escalated image-guided radiotherapy strategies using these CT-PET guided functional volumes have the potential to improve loco-regional control in patients with squamous cell carcinoma of the Head and Neck. CT-PET 18FDG volume delineation is intricately linked to the method and threshold of delineation and the timing of the imaging. 64Cu-ATSM is promising as a hypoxic radio-nuclide and warrants further investigation
    corecore