Real-time droplet monitoring for digital Polymerase Chain Reaction in microfluidic chip

Current cancer diagnosis techniques are often dependent on the collection of tumour tissue, involving invasive processes for the patient. Circulating Tumour DNA (ctDNA) emerges as an alternative resource for cancer detection and monitoring, that can be har vested from simple blood samples. Digital Polymerase Chain Reaction (dPCR) is a fast and sensitive technique for DNA amplification, suitable for low DNA concentrations such as ctDNA. Advances in microfluidics allow the partition of PCR samples into droplets based in water-in-oil emulsions, so that PCR amplification occurs within each droplet. In this way, the PCR reaction is a well controlled process with a low probability of contami nation and allowing a high throughput analysis. The aimed of this work was to develop droplet-based microfluidic device for application to dPCR technique coupled with real-time droplet monitoring. This work focused on the design and fabrication of a microfluidic device capable of producing a large number of uniform droplets with volumes in the nanoliter range and constant frequency. For this, a polydimethylsiloxane (PDMS) droplet generator device was developed, through photo and soft-lithography techniques, and tested with several oil/water flow rates ratios. Then, the droplets generated were characterized in terms of droplet size, velocity and frequency through the implementation of a powerful open-source software for real-time analysis. Several tests on different devices were carried out to evaluate the device reproducibility. Finally, the droplet generator was incorporated with a serpentine design, allowing the PCR cycles to occur in continuous flow. The results revealed that was possible to generate droplets with radius between 22-99 µm and a coefficient of variation bellow 10%. The correspondents volumes ranged between 90 pL-4.18 nL. Moreover, the velocities obtained situated between 0.05 mm/s-7.62 mm/s with droplet generating frequency of 2-50 Hz. Regarding to the droplet monitoring, the results of the workflows developed revealed similarity with the results obtained trough a widely used software for this purposes, with the advantage of allowing real-time analysis for a larger sample of results.As técnicas actuais usadas no diagnóstico de cancro, geralmente dependem da recolha de tecido tumoral, envolvendo processos invasivos para o paciente. O DNA tumoral circu lante (ctDNA) surge como alternativa para a detecção e monitorização do cancro, podendo ser extraído através de amostras de sangue. A reação em cadeia da polimerase de modo digital (dPCR) é uma técnica rápida e sensível para amplificação de DNA, adequado para baixas concentrações de DNA, como o ctDNA. Os avanços na microfluídica permitem a partição das amostras de PCR em gotas com base em emulsões de água em óleo, de modo que a amplificação por PCR ocorra dentro de cada gota. Deste modo, a reação de PCR é um processo bem controlado com baixa probabilidade de contaminação, permitindo uma análise de alto rendimento. Este trabalho teve como objetivo o desenho e a fabricação de um dispositivo de micro fluídica capaz de produzir um elevado número de gotas uniformes, cujos volumes se encontram na gama dos nanolitros, com frequência constante. Para tal, foi desenvolvido um dispositivo para geração de gotas em polidimetilsiloxano (PDMS), através de técnicas de fotolitografia e litografia suave, tendo sido testado com diversas taxas de fluxos entre óleo / água. Posteriormente, as gotas geradas foram caracterizadas em relação ao seu ta manho, velocidade e frequência através do software de análise de vídeo Bonsai. Diversos testes em diferentes dispositivos foram realizados de modo a avaliar a reprodutibilidade do dispositivo. Por último, o gerador de gotas foi incorporado com desenho da serpentina, permitindo que os ciclos de PCR ocorram em fluxo contínuo. Os estudos realizados revelaram que foi possível gerar gotas com raios entre 22-99 µm, e coeficiente de variação inferior a 10%. Os volumes correspondentes variaram entre 90 pL 4.18 nL. Além disso, as velocidades obtidas situaram-se entre 0.05 mm/s-7.62 mm/s com frequência de geração de gotas de 2-50 Hz. Relativamente à monitorização das gotas, os resultados dos workflows desenvolvidos revelaram similaridade com os resultados obtidos através de um software amplamente utilizado para estes fins, com a vantagem de permitir a análise em tempo real para uma amostra maior de resultados

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701