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In this chapter, we present the design and fabrication of a device and implementation of a protocol to realize increased efficiency of immunoassays within microtiter plates. The device, WellProbe, is a 3D-structured probe that can be used to deliver precise flows at the bottom of standard well plates to establish concentric areas of shear stress intensities using hydrodynamically confined flows. The protocols involve both operation and data analysis.
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Diseño de Equipo , Inmunoensayo/métodos , Inmunoensayo/instrumentación , Hidrodinámica , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , HumanosRESUMEN
Anorectal malformations (ARMs) are a spectrum of congenital anomalies where there is abnormal development of the anus and rectum. With an incidence of 1:5000 live births and affecting both males and females, these anomalies vary in their appearance and presentation, lack features enabling antenatal detection and should be detected at birth by the examining midwife or within 72 hours through the newborn and infant physical examination (NIPE) screening programme. However, it is recognised that the diagnosis of ARMs can be missed or delayed leading to morbidity and mortality. In the UK, despite the existence of the NIPE screening programme and NICE guidelines, published literature shows that nearly a quarter of ARMs are not diagnosed at birth. This review takes a critical look at the frequency of missed/delayed diagnosis of ARMs at birth, the implications of delayed diagnosis, and the possible reasons for this related to education and training of healthcare professionals involved in newborn examination, focusing on the UK national screening programme for NIPE. We propose a strategy for enhancing detection of ARMs in a timely manner through the existing framework of the NIPE screening programme.
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Malformaciones Anorrectales , Lactante , Recién Nacido , Masculino , Humanos , Femenino , Embarazo , Malformaciones Anorrectales/diagnóstico , Diagnóstico Tardío , Recto/anomalías , Examen Físico , Reino Unido/epidemiologíaRESUMEN
AIM: To review the role of stomas in the initial and long-term management of Hirschsprung disease (HD). METHODS: Patients treated for HD at our institution between January 2004 and August 2021 were identified. Data were collected regarding: demographics, indication/bowel location/type of stomas performed and outcomes, pull-through (PT) procedure, and follow-up duration. RESULTS: Ninety-five patients (78 male) were identified including one early unrelated death. Forty-four of 94 (47%) required a stoma before PT procedure. Of these 44, 38 (86%) had ileostomies and the remaining six (14%) colostomies; one ileostomy remains long-term. The commonest indication for initial stomas was washout failure (41%). Ninety-one patients had undergone primary PT or secondary PT with stoma closure at the time of the study. A further new stoma was required after primary PT or three-stage management in 20/91 (22%). The commonest indications were constipation/soiling (25%) and anastomotic leak (20%). Seven out of 20 (35%) were performed within 30 days of a previous procedure and all were closed; three patients required further long-term stomas. Thirteen (65%) required a stoma >30 days, nine remain long-term. Surgical revision of stomas was required in 14/56 (25%) - prolapse and retraction being the commonest indications. Overall, 56/94 (60%) patients required stomas (pre- and/or post-PT) to manage their condition and 13/94 (14%) have a long-term stoma in place. Mean follow-up was 7.8 years (0.5 - 17.6). CONCLUSIONS: Stomas remain an integral part of HD management both initially (47%) and long-term (14%); they carry a considerable associated morbidity. Ileostomy is preferred for initial management. LEVEL OF EVIDENCE: Level III.
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Enfermedad de Hirschsprung , Estomas Quirúrgicos , Humanos , Masculino , Colostomía , Enfermedad de Hirschsprung/cirugía , Ileostomía , Prolapso , FemeninoRESUMEN
Tumor cells present complex behaviors in their interactions with other cells. This intricate behavior is driving the need to develop new tools to understand these ecosystems. The surge of spatial technologies allows evaluation of the complexity of relationships between cells present in a tumor, giving insights about tumor heterogeneity and the tumor microenvironment while providing clinically relevant metrics for tumor classification. In this review, we describe key results obtained using spatial techniques, present recent advances in methods to uncover spatially relevant biological significance, and summarize their main characteristics. We expect spatial technologies to significantly broaden our understanding of tumor biology and to generate clinically relevant tools that will ultimately impact personalized medicine.
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Ecosistema , Neoplasias , Humanos , Microambiente Tumoral , Medicina de Precisión , Neoplasias/genética , Neoplasias/patología , Genómica/métodosRESUMEN
We present a method for monitoring spatially localized antigen-antibody binding events on physiologically relevant substrates (cell and tissue sections) using fluorescence lifetime imaging. Specifically, we use the difference between the fluorescence decay times of fluorescently tagged antibodies in free solution and in the bound state to track the bound fraction over time and hence deduce the binding kinetics. We make use of a microfluidic probe format to minimize the mass transport effects and localize the analysis to specific regions of interest on the biological substrates. This enables measurement of binding constants (kon) on surface-bound antigens and on cell blocks using model biomarkers. Finally, we directly measure p53 kinetics with differential biomarker expression in ovarian cancer tissue sections, observing that the degree of expression corresponds to the changes in kon, with values of 3.27-3.50 × 103 M-1 s-1 for high biomarker expression and 2.27-2.79 × 103 M-1 s-1 for low biomarker expression.
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Neoplasias Ováricas , Anticuerpos , Reacciones Antígeno-Anticuerpo , Femenino , Humanos , Cinética , Imagen ÓpticaRESUMEN
We present a microfluidic device for selective separation and extraction of molecules based on their diffusivity. The separation relies on electroosmotically driven bidirectional flows in which high-diffusivity species experience a net-zero velocity and lower diffusivity species are advected to a collection reservoir. The device can operate continuously and is suitable for processing low sample volumes. Using several model systems, we show that the extraction efficiency of the system is maintained at more than 90% over tens of minutes with a purity of more than 99%. We demonstrate the applicability of the device to the extraction of genomic DNA from short DNA fragments.
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Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas , ADNRESUMEN
Hydrodynamic phenomena can be leveraged to confine a range of biological and chemical species without needing physical walls. In this review, we list methods for the generation and manipulation of microfluidic hydrodynamic confinements in free-flowing liquids and near surfaces, and elucidate the associated underlying theory and discuss their utility in the emerging area of open space microfluidics applied to life-sciences. Microscale hydrodynamic confinements are already starting to transform approaches in fundamental and applied life-sciences research from precise separation and sorting of individual cells, allowing localized bio-printing to multiplexing for clinical diagnosis. Through the choice of specific flow regimes and geometrical boundary conditions, hydrodynamic confinements can confine species across different length scales from small molecules to large cells, and thus be applied to a wide range of functionalities. We here provide practical examples and implementations for the formation of these confinements in different boundary conditions - within closed channels, in between parallel plates and in an open liquid volume. Further, to enable non-microfluidics researchers to apply hydrodynamic flow confinements in their work, we provide simplified instructions pertaining to their design and modelling, as well as to the formation of hydrodynamic flow confinements in the form of step-by-step tutorials and analytical toolbox software. This review is written with the idea to lower the barrier towards the use of hydrodynamic flow confinements in life sciences research.
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Disciplinas de las Ciencias Biológicas , Técnicas Analíticas Microfluídicas , Hidrodinámica , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Programas InformáticosRESUMEN
We propose microfluidic add-ons that can be easily added onto standard assay labware such as microwells and slides to enhance the kinetics of immunoassays. We design these monolithic devices having structures that leverage the pipetting step to deliver reagents with deterministic, uniform and strong advection close to the reaction surface. This flow-driven mass transport enhances the flux of analytes to the reaction site and reduces the depletion layer. We demonstrate large gains in time-to-result (5 min instead of 1 h) and/or the sensitivity of immunoassays (approx. 1 order of magnitude), high signal homogeneity and low reagent use by recirculating µL volumes. The impact of this approach on standard immunoassay practice is minimal, preserving both assay labware and dispensing/reading equipment. The devices are compatible with mass production in plastic, offering a solution to enhance the results of conventional assays using well-established protocols and automated analyzer platforms.
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Técnicas Analíticas Microfluídicas , Microfluídica , Inmunoensayo/métodos , Indicadores y Reactivos , Cinética , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodosRESUMEN
Lab-on-a-chip devices leverage microfluidic technologies to enable chemical and biological processes at small scales. However, existing microfluidic channel networks are typically designed for the implementation of a single function or a well-defined protocol and do not allow the flexibility and real-time experimental decision-making essential to many scientific applications. In this Perspective, we highlight that reconfigurability and programmability of microfluidic platforms can support new functionalities that are beyond the reach of current lab-on-a-chip systems. We describe the ideal fully reconfigurable microfluidic device that can change its shape and function dynamically, which would allow researchers to tune a microscale experiment with the capacity to make real-time decisions. We review existing technologies that can dynamically control microscale flows, suggest additional physical mechanisms that could be leveraged towards the goal of reconfigurable microfluidics and highlight the importance of these efforts for the broad scientific community.
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A new workflow for protein-based tumor heterogeneity probing in tissues is here presented. Tumor heterogeneity is believed to be key for therapy failure and differences in prognosis in cancer patients. Comprehending tumor heterogeneity, especially at the protein level, is critical for tracking tumor evolution, and showing the presence of different phenotypical variants and their location with respect to tissue architecture. Although a variety of techniques is available for quantifying protein expression, the heterogeneity observed in the tissue is rarely addressed. The proposed method is validated in breast cancer fresh-frozen tissues derived from five patients. Protein expression is quantified on the tissue regions of interest (ROI) with a resolution of up to 100 µm in diameter. High heterogeneity values across the analyzed patients in proteins such as cytokeratin 7, ß-actin and epidermal growth factor receptor (EGFR) using a Shannon entropy analysis are observed. Additionally, ROIs are clustered according to their expression levels, showing their location in the tissue section, and highlighting that similar phenotypical variants are not always located in neighboring regions. Interestingly, a patient with a phenotype related to increased aggressiveness of the tumor presents a unique protein expression pattern. In summary, a workflow for the localized extraction and protein analysis of regions of interest from frozen tissues, enabling the evaluation of tumor heterogeneity at the protein level is presented.
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Neoplasias de la Mama , Biomarcadores de Tumor , Proteínas de Neoplasias , PronósticoRESUMEN
Patterning biomolecules on surfaces provides numerous opportunities for miniaturizing biological assays; biosensing; studying proteins, cells, and tissue sections; and engineering surfaces that include biological components. In this Feature Article, we summarize the themes presented in our recent Langmuir Lecture on patterning biomolecules on surfaces, miniaturizing surface assays, and interacting with biointerfaces using three key technologies: microcontact printing, microfluidic networks, and microfluidic probes.
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Bioensayo , Microfluídica , Humanos , Proteínas , Propiedades de SuperficieRESUMEN
Copper (Cu) and its alloys are prospective materials in fighting covid-19 virus and several microbial pandemics, due to its excellent antiviral as well as antimicrobial properties. Even though many studies have proved that copper and its alloys exhibit antiviral properties, this research arena requires further research attention. Several studies conducted on copper and its alloys have proven that copper-based alloys possess excellent potential in controlling the spread of infectious diseases. Moreover, recent studies indicate that these alloys can effectively inactivate the covid-19 virus. In view of this, the present article reviews the importance of copper and its alloys in reducing the spread and infection of covid-19, which is a global pandemic. The electronic databases such as ScienceDirect, Web of Science and PubMed were searched for identifying relevant studies in the present review article. The review starts with a brief description on the history of copper usage in medicine followed by the effect of copper content in human body and antiviral mechanisms of copper against covid-19. The subsequent sections describe the distinctive copper based material systems such as alloys, nanomaterials and coating technologies in combating the spread of covid-19. Overall, copper based materials can be propitiously used as part of preventive and therapeutic strategies in the fight against covid-19 virus.
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Aleaciones/química , Antivirales/farmacología , Antivirales/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Cobre/química , Animales , Antiinfecciosos Locales , Desinfección , Humanos , Nanopartículas del Metal/química , Nanoestructuras/química , Pandemias , SARS-CoV-2RESUMEN
Surface assays such as ELISA are pervasive in clinics and research and predominantly standardized in microtiter plates (MTP). MTPs provide many advantages but are often detrimental to surface assay efficiency due to inherent mass transport limitations. Microscale flows can overcome these and largely improve assay kinetics. However, the disruptive nature of microfluidics with existing labware and protocols has narrowed its transformative potential. We present WellProbe, a novel microfluidic concept compatible with MTPs. With it, we show that immunoassays become more sensitive at low concentrations (up to 9× signal improvement in 12× less time), richer in information with 3-4 different kinetic conditions, and can be used to estimate kinetic parameters, minimize washing steps and non-specific binding, and identify compromised results. We further multiplex single-well assays combining WellProbe's kinetic regions with tailored microarrays. Finally, we demonstrate our system in a context of immunoglobulin subclass evaluation, increasingly regarded as clinically relevant.
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AIMS: The aim of this study was to review the use of the on-table "doughnut" biopsy for frozen section assessment of bowel in the operative management of Hirschsprung's disease (HD). METHODS: This was a single-center retrospective review of doughnut histopathology reports, operation notes, and slides from 2010 to 2017. Data were assessed for the presence of transition zone (TZ) features and the subsequent decision as to the level of pull-through. RESULTS: Fifty-five patients had a doughnut biopsy taken as part of their intraoperative frozen section histopathology for pull-through for HD during the study period. Forty-eight required a single doughnut, six required a second more proximal doughnut, and one required a third doughnut. Of the 55 first doughnuts, 37 were identified as normal bowel, 17 were TZ, and not defined in the report in one case. Of the 17 TZ doughnuts, 8 were accepted for pull-through and 7 underwent second doughnuts (normal = 4 and TZ = 3). The third doughnut (one case) was normal. TZ was accepted for pull-through in 10/54 (18.5%) patients despite the use of a doughnut. However, TZ was avoided in six (11.1%), where the single-point biopsy was "normal." CONCLUSIONS: The doughnut allows the entire circumference of pull-through level to be assessed, enabling TZ identification that can be missed by single seromuscular biopsies. This allows identification and avoidance of TZ pull-through, although sometimes, it is accepted for other reasons.
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Historically, technology has been central to new discoveries in biology and progress in medicine. Among various technologies, microtechnologies, in particular, have had a prominent role in the revolution experienced by the life sciences in the last few decades, which will surely continue in the years to come. In this Perspective, we illustrate how microtechnologies, with a focus on microfluidics, have evolved in trends/waves to tackle the boundary of knowledge in the life sciences. We provide illustrative examples of technology-enabled biological breakthroughs and their current and future use in clinics. Finally, we take a closer look at the translational process to understand why the incorporation of new micro-scale technologies in medicine has been comparatively slow so far.
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In cancer research, genomic profiles are often extracted from homogenized macrodissections of tissues, with the histological context lost and a large fraction of material underutilized. Pertinently, the spatial genomic landscape provides critical complementary information in deciphering disease heterogeneity and progression. Microscale sampling methods such as microdissection to obtain such information are often destructive to a sizeable fraction of the biopsy sample, thus showing limited multiplexability and adaptability to different assays. A modular microfluidic technology is here implemented to recover cells at the microscale from tumor tissue sections, with minimal disruption of unsampled areas and tailored to interface with genome profiling workflows, which is directed here toward evaluating intratumoral genomic heterogeneity. The integrated workflow-GeneScape-is used to evaluate heterogeneity in a metastatic mammary carcinoma, showing distinct single nucleotide variants and copy number variations in different tumor tissue regions, suggesting the polyclonal origin of the metastasis as well as development driven by multiple location-specific drivers.
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Neoplasias de la Mama , Variaciones en el Número de Copia de ADN , Neoplasias de la Mama/genética , Femenino , Genómica , Humanos , Mutación , Flujo de TrabajoAsunto(s)
Diagnóstico por Imagen/instrumentación , Procesamiento de Imagen Asistido por Computador/métodos , Pautas de la Práctica en Medicina/estadística & datos numéricos , Consulta Remota/estadística & datos numéricos , Toma de Decisiones Clínicas , Seguridad Computacional/ética , Diagnóstico por Imagen/estadística & datos numéricos , Humanos , Lactante , Masculino , Padres , Pediatría/estadística & datos numéricos , Fotograbar , Pautas de la Práctica en Medicina/ética , Desempeño de Papel , Medicina Estatal/organización & administración , Reino Unido/epidemiologíaRESUMEN
AIM: To evaluate the range and scientific nature of oral presentations in the Scientific Program of IAPSCON from 2014-18. METHODS: The oral presentations in Scientific Program of IAPSCON from 2014 - 18 were tabulated and categorised as per: type of presentation, Paediatric Surgery related clinical subject heading, nature of the content and scientific quality as per Oxford levels of clinical evidence. RESULTS: Most oral presentations were in the form of Short papers. The most common clinical section and clinical condition presented were Urology and Hypospadias respectively. Clinical management followed by operative management represent the majority of the presentations when categorized by the type of content. Certain sections of clinical practice were not represented at all, notably anterior abdominal wall defects and gastro-oesophageal reflux. Most presentations (82.5%) are in Level 4 and 5 as per Oxford levels of clinical evidence, with Levels 1-3 accounting for 5% of presentations. Minimally Invasive Surgery (including robotic) accounted for 8.4% presentations. CONCLUSIONS: This qualitative evaluation shows the variety of oral presentations and areas of greater and lesser interest in the conference meetings. It shows that the scientific quality of the presentations can be improved and highlights certain areas of clinical and scientific research for future consideration.
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INTRODUCTION: Unicompartmental knee arthroplasty (UKA) is a procedure used to treat isolated medial or lateral compartmental osteoarthritis of the knee joint. This procedure involves retention of cruciate ligaments which leads to better functional outcome due to preservation of normal kinematics of the knee joint. In the Indian population, due to requirement of squatting and cross leg sitting habits, knee with more range of movement and with good kinematics is a required feature. The study aims to observe the functional outcome, mortality, revision rate, length of hospital stay and satisfaction rate in two-year postoperative patients in a tertiary health care centre. METHODS: A total of 17 knees of 15 patients were recruited for the study after applying strict inclusion and exclusion criteria. These patients were operated during the period from March 2015 to March 2018. Ten female patients (67%) and five males (33%) were included. The average age was 61 years. All operations were performed by a single surgeon, with a similar implant from a single company. Similar protocol was used both for surgical technique (minimal invasive) and postoperative rehabilitation for all the patients. RESULTS: The functional outcome in the form of Oxford Knee Score (OKS) and Euro-Quol (EQ-5D) scores improved significantly in all the patients at one year postoperatively, and the improvement remained significant for two years. Satisfaction rate was 91.7% (SD-12.8) at two years. Except for one patient (5.8%), all patients were able to cross leg and squat easily. At the end of two years, the overall survival rate of the implant was found to be 94.1%. CONCLUSION: The unicondylar arthroplasty provides excellent satisfaction to the appropriately selected patients with good survivorship of implant. It can be a surgery of choice for Indian population as it restores normal kinematics of knee joint and allows the patient to cross leg and squat with a more range of movement.
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We present a novel method for the local retrieval of surface bound species and their rapid in-line separation using an open space microfluidic device. Separation can be performed in less than 30 s using the difference in diffusivities within parallel microfluidic flows. As a proof-of-principle, we report the rapid and efficient filtration of polystyrene beads from small molecules and surface bound red blood cells from dimethyl sulfoxide for antigen typing.