RESUMO
BACKGROUND: Blood typing, donor compatibility testing, and hematocrit analysis are common tests that are important in many clinical applications, including those found in high-stakes settings such as the trauma center. These tests are typically performed in centralized laboratories with sample batching; the minutes that are lost in this mode can lead to adverse outcomes, especially for critical-care patients. As a step toward providing rapid results at the bedside, we developed a point-of-care hemagglutination system relying on digital microfluidics (DMF) and a unique, automated readout tool, droplet agglutination assessment using digital microfluidics (DAAD). METHODS: ABO and Rhesus blood grouping, donor crossmatching, and hematocrit assays were developed on a portable DMF platform that allowed for automated sample processing. The result of each assay could be determined by eye or automatically with the DAAD imaging tool. RESULTS: DMF-DAAD was applied to 109 samples collected from different sources (including commercial samples, pinpricks from volunteers, and a hospital blood bank), with perfect fidelity to gold-standard results. Some of these tests were carried out by a nonexpert in a hospital trauma center. Proof-of-concept results were also collected from smaller sample sets for donor compatibility testing and hematocrit analysis. CONCLUSION: DMF-DAAD shows promise for delivering rapid, reliable results in a format well suited for a trauma center and other settings where every minute counts.
Assuntos
Tipagem e Reações Cruzadas Sanguíneas , Microfluídica , Bancos de Sangue , Hemaglutinação , Hematócrito , Humanos , Microfluídica/métodosRESUMO
This paper introduces a digital microfluidic (DMF) platform for portable, automated, and integrated Zika viral RNA extraction and amplification. The platform features reconfigurable DMF cartridges offering a closed, humidified environment for sample processing at elevated temperatures, as well as programmable control instrumentation with a novel thermal cycling unit regulated using a proportional integral derivative (PID) feedback loop. The system operates on 12 V DC power, which can be supplied by rechargeable battery packs for remote testing. The DMF system was optimized for an RNA processing pipeline consisting of the following steps: 1) magnetic-bead based RNA extraction from lysed plasma samples, 2) RNA clean-up, and 3) integrated, isothermal amplification of Zika RNA. The DMF pipeline was coupled to a paper-based, colorimetric cell-free protein expression assay for amplified Zika RNA mediated by toehold switch-based sensors. Blinded laboratory evaluation of Zika RNA spiked in human plasma yielded a sensitivity and specificity of 100% and 75% respectively. The platform was then transported to Recife, Brazil for evaluation with infectious Zika viruses, which were detected at the 100 PFU mL-1 level from a 5 µL sample (equivalent to an RT-qPCR cycle threshold value of 32.0), demonstrating its potential as a sample processing platform for miniaturized diagnostic testing.
Assuntos
Infecção por Zika virus , Zika virus , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Manejo de Espécimes , Zika virus/genética , Infecção por Zika virus/diagnósticoRESUMO
The Democratic Republic of the Congo (DRC) has a high measles incidence despite elimination efforts and has yet to introduce rubella vaccine. We evaluated the performance of a prototype rapid digital microfluidics powered (DMF) enzyme-linked immunoassay (ELISA) assessing measles and rubella infection, by testing for immunoglobulin M (IgM), and immunity from natural infection or vaccine, by testing immunoglobulin G (IgG), in outbreak settings. Field evaluations were conducted during September 2017, in Kinshasa province, DRC. Blood specimens were collected during an outbreak investigation of suspected measles cases and tested for measles and rubella IgM and IgG using the DMF-ELISA in the field. Simultaneously, a household serosurvey for measles and rubella IgG was conducted in a recently confirmed measles outbreak area. DMF-ELISA results were compared with reference ELISA results tested at DRC's National Public Health Laboratory and the US Centers for Disease Control and Prevention. Of 157 suspected measles cases, rubella IgM was detected in 54% while measles IgM was detected in 13%. Measles IgG-positive cases were higher among vaccinated persons (87%) than unvaccinated persons (72%). In the recent measles outbreak area, measles IgG seroprevalence was 93% overall, while rubella seroprevalence was lower for children (77%) than women (98%). Compared with reference ELISA, DMF-ELISA sensitivity and specificity were 82% and 78% for measles IgG; 88% and 89% for measles IgM; 85% and 85% for rubella IgG; and 81% and 83% for rubella IgM, respectively. Rubella infection was detected in more than half of persons meeting the suspected measles case definition during a presumed measles outbreak, suggesting substantial unrecognized rubella incidence, and highlighting the need for rubella vaccine introduction into the national schedule. The performance of the DMF-ELISA suggested that this technology can be used to develop rapid diagnostic tests for measles and rubella.
Assuntos
Sarampo , Rubéola (Sarampo Alemão) , Criança , Humanos , Feminino , República Democrática do Congo/epidemiologia , Estudos Soroepidemiológicos , Microfluídica , Anticorpos Antivirais , Rubéola (Sarampo Alemão)/diagnóstico , Rubéola (Sarampo Alemão)/epidemiologia , Rubéola (Sarampo Alemão)/prevenção & controle , Sarampo/diagnóstico , Sarampo/epidemiologia , Sarampo/prevenção & controle , Vacina contra Rubéola , Imunoglobulina M , Imunoglobulina G , Técnicas Imunoenzimáticas , Surtos de DoençasRESUMO
Finger-stick blood sampling is convenient for point of care diagnostics, but whole blood samples are problematic for many assays because of severe matrix effects associated with blood cells and cell debris. We introduce a new digital microfluidic (DMF) diagnostic platform with integrated porous membranes for blood-plasma separation from finger-stick blood volumes, capable of performing complex, multi-step, diagnostic assays. Importantly, the samples can be directly loaded onto the device by a finger "dab" for user-friendly operation. We characterize the platform by comparison to plasma generated via the "gold standard" centrifugation technique, and demonstrate a 21-step rubella virus (RV) IgG immunoassay yielding a detection limit of 1.9 IU mL-1, below the diagnostic cut-off. We propose that this work represents a critical next step in DMF based portable diagnostic assays-allowing the analysis of whole blood samples without pre-processing.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Imunoensaio , Imunoglobulina G , Microfluídica , PlasmaRESUMO
We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies.
Assuntos
Separação Celular/instrumentação , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Animais , Antígeno CD47/genética , Linhagem Celular Tumoral , Separação Celular/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Dispositivos Lab-On-A-Chip , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Redes Neurais de Computação , Proteômica/métodosRESUMO
Serosurveys are useful for assessing population susceptibility to vaccine-preventable disease outbreaks. Although at-risk populations in remote areas could benefit from this type of information, they face several logistical barriers to implementation, such as lack of access to centralized laboratories, cold storage, and transport of samples. We describe a potential solution: a compact and portable, field-deployable, point-of-care system relying on digital microfluidics that can rapidly test a small volume of capillary blood for disease-specific antibodies. This system uses inexpensive, inkjet-printed digital microfluidic cartridges together with an integrated instrument to perform enzyme-linked immunosorbent assays (ELISAs). We performed a field validation of the system's analytical performance at Kakuma refugee camp, a remote setting in northwestern Kenya, where we tested children aged 9 to 59 months and caregivers for measles and rubella immunoglobulin G (IgG). The IgG assays were determined to have sensitivities of 86% [95% confidence interval (CI), 79 to 91% (measles)] and 81% [95% CI, 73 to 88% (rubella)] and specificities of 80% [95% CI, 49 to 94% (measles)] and 91% [95% CI, 76 to 97% (rubella)] (measles, n = 140; rubella, n = 135) compared with reference tests (measles IgG and rubella IgG ELISAs from Siemens Enzygnost) conducted in a centralized laboratory. These results demonstrate a potential role for this point-of-care system in global serological surveillance, particularly in remote areas with limited access to centralized laboratories.
Assuntos
Imunoensaio/métodos , Microfluídica/métodos , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Lactente , Masculino , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Nanostructured microelectrodes (NMEs) are three-dimensional electrodes that have superb sensitivity for electroanalysis. Here we report the integration of NMEs with the versatile fluid-handling system digital microfluidics (DMF), for eventual application to distributed diagnostics outside of the laboratory. In the new methods reported here, indium tin oxide DMF top plates were modified to include Au NMEs as well as counter and pseudoreference electrodes. The new system was observed to outperform planar sensing electrodes of the type that are typically integrated with DMF. A rubella virus (RV) IgG immunoassay was developed to evaluate the diagnostic potential for the new system, relying on magnetic microparticles coated with RV particles and analysis by differential pulse voltammetry. The limit of detection of the assay (0.07 IU mL(-1)) was >100× below the World Health Organization defined cut-off for rubella immunity. The sensitivity of the integrated device and its small size suggest future utility for distributed diagnostics.