RESUMEN
Electrophoretic exclusion, a technique that differentiates species in bulk solution near a channel entrance, has been demonstrated on benchtop and microdevice designs. In these systems, separation occurs when the electrophoretic velocity of one species is greater than the opposing hydrodynamic flow, while the velocity of the other species is less than that flow. Although exclusion has been demonstrated in multiple systems for a range of analytes, a theoretical assessment of resolution has not been addressed. To compare the results of these calculations to traditional techniques, the performance is expressed in terms of smallest difference in electrophoretic mobilities that can be completely separated (R = 1.5). The calculations indicate that closest resolvable species (Δµmin ) differ by approximately 10(-13) m(2) /Vs and peak capacity (nc ) is 1000. Published experimental data were compared to these calculated results.
Asunto(s)
Electroforesis/instrumentación , Electroforesis/métodos , Modelos Teóricos , Diseño de Equipo , Hidrodinámica , Tamaño de la PartículaRESUMEN
Determination of the physicochemical properties of protein therapeutics and their aggregates is critical for developing formulations that enhance product efficacy, stability, safety and manufacturability. Analytical challenges are compounded for materials: (1) that are formulated at high concentration, (2) that are formulated with a variety of excipients, and (3) that are available only in small volumes. In this article, a new instrument is described that measures protein secondary and tertiary structure, as well as molecular size, over a range of concentrations and formulation conditions of low volume samples. Specifically, characterization of colloidal and conformational stability is obtained through a combination of two well-established analytical techniques: dynamic light scattering (DLS) and Raman spectroscopy, respectively. As the data for these two analytical modalities are collected on the same sample at the same time, the technique enables direct correlation between them, in addition to the more straightforward benefit of minimizing sample usage by providing multiple analytical measurements on the same aliquot non-destructively. The ability to differentiate between unfolding and aggregation that the combination of these techniques provides enables insights into underlying protein aggregation mechanisms. The article will report on mechanistic insights for aggregation that have been obtained from the application of this technique to the characterization of lysozyme, which was evaluated as a function of concentration and pH.
Asunto(s)
Espectrometría Raman/instrumentación , Animales , Proteínas Aviares/química , Pollos , Coloides , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Luz , Muramidasa/química , Agregado de Proteínas , Desnaturalización Proteica , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura de TransiciónRESUMEN
Lyme borreliosis or Lyme disease (LD) is the most prevalent tick-borne illness in the United States. Diagnosing LD can be challenging as symptoms can be nonspecific and the ability of laboratory tests to detect infection varies based on the duration of infection and the methodology used. To date, serology testing is the primary laboratory tool employed to aid in diagnosing LD. Since the mid-1990's, a two-tiered algorithm has been recommended for the optimization of specificity while maintaining high sensitivity. This mini-review aims to provide an overview of LD diagnostic testing in North America, with an emphasis on serologic algorithms, in particular the modified two-tiered testing (MTTT) algorithm, along with a discussion on provider ordering patterns and practical considerations for implementation of MTTT.
Asunto(s)
Anticuerpos Antibacterianos , Enfermedad de Lyme , Humanos , Estados Unidos , Pruebas Serológicas/métodos , Sensibilidad y Especificidad , Enfermedad de Lyme/diagnóstico , AlgoritmosRESUMEN
Electrophoretic exclusion, a novel separations technique that differentiates species in bulk solution using the opposing forces of electrophoretic velocity and hydrodynamic flow, has been adapted to a microscale device. Proof-of-principle experiments indicate that the device was able to exclude small particles (1 µm polystyrene microspheres) and fluorescent dye molecules (rhodamine 123) from the entrance of a channel. Additionally, differentiation of the rhodamine 123 and polystyrene spheres was demonstrated. The current studies focus on the direct observation of the electrophoretic exclusion behavior on a microchip.
Asunto(s)
Electroforesis por Microchip/instrumentación , Electroforesis por Microchip/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Micromanipulación/instrumentación , Micromanipulación/métodos , Diseño de Equipo , Microesferas , Tamaño de la Partícula , Poliestirenos/aislamiento & purificación , Rodamina 123/aislamiento & purificaciónRESUMEN
BACKGROUND: Autoimmune serology tests are central to the classification, screening, diagnosis, and monitoring of a variety of autoimmune disorders. To improve the appropriateness of serologic evaluation and support laboratory resource utilization, reflex testing approaches have been proposed and implemented across clinical laboratories. Reflex testing involves a staged approach where an initial test result triggers subsequent tests based on prespecified rules. CONTENT: Various reflex testing approaches in the context of antinuclear antibody-associated rheumatic disease, antineutrophil cytoplasmic autoantibody-associated vasculitis, celiac disease, and myasthenia gravis are reviewed here. Clinical, analytical, and practical considerations of reflex testing implementation are addressed as well as associated limitations and challenges. SUMMARY: Serology reflex testing algorithms for the evaluation of autoimmune diseases can support clinical diagnosis and laboratory resource use but may be challenging to implement and are often applied variably across institutions. Assessments of evidence-driven guidelines, clinical impact, and impact on laboratory workflow are essential to this task.
Asunto(s)
Enfermedades Autoinmunes , Enfermedades Reumáticas , Anticuerpos Antinucleares , Autoanticuerpos , Enfermedades Autoinmunes/diagnóstico , Humanos , Laboratorios ClínicosRESUMEN
Research combining the areas of separation science and microfluidics has gained popularity, driven by the increasing need to create portable, fast, and low analyte-consumption devices. Much of this research has focused on the developments in electrophoretic separations, which use the electrokinetic properties of analytes to overcome many of the problems encountered during system scale-down. In addition, new physical phenomenon can be exploited on the microscale not available in standard techniques. In this study, the innovative developments, including electrophoretic concentration, sample preparation/conditioning, and separation on-chip are reviewed, along with some introductory discussions, from January 2008 to July 2010.
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Fraccionamiento Químico , Electroforesis , Técnicas Analíticas Microfluídicas , Focalización IsoeléctricaRESUMEN
A novel method capable of differentiating and concentrating small molecules in bulk solution termed "electrophoretic exclusion" is described and experimentally investigated. In this technique, the hydrodynamic flow of the system is countered by the electrophoretic velocity to prevent a species from entering into the channel. The separation can be controlled by changing the flow rate or applied electric field in order to exclude certain species selectively while allowing others to pass through the capillary. Proof of principle studies employed a flow injection regime of the method and examined the exclusion of Methyl Violet dye in the presence of a neutral species. Methyl Violet was concentrated almost 40 times the background concentration in 30 s using 6 kV. Additionally, a threshold voltage necessary for exclusion was determined. The establishment of a threshold voltage enabled the differentiation of two similar cationic species: Methyl Green and Neutral Red.
Asunto(s)
Electroforesis Capilar/métodos , Campos Electromagnéticos , Violeta de Genciana/química , Punto Isoeléctrico , Verde de Metilo/química , Peso Molecular , Rojo Neutro/químicaRESUMEN
BACKGROUND AND OBJECTIVE: Leptomeningeal metastasis (LM) can occur as a late manifestation of breast cancer and has traditionally been diagnosed by CSF cytology; however, cytology suffers from low sensitivity and it is believed that many cases of LM go undiagnosed. Some studies have suggested the use of CA 15-3 in CSF (CA 15-3 CSF) to aid in the detection of LM. The purpose of this study was to compare the performance of CA 15-3 CSF to cytology for the detection and treatment monitoring of breast cancer LM. METHODS: CA 15-3 CSF requests between 2014 and 2016 were retrospectively reviewed. Fifty-two measurements from nine patients were from our health system and had corresponding CSF cytology measurements. Concordance between CA 15-3 CSF and CSF cytology was calculated. For patients with quantifiable CA 15-3 CSF, sequential determinations of CA 15-3 and cytology were compared over time to assess correlation of CA 15-3 CSF concentration and cytology with disease status. RESULTS: At the time of initial testing, seven of the nine patients (78%) had positive cytology. Two samples (22%) had quantifiable CA 15-3, both of which were also positive by cytology. The positive concordance between all cytology and CA 15-3 measurements was 9% (2/22), while negative concordance was 100% (30/30). Sequential CA 15-3 and cytology measurements showed a decrease in CA 15-3 that paralleled changes observed with cytology. CONCLUSIONS: In this cohort of patients, CA 15-3 CSF performance was neither superior nor complementary to cytology for the detection of LM, nor did the combination of CA 15-3 CSF and cytology improve performance over cytology alone.
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Neoplasias de la Mama , Neoplasias Meníngeas , Mucina-1/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Histocitoquímica/métodos , Humanos , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patología , Neoplasias Meníngeas/secundario , Metástasis de la Neoplasia , Estudios RetrospectivosRESUMEN
BACKGROUND: Imbalance of bile acids (BA) homeostasis in the gastrointestinal tract can lead to chronic diarrhea or constipation when BA in the colon are in excess or low, respectively. Since both disturbances of bowel function can result from other etiologies, identifying BA imbalance is important to tailor treatment strategies. Serum concentrations of 7-alpha-hydroxy-4-cholesten-3-one (7aC4), a precursor in bile acid synthesis, reflect BA homeostasis. Here we describe a method to accurately measure serum 7aC4 and evaluate the clinical utility in patients with diarrhea or constipation phenotypes. METHODS: Serum 7aC4 is measured after acetonitrile protein precipitation using C18 liquid chromatography, tandem mass spectrometry, and deuterium-labeled 7aC4 internal standard. Assay performance including linearity, precision, and accuracy was assessed using waste serum samples. The reference interval was established in healthy individuals without BA-altering conditions or medications. Clinical performance was assessed in patients with irritable bowel syndrome. RESULTS: The method precisely and accurately measured 7aC4 in human serum from 1.4-338ng/mL with no ion suppression or interference from related 7-keto-cholesterol. Central 95th percentile reference interval was 2.5-63.2ng/mL. Lower serum 7aC4 was found in patients with constipation with sensitivity/specificity of 79%/55% compared to healthy controls. Higher 7aC4 was found in patients with bile acid diarrhea (BAD) compared to those without BAD with sensitivity/specificity of 82%/53%. CONCLUSIONS: We have developed a sensitive and precise assay for measuring the concentration of 7aC4 in serum. The assay can be used to screen for diarrhea caused by bile acid malabsorption.