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The morphology and other phenotypic characteristics of erythrocytes in sickle cell disease (SCD) have been analyzed for decades in patient evaluation. This involves a variety of techniques, including microscopic analysis of stained blood films, flow cytometry, and cell counting. Here, we analyzed SCD blood using imaging flow cytometry (IFC), a technology that combines flow cytometry and microscopy to enable simultaneous rapid-throughput analysis of cellular morphology and cell-surface markers. With IFC, we were able to automate quantification of poikilocytes from SCD blood. An important subpopulation of poikilocytes represented dense cells, although these could not be distinguished from other poikilocytes without first centrifuging the blood through density gradients. In addition, CD71-positive RBCs from SCD patients had two subpopulations: one with high CD71 expression and a puckered morphology and another with lower CD71 expression and biconcave morphology and presumably representing a later stage of differentiation. Some RBCs with puckered morphologies that were strongly positive for DAPI and CD49d were in fact nucleated RBCs. IFC identified more phosphatidylserine-expressing red cells in SCD than did conventional flow cytometry and these could also be divided into two subpopulations. One population had diffuse PS expression and appeared to be composed primarily of RBC ghosts; the other had lower overall PS expression present in intense, punctate dots overlying Howell-Jolly bodies. This study demonstrates that IFC can rapidly reveal and quantify RBC features in SCD that require numerous tedious methods to identify conventionally. Thus, IFC is likely to be a useful technique for evaluating and monitoring SCD.
Assuntos
Anemia Falciforme , Eritrócitos , Anemia Falciforme/metabolismo , Inclusões Eritrocíticas , Citometria de Fluxo/métodos , Humanos , MicroscopiaRESUMO
Immunoassays are important for the detection of proteins to enable disease identification and monitor treatment, but many immunoassays suffer from sensitivity limitations. The development of digital assays has enabled highly sensitive biomarker detection and quantification, but the necessary devices typically require precisely controlled volumes to reduce biases in concentration estimates from compartment size variation. These constraints have led to systems that are often expensive, cumbersome, and challenging to operate, confining many digital assays to centralized laboratories. To overcome these limitations, we have developed a simplified digital immunoassay performed in polydisperse droplets that are prepared without any specialized equipment. This polydisperse digital droplet immunoassay (ddIA) uses proximity ligation to remove the need for wash steps and simplifies the system to a single reagent addition step. Using interleukin-8 (IL-8) as an example analyte, we demonstrated the concept with samples in buffer and diluted whole blood with limits of detection of 0.793 pM and 1.54 pM, respectively. The development of a one-pot, washless assay greatly improves usability compared to traditional immunoassays or digital-based systems that rely heavily on wash steps and can be run with common and readily available laboratory equipment such as a heater and simple fluorescent microscope. We also developed a stochastic model with physically meaningful parameters that can be utilized to optimize the assay and enable quantification without standard curves, after initial characterization of the parameters. Our polydisperse ddIA assay serves as an example of sensitive, lower-cost, and simpler immunoassays suitable for both laboratory and point-of-care applications.
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Imunoensaio/instrumentação , Dispositivos Lab-On-A-Chip , Interleucina-8/análise , Limite de DetecçãoRESUMO
Deleterious changes, collectively termed as storage lesions, alter the characteristics of red blood cell (RBC) morphology during in vitro storage. Due to gradual loss of cellular membrane, RBCs lose their original biconcave disk shape and begin a process of spherical deformation that is characterized by well-defined morphological criteria. At the spheroechinocyte stage, the structure of RBC is irreversibly damaged and lacks the elasticity necessary to efficiently deliver oxygen. Quantifying the prevalence of spheroechinocytes could provide an important morphological measure of the quality of stored blood products. Unlike the conventional RBC morphology characterization assay involving light microscopy, we introduce a label-free assay using imaging flow cytometry (IFC). The technique captures 100,000 images of a sample and calculates a relative measure of spheroechinocyte population in a fraction of the time required by the conventional method. A comparative method study, measuring a morphological index for 11 RCC units through storage, found that the two techniques measured similar trends with IFC reporting the metric at an average of 3.9% higher. We monitored 18 RCC units between Weeks 1 and 6 of storage and found that the spheroechinocyte population increased by an average of 26.2%. The large (3.5-64.1%) variation between the units' spheroechinocyte population percentage at Week 1 suggests a possible dependence of blood product quality on donor characteristics. Given our method's ability to rapidly monitor large samples and refine morphological characterization beyond conventional methods, we believe our technique offers good potential for studying the underlying relationships between RBC morphology and blood storage lesions. © 2019 International Society for Advancement of Cytometry.
Assuntos
Preservação de Sangue , Eritrócitos/citologia , Citometria de Fluxo/métodos , Deformação Eritrocítica , Humanos , Citometria por Imagem/métodos , Processamento de Imagem Assistida por Computador , MicroscopiaRESUMO
In digital assays, devices are typically considered to require precisely controlled volumes since variation in compartment volumes causes biases in concentration estimates. To enable more possibilities in device design, we derived two methods to accurately calculate target concentrations from raw results when the compartment volume may vary and may not follow known parametrically described distributions. The Digital Variable Volume (dvv) method uses volumes of ON compartments (those with positive signals) and the total sample volume, while the Digital Variable Volume Approximation (dvva) method uses the number of ON compartments, the total number of compartments, and a set of separately measured volumes. We verified the trueness of the dvv and dvva methods using simulated assays where volumes followed an empirical distribution (based on measured droplet volumes) and well known distributions with a wide range of standard deviations. We applied both methods to digital PCR experiments with polydisperse volumes, and also derived equations to estimate standard errors and limits of detection. The dvv method allows the compartment volume to follow any distribution in each assay run, the dvva method allows for quantification without in-assay volume measurements, and both methods potentially enable new designs of digital assays.
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Nucleic acid amplification technology, such as polymerase chain reaction (PCR), has enabled highly sensitive and specific disease detection and quantification, leading to more accurate diagnosis and treatment regimens. Lab-on-a-chip applications have developed methods to partition single biomolecules, such as DNA and RNA, into picoliter-sized droplets. These individual reaction vessels lead to digitization of PCR enabling improved time to detection and direct quantification of nucleic acids without a standard curve, therefore simplifying assay analysis. Though impactful, these improvements have generally been restricted to centralized laboratories with trained personnel and expensive equipment. To address these limitations and make this technology more applicable for a variety of settings, we have developed a statistical framework to apply to droplet PCR performed in polydisperse droplets prepared without any specialized equipment. The polydisperse droplet system allows for accurate quantification of droplet digital PCR (ddPCR) and reverse transcriptase droplet digital PCR (RT-ddPCR) that is comparable to commercially available systems such as BioRad's ddPCR. Additionally, this approach is compatible with a range of input sample volumes, extending the assay dynamic range beyond that of commercial ddPCR systems. In this work, we show that these ddPCR assays can reduce overall assay time while still providing quantitative results. We also report a multiplexed ddPCR assay and demonstrate proof-of-concept methods for rapid droplet preparation in multiple samples simultaneously. Our simple polydisperse droplet preparation and statistical framework can be extended to a variety of settings for the quantification of nucleic acids in complex samples.
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Dispositivos Lab-On-A-Chip , Reação em Cadeia da Polimerase/métodos , DNA/análise , Emulsões , RNA/análiseRESUMO
Tumor-derived extracellular vesicles (EVs) are emerging as an important mode of intercellular communication, capable of transferring biologically active molecules that facilitate the malignant growth and metastatic process. CD133 (Prominin-1), a stem cell marker implicated in tumor initiation, differentiation and resistance to anti-cancer therapy, is reportedly associated with EVs in various types of cancer. However, little is known about the factors that regulate the release of these CD133+ EVs. Here, we report that the HDAC6 inhibitor tubacin promoted the extracellular release of CD133+ EVs from human FEMX-I metastatic melanoma and Caco-2 colorectal carcinoma cells, with a concomitant downregulation of intracellular CD133. This effect was specific for tubacin, as inhibition of HDAC6 deacetylase activity by another selective HDAC6 inhibitor, ACY-1215 or the pan-HDAC inhibitor trichostatin A (TSA), and knockdown of HDAC6 did not enhance the release of CD133+ EVs. The tubacin-induced EV release was associated with changes in cellular lipid composition, loss of clonogenic capacity and decrease in the ability to form multicellular aggregates. These findings indicate a novel potential anti-tumor mechanism for tubacin in CD133-expressing malignancies. J. Cell. Biochem. 118: 4414-4424, 2017. © 2017 Wiley Periodicals, Inc.
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Antígeno AC133/metabolismo , Anilidas/farmacologia , Micropartículas Derivadas de Células/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Linhagem Celular Tumoral , Desacetilase 6 de Histona/metabolismo , HumanosRESUMO
Background and Purpose: Classically, abdominal X-ray (KUB), ultrasound, or a combination of both have been routinely used for ureteral stone surveillance after initial diagnosis. More recently, ultra-low-dose CT (ULD CT) has emerged as a CT technique that reduces radiation dose while maintaining high sensitivity and specificity for urinary stone detection. We aim to evaluate our initial experience with ULD CT for patients with ureterolithiasis, measuring real-world radiation doses and stone detection performance. Methods: We reviewed all ULD CT scans performed at the Veterans Affairs Palo Alto Health Care System between 2016 and 2018. We included patients with ureteral stones and calculated the mean effective radiation dose per scan. We determined stone location and size, if the stone was visible on the associated KUB or CT scout film, and if hydronephrosis was present. We performed logistic regression to identify variables associated with visibility on KUB or CT scout film and hydronephrosis. Results: One hundred eighteen ULD scans were reviewed, of which 50 detected ureteral stones. The mean effective radiation dose was 1.04 ± 0.41 mSv. Of the ULD CTs that detected ureterolithiasis, 38% lacked visibility on KUB/CT scout film and had no associated hydronephrosis, suggesting that they would be missed with a combination of KUB and ultrasound. Larger stones (odds ratio [OR]: 1.40, 95% confidence interval [CI]: 1.08, 1.96 for every 1 mm increase in stone size) were more likely to be detected by KUB/CT scout film or ultrasound, while stones in the distal ureter (OR: 0.18, 95% CI: 0.03, 0.81) were more likely to be missed by KUB/CT scout film or hydronephrosis. Conclusions: Based on our institutions' initial experience, ULD CT detects small and distal ureteral stones that would likely be missed by KUB or ultrasound, while maintaining a low effective radiation dose. An ULD CT protocol should be considered when reimaging for ureteral stones is necessary.
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Hidronefrose/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Cálculos Ureterais/diagnóstico por imagem , Cálculos Urinários/diagnóstico por imagem , Urolitíase/diagnóstico por imagem , Idoso , Feminino , Seguimentos , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Radiografia Abdominal , Sensibilidade e Especificidade , Ureter/diagnóstico por imagem , Cálculos Ureterais/terapia , Urolitíase/terapiaRESUMO
Microfluidic devices can deliver soluble factors to cell and tissue culture microenvironments with precise spatiotemporal control. However, enclosed microfluidic environments often have drawbacks such as the need for continuous culture medium perfusion which limits the duration of experiments, incongruity between microculture and macroculture, difficulty in introducing cells and tissues, and high shear stress on cells. Here, we present an open-chamber microfluidic device that delivers hydrodynamically focused streams of soluble reagents to cells over long time periods (i.e., several hours). We demonstrate the advantage of the open chamber by using conventional cell culture techniques to induce the differentiation of myoblasts into myotubes, a process that occurs in 7-10 days and is difficult to achieve in closed chamber microfluidic devices. By controlling the flow rates and altering the device geometry, we produced sharp focal streams with widths ranging from 36 µm to 187 µm. The focal streams were reproducible (â¼12% variation between units) and stable (â¼20% increase in stream width over 10 h of operation). Furthermore, we integrated trenches for micropatterning myoblasts and microtraps for confining single primary myofibers into the device. We demonstrate with finite element method (FEM) simulations that shear stresses within the cell trench are well below values known to be deleterious to cells, while local concentrations are maintained at â¼22% of the input concentration. Finally, we demonstrated focused delivery of cytoplasmic and nuclear dyes to micropatterned myoblasts and myofibers. The open-chamber microfluidic flow-focusing concept combined with micropatterning may be generalized to other microfluidic applications that require stringent long-term cell culture conditions.
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Microfluidic automation - the automated routing, dispensing, mixing, and/or separation of fluids through microchannels - generally remains a slowly-spreading technology because device fabrication requires sophisticated facilities and the technology's use demands expert operators. Integrating microfluidic automation in devices has involved specialized multi-layering and bonding approaches. Stereolithography is an assembly-free, 3D-printing technique that is emerging as an efficient alternative for rapid prototyping of biomedical devices. Here we describe fluidic valves and pumps that can be stereolithographically printed in optically-clear, biocompatible plastic and integrated within microfluidic devices at low cost. User-friendly fluid automation devices can be printed and used by non-engineers as replacement for costly robotic pipettors or tedious manual pipetting. Engineers can manipulate the designs as digital modules into new devices of expanded functionality. Printing these devices only requires the digital file and electronic access to a printer.
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Dispositivos Lab-On-A-Chip , Impressão Tridimensional , Animais , Automação , Células CHO , Cálcio/metabolismo , Cricetinae , Cricetulus , Membranas Artificiais , Imagem MolecularRESUMO
We developed micromolded microwell arrays to study the proliferation and senescence of single cells. Microwell arrays were designed to be compatible with conventional cell culture protocols to simplify cell loading, cell culture, and imaging. We demonstrated the utility of these arrays by measuring the proliferation and senescence of isogenic cells which expressed or had been depleted of the human Werner syndrome protein. Our results allowed us to reveal cell-to-cell heterogeneity in proliferation in WRN+ and WRN-depleted fibroblasts during clonal growth.
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There is a critical unmet need to tailor chemotherapies to individual patients. Personalized approaches could lower treatment toxicity, improve the patient's quality of life, and ultimately reduce mortality. However, existing models of drug activity (based on tumor cells in culture or animal models) cannot accurately predict how drugs act in patients in time to inform the best possible treatment. Here we demonstrate a microfluidic device that integrates live slice cultures with an intuitive multiwell platform that allows for exposing the slices to multiple compounds at once or in sequence. We demonstrate the response of live mouse brain slices to a range of drug doses in parallel. Drug response is measured by imaging of markers for cell apoptosis and for cell death. The platform has the potential to allow for identifying the subset of therapies of greatest potential value to individual patients, on a timescale rapid enough to guide therapeutic decision-making.