Manual Steps Research Articles

A multi-depth spiral milli fluidic device for whole mount zebrafish antibody staining

Whole mount zebrafish antibody staining (ABS) is a common staining technique used to localize protein information in a zebrafish embryo or larva. Like most biological assays, the whole mount zebrafish ABS is still largely conducted manually through labor intensive and time-consuming steps which affect both consistency and throughput of the assay. In this work, we develop a milli fluidic device that can automatically trap and immobilize the fixed chorion-less zebrafish embryos for the whole mount ABS. With just a single loading step, the zebrafish embryos can be trapped by the milli fluidic device through a chaotic hydrodynamic trapping process. Moreover, a consistent body orientation (i.e., head point inward) for the trapped zebrafish embryos can be achieved without additional orientation adjustment device. Furthermore, we employed a consumer-grade SLA 3D printer assisted method for device prototyping which is ideal for labs with limited budgets. Notably, the milli fluidic device has enabled the optimization and successful implementation of whole mount zebrafish Caspase-3 ABS. We demonstrated our device can accelerate the overall procedure by reducing at least 50% of washing time in the standard well-plate-based manual procedure. Also, the consistency is improved, and manual steps are reduced using the milli fluidic device. This work fills the gap in the milli fluidic application for whole mount zebrafish immunohistochemistry. We hope the device can be accepted by the zebrafish community and be used for other types of whole mount zebrafish ABS procedures or expanded to more complicated in situ hybridization (ISH) procedure.Graphical

Easy, Robust, and Repeatable Online Acid Cleavage of Proteins in Mobile Phase for Fast Quantitative LC-MS Bottom-Up Protein Analysis─Application for Ricin Detection.

Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.

Pointing It Out! Comparing Manual Segmentation of 3D Point Clouds between Desktop, Tablet, and Virtual Reality

Scanning everyday objects with depth sensors is the state-of-the-art approach to generating point clouds for realistic 3D representations. However, the resulting point cloud data suffers from outliers and contains irrelevant data from neighboring objects. To obtain only the desired 3D representation, additional manual segmentation steps are required. In this paper, we compare three different technology classes as independent variables (desktop vs. tablet vs. virtual reality) in a within-subject user study (N = 18) to understand their effectiveness and efficiency for such segmentation tasks. We found that desktop and tablet still outperform virtual reality regarding task completion times, while we could not find a significant difference between them in the effectiveness of the segmentation. In the post hoc interviews, participants preferred the desktop due to its familiarity and temporal efficiency and virtual reality due to its given three-dimensional representation.

Evaluation of two detergent-disinfectants and a detergent on a Klebsiella pneumoniae biofilm formed within Tygon tubes

Transmission of infections via contaminated endoscopes is a common problem. Manual cleaning, using at least a detergent, is an important step in endoscope processing and should be performed as soon as possible to avoid drying of organic residues that might interfere with high-level disinfection and promote biofilm formation. To assess the efficacy of two detergent-disinfectants, enzymatic and non-enzymatic, and of an enzymatic detergent used during the manual cleaning against a Klebsiella pneumoniae biofilm. A 24h biofilm statically formed in a Tygon tube was exposed to detergent-disinfectants at 20°C and 35°C for 10mn, and to enzymatic detergent at 45°C for 60mn. The logarithmic reduction in bacteria in the Tygon tube and the number of bacteria in the product supernatant were calculated. Biofilm formation was reproducible between assays. After exposure to detergent-disinfectants, the logarithmic reduction was between 6.32 and 6.71 log10cfu/cm2 in the Tygon tubes. No bacteria were found in their supernatants. Results in the detergent-disinfectant group were not affected by the exposure temperature or the addition of enzymes. No decrease in the bacterial load was observed in the Tygon tubes after exposure to the enzymatic detergent. Bacteria were found in its supernatant. These results show the importance of the choice of products used duringthemanual cleaning phase. They also show the potential benefit of combining detergent and disinfectant activity to decrease the bacterial load during the manual cleaning step of endoscope processing.

SURVEY TOOL ALIGNMENT FOR REAL-TIME MIXED REALITY INFORMATION MODEL INTERACTION IN HERITAGE RECORDING

Abstract. While heritage documentation today is largely based on three-dimensional models as key element, the easy access to tools for creating such models as well as quick and easier to use workflows lead to a stark increase in data creation while being followed by less structured and standardized methodology. Increasingly information models are applied to structure all available data regarding an object and reference it within a digital twin of the physical object. Nevertheless, various approaches reviewed all show a process of information model creating as post-processing task, undoubtedly leading to parallel systems of captured data, and disseminated data.The following research proposes to encounter the mentioned issues by developing a strongly interwoven integrative building documentation workflow with including GNSS-RTK based survey and augmented reality tools, to start compiling coordinate-based information models right on site during the first acquirement of data. As much data as possible should be included into this process, covering geometry, visual observations, and semantic information. Making data available right after inclusion into the information model should then allow to give real-time feedback on all data available (old and new) right on site or remotely within a web-platform, virtual reality interface or else. Detaching the work of curating and information model from a manual processing step, but rather being able to link information through their position right at the time of origin, would allow models to be created, enhanced, and expanded collaboratively further giving them a fourth dimension of time and collective knowledge.

AMEBaS: Automatic Midline Extraction and Background Subtraction of Ratiometric Fluorescence Time-Lapses of Polarized Single Cells

Cell polarity is a macroscopic phenomenon established by a collection of spatially concentrated molecules and structures that culminate in the emergence of specialized domains at the subcellular level. It is associated with developing asymmetric morphological structures that underlie key biological functions such as cell division, growth, and migration. In addition, the disruption of cell polarity has been linked to tissue-related disorders such as cancer and gastric dysplasia. Current methods to evaluate the spatiotemporal dynamics of fluorescent reporters in individual polarized cells often involve manual steps to trace a midline along the cells' major axis, which is time consuming and prone to strong biases. Furthermore, although ratiometric analysis can correct the uneven distribution of reporter molecules using two fluorescence channels, background subtraction techniques are frequently arbitrary and lack statistical support. This manuscript introduces a novel computational pipeline to automate and quantify the spatiotemporal behavior of single cells using a model of cell polarity: pollen tube/root hair growth and cytosolic ion dynamics. A three-step algorithm was developed to process ratiometric images and extract a quantitative representation of intracellular dynamics and growth. The first step segments the cell from the background, producing a binary mask through a thresholding technique in the pixel intensity space. The second step traces a path through the midline of the cell through a skeletonization operation. Finally, the third step provides the processed data as a ratiometric timelapse and yields a ratiometric kymograph (i.e., a 1D spatial profile through time). Data from ratiometric images acquired with genetically encoded fluorescent reporters from growing pollen tubes were used to benchmark the method. This pipeline allows for faster, less biased, and more accurate representation of the spatiotemporal dynamics along the midline of polarized cells, thus advancing the quantitative toolkit available to investigate cell polarity. The AMEBaS Python source code is available at: https://github.com/badain/amebas.git

Novel 3D printed probe for bioimpedance spectroscopic measurement of oral mucosa: design and testing with ex vivo porcine oral tissues

Three-dimensional (3D) printing has a high potential in various biomedical applications. We hypothesize that 3D printing could be a viable option to construct novel bioimpedance spectroscopic (BIS) sensors suitable for electrochemical characterization of oral mucosal tissues. Previous BIS studies have relied on hand-made probes possessing significant limitations related to their single patient disposable use, large inter-probe differences, and weak reproducibility of measurement. There is also uncertainty related to the effect of varying loading pressure between the probe and biological tissue. Here, we introduce three differently sized rectangular shaped 3D printed probes and test those using a four-terminal measurement principle on various porcine oral tissue samples. We find that constructing a fully 3D printed probe is a challenging task, prone to issues relating to short-circuiting or electrochemical corrosion. However, our final prototype version, constructed with silver-coated copper electrodes, showed favorable characteristics in BIS experiments. All three differently sized probes were able to differentiate between different tissue types with excellent reproducibility. The effect of loading pressure was found to be almost negligible when using small- and medium-sized probes. However, further studies are needed to measure tissues with uneven surfaces, such as palatinum, and to avoid manual or (electro)chemical surface-finishing steps.

UV-Assisted Hyperbranched Poly(β-amino ester) Modification of a Silica Membrane for Two-Step Microfluidic DNA Extraction from Blood.

Integrating nucleic acid extraction in amplification-based point-of-care diagnostics will be a significant feature for next-generation point-of-care virus detection devices. However, extracting DNA efficiently on a microfluidic chip poses many technological and commercialization challenges, including manual steps, multiple instruments, pretreatment processes, and the use of organic solvents (ethanol, IPA) that inhibit detection, which is not viable with routine testing such as viral load monitoring of transplant patients for post-operative care. This paper presents a microfluidic system capable of two-step DNA extraction from blood using a UV-assisted hyperbranched poly(β-amino ester) (HPAE)-modified silica membrane for cytomegalovirus (CMV) detection in a rapid and instrument-free manner without the presence of amplification inhibitors. HPAEs of varying branch ratios were synthesized, screened, and coated on a silica membrane and bonded between two layers of poly(methyl methacrylate) (PMMA) substrates. Our system could selectively extract DNA from blood with an efficiency of 94% and a lower limit viral load of 300 IU/mL in 20 min. The extracted DNA was used as the template for real-time loop-mediated isothermal amplification (LAMP)-based detection of CMV and was found to produce a fluorescent signal intensity that was comparable with commercially extracted templates. This system can be integrated easily with a nucleic acid amplification system and used for routine rapid testing of viral load in patient blood samples.

When Automatic Filtering Comes to the Rescue: Pre-Computing Company Competitor Pairs in Owler

Competitor data constitutes information significantly valuable for many business applications. Meltwater provides users with access to a large Company Information System (CIS), Owler, which contains competitor pairs and other useful information about companies. Meltwater has been seeking a practical solution to discover more competitor pairs in Owler. The first attempt, a fully-manual workflow (called MW_Manual) for finding more competitor pairs in Owler consisted of two manual steps: a filtering step that excludes obvious non-competitor company pairs, and a further inspection process that inspects each left company pair after the filtering step. MW_Manual was cost prohibitive because the results of the filtering step contained too many non-competitor pairs. Inspecting such non-competitor pairs caused an overhead to the overall workload. To reduce the manual workload, especially the required human effort in the manual inspection process, Meltwater has transformed MW_Manual into a semi-automatic workflow (called MW_CPFilter) by replacing the manual filtering with an automatic yet more precise process that adopts a system called CPFilter. This paper presents CPFilter, a system used in the filtering process of MW_CPFilter. CPFilter automatically pre-computes likely competitor pairs from existing competitor pairs in Owler. CPFilter combines (i) the generation of new competitor candidate pairs by inference from existing competitors and other company-specific knowledge, with (ii) the validation of each candidate competitor pair of two companies by checking whether or not empirical evidence that indicates the competitor relationships of these two companies can be found. CPFilter has three key advantages compared with the manual filtering process and previous works: (i) it resulted in a high workload reduction rate of 0.81, (ii) it is domain-independent so that it can be applied to different sectors in Owler, and (iii) its results are explainable so that humans can easily understand its results.

Development of a high-throughput minimum inhibitory concentration (HT-MIC) testing workflow.

The roots of the minimum inhibitory concentration (MIC) determination go back to the early 1900s. Since then, the test has undergone modifications and advancements in an effort to increase its dependability and accuracy. Although biological investigations use an ever-increasing number of samples, complicated processes and human error sometimes result in poor data quality, which makes it challenging to replicate scientific conclusions. Automating manual steps using protocols decipherable by machine can ease procedural difficulties. Originally relying on manual pipetting and human vision to determine the results, modern broth dilution MIC testing procedures have incorporated microplate readers to enhance sample analysis. However, current MIC testing procedures are unable to simultaneously evaluate a large number of samples efficiently. Here, we have created a proof-of-concept workflow using the Opentrons OT-2 robot to enable high-throughput MIC testing. We have further optimized the analysis by incorporating Python programming for MIC assignment to streamline the automation. In this workflow, we performed MIC tests on four different strains, three replicates per strain, and analyzed a total of 1,152 wells. Comparing our workflow to a conventional plate MIC procedure, we find that the HT-MIC method is 800% faster while simultaneously boasting a 100% accuracy. Our high-throughput MIC workflow can be adapted in both academic and clinical settings since it is faster, more efficient, and as accurate than many conventional methods.

Large-scale fabrication of ion-selective electrodes for simultaneous detection of Na+, K+, and Ca2+ in biofluids using a smartphone-based potentiometric sensing platform.

A significant bottleneck exists for mass-production of ion-selective electrodes despite recent developments in manufacturing technologies. Here, we present a fully-automated system for large-scale production of ISEs. Three materials, including polyvinyl chloride, polyethylene terephthalate and polyimide, were used as substrates for fabricating ion-selective electrodes (ISEs) using stencil printing, screen-printing and laser engraving, respectively. We compared sensitivities of the ISEs to determine the best material for thefabrication process of the ISEs. The electrode surfaces were modified with various carbon nanomaterials including multi-walled carbon nanotubes, graphene, carbon black, and their mixed suspensions as the intermediate layer to enhance sensitivities of the electrodes. An automated 3D-printed robot was used for the drop-cast procedure during ISE fabrication to eliminate manual steps. The sensor array was optimized, and the detection limits were 10-5M, 10-5M and 10-4M for detection of K+, Na+ and Ca2+ ions, respectively. The sensor array integrated with aportable wireless potentiometer was used to detect K+, Na+ and Ca2+ in real urine and simulated sweat samples and results obtained werein agreement with ICP-OES with good recoveries. The developed sensing platform offers low-cost detection of electrolytes for point-of-care applications.

Toward Retinal Organoids in High-Throughput.

Human retinal organoids recapitulate the cellular diversity, arrangement, gene expression, and functional aspects of the human retina. Protocols to generate human retinal organoids from pluripotent stem cells are typically labor intensive, include many manual handling steps, and the organoids need to be maintained for several months until they mature. To generate large numbers of human retinal organoids for therapy development and screening purposes, scaling up retinal organoid production, maintenance, and analysis is of utmost importance. In this review, we discuss strategies to increase the number of high-quality retinal organoids while reducing manual handling steps. We further review different approaches to analyze thousands of retinal organoids with currently available technologies and point to challenges that still await to be overcome both in culture and analysis of retinal organoids.

A review of open-source image analysis tools for mammalian cell culture: algorithms, features and implementations.

Cell culture is undeniably important for multiple scientific applications, including pharmaceuticals, transplants, and cosmetics. However, cell culture involves multiple manual steps, such as regularly analyzing cell images for their health and morphology. Computer scientists have developed algorithms to automate cell imaging analysis, but they are not widely adopted by biologists, especially those lacking an interactive platform. To address the issue, we compile and review existing open-source cell image processing tools that provide interactive interfaces for management and prediction tasks. We highlight the prediction tools that can detect, segment, and track different mammalian cell morphologies across various image modalities and present a comparison of algorithms and unique features of these tools, whether they work locally or in the cloud. This would guide non-experts to determine which is best suited for their purposes and, developers to acknowledge what is worth further expansion. In addition, we provide a general discussion on potential implementations of the tools for a more extensive scope, which guides the reader to not restrict them to prediction tasks only. Finally, we conclude the article by stating new considerations for the development of interactive cell imaging tools and suggesting new directions for future research.

Semi-automatic 3D reconstruction of middle and inner ear structures using CBCT

ABSTRACT We present a semi-automatic reconstruction approach of middle and inner ear structures using generic 3D deformable surface models from Cone Beam CT (CBCT) examination. First, the user must position a set of control points in the CBCT volume for each of the 4 structures of the inner and middle ear. These points are used to position the deformable surface models and to customize them so that they are as close to the boundaries as possible. Finally, each mesh is refined iteratively segmenting the limits of the structure while taking into account neighbouring structures as boundary constraints. Our method is tested on left and right ears of 20 scans of patients analysed retrospectively. The results show the efficiency and reliability of this approach with an average Dice Similarity Coefficient of 91.8% for the inner ear model and 89.9% for the ossicular chain and a total reconstruction time of 5 minutes. The implementation of our method in a clinical setting could provide clinicians with distinct and accurate 3D models of the ear structures without requiring a tedious manual segmentation step, in order to give them a better understanding of the auditory system in vivo and help them in diagnosis and follow-up.

Automation in flow cytometry: Guidelines and review of systems.

Automation in flow cytometry has recently advanced from the partial laboratory automation and robotics islets, to more fully integrated systems. This article reviews three manufacturers' newest sample preparation systems: the Beckman CellMek, the Sysmex PS-10, and the BD FACSDuet. These three instruments are capable of performing many of the manual steps in flow cytometry sample processing (pipetting, staining, lysing, washing, fixing). General description, capabilities, advantages, and disadvantages of each system are compared. Overall, these systems have the potential to become mainstay items in today's busy clinical flow cytometry laboratories, and save a significant amount of hands-on time for laboratory staff.

Electrochemical capillary-driven microfluidic DNA sensor for HIV-1 and HCV coinfection analysis

Electrochemical DNA sensors can be operated in either static or flow-based detection schemes. In static schemes, manual washing steps are still necessary, resulting in a tedious and time-consuming process. In contrast, in flow-based electrochemical sensors, the current response is collected when the solution flows through the electrode continuously. However, the drawback of such a flow system is the low sensitivity due to the limited time for the interaction between the capturing element and the target. Herein, we propose a novel electrochemical capillary-driven microfluidic DNA sensor to combine the advantages of static and flow-based electrochemical detection systems into a single device by incorporating burst valve technology. The microfluidic device with a two-electrode configuration was applied for the simultaneous detection of two different DNA markers, human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) cDNA, via the specific interaction between pyrrolidinyl peptide nucleic acids (PNA) probes and the DNA target. The integrated system, while requiring a small sample volume (7 μL for each sample loading port) and less analysis time, achieved good performance in terms of the limits of detection (LOD) (3SDblank/slope) and quantification (LOQ) (10SDblank/slope) at 1.45 nM and 4.79 nM for HIV and 1.20 nM and 3.96 nM for HCV, respectively. The simultaneous detection of HIV-1 and HCV cDNA prepared from human blood samples showed results that are in complete agreement with the RT‒PCR assay. The results qualify this platform as a promising alternative for the analysis of either HIV-1/HCV or coinfection that can be easily adapted for other clinically important nucleic acid-based markers.

All-in-one microfluidic chip for 30-min quantitative point-of-care-testing of nucleic acids

Nucleic acid testing (NAT) is directly oriented to determining the genetic material of pathogens and is characterized by its high sensitivity and specificity, which are indispensable qualities in disease diagnosis. However, standard laboratory NAT methods require joint testing by highly trained inspectors using multiple instruments in professional laboratories. The entire process requires many manual steps, and the total testing time may range from 3 to 5 h, indicating that these methods cannot be used to realize the demands of on-site rapid testing. In this study, we propose a microfluidic chip for the on-site and rapid detection of nucleic acids. We utilize dynamic sealing, ultrasound, and advanced control methods and integrate the entire process of reagent pre-storage, extraction, Real-time Quantitative polymerase chain reaction (qPCR), and fluorescence detection. The sensitivity of this system is in line with current clinical standards, and the nucleic acid quantification process is completed fully automated within 30 min. Compared with conventional microfluidic chips, the proposed system has the advantages of high integration, low cost, and it may be produced at a high volume. Moreover, it can be used in a wide range of screening cases in the context of the COVID-19 pandemic and exhibits broad clinical application prospects.

Investigating online activity in UK adolescent mental health patients: a feasibility study using a natural language processing approach for electronic health records

ObjectivesTo assess the feasibility of using a natural language processing (NLP) application for extraction of free-text online activity mentions in adolescent mental health patient electronic health records (EHRs).SettingThe Clinical Records...

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

2023 TERMIS – AMERICAS Conference & Exhibition Boston Marriott Copley Place April 11–14, 2023

Abstract 3794: Sensitive detection of microRNA biomarkers of pancreatic cancer using one-pot endonucleolytically exponentiated rolling circle amplification by CRISPR-Cas12a

Abstract While miRNAs offer a promising source for developing diagnostic cancer biomarkers, the progress towards clinical utilities remains largely limited, due in part to the long-standing challenge in sensitive, specific, and robust detection of miRNAs in human biofluids. Emerging next-generation molecular technologies, such as the CRISPR-based methods, promise to transform nucleic acid testing. The prevailing strategy used in existing CRISPR-based methods is to orthogonally hyphenate two separate reactions for pre-amplification, e.g., rolling circle amplification (RCA), and amplicon detection by Cas12a/13a trans-cleavage in tandem. Thus, existing CRISPR-based miRNA assays require multiple manual steps and lack the analytical performance of the gold standard, RT-qPCR. Radically deviating from the existing strategies, we developed a one-step, one-pot isothermal miRNA assay termed “Endonucleolytically eXponenTiated Rolling circle Amplification with the dual-functional CRISPR-Cas12a” (EXTRA-CRISPR) for miRNA detection with RT-qPCR-like analytical performance, including high sensitivity with a single-digit fM detection limit, single-nucleotide specificity, and rapid and flexible turnaround (from 20 min to 3 h for the entire analysis depending on targets and samples). We adapted the EXTRA-CRISPR assay to quantifying miRNA biomarkers in extracellular vesicles (EVs), a major carrier of miRNAs in human biofluids, for liquid biopsy diagnosis of pancreatic ductal adenocarcinoma (PDAC). Using the EXTRA-CRISPR, we demonstrated highly sensitive and specific profiling of a panel of four miRNA markers (miR-21, miR-196a, miR-451a, and miR-1246) in plasma EVs (PDAC patients (n = 20) and healthy donors (n = 15)). Based on the individual EV-miRNA tests (AUC values ranging from 0.677 for miR-196a to 0.793 for miR-451a), an EV signature (EV-Sig) combining miR-21, miR-451a and miR-1246 was devised with a machine learning method to improve the diagnostic performance for PDAC (AUC = 0.853). The diagnostic performance of the EXTRA-CRSPR tests were rigorously validated by parallel RT-qPCR analysis of the same clinical samples, and the RT-qPCR signature combining the three markers confers a similar AUC of 0.874 for PDAC detection. Overall, the analytical and diagnostic performance of our method were shown to be comparable with that of the commercial RT-qPCR assays, while greatly simplifying the analysis workflow. Therefore, we envision that our technology provides a promising tool to advance miRNA analysis and clinical marker development for liquid biopsy-based cancer diagnosis and prognosis. Moreover, this simple and robust assay permitted direct coupling with a 3D printed smartphone-based portable device and the lateral flow assay for signal readout, demonstrating its potential adaptability to low-cost point-of-care diagnostics. Citation Format: He Yan, Song Han, Steven Hughes, Yong Zeng. Sensitive detection of microRNA biomarkers of pancreatic cancer using one-pot endonucleolytically exponentiated rolling circle amplification by CRISPR-Cas12a. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3794.