Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection.

BACKGROUND: Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated. METHODS: One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control. RESULTS: No significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350€ and 400€ for every 96 extracted samples compared to the commercial kit. CONCLUSION: The protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities.

Application of a fast and cost-effective 'three-in-one' MMR ELISA as a tool for surveying anti-MMR humoral immunity: the Hungarian experience.

In Hungary, between February 2017 and July 2019, 70 confirmed measles cases were reported, raising questions about the adequacy of population-level immunity. Although the assumed vaccination coverage is ≥99%, in a recent study, we detected potential gaps in the anti-measles humoral immunity. In Hungary, according to a decree by the Ministry of Public Welfare, beginning from 2021, the healthcare provider should conduct a serosurvey of anti-measles protection levels of healthcare professionals. To facilitate the compliance with this requirement, we developed a quick 'three-in-one' or 'triple' MMR (measles, mumps and rubella) indirect ELISA (IgG); an assay format that is currently not available commercially. High throughput applicability of the 'three-in-one' ELISA was verified using 1736 sera from routine laboratory residual samples, using an automated platform (Siemens BEP 2000 Advance). Assay verification was performed by comparing the full antigen repertoire-based 'target' assay with in-house 'control' assays using recombinant viral antigen coatings, and by validated commercially available kits. Indirect immunofluorescence was used as an independent reference method. Data were analysed using OriginLab, IBM SPSS, RStudio and MedCalc. In case of measles, we combined our current results with previously published data (Ntotal measles = 3523). Evaluation of anti-mumps and anti-rubella humoral antibody levels was based on the measurement of 1736 samples. The lowest anti-measles seropositivity (79.3%) was detected in sera of individuals vaccinated between 1978 and 1987. Considering the antigen-specific seropositivity ratios of all samples measured, anti-measles, -mumps and -rubella IgG antibody titres were adequate in 89.84%, 91.82% and 92.28%, respectively. Based on the virus-specific herd immunity threshold (HIT) values (HITMeasles = 92-95%, HITMumps = 75-86%, HITRubella = 83-86), it can be stated that regarding anti-measles immunity, certain age clusters of the population may have inadequate levels of humoral immunity. Despite the potential gaps in herd immunity, the use of MMR vaccine remains an effective and low-cost approach for the prevention of measles, mumps and rubella infections.

Benefits of VISION Max automated cross-matching in comparison with manual cross-matching: A multidimensional analysis.

BACKGROUND: VISION Max (Ortho-Clinical Diagnostics, Raritan, NJ, USA) is a newly introduced automated blood bank system. Cross-matching (XM) is an important test confirming safety by simulating reaction between packed Red Blood Cells (RBCs) and patient blood in vitro before transfusion. We assessed the benefits of VISION Max automated XM (A-XM) in comparison with those of manual XM (M-XM) by using multidimensional analysis (cost-effectiveness and quality improvement). MATERIALS AND METHODS: In a total of 327 tests (130 patients), results from A-XM and M-XM were compared. We assessed the concordance rate, risk priority number (RPN), turnaround time, hands-on time, and the costs of both methods. We further simulated their annual effects based on 37,937 XM tests in 2018. RESULTS: The concordance rate between A-XM and M-XM was 97.9% (320/327, kappa = 0.83), and the seven discordant results were incompatible for transfusion in A-XM, while compatible for transfusion in M-XM. None of the results was incompatible for transfusion in A-XM, while compatible for transfusion in M-XM, meaning A-XM detect agglutination more sensitively and consequently provides a more safe result than M-XM. A-XM was estimated to have a 6.3-fold lower risk (229 vs. 1,435 RPN), shorter turnaround time (19.1 vs. 23.3 min, P < 0.0001), shorter hands-on time (1.1 vs. 5.3 min, P < 0.0001), and lower costs per single test than M-XM (1.44 vs. 2.70 USD). A-XM permitted annual savings of 46 million RPN, 15.1 months of daytime workers' labor, and 47,042 USD compared with M-XM. CONCLUSION: This is the first attempt to implement A-XM using VISION Max. VISION Max A-XM appears to be a safe, practical, and reliable alternative for pre-transfusion workflow with the potential to improve quality and cost-effectiveness in the blood bank.

Total Laboratory Automation: What Is Gained, What Is Lost, and Who Can Afford It?

The first clinical microbiology laboratory in the United States adopted total automation for bacteriology processing in 2014. Since then, others have followed with installation of either the BD Kiestra TLA or the Copan WASPLab. This article discusses commercially available automated systems in the United States; why automation is needed; and quality improvements, efficiency, and cost savings associated with automation. After learning how these systems are used, gains and losses experienced, and how one can afford the most expensive equipment ever purchased for clinical microbiology laboratories, the question is, how can one afford not to purchase one of these microbiology automation systems?

Cost-Effective Automated Preparation of Serum Samples for Reproducible Quantitative Clinical Proteomics.

Reproducible sample preparation remains a significant challenge in large-scale clinical research using selected reaction monitoring-mass spectrometry (SRM-MS), which enables a highly sensitive multiplexed assay. Although automated liquid-handling platforms have tremendous potential for addressing this issue, the high cost of their consumables is a drawback that renders routine operation expensive. Here we evaluated the performance of a liquid-handling platform in preparing serum samples compared with a standard experiment while reducing the outlay for consumables, such as tips, wasted reagents, and reagent stock plates. A total of 26 multiplex assays were quantified by SRM-MS using four sets of 24 pooled human serum aliquots; the four sets used a fixed number (1, 4, 8, or 24) of tips to dispense digestion reagents. This study demonstrated that the use of 4 or 8 tips is comparable to 24 tips (standard experiment), as evidenced by their coefficients of variation: 13.5% (for 4 and 8 tips) versus 12.0% (24 tips). Thus we can save 37% of the total experimental cost compared with the standard experiment, maintaining nearly equivalent reproducibility. The routine operation of cost-effective liquid-handling platforms can enable researchers to process large-scale samples with high throughput, adding credibility to their findings by minimizing human error.

Advantages and limitations of total laboratory automation: a personal overview.

Automation is considered one of the most important breakthroughs in the recent history of laboratory diagnostics. In a model of total laboratory automation (TLA), many analyzers performing different types of tests on different sample matrices are physically integrated as modular systems or physically connected by assembly lines. The opportunity to integrate multiple diagnostic specialties to one single track seems effective to improve efficiency, organization, standardization, quality and safety of laboratory testing, whilst also providing a significant return of investment on the long-term and enabling staff requalification. On the other hand, developing a model of TLA also presents some potential problems, mainly represented by higher initial costs, enhanced expenditure for supplies, space requirements and infrastructure constraints, staff overcrowding, increased generation of noise and heat, higher risk of downtime, psychological dependence, critical issues for biospecimen management, disruption of staff trained in specific technologies, along with the risk of transition toward a manufacturer's-driven laboratory. As many ongoing technological innovations coupled with the current scenario, profoundly driven by cost-containment policies, will promote further diffusion of laboratory automation in the foreseeable future, here we provide a personal overview on some potential advantages and limitations of TLA.

Implementation of total laboratory automation at a tertiary care hospital in Saudi Arabia: effect on turnaround time and cost efficiency.

BACKGROUND: Total laboratory automation (TLA) is a relatively new way of improving the management of high volume clinical laboratories. TLA may reduce staff, reduce operating costs, decrease testing time and provide enhanced process control. OBJECTIVES: Establish a cost efficient TLA that is less labor intensive, improves productivity and reduces turnaround time (TAT). DESIGN: Implementation of TLA for random glucose and troponin-T as sentinel tests to compare change in TAT. SETTING: Tertiary hospital with high volume of laboratory tests. METHODS: Routine patient samples for random glucose and troponin-T were used to capture TAT. Information on staff grades and schedules before and after implementing the TLA, and cost of contracts to deliver the service were collected. MAIN OUTCOME MEASURES: TAT, cost efficiency, and reduction in labor. RESULTS: The consolidation of contracts resulted in a reduction of 28.8 million SAR in direct costs. Staffing cost was reduced by 1.14 million SAR with less senior staff required; there were reductions in staff at both senior and junior level. The overall TAT for all tests was reduced by 32% in 2016 (after TLA implementation) compared to 2012 (before TLA implementation). The median TAT for random glucose tests was reduced by 21% (to 55.7 minutes in 2016 from 70.1 minutes in 2012). Evidence of test optimization by exploring the impact of stat tests, auto-dilutions and reruns on the overall TAT of the TLA is shown by comparing troponin T TATs after reclassifying stat tests (in 2016) to routine (in 2017). At the 75th percentile, there was a 27% reduction in TAT when comparing August 2016 to March 2017 with a 19% reduction in median TAT. CONCLUSION: By moving from stat to routine assays, the TAT was reduced, which is counter-intuitive. The use of stat assays slowed down the performance of the TLA. A careful review of the mix of assays should be conducted to maximize performance and to ensure that the system delivers what is required. LIMITATIONS: Room for improvement by systematically analyzing and reviewing the impact of making minor changes that could have significant impact on TAT. CONFLICT OF INTEREST: None.

Development of Fully Automated Low-Cost Immunoassay System for Research Applications.

Enzyme-linked immunosorbent assay (ELISA) automation for routine operation in a small research environment would be very attractive. A portable fully automated low-cost immunoassay system was designed, developed, and evaluated with several protein analytes. It features disposable capillary columns as the reaction sites and uses real-time calibration for improved accuracy. It reduces the overall assay time to less than 75 min with the ability of easy adaptation of new testing targets. The running cost is extremely low due to the nature of automation, as well as reduced material requirements. Details about system configuration, components selection, disposable fabrication, system assembly, and operation are reported. The performance of the system was initially established with a rabbit immunoglobulin G (IgG) assay, and an example of assay adaptation with an interleukin 6 (IL6) assay is shown. This system is ideal for research use, but could work for broader testing applications with further optimization.

Cost effective raspberry pi-based radio frequency identification tagging of mice suitable for automated in vivo imaging.

BACKGROUND: Automation of animal experimentation improves consistency, reduces potential for error while decreasing animal stress and increasing well-being. Radio frequency identification (RFID) tagging can identify individual mice in group housing environments enabling animal-specific tracking of physiological parameters. NEW METHOD: We describe a simple protocol to radio frequency identification (RFID) tag and detect mice. RFID tags were injected sub-cutaneously after brief isoflurane anesthesia and do not require surgical steps such as suturing or incisions. We employ glass-encapsulated 125kHz tags that can be read within 30.2±2.4mm of the antenna. A raspberry pi single board computer and tag reader enable automated logging and cross platform support is possible through Python. RESULTS: We provide sample software written in Python to provide a flexible and cost effective system for logging the weights of multiple mice in relation to pre-defined targets. COMPARISON WITH EXISTING METHODS: The sample software can serve as the basis of any behavioral or physiological task where users will need to identify and track specific animals. Recently, we have applied this system of tagging to automated mouse brain imaging within home-cages. CONCLUSIONS: We provide a cost effective solution employing open source software to facilitate adoption in applications such as automated imaging or tracking individual animal weights during tasks where food or water restriction is employed as motivation for a specific behavior.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

Feeding Experimentation Device (FED): A flexible open-source device for measuring feeding behavior.

BACKGROUND: Measuring food intake in rodents is a conceptually simple yet labor-intensive and temporally-imprecise task. Most commonly, food is weighed manually, with an interval of hours or days between measurements. Commercial feeding monitors are excellent, but are costly and require specialized caging and equipment. NEW METHOD: We have developed the Feeding Experimentation Device (FED): a low-cost, open-source, home cage-compatible feeding system. FED utilizes an Arduino microcontroller and open-source software and hardware. FED dispenses a single food pellet into a food well where it is monitored by an infrared beam. When the mouse removes the pellet, FED logs the timestamp to a secure digital (SD) card and dispenses a new pellet into the well. Post-hoc analyses of pellet retrieval timestamps reveal high-resolution details about feeding behavior. RESULTS: FED is capable of accurately measuring food intake, identifying discrete trends during light and dark-cycle feeding. Additionally, we show the utility of FED for measuring increases in feeding resulting from optogenetic stimulation of agouti-related peptide neurons in the arcuate nucleus of the hypothalamus. COMPARISON TO EXISTING METHODS: With a cost of ∼$350 per device, FED is >10× cheaper than commercially available feeding systems. FED is also self-contained, battery powered, and designed to be placed in standard colony rack cages, allowing for monitoring of true home cage feeding behavior. Moreover, FED is highly adaptable and can be synchronized with emerging techniques in neuroscience, such as optogenetics, as we demonstrate here. CONCLUSIONS: FED allows for accurate, precise monitoring of feeding behavior in a home cage setting.

Repository Planning, Design, and Engineering: Part II-Equipment and Costing.

Part II of this article discusses and provides guidance on the equipment and systems necessary to operate a repository. The various types of storage equipment and monitoring and support systems are presented in detail. While the material focuses on the large repository, the requirements for a small-scale startup are also presented. Cost estimates and a cost model for establishing a repository are presented. The cost model presents an expected range of acquisition costs for the large capital items in developing a repository. A range of 5,000-7,000 ft(2) constructed has been assumed, with 50 frozen storage units, to reflect a successful operation with growth potential. No design or engineering costs, permit or regulatory costs, or smaller items such as the computers, software, furniture, phones, and barcode readers required for operations have been included.

Adapting a Low-Cost Selective Compliant Articulated Robotic Arm for Spillage Avoidance.

Flexible automation systems provide the needed adaptability to serve shorter-term projects and specialty applications in biochemical analysis. A low-cost selective compliant articulated robotic arm designed for liquid spillage avoidance is developed here. In the vertical-plane robotic arm movement test, the signals from an inertial measurement unit (IMU) and accelerometer were able to sense collisions. In the horizontal movement test, however, only the signals from the IMU enabled collision to be detected. Using a calculation method developed, it was possible to chart the regions where the obstacle was likely to be located when a collision occurred. The low cost of the IMU and its easy incorporation into the robotic arm offer the potential to meet the pressures of lowering operating costs, apply laboratory automation in resource-limited venues, and obviate human intervention in response to sudden disease outbreaks.

A fully automated Drosophila olfactory classical conditioning and testing system for behavioral learning and memory assessment.

BACKGROUND: Aversive olfactory classical conditioning has been the standard method to assess Drosophila learning and memory behavior for decades, yet training and testing are conducted manually under exceedingly labor-intensive conditions. To overcome this severe limitation, a fully automated, inexpensive system has been developed, which allows accurate and efficient Pavlovian associative learning/memory analyses for high-throughput pharmacological and genetic studies. NEW METHOD: The automated system employs a linear actuator coupled to an odorant T-maze with airflow-mediated transfer of animals between training and testing stages. Odorant, airflow and electrical shock delivery are automatically administered and monitored during training trials. Control software allows operator-input variables to define parameters of Drosophila learning, short-term memory and long-term memory assays. RESULTS: The approach allows accurate learning/memory determinations with operational fail-safes. Automated learning indices (immediately post-training) and memory indices (after 24h) are comparable to traditional manual experiments, while minimizing experimenter involvement. COMPARISON WITH EXISTING METHODS: The automated system provides vast improvements over labor-intensive manual approaches with no experimenter involvement required during either training or testing phases. It provides quality control tracking of airflow rates, odorant delivery and electrical shock treatments, and an expanded platform for high-throughput studies of combinational drug tests and genetic screens. The design uses inexpensive hardware and software for a total cost of ∼$500US, making it affordable to a wide range of investigators. CONCLUSIONS: This study demonstrates the design, construction and testing of a fully automated Drosophila olfactory classical association apparatus to provide low-labor, high-fidelity, quality-monitored, high-throughput and inexpensive learning and memory behavioral assays.

Consolidating a Distributed Compound Management Capability into a Single Installation: The Application of Overall Equipment Effectiveness to Determine Capacity Utilization.

Compound management (CM) is a critical discipline enabling hit discovery through the production of assay-ready compound plates for screening. CM in pharma requires significant investments in manpower, capital equipment, repairs and maintenance, and information technology. These investments are at risk from external factors, for example, new technology rendering existing equipment obsolete and strategic site closures. At AstraZeneca, we faced the challenge of evaluating the number of CM sites required to support hit discovery in response to site closures and pressure on our operating budget. We reasoned that overall equipment effectiveness, a tool used extensively in the manufacturing sector, could determine the equipment capacity and appropriate number of sites. We identified automation downtime as the critical component governing capacity, and a connection between automation downtime and the availability of skilled staff. We demonstrated that sufficient production capacity existed in two sites to meet hit discovery demand without the requirement for an additional investment of $7 million in new facilities. In addition, we developed an automated capacity model that incorporated an extended working-day pattern as a solution for reducing automation downtime. The application of this solution enabled the transition to a single site, with an annual cost saving of $2.1 million.

A novel experimental approach to investigate radiolysis processes in liquid samples using collimated radiation sources.

Here is detailed a novel and low-cost experimental method for high-throughput automated fluid sample irradiation. The sample is delivered via syringe pump to a nozzle, where it is expressed in the form of a hanging droplet into the path of a beam of ionising radiation. The dose delivery is controlled by an upstream lead shutter, which allows the beam to reach the droplet for a user defined period of time. The droplet is then further expressed after irradiation until it falls into one well of a standard microplate. The entire system is automated and can be operated remotely using software designed in-house, allowing for use in environments deemed unsafe for the user (synchrotron beamlines, for example). Depending on the number of wells in the microplate, several droplets can be irradiated before any human interaction is necessary, and the user may choose up to 10 samples per microplate using an array of identical syringe pumps, the design of which is described here. The nozzles consistently produce droplets of 25.1 ± 0.5 µl.

A Low-Cost and High-Resolution Droplet Position Detector for an Intelligent Electrowetting on Dielectric Device.

A low-cost and high-resolution capacitive-to-digital converter integrated circuit is used for droplet position detection in a digital microfluidic system. A field-programmable gate array FPGA is used as the integrated logic hub of the system for a highly reliable and efficient control of the circuit. A fast-fabricating PCB (printed circuit board) substrate microfluidic system is proposed. Smaller actuation threshold voltages than those previously reported are obtained. Droplets (3 µL) are actuated by using a 200 V, 500 Hz modulating pulsed voltage. Droplet positions can be detected and displayed on a PC-based 3D animation in real time. The actuators and the capacitance sensing circuits are implemented on one PCB to reduce the size of the system. With the capacitive droplet position detection system, the PCB-based electrowetting on dielectric device (EWOD) reported in this work has promise in automating immunohistochemistry experiments.