Establishment of low-cost laboratory automation processes using AutoIt and 4-axis robots.

In most small laboratories, many processes are not yet automated because existing laboratory automation solutions are usually expensive and inflexible to use. Examples of this are autosamplers that are only compatible with one specific laboratory instrument or larger liquid handling stations that are expensive and usually self-contained. A flexible and inexpensive way to automate laboratory processes would be to automate existing laboratory equipment with the help of suitable robotic arms. In this study, we investigate the feasibility of such a strategy based on a low-cost 4-axis robot and freely available software. We used the scripting language AutoIt that automates any Windows-based instrument control software. Using these tools, we automated three fundamentally different laboratory processes: a pipetting process, a use as an autosampler for an atomic absorption spectroscopy instrument, and a more complex process involving the inoculation of bacterial cultures. We also integrated a conventional webcam for 2D barcode recognition. Compared to a trained professional who performed all experiments manually, all setups showed no significant differences in accuracy and precision. In summary, the tested system consisting of a 4-axis robot and freely available software is suitable for flexible automation and has potential for even more complex laboratory processes. Limitations such as a lack of collaboration and speed will be addressed in follow-up studies. The system thus represents a well-suited flexible laboratory automation system for both research and teaching purposes.

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.

Well-Based Antibody Arrays.

Multiplex immunoassays are important tools in basic research and diagnostics. The ability to accurately quantify the presence of several antigens within an individual sample all at once has been useful in developing a proteomics view of biology. This in turn has enabled the development of disease-associated immunodiagnostic panels for better prognosis and well-being. Moreover, it is well understood that such multiplexing approaches lend themselves to automation, thereby reducing labor while providing the ability to dramatically conserve both reagent and sample all of which will reduce the cost per test. Here we describe various methods to create and use multiplex immunoassays in the wells of microtiter plates or similar formats.

Assessment of SARS-CoV-2 serological tests for the diagnosis of COVID-19 through the evaluation of three immunoassays: Two automated immunoassays (Euroimmun and Abbott) and one rapid lateral flow immunoassay (NG Biotech).

BACKGROUND: The emergence of new SARS-CoV-2 has promoted the development of new serological tests that could be complementary to RT-PCR. Nevertheless, the assessment of clinical performances of available tests is urgently required as their use has just been initiated for diagnose. OBJECTIVES: The aim of this study was to assess the performance of three immunoassays for the detection of SARS-CoV-2 antibodies. METHODS: Two automated immunoassays (Abbott SARS-CoV-2 CLIA IgG and Euroimmun Anti-SARS-CoV-2 ELISA IgG/IgA assays) and one lateral flow immunoassay (LFIA NG-Test® IgG-IgM COVID-19) were tested. 293 specimens were analyzed from patients with a positive RT-PCR response, from patients with symptoms consistent with COVID-19 but exhibiting a negative response to the RT-PCR detection test, and from control group specimens. Days since symptoms onset were collected from clinical information sheet associated with respiratory tract samples. RESULTS: Overall sensitivity for IgG was equivalent (around 80 %) for CLIA, ELISA and LFIA. Sensitivity for IgG detection, >14 days after onset of symptoms, was 100.0 % for all assays. Overall specificity for IgG was greater for CLIA and LFIA (more than 98 %) compared to ELISA (95.8 %). Specificity was significantly different between IgA ELISA (78.9 %) and IgM LFIA (95.8 %) (p < 0.05). The best agreement was observed between CLIA and LFIA assays (97 %; k = 0.936). CONCLUSION: Excellent sensitivity for IgG detection was obtained >14 days after onset of symptoms for all immunoassays. Specificity was also excellent for IgG CLIA and IgG LFIA. Our study shows that NG-Test® is reliable and accurate for routine use in clinical laboratories.

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.

Prognostic value of DNA ploidy and automated assessment of stroma fraction in prostate cancer.

The combination of DNA ploidy and automatically estimated stroma fraction has been shown to correlate with recurrence and cancer death in colorectal cancer. We aimed to extend this observation and evaluate the prognostic importance of this combined marker in prostate cancer. DNA ploidy status was determined by image cytometry and the stroma fraction was estimated automatically on hematoxylin and eosin stained sections in three tumor samples from each patient to account for tumor heterogeneity. The optimal threshold for low (≤56%) and high (>56%) stroma fraction was identified in a discovery cohort (n = 253). The combined marker was validated in an independent patient cohort (n = 259) with biochemical recurrence as endpoint. The combined marker predicted biochemical recurrence independently in the validation cohort. Multivariable analysis showed that the highest risk of recurrence was observed for patients with samples that had both non-diploid ploidy status and a high stroma fraction (hazard ratio: 2.51, 95% confidence interval: 1.18-5.34). In conclusion, we suggest the combination of DNA ploidy and automatically estimated stroma fraction as a prognostic marker for the risk stratification of prostate cancer patients. It may also be a potential generic marker as concurrent results have been described in colorectal cancer.

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.

Verification of the Performance of a Fully Automated Coagulation Analyzer and Ongoing Assessment of External Quality Assurance Results in an Academic Laboratory in South Africa.

BACKGROUND: Verification of the performance of analytical platforms is indicated prior to adoption of new Technology for patient sample analysis. Acceptance criteria for the performance of coagulation analytical platforms are not always readily available and is complicated by the multiple assays and test principles in this section of the clinical laboratory. Coagulation samples are also prone to pre-analytical, post-sample collection variables potentially interfering with accuracy analysis. METHODS: This verification study assessed the accuracy of the automated STAGO STA-R Max® coagulation analyzers by means of a comparison study of results obtained on the previously validated STAGO STA-R Evolution® analyzer for 22 coagulation parameters on 40 individual patient samples for each parameter. Within- and between- run reproducibility on commercial control material, carry-over from abnormal to normal samples and the interference of bilirubin, hemoglobin and lipids on the chromogenic analytical channel were also assessed. Ongoing evaluation of the analyzer performance was assessed by External Quality Assurance (EQA) scheme participation. RESULTS: The reproducibility (precision) on 2 levels (Normal and Pathological) commercial control material was acceptable with co-efficient of variance (CV) results below the manufacturer target % CVs. The correlation study demonstrated accuracy of results obtained on the analyzers for all parameters except for D-dimers and coagulation Factor VII. Subsequent EQA performance for these two parameters were however satisfactory. Interference from bilirubin, hemoglobin and lipids did occur in the chromogenic channel. No clinically significant carry over from abnormal to normal samples were observed. CONCLUSIONS: The performance of the STAGO STA-R Max® analyzer is acceptable across the full coagulation test repertoire with the exception of the von Willebrand activity assay. Participation in EQA scheme assessments will be an integral part of ongoing monitoring of the performance of this automated analyzer.

Intelligent liver function testing (iLFT): A trial of automated diagnosis and staging of liver disease in primary care.

BACKGROUND & AIMS: Liver function tests (LFTs) are frequently requested blood tests which may indicate liver disease. LFTs are commonly abnormal, the causes of which can be complex and are frequently under investigated. This can lead to missed opportunities to diagnose and treat liver disease at an early stage. We developed an automated investigation algorithm, intelligent liver function testing (iLFT), with the aim of increasing the early diagnosis of liver disease in a cost-effective manner. METHODS: We developed an automated system that further investigated abnormal LFTs on initial testing samples to generate a probable diagnosis and management plan. We integrated this automated investigation algorithm into the laboratory management system, based on minimal diagnostic criteria, liver fibrosis estimation, and reflex testing for causes of liver disease. This algorithm then generated a diagnosis and/or management plan. A stepped-wedged trial design was utilised to compare LFT outcomes in general practices in the 6 months before and after introduction of the iLFT system. Diagnostic outcomes were collated and compared. RESULTS: Of eligible patients with abnormal LFTs, 490 were recruited to the control group and 64 were recruited to the intervention group. The primary diagnostic outcome was based on the general practitioner diagnosis, which agreed with the iLFT diagnosis in 67% of cases. In the iLFT group, the diagnosis of liver disease was increased by 43%. Additionally, there were significant increases in the rates of GP visits after diagnosis and the number of referrals to secondary care in the iLFT group. iLFT was cost-effective with a low initial incremental cost-effectiveness ratio of £284 per correct diagnosis, and a saving to the NHS of £3,216 per patient lifetime. CONCLUSIONS: iLFT increases liver disease diagnoses, improves quality of care, and is highly cost-effective. This can be achieved with minor changes to working practices and exploitation of functionality existing within modern laboratory diagnostics systems. LAY SUMMARY: There is a growing epidemic of advanced liver disease, this could be offset by early detection and management. Checking liver blood tests (LFTs) should be an opportunity to diagnose liver problems, but abnormal results are often incompletely investigated. In this study we were able to substantially increase the diagnostic yield of the abnormal LFTs using the automated intelligent LFT system. With the addition of referral recommendations and management plans, this strategy provides optimum investigation and management of LFTs and is cost saving to the NHS.

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.

Automated monitoring of mouse feeding and body weight for continuous health assessment.

Routine health assessment of laboratory rodents can be improved using automated home cage monitoring. Continuous, non-stressful, objective assessment of rodents unaware that they are being watched, including during their active dark period, reveals behavioural and physiological changes otherwise invisible to human caretakers. We developed an automated feeder that tracks feed intake, body weight, and physical appearance of individual radio frequency identification-tagged mice in social home cages. Here, we experimentally induce illness via lipopolysaccharide challenge and show that this automated tracking apparatus reveals sickness behaviour (reduced food intake) as early as 2-4 hours after lipopolysaccharide injection, whereas human observers conducting routine health checks fail to detect a significant difference between sick mice and saline-injected controls. Continuous automated monitoring additionally reveals pronounced circadian rhythms in both feed intake and body weight. Automated home cage monitoring is a non-invasive, reliable mode of health surveillance allowing caretakers to more efficiently detect and respond to early signs of illness in laboratory rodent populations.

Confirmation of ovulation from urinary progesterone analysis: assessment of two automated assay platforms.

Urinary concentrations of the major progesterone (P4) metabolite pregnanediol-3-glucuronide (PDG) are used to confirm ovulation. We aimed to determine whether automated immunoassay of urinary P4 was as efficacious as PDG to confirm ovulation. Daily urine samples from 20 cycles in 14 healthy women in whom ovulation was dated by ultrasound, and serial weekly samples from 21 women in whom ovulation was unknown were analysed. Daily samples were assayed by two automated P4 immunoassays (Roche Cobas and Abbott Architect) and PDG ELISA. Serial samples were assayed for P4 by Architect and PDG by ELISA. In women with detailed monitoring of ovulation, median (95% CI) luteal phase increase was greatest for PDG, 427% (261-661), 278% (187-354) for P4 Architect and least for P4 Cobas, 146% (130-191), p < 0.0001. Cobas P4 also showed marked inaccuracy in serial dilution. Similar ROC AUCs were observed for individual threshold values and two-sample percent rise analyses for P4 Architect and PDG (both >0.92). In serial samples classified as (an)ovulatory by PDG, P4 Architect gave ROC AUC 0.95 (95% CI 0.89 to 1.01), with sensitivity and specificity for confirmation of ovulation of 0.90 and 0.91 at a cutoff of 1.67 µmol/mol. Automated P4 may potentially be as efficacious as PDG ELISA but research from a range of clinical settings is required.

Automated assessment of steatosis in murine fatty liver.

Although mice are commonly used to study different aspects of fatty liver disease, currently there are no validated fully automated methods to assess steatosis in mice. Accurate detection of macro- and microsteatosis in murine models of fatty liver disease is important in studying disease pathogenesis and detecting potential hepatotoxic signature during drug development. Further, precise quantification of macrosteatosis is essential for quantifying effects of therapies. Here, we develop and validate the performance of automated classifiers built using image processing and machine learning methods for detection of macro- and microsteatosis in murine fatty liver disease and study the correlation of automated quantification of macrosteatosis with expert pathologist's semi-quantitative grades. The analysis is performed on digital images of 27 Hematoxylin & Eosin stained murine liver biopsy samples. An expert liver pathologist scored the amount of macrosteatosis and also annotated macro- and microsteatosis lesions on the biopsy images using a web-application. Using these annotations, supervised machine learning and image processing techniques, we created classifiers to detect macro- and microsteatosis. For macrosteatosis prediction, the model's precision, sensitivity and area under the receiver operator characteristic (AUROC) were 94.2%, 95%, 99.1% respectively. When correlated with pathologist's semi-quantitative grade of steatosis, the model fits with a coefficient of determination value of 0.905. For microsteatosis prediction, the model has precision, sensitivity and AUROC of 79.2%, 77%, 78.1% respectively. Validation by the expert pathologist of classifier's predictions made on unseen images of biopsy samples showed 100% and 63% accuracy for macro- and microsteatosis, respectively. This novel work demonstrates that fully automated assessment of steatosis is feasible in murine liver biopsies images. Our classifier has excellent sensitivity and accuracy for detection of macrosteatosis in murine fatty liver disease.

3-D motion capture for long-term tracking of spontaneous locomotor behaviors and circadian sleep/wake rhythms in mouse.

BACKGROUND: Locomotor activity provides an index of an animal's behavioral state. Here, we report a reliable and cost-effective method that allows long-term (days to months) simultaneous tracking of locomotion in mouse cohorts (here consisting of 24 animals). NEW METHOD: The technique is based on a motion capture system used mainly for human movement study. A reflective marker was placed on the head of each mouse using a surgical procedure and labeled animals were returned to their individual home cages. Camera-recorded data of marker displacement resulting from locomotor movements were then analyzed with custom built software. To avoid any data loss, data files were saved every hour and automatically concatenated. Long-term recordings (up to 3 months) with high spatial (<1mm) and temporal (up to 100Hz) resolution of animal movements were obtained. RESULTS: The system was validated by analyzing the spontaneous activity of mice from post-natal day 30-90. Daily motor activity increased up to 70days in correspondence with maturational changes in locomotor performance. The recorded actigrams also permitted analysis of circadian and ultradian rhythms in cohort sleep/wake behavior. COMPARISON WITH EXISTING METHOD(S): In contrast to traditional session-based experimental approaches, our technique allows locomotor activity to be recorded with minimal experimenter manipulation, thereby minimizing animal stress. CONCLUSIONS: Our method enables the continuous long-term (up to several months) monitoring of tens of animals, generating manageable amounts of data at minimal costs without requiring individual dedicated devices. The actigraphic data collected allows circadian and ultradian analysis of sleep/wake behaviors to be performed.

Distinguish self- and hetero-perceived stress through behavioral imaging and physiological features.

Stress reactivity is a complex phenomenon associated to multiple and multimodal expressions. Response to stressors has an obvious survival function and may be seen as an internal regulation to adapt to threat or danger. The intensity of this internal response can be assessed as the self-perception of the stress response. In species with social organization, this response also serves a communicative function, so-called hetero-perception. Our study presents multimodal stress detection assessment - a new methodology combining behavioral imaging and physiological monitoring for analyzing stress from these two perspectives. The system is based on automatic extraction of 39 behavioral (2D+3D video recording) and 62 physiological (Nexus-10 recording) features during a socially evaluated mental arithmetic test. The analysis with machine learning techniques for automatic classification using Support Vector Machine (SVM) show that self-perception and hetero-perception of social stress are both close but different phenomena: self-perception was significantly correlated with hetero-perception but significantly differed from it. Also, assessing stress with SVM through multimodality gave excellent classification results (F1 score values: 0.9±0.012 for hetero-perception and 0.87±0.021 for self-perception). In the best selected feature subsets, we found some common behavioral and physiological features that allow classification of both self- and hetero-perceived stress. However, we also found the contributing features for automatic classifications had opposite distributions: self-perception classification was mainly based on physiological features and hetero-perception was mainly based on behavioral features.

A versatile and low-cost open source pipetting robot for automation of toxicological and ecotoxicological bioassays.

In the past decades, bioassays and whole-organism bioassay have become important tools not only in compliance testing of industrial chemicals and plant protection products, but also in the monitoring of environmental quality. With few exceptions, such test systems are discontinuous. They require exposure of the biological test material in small units, such as multiwell plates, during prolonged incubation periods, and do not allow online read-outs. It is mostly due to these shortcomings that applications in continuous monitoring of, e.g., drinking or surface water quality are largely missing. We propose the use of pipetting robots that can be used to automatically exchange samples in multiwell plates with fresh samples in a semi-static manner, as a potential solution to overcome these limitations. In this study, we developed a simple and low-cost, versatile pipetting robot constructed partly using open-source hardware that has a small footprint and can be used for online monitoring of water quality by means of an automated whole-organism bioassay. We tested its precision in automated 2-fold dilution series and used it for exposure of zebrafish embryos (Danio rerio)-a common model species in ecotoxicology-to cadmium chloride and permethrin. We found that, compared to conventional static or semi-static exposure scenarios, effects of the two chemicals in zebrafish embryos generally occurred at lower concentrations, and analytically verified that the increased frequency of media exchange resulted in a greater availability of the chemical. In combination with advanced detection systems this custom-made pipetting robot has the potential to become a valuable tool in future monitoring strategies for drinking and surface water.

The Invisible Army.

The "invisible army" of clinical microbiologists is facing major changes and challenges. The rate of change in both the science and technology is accelerating with no end in sight, putting pressure on our army to learn and adapt as never before. Health care funding in the United States is undergoing dramatic change which will require a new set of assumptions about how clinical microbiology is practiced here. A major challenge facing the discipline is the replacement of a generation of clinical microbiologists. In my opinion, it is incumbent on us in the invisible army to continue to work with the American Society for Microbiology (ASM) in meeting the future challenges faced by our discipline. In this commentary, I will first discuss some recent history of clinical microbiology within ASM and then some current challenges we face.

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.