Analytical and Clinical Validation of a Non-Ristocetin Based VWF Assay on 2 Automated Analyzers in a Large Reference Laboratory.

BACKGROUND: Historically, von Willebrand factor (VWF) activity assays utilized ristocetin despite limitations including poor limits of detection and high imprecision. Newer VWF activity assays such as the INNOVANCE® VWF Ac assay, however, do not rely on ristocetin to measure platelet-dependent VWF function. The purpose of this study was to evaluate the analytical and clinical performance of the Siemens Healthineers INNOVANCE VWF Ac Assay on the Siemens BCS® XP and the Sysmex® CS-2500 systems in a large reference laboratory setting. METHODS: Performance indicators for the INNOVANCE VWF Ac assay were the limit of quantitation (LoQ), precision, and method comparison. Method comparison studies were performed using remnant plasma patient samples from routine coagulation tests and analyzed using both the INNOVANCE VWF Ac assay and the Siemens Healthineers ristocetin-dependent BC von Willebrand Reagent. RESULTS: Evaluation of the INNOVANCE VWF Ac assay on the BCS® XP and CS-2500 systems demonstrated good precision and a lower LoQ compared to the BC von Willebrand Reagent. Method comparisons support the use of the INNOVANCE VWF Ac assay on the BCS® XP and CS-2500 systems to measure platelet-dependent VWF function. The INNOVANCE VWF Ac assay was able to further assist in von Willebrand disease classification in 6/7 (86%) samples when the result was below the LoQ for the BC von Willebrand Reagent (ristocetin cofactor activity). CONCLUSIONS: These data are consistent with the 2021 American Society of Hematology/International Society on Thrombosis and Haemostasis/National Hemophilia Foundation/World Federation of Hemophilia von Willebrand disease guidelines that suggest using newer assays such as the INNOVANCE VWF Ac assay in place of ristocetin cofactor activity assays.

Development and transfer of automated methods in neuroscience: The DADTA.

In the second half of the 20th century, neuroscientists across North America developed automated systems for use in their research laboratories. Their decisions to do so were complex and contingent, partly a result of global reasons, such as the need to increase efficiency and flexibility, and partly a result of local reasons, such as the need to amend perceived biases of earlier research methodologies. Automated methods were advancements but raised several challenges. Transferring a system from one location to another required that certain components of the system be standardized, such as the hardware, software, and programming language. This proved difficult as commercial manufacturers lacked incentives to create standardized products for the few neuroscientists working towards automation. Additionally, investing in automated systems required massive amounts of time, labor, funding, and computer expertise. Moreover, neuroscientists did not agree on the value of automation. My brief history investigates Karl Pribram's decisions to expand his newly created automated system by standardizing equipment, programming, and protocols. Although he was an eminent Stanford neuroscientist with strong institutional support and computer know-how, the development and transfer of his automated behavioral testing system was riddled with challenges. For Pribram and neuroscience more generally, automation was not so automatic.

Achieving near-zero particle generation by simplicity of design-A compliant-mechanism-based gripper for clean-room environments.

Lab Automation facilitates high-throughput processes and improves reproducibility and efficiency while removing human action, primary source of contaminating particles. Handling poses a risk of contamination due to close contact with the objects. We propose a novel gripper (CrocoGrip) relying on compliant mechanisms to reduce the amount of contaminating particles generated by the gripper rather than preventing their emission, the latter being the common approach in current grippers. Our novel gripper is actuated by linear solenoids and purely relies on deformation for its motion. As a result, abrasive behavior and, therefore, the generation of particles is reduced without the need for additional sealing. We experimentally proved that only particles smaller than 3.0µm are emitted by the gripper, with a large proportion of the particles being generated by the actuation. The CrocoGrip fulfills the demands of ISO14644 class 5. The gripping relies on the deformation energy of the compliant mechanism, making the gripping energy-efficient and safe. The maximum gripping force achieved by the CrocoGrip was 5.5N. Because the force transmitted to the handling object depends on the design of the gripping jaws, which are interchangeable, the force can be reduced for more sensible handling objects. Using three different sets of jaws, CrocoGrip was able to handle a microplate in SBS-standard, a 50mL Falcon tube, and a Ø60mm Petri dish using a robotic arm. Due to the monolithic design of the CrocoGrip and, as a result, the need for few components, we achieve a simplicity of design, making cleaning, sterilization and maintenance easy, even for nonexperts. The CrocoGrip exploits the advantages of compliant mechanisms, especially for applications requiring clean-room environments. This approach of compliant-mechanism-based grippers enables an increase in the cleanliness of handling processes without an increase in system complexity of the gripper to facilitate the lab automation of highly sensible processes, such as in tissue engineering.

AI Driven Lab-on-Chip Cartridge for Automated Urinalysis.

After haematology, urinalysis is the most common biological test performed in clinical settings. Hence, simplified workflow and automated analysis of urine elements are of absolute necessities. In the present work, a novel lab-on-chip cartridge (Gravity Sedimentation Cartridge) for the auto analysis of urine elements is developed. The GSC consists of a capillary chamber that uptakes a raw urine sample by capillary force and performs particles and cells enrichment within 5 min through a gravity sedimentation process for the microscopic examination. Centrifugation, which is necessary for enrichment in the conventional method, was circumvented in this approach. The AI100 device (Image based autoanalyzer) captures microscopic images from the cartridge at 40x magnification and uploads them into the cloud. Further, these images were auto-analyzed using an AI-based object detection model, which delivers the reports. These reports were available for expert review on a web-based platform that enables evidence-based tele reporting. A comparative analysis was carried out for various analytical parameters of the data generated through GSC (manual microscopy, tele reporting, and AI model) with the gold standard method. The presented approach makes it a viable product for automated urinalysis in point-of-care and large-scale settings.

Piston-driven automated liquid handlers.

Laboratory capacities are often limited by time-consuming manual repetitive procedures rather than analysis time itself. While modern instruments are typically equipped with an autosampler, sample preparation often follows manual procedures including many labor-intensive, monotonous tasks. Particularly, for a high number of samples, well plates, and low microliter pipetting, manual preparation is error-prone often requiring repeated experiments. Sampling and sample preparation can account for greater analytical variability than instrument analysis. Repetitive tasks such as liquid handling benefit strongly from technological advances and led to the increasing applications of various automated liquid handlers (ALHs). In this review, we discuss the considerations for ALHs in the microliter range and highlight advantages and challenges when transforming from manual to automated workflows. We strongly focused on differences in liquid handling and outlined advantages due to sensor-controlled pipetting. ALHs can substantially improve costs-effectiveness and laboratory capacity. This is a consequence of increased efficiency, and throughput of laboratories while simultaneously raising data quality. Additionally, ALHs can improve safety, documentation of data, and sustainability. While automation requires careful consideration and resource demanding implementation, we believe it offers numerous advantages and can help to transform modern laboratories.

High-Throughput Selection and Characterisation of Aptamers on Optical Next-Generation Sequencers.

Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process. 'High-throughput sequencing fluorescent ligand interaction profiling' (HiTS-FLIP) significantly increases the selection efficiency and is consequently a very powerful and versatile technology for the selection of high-performance aptamers. It is the first experiment to allow the direct and quantitative measurement of the affinity and specificity of millions of aptamers simultaneously by harnessing the potential of optical next-generation sequencing platforms to perform fluorescence-based binding assays on the clusters displayed on the flow cells and determining their sequence and position in regular high-throughput sequencing. Many variants of the experiment have been developed that allow automation and in situ conversion of DNA clusters into base-modified DNA, RNA, peptides, and even proteins. In addition, the information from mutational assays, performed with HiTS-FLIP, provides deep insights into the relationship between the sequence, structure, and function of aptamers. This enables a detailed understanding of the sequence-specific rules that determine affinity, and thus, supports the evolution of aptamers. Current variants of the HiTS-FLIP experiment and its application in the field of aptamer selection, characterisation, and optimisation are presented in this review.

Cytomegalovirus Viral Load in Transplanted Patients Using the NeuMoDx™ (Qiagen) Automated System: A 1-Month Experience Feedback.

Cytomegalovirus (CMV) reactivations represent a significant morbidity and mortality problem in transplant patients. Reliable and rapid measurement of CMV viral load is a key issue for optimal patient management. We report here the evaluation of NeuMoDx™ (Qiagen) in a routine hospital setting (University Hospitals of Marseille, France) in comparison with our classical reference technique R-GENE. During one month, 719 CMV viral loads from 507 patients were measured in parallel in both techniques. Using the ROC (receiver operating characteristic) curve and our biological experience we suggest that values <52 IU/mL (geometric mean) correspond to negative samples, values >140 IU/mL (Fowlkes-Mallows index) correspond to quantifiable positive results and values ranging from 52 to 140 IU/mL represent non-quantifiable positive results. Follow-up of 15 transplant patients who developed CMV reactivation during the study showed that NeuMoDx™ provided higher viral load measurement during the first two weeks of follow-up for three patients. These important intra-individual variations resulted in a significant median increase considering the whole data set (6.7 points of difference expressed as a percentage of the initial viral load). However, no difference between the two techniques was noticeable after two weeks of treatment. Subsequent to this first study we conclude that NeuMoDx™, used with optimized logistics and an adapted threshold, allows a rapid CMV viral load measurement and that its use does not lead to any difference in patient management compared to the reference technique R-GENE®.

GMP compliant isolation of mucosal epithelial cells and fibroblasts from biopsy samples for clinical tissue engineering.

Engineered epithelial cell sheets for clinical replacement of non-functional upper aerodigestive tract mucosa are regulated as medicinal products and should be manufactured to the standards of good manufacturing practice (GMP). The current gold standard for growth of epithelial cells for research utilises growth arrested murine 3T3 J2 feeder layers, which are not available for use as a GMP compliant raw material. Using porcine mucosal tissue, we demonstrate a new method for obtaining and growing non-keratinised squamous epithelial cells and fibroblast cells from a single biopsy, replacing the 3T3 J2 with a growth arrested primary fibroblast feeder layer and using pooled Human Platelet lysate (HPL) as the media serum supplement to replace foetal bovine serum (FBS). The initial isolation of the cells was semi-automated using an Octodissociator and the resultant cell suspension cryopreservation for future use. When compared to the gold standard of 3T3 J2 and FBS containing medium there was no reduction in growth, viability, stem cell population or ability to differentiate to mature epithelial cells. Furthermore, this method was replicated with Human buccal tissue, providing cells of sufficient quality and number to create a tissue engineered sheet.

The 3D reconstructed skin micronucleus assay using imaging flow cytometry and deep learning: A proof-of-principle investigation.

The reconstructed skin micronucleus (RSMN) assay was developed in 2006, as an in vitro alternative for genotoxicity evaluation of dermally applied chemicals or products. In the years since, significant progress has been made in the optimization of the assay, including publication of a standard protocol and extensive validation. However, the diverse morphology of skin cells makes cell preparation and scoring of micronuclei (MN) tedious and subjective, thus requiring a high level of technical expertise for evaluation. This ultimately has a negative impact on throughput and the assay would benefit by the development of an automated method which could reduce scoring subjectivity while also improving the robustness of the assay by increasing the number of cells that can be scored. Imaging flow cytometry (IFC) with the ImageStream®X Mk II can capture high-resolution transmission and fluorescent imagery of cells in suspension. This proof-of-principle study describes protocol modifications that enable such automated measurement in 3D skin cells following exposure to mitomycin C and colchicine. IFC was then used for automated image capture and the Amnis® Artificial Intelligence (AAI) software permitted identification of binucleated (BN) cells with 91% precision. On average, three times as many BN cells from control samples were evaluated using IFC compared to the standard manual analysis. When IFC MNBN cells were visually scored from within the BN cell images, their frequency compared well with manual slide scoring, showing that IFC technology can be applied to the RSMN assay. This method enables faster time to result than microscope-based scoring and the initial studies presented here demonstrate its capability for the detection of statistically significant increases in MNBN frequencies. This work therefore demonstrates the feasibility of combining IFC and AAI to automate scoring for the RSMN assay and to improve its throughput and statistical robustness.

Analytical Performance Evaluation of Automated Coagulation Analyzer CP3000 for Routine and Special Coagulation Assays.

CP3000 coagulation analyzer is a high-throughput, fully automated coagulation analyzer. The objective of this study was to evaluate the analytical performance of CP3000 coagulation system for general and special coagulation analyses. Quality control materials and patient samples were used to evaluate the analytical performance of CP3000 coagulation system. Precision, carryover, linearity, comparability with ACL-TOP 700 coagulation system, and verification of reference range were evaluated or performed according to Clinical and Laboratory Standards Institute guidelines. Within-run and between-run precisions were below 5% for both normal and abnormal ranges. There was no detectable carryover. The linearity of antithrombin and fibrinogen were excellent. The comparability between CP3000 and ACL-TOP 700 coagulation systems was acceptable except for activated partial thromboplastin time and thrombin time due to differences in reagent composition. Reference ranges proposed by the manufacturer were verified to be acceptable. CP3000 coagulation system is a reliable system that can be used to perform routine and special coagulation tests rapidly and accurately. Because of its small footprint as an additional advantage, the implementation of CP3000 coagulation system can be efficient in hospital laboratories of various sizes.

High diversity droplet microfluidic libraries generated with a commercial liquid spotter.

Droplet libraries consisting of many reagents encapsulated in separate droplets are necessary for applications of microfluidics, including combinatorial chemical synthesis, DNA-encoded libraries, and massively multiplexed PCR. However, existing approaches for generating them are laborious and impractical. Here, we describe an automated approach using a commercial array spotter. The approach can controllably emulsify hundreds of different reagents in a fraction of the time of manual operation of a microfluidic device, and without any user intervention. We demonstrate that the droplets produced by the spotter are similarly uniform to those produced by microfluidics and automate the generation of a ~ 2 mL emulsion containing 192 different reagents in ~ 4 h. The ease with which it can generate high diversity droplet libraries should make combinatorial applications more feasible in droplet microfluidics. Moreover, the instrument serves as an automated droplet generator, allowing execution of droplet reactions without microfluidic expertise.

Rapid Throughput Concentration-Response Analysis of Human Taste Discrimination.

Human taste threshold measurements often are used to infer tastant receptor functionality. However, taste thresholds can be influenced by receptor-independent variables. Examination of the full range of taste-active concentrations by taste discrimination has been hampered by logistics of testing multiple concentrations in replicate with human subjects. We developed an automated rapid throughput operant methodology for taste discrimination and applied it to concentration-response analysis of human taste. Tastant solutions (200 µl) drawn from a 96-well plate and self-administered to the tongue served as discriminative stimuli for money-reinforced responses on a touch-sensitive display. Robust concentration-response functions for "basic taste" stimuli were established, with particular focus on agonists of the taste 1 receptor member 2-taste 1 receptor member 3 heterodimer receptor (TAS1R2/R3). With a training cue of 100 mM sucrose, EC50 values of 56, 79, and 310 µM and 40 mM were obtained for rebaudioside A, sucralose, acesulfame potassium, and sucrose, respectively. Changing the sucrose training cue to 300 mM had no impact, but changing to 30 mM resulted in slight leftward shifts in potencies. A signal detection method also was used to determine values of d', a probabilistic value for discriminability, which indicated that 5 mM was near the limits of detection for sucrose. With repeated testing, both EC50 values and 5 mM sucrose d' values were established for each individual subject. The results showed little correspondence between threshold sensitivities and EC50 values for sucrose. We conclude that concentration-response analysis of taste discrimination provides a more reliable means of inferring receptor function than measurement of discriminability at the lowest detectable tastant concentrations. SIGNIFICANCE STATEMENT: Many inferences about human tastant receptor functionality have been made from taste threshold measurements, which can be influenced by variables unrelated to receptors. We herein report a new methodology that enables rigorous concentration-response analysis of human taste discrimination and its use toward quantitative characterization of tastant agonist activity. Our data suggest that taste discrimination concentration-response functions are a more reliable reflection of underlying receptor activity than threshold measures obtained at the lowest detectable tastant concentrations.

Leveraging Automation toward Development of a High-Throughput Gene Expression Profiling Platform.

We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel.

The design and application of an automated microscope developed based on deep learning for fungal detection in dermatology.

BACKGROUND: Light microscopy to study the infection of fungi in skin specimens is time-consuming and requires automation. OBJECTIVE: We aimed to design and explore the application of an automated microscope for fungal detection in skin specimens. METHODS: An automated microscope was designed, and a deep learning model was selected. Skin, nail and hair samples were collected. The sensitivity and the specificity of the automated microscope for fungal detection were calculated by taking the results of human inspectors as the gold standard. RESULTS: An automated microscope was built, and an image processing model based on the ResNet-50 was trained. A total of 292 samples were collected including 236 skin samples, 50 nail samples and six hair samples. The sensitivities of the automated microscope for fungal detection in skin, nails and hair were 99.5%, 95.2% and 60%, respectively, and the specificities were 91.4%, 100% and 100%, respectively. CONCLUSION: The automated microscope we developed is as skilful as human inspectors for fungal detection in skin and nail samples; however, its performance in hair samples needs to be improved.

Hemoglobin point-of-care testing in rural Gambia: Comparing accuracy of HemoCue and Aptus with an automated hematology analyzer.

BACKGROUND: Anemia is one of the most impactful nutrient deficiencies in the world and disproportionately affects children in low-resource settings. Point-of-care devices (PoCDs) measuring blood hemoglobin (Hb) are widely used in such settings to screen for anemia due to their low cost, speed, and convenience. Here we present the first iteration of Aptus, a new PoCD which measures Hb and hematocrit (HCT). AIM: To evaluate the accuracy of Aptus and HemoCue® Hb 301 against an automated hematology analyzer (Medonic®) in Gambian children aged 6-35 months and the Aptus' usage in the field. METHODS: Aptus, HemoCue® and Medonic® were compared using venous blood (n = 180), and Aptus and HemoCue® additionally using capillary blood (n = 506). Agreement was estimated using Bland-Altman analysis and Lin's concordance. Usage was assessed by error occurrence and user experience. RESULTS: Mean Hb values in venous blood did not significantly differ between Aptus and HemoCue® (10.44±1.05 vs 10.56±0.93g/dl, p>0.05), but both measured higher Hb concentrations than Medonic® (9.75±0.99g/dl, p<0.0001). Lin's coefficient between Aptus and Medonic® was rc = 0.548, between HemoCue® and Medonic® rc = 0.636. Mean bias between the PoCDs venous measurements was -0.11g/dl with limits of agreement (LoA) -1.63 and 1.40g/dl. The bias was larger for the comparisons between the Medonic® and both Aptus (0.69g/dl, LoA 0.92 and 2.31g/dl) and HemoCue® (0.81g/dl, LoA 0.17 and 1.78g/dl). ROC curves showed an AUC of 0.933 in HemoCue® and 0.799 in Aptus. Capillary Hb was higher with Aptus than HemoCue® (10.33±1.11g/dl vs 10.01±1.07g/dl, p<0.0001). Mean bias was 0.32g/dl with LoA of -1.91 and 2.54g/dl. Aptus' usage proved intuitive, yet time-to-results and cuvettes could be improved. CONCLUSION: Both PoCDs showed a relatively limited bias but large LoA. Aptus and HemoCue® showed similar accuracy, while both overestimated Hb levels. Aptus showed promise, with its operation unimpaired by field conditions as well as being able to show HCT values.

[Digital morphology analyzers in hematology: a French survey]. / État des lieux des pratiques d'utilisation du microscope automatisé en hématologie en France.

Digital morphology hematology analyzers are becoming more prevalent in laboratories Aims: investigate practices and assess the benefits and limits of digital automated microscopy in hematology. METHODS: questionnaire sent by e-mail in 2018 to French public and private laboratories. RESULTS: out of 118 responses (56 private, 62 public), 117 participants had a CellaVision® microscope, 1 had a West Medica®. Practices were sometimes different, especially in the choice of smears to be digitized or for quality controls (16.1% had internal quality controls, 48.3% external quality controls); 62.1% never used the red blood cell (RBC) characterization tool; the number of cells counted varied from 100 to 400. The study reported a high rate of agreement for these benefits: traceability (95.7%), staff training (94.1%), eye strain (91.4%), risk of error (87.2%), time saving (83.6%). Among the disadvantages, apart from the inadequate search for platelets clumps (93.2%), the agreement rates were often lower: adaptation to digital images (61.2%), difficult assessment of atypical morphologies (49.6%) or RBC morphology (49.6%). CONCLUSION: despite well-established benefits, standardization of practices and technical improvement are still needed.

[Use of « Reference change value ¼ in medical biology: about two examples: total PSA and hemoglobin]. / Utilisation de la « Reference change value ¼ en biologie médicale : à propos de deux exemples : PSA total et hémoglobine.

The interpretation of the variation between the results of two dosages performed on the same patient is generally quite empirical. It is usually based on the experience of the biologist or physician. Through two examples, total PSA and hemoglobin, we hoped to set up an indicator of the significance variation between results: The Reference change value or RCV to provide assistance to the validator biologist and prescriber based on measured statistical arguments. This article describes the methodology used for the RCV calculation, the formatting on analysis reports and the limitations of the system.