The Rocky Road to a Digital Lab.

The pharmaceutical industry has begun incorporating continuous manufacturing technology in synthetic routes toward active pharmaceutical ingredients (APIs). The development of smart manufacturing routes can be accelerated by utilizing digitalization, process analytical technology (PAT), and data-rich experimentation from an early stage. Here, we present the key aspects of implementing automated flow chemistry reactor platforms with real-time process analytics. Based on our experiences in this field, we aim to highlight the potential of these platforms to conduct self-optimization, automated reaction model building, dynamic experiments and to implement advanced process control strategies.

An Optimized Melanin Depigmentation Method for Histopathological and Immunohistochemical Staining of Formalin-Fixed Paraffin-Embedded Tissues.

Introduction. The difficulty in diagnosis of severe melanocytic lesions is a problem to be overcome in pathological practice. Melanin bleaching is an effective approach to ameliorate melanin disturbances in severely pigmented lesions. Although various methods for improving melanin pigmentation in immunohistochemical staining have been reported, these depigmentation methods still need to be optimized and standardized. In this study, the coloring efficiency of 3,3'-diaminobenzidine (DAB) and alkaline phosphatase (AP) after melanin depigmentation was compared under the automatic immunohistochemical staining platform. Methods. The applicability of the optimized depigmentation method was validated in 10 formalin-fixed paraffin-embedded (FFPE) blocks of ocular melanoma tissues. Specimens were demelaninized with 10% hydrogen peroxide at 60°C for immunohistochemical staining (Melan-A and SOX10), and tissue chromogenic staining was performed with DAB and AP detection systems, respectively. Results. The optimized depigmentation method including immunohistochemistry (IHC) could be completed in 3 h, effectively preserving cell morphology and immunoreactivity. Among these, the color-rendering effect and contrast of AP are better than DAB. Conclusion. This optimized method can effectively remove melanin and improve the accuracy of IHC staining interpretation. AP staining has better visibility and readability without the interference of residual melanin. The comparison results showed that after melanin depigmentation, the immunohistochemical staining agent was replaced with red AP, which avoided the misjudgment caused by brown DAB when melanin depigmentation was incomplete. This improved method can be applied to future histopathological and immunohistochemical staining of melanin-deposited tissues.

Development and clinical applications of an enclosed automated targeted NGS library preparation system.

The rapid development of next-generation sequencing (NGS) technology has promoted its wide clinical application in precision medicine for oncology. However, laborious and time-consuming manual operations, highly skilled personnel requirements, and cross-contamination are major challenges for the clinical implementation of NGS technology-based tests. The Automated NGS Diagnostic Solutions (ANDiS) 500 system is a fully enclosed cassette-dependent automated NGS library preparation system. This platform could produce qualified targeted amplicon library in three steps with only 15 min of hands-on time. Rigorous cross-contamination test using simulated contaminant plasmids confirmed that the design of disposable cassette guarantees zero sample cross-contamination. The BRCA1 and BRCA2 mutation detection panel and gastrointestinal cancer-related gene analysis panel for the ANDiS 500 platform showed 100% accuracy and precision in detecting germ-line mutations and somatic mutations respectively. Furthermore, those panels showed 100% concordance with verified methods in a prospective cohort study enrolling 363 patients and a cohort of 45 pan-cancer samples. In conclusion, the ANDiS 500 automated platform could overcome major challenges for implementing NGS assays clinically and is eligible for routine clinical tests.

Automated Mobile Hot Mist Generator: A Quest for Effectiveness in Fruit Horticulture.

The study relates to the use of automated plant protection systems in agriculture. The article presents a proprietary automated mobile platform with an aerosol generator of hot mist. Furthermore, the cause of the loss of a chemical preparation in the spraying of plant protection products on the tree crown was determined in the course of field research. A statistical analysis of the results of experiment was carried out and the effect of droplet size on leaf coating density was determined. The manuscript presents a diagram of the degree of penetration of the working solution as it drops into the crown of the tree, as well as a cross-sectional graph of the permeability of the spray from the projection of the fruit tree crown. The most effective modes of operation of the automated mobile platform for spraying plant protection products with a mist generator aggregate were established. Analysis of the results shows that the device meets the spraying requirements of the procedure for spraying plant protection products. The novelty of this research lies in the optimal modes identified by movement of the developed automated mobile platform and the parameters of plant treatment with protective equipment when using a hot mist generator. The following mode parameters were established: the speed of the automated platform was 3.4 km/h, the distance to the crown of the tree was 1.34 m, and the flow rate of the working fluid was 44.1 L/h. Average fuel consumption was 2.5 L/h. Effective aerosol penetration reduced the amount of working fluid used by up to 50 times.

Integrated Single Microbead-Arrayed µ-Fluidic Platform for the Automated Detection of Multiplexed Biomarkers.

An automated, single microbead-arrayed µ-fluidic immunoassay (AMIA) device is innovatively devised in this study, which enables the highly sensitive and simultaneous detection of multiplex biomarkers with fully automatic operations. The AMIA platform not only achieves automated assay processing and multiplexed target detection by integrating single microbead manipulation, sample loading, multistep washing, and immunoreaction on a microfluidic chip but also confers high sensitivity due to the highly efficient signal enriching effect on a single microbead by the use of only a routine sandwich immunoreaction. As such, as low as the pg/mL level of multiplexed protein biomarkers can be simultaneously determined in a quite small volume of serum (∼20 µL is enough), which can well meet the clinical demand for disease screening and prognosis. What is more, the detection results of several clinically important biomarkers in clinical samples with the AMIA platform exhibit excellent consistency with those obtained by using a standard clinical test. Thus, in virtue of the excellent features in terms of high sensitivity, multiplexing capability, generality, and high degree of automation, the AMIA provides a practical and user-friendly platform for assaying different biomarkers in clinical diagnostics and point-of-care testing.

18F[AlF]-radiolabelled Peptides on the Automated Synthesis Platform: Translating the Laboratory Bench Work to Bedside.

Using radiolabelled peptides that bind, with high affinity and specificity, to receptors on tumour cells is one of the most promising fields in modern molecular imaging and targeted radionuclide therapy (1). In the emergence of molecular imaging and nuclear medicine diagnosis and therapy, albeit theranostic, radiolabelled peptides have become vital tools for in vivo visualisation and monitoring physiological and biochemical processes on molecular and cellular levels (2). This approach may benefit patients in the era of personalised medicine.

Improved assay protocol for measurement of ammonia on the Roche Cobas 8000 automated platform.

INTRODUCTION: Ammonia is a metabolite of protein catabolism that, when elevated, may be toxic for tissues, especially for the central nervous system. Elevated ammonia in blood is an indicator and a prognostic factor for hepatic and kidney disease or inherited metabolic disorders in nitrogen metabolism. The accuracy of ammonia determination is influenced by sampling condition, handling, storage and assay itself. Our and other laboratories have been experiencing high frequencies sample error flags while measuring ammonia with glutamate dehydrogenase method on Roche Cobas 8000 platform. To reduce the number of error flags we adapted Roche NH3L protocol by incorporation of an additional onboard routine step for sample pre-dilution. MATERIAL AND METHODS: The AMC NH3L is an adaptation of Roche protocol that uses four fold pre-dilution of the sample in the rerun prior to the analysis. It was assessed for 1.occurrence of absorbance error flags, 2.precision, 3.correlation with Roche method and 4.interference by hemolysis, icterus and lipemia. RESULTS: The AMC NH3L adaptation demonstrates acceptable within-run and total precision. Comparison studies show no differences between the Roche rerun application and AMC NH3L adaptation. The AMC NH3L adaptation solves 78% of absorbance errors and for samples with high ammonia concentration is less affected by interferences from icterus and hemolysis than the Roche rerun application. CONCLUSION: The AMC NH3L adaptation is less prone to instrument error flags and for samples with high ammonia concentration, is more robust to endogenous interferences. The AMC NH3L adaptation is viable alternative to the Roche protocol for the ammonia measurement.

Spatio-Temporal Optimization of Perishable Goods' Shelf Life by a Pro-Active WSN-Based Architecture.

The waste in the perishable goods supply-chain has prompted many global organizations (e.g., FAO and WHO), to develop the Hazard Analysis and Critical Control Points (HACCP) protocol that ensures a high degree of food quality, minimizing the losses in all the stages of the farm-to-fork chain. It has been proven that good warehouse management practices improve the average life of perishable goods. The advances in wireless sensors network (WSN) technology offers the possibility of a "smart" storage organization. In this paper, a low cost reprogrammable WSN-based architecture for functional warehouse management is proposed. The management is based on the continuous monitoring of environmental parameters (i.e., temperature, light exposure and relative humidity), and on their combination to extract a spatial real-time prediction of the product shelf life. For each product, the quality decay is computed by using a 1st order kinetic Arrhenius model to the whole storage site area. It strives to identify, in a way compatible with the other products' shelf lives, the position within the warehouse that maximizes the food expiration date. The shelf life computing and the "first-expired first-out" logistic problem are entrusted to a Raspberry Pi-based central unit, which manages a set of automated pallet transporters for the displacement of products, according to the computed shelf lives. The management unit supports several commercial light/temperature/humidity sensor solutions, implementing ZigBee, Bluetooth and HTTP-request interfaces. A proof of concept of the presented pro-active WSN-based architecture is also shown. Comparing the proposed monitoring system for the storage of e.g., agricultural products, with a typical one, the experimental results show an improvement of the expected expiration date of about 1.2 ± 0.5 days, for each pallet, when placed in a non-refrigerated environment. In order to stress the versatility of the WSN solution, a section is dedicated to the implemented system user interfaces that highlight detecting critical situations and allow timely automatic or human interventions, minimizing the latter.

Multiparametric Profiling for Identification of Chemosensitizers against Gram-Negative Bacteria.

Antibiotic resistance is now a worldwide therapeutic problem. Since the beginning of anti-infectious treatment bacteria have rapidly shown an incredible ability to develop and transfer resistance mechanisms. In the last decades, the design variation of pioneer bioactive molecules has strongly improved their activity and the pharmaceutical companies partly won the race against the clock. Since the 1980s, the new classes of antibiotics that emerged were mainly directed to Gram-positive bacteria. Thus, we are now facing to multidrug-resistant Gram-negative bacteria, with no therapeutic options to deal with them. These bacteria are mainly resistant because of their double membrane that conjointly impairs antibiotic accumulation and extrudes these molecules when entered. The main challenge is to allow antibiotics to cross the impermeable envelope and reach their targets. One promising solution would be to associate, in a combination therapy, a usual antibiotic with a non-antibiotic chemosensitizer. Nevertheless, for effective drug discovery, there is a prominent lack of tools required to understand the rules of permeation and accumulation into Gram-negative bacteria. By the use of a multidrug-resistant enterobacteria, we introduce a high-content screening procedure for chemosensitizers discovery by quantitative assessment of drug accumulation, alteration of barriers, and deduction of their activity profile. We assembled and analyzed a control chemicals library to perform the proof of concept. The analysis was based on real-time monitoring of the efflux alteration and measure of the influx increase in the presence of studied compounds in an automatized bio-assay. Then, synergistic activity of compounds with an antibiotic was studied and kinetic data reduction was performed which led to the calculation of a score for each barrier to be altered.

Melanin Bleaching With Warm Hydrogen Peroxide and Integrated Immunohistochemical Analysis: An Automated Platform.

OBJECTIVE: Diagnosing melanocytic lesions is among the most challenging problems in the practice of pathology. The difficulty of physically masking melanin pigment and the similarity of its color to commonly used chromogens often complicate examination of the cytomorphology and immunohistochemical staining results for tumor cells. Melanin bleach can be very helpful for histopathological diagnosis of heavily pigmented melanocytic lesions. Although various depigmentation methods have been reported, no standardized methods have been developed. This study developed a fully automated platform that incorporates hydrogen peroxide-based melanin depigmentation in an automated immunohistochemical analysis. METHODS AND MATERIALS: The utility of the method was tested in 1 cell block of malignant melanoma cells in pleural effusion, 10 ocular melanoma tissue samples, and 10 cutaneous melanoma tissue samples. Our results demonstrated that the proposed method, which can be performed in only 3 hours, effectively preserves cell cytomorphology and immunoreactivity. RESULTS: The method is particularly effective for removing melanin pigment to facilitate histopathological examination of cytomorphology and for obtaining an unmasked tissue section for immunohistochemical analysis.

A rapid automatic processing platform for bead label-assisted microarray analysis: application for genetic hearing-loss mutation detection.

Molecular diagnostics using microarrays are increasingly being used in clinical diagnosis because of their high throughput, sensitivity, and accuracy. However, standard microarray processing takes several hours and involves manual steps during hybridization, slide clean up, and imaging. Here we describe the development of an integrated platform that automates these individual steps as well as significantly shortens the processing time and improves reproducibility. The platform integrates such key elements as a microfluidic chip, flow control system, temperature control system, imaging system, and automated analysis of clinical results. Bead labeling of microarray signals required a simple imaging system and allowed continuous monitoring of the microarray processing. To demonstrate utility, the automated platform was used to genotype hereditary hearing-loss gene mutations. Compared with conventional microarray processing procedures, the platform increases the efficiency and reproducibility of hybridization, speeding microarray processing through to result analysis. The platform also continuously monitors the microarray signals, which can be used to facilitate optimization of microarray processing conditions. In addition, the modular design of the platform lends itself to development of simultaneous processing of multiple microfluidic chips. We believe the novel features of the platform will benefit its use in clinical settings in which fast, low-complexity molecular genetic testing is required.