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Background The tetrazolium-based MTT assay has long been regarded as the gold standard of cytotoxicity assays as it is highly sensitive and has been miniaturised for use as a high-throughput screening assay. However, various reports refer to interference by different test compounds, including the glycolysis inhibitor 3-bromopyruvate, with the conversion of the dye to coloured formazan crystals. This study assessed the linear range and reproducibility of three commonly used cell enumeration assays; the neutral red uptake (NRU), resazurin reduction (RES) and sulforhodamine B (SRB) assays, in comparison to the MTT assay. Interference between the MTT assay and three glycolysis inhibitors, 2-deoxyglucose, 3-bromopyruvate and lonidamine, was investigated. Results Data indicate that the NRU, RES and SRB assays showed the smallest variability across the linear range, while the largest variation was observed for the MTT assay. This implies that these assays would more accurately detect small changes in cell number than the MTT assay. The SRB assay provided the most reproducible results as indicated by the coefficient of determination after a limited number of experiments. The SRB assay also produced the lowest variance in the derived 50% inhibitory concentration (IC 50 ), while IC 50 concentrations of 3-bromopyruvate could not be detected using either the MTT or RES assays after 24 hours incubation. Interference in the MTT assay was observed for all three tested glycolysis inhibitors in a cell-free environment. No interferences were observed for the NRU, SRB or RES assays. Conclusions This study demonstrated that the MTT assay was not the best assay in a number of parameters that must be considered when a cell enumeration assay is selected: the MTT assay was less accurate in detecting changes in cell number as indicated by the variation observed in the linear range, had the highest variation when the IC 50 concentrations of the glycolysis inhibitors were determined, and interference between the MTT assay and all the glycolysis inhibitors tested were observed. The SRB assay performed best overall considering all of the parameters, suggesting that it is the most suitable assay for use in preclinical screening of novel therapeutic compounds with oxido-reductive potential.

Cancer drug screening in patient‐derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix‐dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro , an automated microscopy‐based assay to resolve drug‐induced cell death and proliferation inhibition. Using DeathPro , we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug‐induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests. Synopsis DeathPro , an automated microscopy‐based assay resolves cell death and proliferation inhibition in 2D and 3D cultures. Drug screens using DeathPro provide insights into the impact of culture systems on drug effects and their links to genomic features. DeathPro resolves cytotoxic and cytostatic effects in drug screens with patient‐derived ovarian and lung cancer cells, organoids and co‐cultures with fibroblasts. Drug responses in cancer organoids are more diverse than in 2D cultured cells. Cytostatic drugs depend on culture systems, cytotoxic effects are independent of the culture format. Genomic analysis of cancer patients links DNA repair deficiency to drug sensitivity in organoids. Graphical Abstract DeathPro , an automated microscopy‐based assay resolves cell death and proliferation inhibition in 2D and 3D cultures. Drug screens using DeathPro provide insights into the impact of culture systems on drug effects and their links to genomic features.

The universal definition of myocardial infarction (UDMI) standardizes the approach to the diagnosis and management of myocardial infarction. High-sensitivity cardiac troponin testing is recommended because these assays have improved precision at low concentrations, but concerns over specificity may have limited their implementation. We undertook a global survey of 1902 medical centers in 23 countries evenly distributed across 5 continents to assess adoption of key recommendations from the UDMI. Respondents involved in the diagnosis and management of patients with suspected acute coronary syndrome completed a structured telephone questionnaire detailing the primary biomarker, diagnostic thresholds, and clinical pathways used to identify myocardial infarction. Cardiac troponin was the primary diagnostic biomarker at 96% of surveyed sites. Only 41% of centers had adopted high-sensitivity assays, with wide variation from 7% in North America to 60% in Europe. Sites using high-sensitivity troponin more frequently used serial sampling pathways (91% vs 78%) and the 99th percentile diagnostic threshold (74% vs 66%) than sites using previous-generation assays. Furthermore, high-sensitivity institutions more often used earlier serial sampling (≤3 h) and accelerated diagnostic pathways. Fewer than 1 in 5 high-sensitivity sites had adopted sex-specific thresholds (18%). There has been global progress toward the recommendations of the UDMI, particularly in the use of the 99th percentile diagnostic threshold and serial sampling. However, high-sensitivity assays are still used by a minority of sites, and sex-specific thresholds by even fewer. Additional efforts are required to improve risk stratification and diagnosis of patients with myocardial infarction.

NRC publication: Yes

The Third AAPS/FDA Bioanalytical Workshop, entitled \"Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays\" was held on May 1-3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions. The proceedings from each day were reviewed and summarized in the evening sessions among the speakers and moderators of the day. The consensus summary was presented back to the workshop on the last day and was further debated. This communication represents the distillate of the workshop proceedings and provides the summary of consensus reached and also contains the validation topics where no consensus was reached.

Cell-based assays for compound screening and profiling are fundamentally important in life sciences, chemical biology and pharmaceutical research. Most cell assays measure the amount of a single reporter molecule or cellular endpoint, and require the use of fluorescence or other labeled materials. Consequently, there is high demand for label-free technologies that enable multiple biomolecules or endpoints to be measured simultaneously. Here, we describe how to develop, optimize and validate MALDI-TOF mass spectrometry (MS) cell assays that can be used to measure cellular uptake of transporter substrates, to monitor cellular drug target engagement or to discover cellular drug-response markers. In uptake assays, intracellular accumulation of a transporter substrate and its inhibition by test compounds is measured. In drug response assays, changes to multiple cellular metabolites or to abundant posttranslational protein modifications are monitored as reporters of drug activity. We detail a ten-part optimization protocol with every part taking 1–2 d that leads to a final 2 d optimized procedure, which includes cell treatment, transfer, MALDI MS-specific sample preparation, quantification using stable-isotope-labeled standards, MALDI-TOF MS data acquisition, data processing and analysis. Key considerations for validation and automation of MALDI-TOF MS cell assays are outlined. Overall, label-free MS cell-based assays offer speed, sensitivity, accuracy and versatility in drug research. MALDI-TOF mass spectrometry (MS) can detect multiple compounds simultaneously. This protocol describes how to develop and optimize high-throughput, cell-based assays that use MALDI-TOF MS to detect drug uptake or biochemical markers of drug activity.

Prion real-time quaking-induced conversion (RT-QuIC) is emerging as the most potent assay for the in vivo diagnosis of Creutzfeldt–Jakob disease (CJD), but its full application, especially as a screening test, is limited by suboptimal substrate availability, reagent costs, and incomplete assay standardization. Therefore, the search for the most informative cerebrospinal fluid (CSF) surrogate biomarker is still of primary importance. We compared the diagnostic accuracy of CSF protein 14-3-3, measured with both western blot (WB) and enzyme-linked immunosorbent assay (ELISA), total (t)-tau and neurofilament light chain protein (NfL) alone or in combination with RT-QuIC in 212 subjects with rapidly progressive dementia in which we reached a highly probable clinical diagnosis at follow-up or a definite neuropathological diagnosis. T-tau performed best as surrogate CSF biomarker for the diagnosis of CJD (91.3% sensitivity and 78.9% specificity). The 14-3-3 ELISA assay demonstrated a slightly higher diagnostic value compared to the WB analysis (76.9% vs. 72.2%), but both methods performed worse than the t-tau assay. NfL was the most sensitive biomarker for all sCJD subtypes (> 95%), including those with low values of t-tau or 14-3-3, but showed the lowest specificity (43.1%). When ELISA-based biomarkers were adopted as screening tests followed by RT-QuIC, t-tau correctly excluded a higher number of non-CJD cases compared to NfL and 14-3-3 ELISA. Our study showed that among the CSF surrogate biomarkers of potential application for the clinical diagnosis of CJD, t-tau performs best either alone or as screening test followed by RT-QuIC as a second-level confirmatory test.

We present a versatile tool for the generation of monodisperse water-in-fluorinated-oil droplets in standard reaction tubes by centrifugal step emulsification. The microfluidic cartridge is designed as an insert into a standard 2 mL reaction tube and can be processed in standard laboratory centrifuges. It allows for droplet generation and subsequent transfer for any downstream analysis or further use, does not need any specialized device, and manufacturing is simple because it consists of two parts only: A structured substrate and a sealing foil. The design of the structured substrate is compatible to injection molding to allow manufacturing at large scale. Droplets are generated in fluorinated oil and collected in the reaction tube for subsequent analysis. For sample sizes up to 100 µL with a viscosity range of 1 mPa·s–4 mPa·s, we demonstrate stable droplet generation and transfer of more than 6 × 105 monodisperse droplets (droplet diameter 66 µm ± 3 µm, CV ≤ 4%) in less than 10 min. With two application examples, a digital droplet polymerase chain reaction (ddPCR) and digital droplet loop mediated isothermal amplification (ddLAMP), we demonstrate the compatibility of the droplet production for two main amplification techniques. Both applications show a high degree of linearity (ddPCR: R2 ≥ 0.994; ddLAMP: R2 ≥ 0.998), which demonstrates that the cartridge and the droplet generation method do not compromise assay performance.

Approximately 3.6 million cases of active tuberculosis (TB) go potentially undiagnosed annually, partly due to limited access to confirmatory diagnostic tests, such as molecular assays or mycobacterial culture, in community and primary healthcare settings. This article provides guidance for TB triage test evaluations. A TB triage test is designed for use in people with TB symptoms and/or significant risk factors for TB. Triage tests are simple and low-cost tests aiming to improve ease of access and implementation (compared with confirmatory tests) and decrease the proportion of patients requiring more expensive confirmatory testing. Evaluation of triage tests should occur in settings of intended use, such as community and primary healthcare centers. Important considerations for triage test evaluation include study design, population, sample type, test throughput, use of thresholds, reference standard (ideally culture), and specimen flow. The impact of a triage test will depend heavily on issues beyond accuracy, primarily centered on implementation.