Evaluation of three different assays for the assessment of Epstein Barr Virus immunological status

Various attempts have been made to improve Epstein Barr Virus serodiagnosis by developing convenient methods. The present study evaluated the performance of multiplexed bead assays and immunoblot based assays on automated platforms by comparing them with immunofluorescence based assays for the determination of EBV immune status. A total of 45 serum samples were included in the study. Serum samples were tested by multiplexed bead EBV assays (AtheNA Multi-Lyte, Zeus Scientific,USA) and immunoblot based assays (Euroline, Euroimmun AG, Germany) on automated platforms. Assay systems were evaluated by comparing them with immunofluorescence based assays (Zeus Scientific, USA). For EBV anti-VCA IgM, anti-VCA IgG, anti-EA and anti-EBNA, the kappa values reflecting agreements of AtheNA and IFA were 0.20, 0.54, 0.92 and 0.95 for anti-EA, anti-VCA IgG, anti-VCA IgM and anti-EBNA respectively and the agreements of Euroline and IFA were 0.53, 0.67, 0.81 and 1.000 for anti-VCA IgG, anti-EA, anti-VCA IgM and anti-EBNA respectively. The results of the study performed on a limited number of serum samples demonstrated that the multiplexed bead assays and immunoblot assays agree with the standard IFA assay for anti-EBNA IgG and anti-VCA IgM detection while the agreement is less for anti-EA and anti-VCA IgG.     

Evaluation of a scanner-assisted colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria

We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within +/-1 log(2) dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria.     

Evaluation of a Scanner-Assisted Colorimetric MIC Method for Susceptibility Testing of Gram-Negative Fermentative Bacteria

We describe the ScanMIC method, a colorimetric MIC method for susceptibility testing of gram-negative fermentative bacteria. The method is a slight modification of the National Committee for Clinical Laboratory Standards (NCCLS) recommended broth microdilution method that uses a redox indicator 2,3,5-triphenyltetrazolium chloride (TTC) to enhance the estimate of bacterial growth inhibition in a microplate and a flatbed scanner to capture the microplate image. In-house software was developed to transform the microplate image into numerical values based on the amount of bacterial growth and to generate the MICs automatically. The choice of indicator was based on its low toxicity and ease of reading by scanner. We compared the ScanMIC method to the NCCLS recommended broth microdilution method with 197 coliform strains against seven antibacterial agents. The interpretative categorical agreement was obtained in 92.4% of the assays, and the agreement for MIC differences (within ±1 log₂ dilution) was obtained in 96% for ScanMIC versus broth microdilution and 97% for a two-step incubation colorimetric broth microdilution versus the broth microdilution method. The method was found to be labor-saving, not to require any initial investment, and to show reliable results. Thus, the ScanMIC method could be useful for epidemiological surveys that include susceptibility testing of bacteria.     

Modification of the fluorescein diacetate assay for screening of antifungal agents against Candida albicans: comparison with the NCCLS methods

A modified fluorescein diacetate (FDA) assay has been compared with standard NCCLS broth macrodilution and broth microdilution methods for the detection of antifungal activity. The FDA assay was performed in a medium containing bacteriological peptone, NaCl, yeast extract and glucose (0.2%, 0.1%, 0.1% and 1% w/v, respectively) and buffered with 10 mM BES buffer. The MICs of amphotericin B, fluconazole, miconazole and flucytosine (representing three major classes of antifungal agents) obtained by the three methods were compared. The results obtained with the FDA assays correlated well with the NCCLS macrodilution method for MICs of amphotericin B, miconazole and fluconazole, but not for flucytosine. However, the MIC values of flucytosine obtained with the FDA assay were well within the quality control range for the two reference strains recommended by the NCCLS. The FDA assay described is an attractive alternative to the NCCLS methods for screening for antifungal agents, with the added advantage of objectivity of fluorescence measurement.     

Semiautomated Method for Microbiological Vitamin Assays

A semiautomated method for microbiological vitamin assays is described, which includes separate automated systems for the preparation of the cultures and for the measurement of turbidity. In the dilution and dosage unit based on the continuous-flow principle, vitamin samples were diluted to two different dose levels at a rate of 40 per hr, mixed with the inoculated test broth, and dispensed into culture tubes. After incubation, racks with culture tubes were placed on the sampler of an automatic turbidimeter. This unit, based on the discrete-sample system, measured the turbidity and printed the extinction values at a rate of 300 per hr. Calculations were computerized and the results, including statistical data, are presented in an easily readable form. The automated method is in routine use for the assays of thiamine, riboflavine, pyridoxine, cyanocobalamin, calcium pantothenate, nicotinic acid, pantothenol, and folic acid. Identical vitamin solutions assayed on different days gave variation coefficients for the various vitamin assays of less than 10%.     

Automated triplexed hepatocyte-based viability and CYP1A and -3A induction assays

Cytochrome P450 (CYP) enzymes are key players in drug metabolism. Therefore, it is essential to understand how these enzymes can be affected by xenobiotics with regards to induction and toxicity to avoid potential drug-drug interactions. Typically, information has been gathered by combining data from multiple experiments, which is time-consuming and labor intensive, and interassay variability may lead to misinterpretation. Monitoring CYP induction and cytotoxicity by xenobiotics using an automated, multiplexed format can decrease workload and increase data confidence. Here the authors demonstrate the ability to monitor CYP1A and CYP3A4 induction, combined with a cytotoxicity measurement, from a single microplate well using cryopreserved human hepatocytes. The assay procedure was automated in a 384-well format, including cell manipulations, compound titration and transfer, and reagent dispensing, using simple robotic instrumentation. EC(50) and E(max) values were derived for multiple known CYP1A and -3A4 inducers. Induction and toxicological responses in the triplex system were validated based on literature values from conventional single-parameter assays. Validation and pharmacology data confirm that multiplexed cell-based CYP assays can simplify workload, save time and effort, and generate biologically relevant data.     

Quantification of the proliferation index of human dermal fibroblast cultures with the ArrayScan high-content screening reader

High-throughput cell-based assays are becoming a powerful approach in the drug discovery process. The ArrayScan high-content screening (HCS) reader is a cytometer based on a fully automated fluorescence microscope that is able to obtain quantitative information on the intensity and localization of fluorescence signals within single cells over a wide cell population. The aim of this work was to set up an automated HCS multiparameter analysis for the quantification of the in vitro proliferation index of normal human dermal fibroblast (NHDF) cultures. The authors stimulated starved NHDF with insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, or serum, and they quantified the proliferation index by measuring the expression of Ki-67 antigen, the incorporation of bromodeoxyuridine (BrdU), and the phosphorylation of the retinoblastoma protein (pRb). This approach also allowed quantification of the mitotic index by phospho-histone H3 staining and the percentage of cells in the S-phase by BrdU incorporation. The proliferation data from the ArrayScan assays were validated by comparison with a reference enzyme-linked immunosorbent assay (ELISA) and by flow cytometry. The measured proliferation indices were highly reproducible in repeated measures and independent experiments. The authors therefore propose that the ArrayScan HCS system could be used for high-throughput multiparameter analysis and quantification of the proliferation of cellular cultures.     

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.     

Determination of amphetamine and methamphetamine enantiomers by chiral derivatization and gas chromatography–mass spectrometry as a test case for an automated sample preparation system

An automated sample preparation system has been applied to the chiral analysis of amphetamine and methamphetamine using derivatization with trifluoracetyl-L -prolyl chloride (L -TPC) and subsequent separation on a gas chromatography–mass spectrometry (GC-MS) system. Tasks automated were the dilution of standards and the off-line preparation of the diastereoisomer derivatives. Chromatographic performance, sensitivity, and reproducibility of the automated procedure were compared to the equivalent values obtained with two existing assays methods which employ manual derivatiation, either on-column or off-line. Chromatographic performance was unaffected by the derivatization procedure and sensitivity was better for both automated and manual off-line derivatization. Qualitative reproducibility as based on enantiomeric composition was equivalent for all three approaches, while quantitative reproducibility as based on peak areas was best for the automated procedure. Considering the fact that the diastereoisomer derivatives are unstable over time, automated sample preparation with “just-in-time” derivatization can increase the overall precision of the analytical method. The procedures described here are general enough in nature that they could be applied to other chiral or even achiral analytes. © 1994 Wiley-Liss, Inc.     

Automated assays for superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activity

Automated assays for catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase are presented. The assay for catalase is based on the peroxidatic activity of the enzyme. The glutathione peroxidase and reductase assays measure the consumption of NADPH following the reduction of t-butyl hydroperoxide and oxidized glutathione, respectively. The assay for superoxide dismutase is based on the reduction of cytochrome c. All assays utilize the Cobas FARA clinical automated analyzer and provide considerable time savings over the manual assays.     

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.     

Evaluation of leukocyte differential counts on the QBC centrifugal hematology analyzer according to NCCLS standard H20-T

A performance study of the QBC centrifugal hematology system was conducted according to reference and analytical procedures defined in NCCLS Tentative Standard for Leukocyte Differential Counting, H20-T. A complex mathematical transformation and analysis of variance (ANOVA) were applied to the test data. This statistical technique is intended to segregate and emphasize differences in samples and methods. The comparative study of leukocyte differential counts show that QBC is equivalent in performance to the NCCLS reference method when the latter counts are grouped according to the WBC subpopulations reported by QBC. Within these grouped subpopulations, QBC counts were more precise than manual reference counts and otherwise comparable in terms of results. Similarly, QBC compared favorably with the reference method in its clinical sensitivity to abnormals, based on between-method versus within-method studies of hospital specimens.     

Cellular assay optimization: part II: the use of a simple integrated robotic work cell to allow the multiplexed batching of cellular assays

This report describes the implementation of an automated work cell with commercially available hardware and software, capable of handling up to 15 separate reagents for performing 96-well or 384-well assays but with a small footprint and only a single liquid dispenser and two plate washers. Extremely flexible software was used to enable this simple work cell to perform processes that would traditionally require a much larger, more expensive automation platform. With the development of the C-Myc assays for the targets DYRK, BMX, PERK, and FAK, the authors describe a software solution to multibatch assays to run simultaneously, reducing reagent dead volume and increasing the efficiency of running multiple assays such that the time to generate data across multiple targets was significantly shortened. Although a larger automated system with multiple robotic arms and extensive equipment would also be able to process multiple assays simultaneously, the work cell we have described represents an inexpensive and flexible, easily upgradable option suitable for a wider range of labs.     

Object-oriented image analysis for high content screening: detailed quantification of cells and sub cellular structures with the Cellenger software.

BACKGROUND: Detailed image analysis still is a considerable bottleneck for many cellular assays, and automated solutions to the problem are desirable. However, dealing with the complexity and variability of structures in cellular images makes detailed and reliable analysis a nontrivial task. METHODS: Therefore, based on the object-oriented image analysis approach, a novel image analysis technology, a flexible and reliable system for image analysis in cellular assays was developed. It contains a library of predefined, adaptable modules, each of them developed for a specific analysis task. The system can be configured easily by combining appropriate modules and adapting them interactively to the specific image data, if necessary. By representing cells and sub cellular structures within a network of interlinked image objects, a large number of parameters can be derived that describe shape, intensity, and relevant structural and relational aspects of any chosen class of structures. RESULTS: Thus, multi-parameter analysis and multiplexing are supported. A sample application based on this approach demonstrates that GFP signals can be distinguished based on their properties and the relative location within the cell.     

A generic hierarchical screening method for the analysis of microscale refolds using an automated robotic platform

The refolding of protein derived from inclusion bodies is often characterized by low yields of active protein. The optimization of the refolding step is achieved empirically and consequently is time-consuming slowing process development. An automated robotic platform has been used to develop a dilution refold process-screening platform upon which a hierarchical set of assays rapidly determine optimal refolding conditions at the microscale. This hierarchy allows the simplest, cheapest, and most generic high-throughput assays to first screen for a smaller subset of potentially high-yielding conditions to take forward for analysis by slower, more expensive, or protein specific assays, thus saving resources whilst maximizing information output. An absorbance assay was used to initially screen out aggregating conditions, followed by an intrinsic fluorescence assay of the soluble protein to identify the presence of native-like tertiary structure, which was then confirmed by an activity assay. Results show that fluorescence can be used in conjunction with absorbance to eliminate low-yielding conditions, leaving a significantly reduced set of conditions from which the highest yielding ones can then be identified with slower and often more costly activity or RP-HPLC assays, thus reducing bottlenecks in high-throughput analysis. The microwell-based automated process sequence with generic hierarchical assays was also used to study and minimize the effect on redox potential or misfolding, of oxygenation due to agitation, before demonstrating that the platform can be used to rapidly collect data and evaluate different refolding conditions to speed up the acquisition of process development data in a resource efficient manner.     

Development of multigene expression signature maps at the protein level from digitized immunohistochemistry slides

Molecular classification of diseases based on multigene expression signatures is increasingly used for diagnosis, prognosis, and prediction of response to therapy. Immunohistochemistry (IHC) is an optimal method for validating expression signatures obtained using high-throughput genomics techniques since IHC allows a pathologist to examine gene expression at the protein level within the context of histologically interpretable tissue sections. Additionally, validated IHC assays may be readily implemented as clinical tests since IHC is performed on routinely processed clinical tissue samples. However, methods have not been available for automated n-gene expression profiling at the protein level using IHC data. We have developed methods to compute expression level maps (signature maps) of multiple genes from IHC data digitized on a commercial whole slide imaging system. Areas of cancer for these expression level maps are defined by a pathologist on adjacent, co-registered H&E slides, allowing assessment of IHC statistics and heterogeneity within the diseased tissue. This novel way of representing multiple IHC assays as signature maps will allow the development of n-gene expression profiling databases in three dimensions throughout virtual whole organ reconstructions.     

Comparison of four different assays for determination of serum S-100B

BACKGROUND: S-100B determination has been shown to be clinically useful in the management of melanoma patients. After the development of a test for determination of the isoforms S100-A1B and S100-BB in serum (S-100B), several sensitive assays for the detection of serum S-100B have become available. We compared four S-100B assays, two automated (LIAISON Sangtec 100 and Elecsys S100) and two manual ones (Sangtec 100 ELISA and CanAg S100 EIA), with respect to clinical data, reference values and correlation. METHODS: In a total of 280 samples from 155 melanoma patients and 98 healthy individuals S-100B values were measured simultaneously with the different assays. RESULTS: The inter and intra coefficients of variation were best for the automated assays. The functional sensitivity of both manual assays was 0.15 microg/L. Method comparison revealed satisfactory correlation coefficients of 0.9 or higher, but the slopes ranged from 0.29 to 3.36. Except for the Sangtec 100 ELISA, the linearity between the assays was acceptable. The overall sensitivity for melanoma ranged from 37% (Elecsys S100) to 47% (LIAISON Sangtec 100) and the sensitivity increased with stage. ROC curves showed the best accuracy for the LIAISON Sangtec 100 assay. CONCLUSIONS: All assays gave satisfactory results, but it is advisable to improve the performance of the manual assays for better sensitivity. Agreement about an international reference standard is needed.     

Quantification of Dynamic Morphological Drug Responses in 3D Organotypic Cell Cultures by Automated Image Analysis

Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation.