HTS Automation Study: Results from a 2001 Survey of the Current vs. Desired State of HTS Automation

A survey of directors of screening organizations was conducted in 2001 to evaluate their perceptions of the current vs. desired state of high-throughput screening (HTS) automation. The survey encompassed attributes such as automation flexibility, throughput and operation. These and other automation attributes were ranked based on importance to the respondent and/or the limitations these attributes imposed on the screening organization.     

Determining Feasibility and Parameter Values for Compound Orders Based on Transfer Amounts, Liquid Handler Capabilities, and Container Volume Capacities

An algorithm is described for determining feasibility and calculating parameter values for one or more compound processing orders. Feasibility is determined primarily by applying a series of inequality constraints that result in a valid target concentration range within which orders can be processed. These inequality constraints are obtained from initial compound amounts, source and destination container volume capacities, the liquid handler transfer volume range, and the amount of compound to be processed. If feasible, calculated parameter values include the dilution, transfer and top-off volumes necessary to fulfill the order. Sample code implementing the algorithm is given in Microsoft Visual Basic®.     

The μFill: A New 96-/384- Well Microplate Reagent Dispenser for HTS and Drug Discovery

Today's biomedical research requires instrumentation that is both functional and versatile. While high throughput screening (HTS) and drug discovery laboratories require instrumentation that can be automated, pilot assay laboratories may not necessarily need total automation. Towards that end, Bio-Tek has developed the μFill, a 96-/384- well microplate reagent dispenser capable of running stand-alone or computer-controlled as part of a robotics system. The μFill is compatible with both 96- and 384-well microplates, and using a specially designed adaptor can also dispense to deep-well microplates. It is capable of dispensing from 10-3000 μl for 96- well plates and 5-1500 μl for 384-well plates in 1-μl increments. The μFill can dispense 20 μl into a 96-well plate in four seconds and into a 384-well microplate in 12 seconds. The microprocessor-controlled syringe pump is based on a tested, low-maintenance design that requires no calibration, yet provides a high degree of accuracy and precision. The accuracy with an 80 μl dispense is within 1 μl with a percentage CV of less than 2%, and with a dispense volume of 20 μl, the percentage CV is still less than 5%. For those needing to dispense organic solvents or sterile aqueous solutions, a model that is autoclavable and has increased solvent resistance is also available. The programming allows for the control of flow rates from 225 μl/well/sec, for dispensing to cell cultures, and to 1000 μl/well/sec for rapid and vigorous reagent dispensing. The flexible software provides complete programming capabilities from the keypad. For more complete automation, robotics interfaces can be developed using ActiveX® software commands. The μFill's size, with a 14 x 14-inch footprint and a height of seven inches, allows it to be used almost anywhere.     

Automated High Throughput Screening of Fluorescent Imaging Plate Reader (FLIPR) Assays Using Assay Platform™ Integrated with Activity Base for ‘on the fly’ Compound Reconfirmation

Advances in the field of automation have meant hitherto complex manual cell-based assays can now be automated. These improvements have brought significant enhancements in throughput, data fidelity and consistency, and allowed a reallocation of constrained resources.Building upon these improvements, we have linked our automated cell-based screening system, Assay Platform™, to Activity Base (IDBS), a software package designed to automate the analysis of HTS data. Customisation of this package has resulted in software that can identify ‘active’ compounds and re-pick them ‘on the fly’ from the original compound plates for triplicate re-testing without operator intervention.Based on an operator initially defining ‘normal’ parameters for assay activity in Activity Base, combined with an automated quality control software module that checks data fidelity, wells containing ‘active’ compounds can be re-picked and re-tested at the end of an automated screening run. Automating cell-based assays has significantly improved productivity, and, with the synergism of Activity Base, has given us greater power to complete each screening run and report ‘active’ compounds to Chemistry more rapidly. This article presents our approach to the automation of cell-based Fluorescent Imaging Plate Reader (FLIPR) screening together with automated active re-test confirmation using Activity Base.     

Developing an Automated Enzyme Immunoassay for High-Throughput Screening of Bovine Serum Antibodies to Neospora Caninum

An enzyme-linked immunosorbent assay (ELISA) for Neospora caninum antibodies was automated with a robotic workstation, the Beckman Coulter Biomek 2000, to screen 200 bovine sera. Comparing these results with manually run ELISA data, a 95.92% agreement (K = 0.9592) between the two assays was obtained. The automated assay was specific and sensitive with excellent positive and negative predictive values. The results were repeatable and reproducible. The automation flexibility was high and the operation complexity was minimal. High-throughput screening (HTS) for bovine antibodies to Neospora caninum was achieved. The assay was developed according to the internationally recognized ISO17025 standard requirements.     

Transferring Industrial Automation Technology to the Laboratory

Over the past few years, there has been much discussion about transferring industrial technology to laboratories. While it is easy to look at the superficial similarities, it is more important to examine the different requirements of different industries. In this way, it is possible to identify the technologies and techniques that can be successfully transferred to the laboratory to improve performance.This paper takes three very different industries - the bakery, High Throughput Screening (HTS) and mobile phone assembly and examines their different requirements. These industries have been selected from among the many sectors where the RTS Group operates - thus allowing real data from a number of situations to be used.One of the most important areas in automation design is the relationship between flexibility and throughput. This paper focuses on this relationship and its influence over machine configuration when comparing the requirements of the different industries.     

An Automated System for Processing Sample Vials Stored at Ultra-Low Temperatures

The BIOPHILE Individual Vial Retriever (IVR) system has been developed to provide automated access to vials stored at ultra-low temperatures. The IVR performs storage, retrieval, sorting, cataloging, volume estimation (weighing), barcode reading, and re-racking. All racking operations are performed in an environment designed to keep samples frozen at their optimal temperature. Operating temperatures are −80°C, −40°C, −20°C, and room temperature. Laboratory Information Management System (LIMS) integration, automation integration, chain of custody tracking, and FDA 21CFR Part 11 compliance are supported. This article introduces the IVR and provides information on its characteristics.     

Real-time implementation of multi-linear model-based control strategies––an application to a bench-scale pH neutralization reactor

The real-time implementation of a set of multi-linear model-based control design methodologies is studied using a bench-scale pH neutralization system that exhibits nonlinear dynamics. It is envisaged that advanced model-based control strategy based on the multi-linear models presents a promising paradigm to design controllers for complex nonlinear plants. The multi-linear modeling philosophy is based on the selection of a set of linear models, complemented with an adaptation mechanism to explain the nonlinear plant behavior in the whole operating range. Practical implications of each control strategy are evaluated and discussed.     

ISEC: a program to calculate insulin secretion

ISEC (Insulin SECretion) is a computer program which calculates pre-hepatic insulin secretion from plasma C-peptide measurements. The program uses a regression (population) model to derive parameters of C-peptide kinetics from subject's gender, type (normal, obese, non-insulin dependent diabetes mellitus), age, weight, and height. Insulin secretion is calculated as a piece-wise constant (step) function with flexible step length allowing for a fine resolution of the secretion profile between measurements. A constrained regularisation method of deconvolution is employed to carry out the calculations. The calculated profile satisfies three properties: (i) it fits the measurement within the given level of the measurement error, (ii) it is non-negative, and (iii) it has a minimum value of a regularisation criterion (norm of second differences) which quantifies the degree of deviation of the secretion profile from a straight line. Both theoretical aspects and specific features related to ISEC are considered. To exemplify the use of ISEC, pre-hepatic insulin secretion is calculated during meal tolerance test, frequently sampled intravenous glucose tolerance test, hyperinsulinaemic euglycaemic glucose clamp, and basal conditions with frequent sampling.     

Selective Ion Extraction for High Throughput Screening with a Microfluidic Enzyme Assay

This proof-of-concept paper describes the application of selective ion extraction to an assay of protein kinase A on a microfluidic chip platform. Selective ion extraction is a flux balance technique, where a combination of independent pressure control and voltage are used to selectively extract one ion from a mixture. The assay product is completely separated and diverted into a separate channel from the waste stream containing the unconverted substrate and enzyme. By detecting only product, background noise generated by the substrate is removed which increases the signal to noise ratio and assay sensitivity. This technique is intended for adapting kinase or protease assays with low conversion rates to an on-chip reaction format for HTS screening.     

Automated RNA Isolation Using the Ambion RNAqueous™-96 Kit with the Packard MutiPROBEII HT Liquid Handling System

Automated isolation of total RNA from cultured cells was accomplished by adapting Ambion's solid-phase total RNA isolation kit, RNAqueous-96, for use with the MultiPROBE II liquid handling system from Packard Instruments.Cultured cells (HeLa S3 adherent cells and K562 cells in suspension) were lysed, DNase treated, washed, and then eluted from the 96-well filter plate.Total RNA recovered was analyzed for yield, intactness, purity, and ability to serve as a substrate for real-time RT-PCR.     

Detection of Cellular Parameters Using a Microfluidic Chip-Based System

Lab-on-a-chip technology achieves a reduction of sample and reagent volume and automates complex laboratory processes. Here, we present the implementation of cell assays on a microfluidic platform using disposable microfluidic chips. The applications are based on the controlled movement of cells by pressure-driven flow inside networks of microfluidic channels. Cells are hydrodynamically focused and pass the fluorescence detector in single file. Initial applications are the determination of protein expression and apoptosis parameters. The microfluidic system allows unattended measurement of six samples per chip. Results obtained with the microfluidic chips showed good correlation with data obtained using a standard flow cytometer.     

Assay on FAQ''s on Total Lab Automation

The purpose of the introduction of an automated laboratory system in one format or another is reducing the costs of the laboratory by cutting staff. Also speeding up the performance often is a goal. At the same time it is planned to increase the quality of the work done by using automated labelling and reading of documents. The use of vacuum tubes and the handling of these by the apparatus are raising the safety of the labwork.

In this paper many aspects of the daily routines when using a TLA system are discussed. The aspects are collected during the discussions before and after the implementation of the Roche CLAS system in our laboratory. Some consequences were foreseen, and others came unexpectedly. But still the subjects form a point of discussion around the TLA systems. The most important message to be picked up is that one really has to consider all possible problems that could arise from the implementation of TLA.     

Development of a μ-concentrator Using Dielectrophoretic Forces

We present the simulation, development and experimental validation of a μ-concentrator based on dielectrophoresis, DEP.In a first step dielectrophoretic force fields of various electrodes are computed and compared. The simulation results for various electrode dimensions may serve as a general design rule for DEP devices. Favorable electrode designs were realized in gold on glass substrates. The performance of the DEP chips is validated by concentration of E.-Coli bacteria, a separation efficiency of 99.93% was achieved. Furthermore, we outline how the combination of forced convection and DEP allows for bacteria separation at increased flow rates.     

The First Automated High Content Screening System

With biological discovery and technology advancing in parallel, AstraZeneca (AZ) announced that it would soon take delivery of what is thought to be the first fully automated high-throughput high-content screening system. This custom designed assay platform from RTS Life Science International (RTS) automates the IN Cell Analyzer 3000 sub cellular analysis system from Amersham Biosciences. RTS has integrated its advanced scheduling system with the imaging tool to enable AZ to evaluate the effect of drug compounds on cellular processes.     

Dual series representation and its applications to a string subjected to support motions

Dual series representation (DSR) for the dynamic response of a finite elastic body subjected to boundary traction and boundary support excitations is proposed in this paper. To confirm the validity of the present model, a string subjected to support motions is solved. Four analytical methods including (1) a diamond rule, (2) a series solution with the quasi-static decomposition method, (3) DSR by the Cesáro sum technique, and (4) DSR by the Stokes' transformation method are presented. It is found that the numerical results obtained by using these four methods are in good agreement, and that both the Cesáro sum and Stokes' transformation regularization techniques can extract the finite part of the divergent series. The advantages and disadvantages of these four methods are discussed. In comparison with the quasi-static decomposition method and the Cesáro sum technique, the Stokes' transformation is the best way not only because it is free from calculation of the quasi-static solution, but also because its convergence rate is as fast as that of the mode acceleration method.     

Customization of a Commercially Available Prep Scale SFC System to Provide Enhanced Capabilities

Preparative Scale Supercritical Fluid Chromatography is emerging as a powerful alternative to HPLC for the purification and separation of complex chemical reaction mixtures. Advantages include greatly reduced solvent usage (and thus lower cost and environmental impact), higher throughput, and in some cases higher resolution. While there are commercially available prep SFC instruments, none currently offer all the features desired by many medicinal chemists engaged in the drug discovery process. These include: the ability to collect an unlimited number of fractions per sample with high recovery and negligible carryover, fully automated capacity to collect several hundred fractions, and the ability to collect fractions into the same disposable test tubes and racks which are already employed in HPLC. This article describes the customization of a preparatory scale SFC system purchased from Berger Instruments, Inc., Newark, DE. (a subsidiary Mettler-Toledo International, Inc., of Greifensee, Switzerland) in order to provide these capabilities.     

“The Spotting Accelerator™”, Customizable Head Assembly For Advanced Microarraying

Microarraying technologies have emerged as key tools for determining genomic expression. The quality of gene arrays is reliant on spotting pins for production of uniform spots, which are consistent in volume, shape, size and alignment.Point Technologies, Inc., has developed an advanced spotting technology. Emphasis has been placed on producing the “perfect spots” customized to each microarraying application. Uniform surface texturing such as highly polished or matte finishes, and hydrophilic/hydrophobic zonal coating is produced using Point Technologies' proprietary micro-machining technology. Microfabrication also permits the production of advanced pin designs, and high density, high throughput print-head assemblies of 1536 pins and greater. Point Technologies specializes in the micro-electrochemical machining of spotting pins in any size, shape and configuration.Presented data will demonstrate spotting pin design solutions, such as: variety of tip dimensions, surface finishes, specialized coatings, and material options instrumental in producing the ideal spot size, shape and spot density during extended usage.     

A Study of a High-Throughput Plasmid DNA Purification System

An automated process that incorporates Millipore's Plasmid Miniprep₉₆ Montáge™ Kit with the Apogent Discoveries PlateMate Plus® and Tango™ automated high-throughput dispensing systems has been developed for purifying plasmid DNA. To test the efficacy of this process, parameters such as the reproducibility and consistency of the purified DNA quantity and quality as well as the purification speed were analyzed. The purification time for two plates of the Plasmid Miniprep₉₆ Kit (192 samples) was approximately 60 minutes using a PlateMate Plus equipped with 96 disposable tips and the Tango system equipped with 96 RB (resin bead) syringes. High uniformity and consistency in DNA yields (determined by spectrophotometric analysis) and quality (determined by gel electrophoresis analysis) among the different wells were observed. The purified plasmid DNA samples sequenced at an exceptional level with an average PHRED Q > 20 of 819 ± 25.*Millipore and Montage are the trademarks of Millipore Corporation     

Global observability of a class of nonlinear discrete time systems

It is shown that discrete time systems whose dynamics are governed by piecewise monotone maps of the interval are globally observable under a general class of observations. The results rest on the fact that the dynamics are ergodic.