This case study examines the automation and process change options available to emerging discovery/development stage pharmaceutical companies when considering implementing sophisticated high-throughput screens. Generally there are both financial and personnel constraints that have to be addressed when implementing state-of-the-art screening technology in smaller companies which generally are not as significant as in large pharmaceutical companies. When NPS Pharmaceuticals considered installing a Molecular Devices FLIPR&#153; for high-throughput cell based screening it became clear that, to make the best decision, the whole screening process at NPS Pharmaceuticals from screen development and validation, tissue culture, compound distribution, data handling and screening had to be re-examined to see what automation options were possible and which, if any, made sense to implement. Large scale automated systems were not considered due to their cost and the lack of in-house engineering infrastructure to support such systems. The current trend towards workstation based laboratory automation suggested that a minimalist approach to laboratory automation, coupled with improved understanding of the physical process of screening, would yield the best approach. Better understanding of the work flow within the Biomolecular Screening team enabled the group to optimize the process and decide what support equipment was needed. To install the FLIPR&#153;, train users, set up the tissue culture protocols for cell supply, establish high-throughput screening database protocols, integrate compound distribution and re-supply and validate the pharmacology on four cell based screens took the team 3 months. The integration of the screening team at the primary, secondary and tertiary screening stages of the target discovery project teams at NPS has enabled us to incorporate minimal automation into the Biomolecular Screening Group whilst retaining an enriching work environment. This is reflected in our current consistent throughput of 64 96-well microplates per day on the FLIPR&#153;, a figure that is comparable with that achieved within most major pharmaceutical companies. This case study suggests that process optimization coupled with modern stand alone automated workstations can achieve significant throughput in a resource constrained environment. Significantly greater throughput could be achieved by coupling the process improvement techniques described above with 384-well microplate technology.

Derek J. Hook

Case study of the automation options and decisions made in implementing a high-throughput cell based screen using the FLIPR&#153;

Laboratories that once viewed automation as an expensive luxury are now looking to automation as a solution to increase sample throughput, to help ensure data integrity and to improve laboratory safety. The question is no longer, &#145;Should we automate?&#146;, but &#145;How should we approach automation?&#146; A laboratory may choose from three approaches when deciding to automate: (1) contract with a third party vendor to produce a turnkey system, (2) develop and fabricate the system in-house or (3) some combination of approaches (1) and (2). The best approach for a given laboratory depends upon its available resources. The first lesson to be learned in automation is that no matter how straightforward an idea appears in the beginning, the solution will not be realized until many complex problems have been resolved. Issues dealing with sample vessel manipulation, liquid handling and system control must be addressed before a final design can be developed. This requires expertise in engineering, electronics, programming and chemistry. Therefore, the team concept of automation should be employed to help ensure success. This presentation discusses the advantages and disadvantages of the three approaches to automation. The development of an automated sample handling and control system for the STAR&#153; System focused microwave will be used to illustrate the complexities encountered in a seemingly simple project, and to highlight the importance of the team concept to automation no matter which approach is taken. The STAR&#153; System focused microwave from CEM Corporation is an open vessel digestion system with six microwave cells. This system is used to prepare samples for trace metal determination. The automated sample handling was developed around a XYZ motorized gantry system. Grippers were specially designed to perform several different functions and to provide feedback to the control software. Software was written in Visual Basic 5.0 to control the movement of the samples and the operation and monitoring of the STAR&#153; microwave. This software also provides a continuous update of the system's status to the computer screen. The system provides unattended preparation of up to 59 samples per run.

Robert J. Fix Sr

Jannell M. Rowe

Bain C. McConnell

Automation &#8212; down to the nuts and bolts

Recent clinical data indicate that the measurement of the concentration of C-reactive protein (CRP) requires a higher sensitivity and wider dynamic range than most of the current methods can offer. Our goal was to develop a totally automated and highly sensitive CRP assay with an extended range on the Dimension® clinical chemistry system based on particle-enhanced turbidimetric-immunoassay (PETIA) technology. The improved method was optimized and compared to the Binding Site's radial immunodiffusion assay using disease state specimens to minimize interference. Assay performance was assessed on the Dimension® system in a 12-instrument inter-laboratory comparison study. A split-sample comparison (n = 622) was performed between the improved CRP method on the Dimension® system and the N Latex CRP mono method on the Behring Nephelometer, using a number of reagent and calibrator lots on multiple instruments. The method was also referenced to the standard material, CRM470, provided by the International Federation of Clinical Chemistry (IFCC). The improved CRP method was linear to 265.1mg/l with a detection limit between 0.2 and 0.5mg/l. The method detects antigen excess from the upper assay limit to 2000mg/l, thereby allowing users to retest the sample with dilution. Calibration was stable for 60 days. The within-run reproducibility (CV) was less than 5.1% and total reproducibility ranged from 1.1 to 6.7% between 3.3 and 265.4mg/l CRP. Linear regression analysis of the results on the improved Dimension® method (DM) versus the Behring Nephelometer (BN) yielded the following equation: DM = 0.99 × BN − 0.37; r = 0.992. Minimal interference was observed from sera of patients with elevated IgM, IgG and IgA. The recovery of the IFCC standard was within 100 ± 7 % across multiple lots of reagent and calibrator. The improved CRP method provided a sensitive, accurate and rapid approach to quantify CRP in serum and plasma on the Dimension® clinical chemistry system. The ability to detect antigen excess eliminated reporting falsely low results caused by the prozone effect.

Tie Q. Wei

Steve Kramer

Victor P. Chu

Dave Hudson

Daniel Kilgore

Sue Salyer

Grace Parker

Amy Eyberger

Rene Arentzen

Heikki Koiv

An improved automated immunoassay for C-reactive protein on the Dimension® clinical chemistry system

Hirudin, an extract from the leech, has powerful antithrombin activity affecting the blood coagulation pathway. We evaluated the usefulness of hirudin in anticoagulating specimens for routine laboratory tests. Results using blood anticoagulated with hirudin corresponded well with results with blood treated with ethylenediamine tetraacetic acid (EDTA) in the complete blood count (CBC), including white blood cell (WBC) differential count and morphology of blood cells, when CBC was performed within 2 h of blood collection. Clinical chemistry results from hirudin-treated samples were similar to results obtained with serum specimens. Thus, hirudin may be a useful anticoagulant for emergency laboratory medicine.

Takeo  Kumura

Masayuki  Hino

Takahisa  Yamane

Noriyuki  Tatsumi

Hirudin as an anticoagulant for both haematology and chemistry tests

A method for the determination of total mercury in rat adipose tissue by cold vapour atomic fluorescence spectrometry (CVAFS) has been developed. Adipose samples were initially subjected to a lyophilization procedure in order to facilitate the homogenization and accurate weighing of small tissue aliquots (~50 mg). A closed vessel microwave digestion procedure using a mixture of sulphuric and nitric acids was used to liberate mercury from the adipose matrix. All mercury species were quantitatively oxidized to Hg(II) by a potassium bromate/bromide oxidation, then reduced to Hg(0) vapour by stannous chloride prior to fluorescence detection. The CVAFS exhibited a linear range of 10 pg Hg/ml to 120 pg Hg/ml. The method detection limit in solution was 2 pg Hg/ml, or 1 ng Hg/g adipose tissue, based on a nominal 50 mg sample and a final volume of 25 ml. A reference material from the National Research Council of Canada (DOLT-2, trace metals in dogfish liver) was prepared in quadruplicate in order to assess the accuracy and precision of the method. Mercury in this material was recovered at 2.22 ± 0.08 μ g/g, which is 104% of the certified level (2.14 ± 0.10 μ g/g).

Keith E. Levine

Reshan A. Fernando

M. Lang

Amal Essader

Robert W. Handy

Bradley J. Collins

Development of a method for the determination of ultra-trace level mercury in adipose tissue by cold vapour atomic fluorescence spectrometry

Miniature spectrometers incorporating array detectors are becoming a viable, low-cost option for field and process deployments. The performance characteristics of one such instrument are reported and compared with those of a conventional benchtop instrument. The parameters investigated were wavelength repeatability, photometric linearity, instrumental noise (photometric precision) and instrumental drift.

Simon Coles

Malcolm Nimmo

Paul J. Worsfold

Performance characteristics of a low-cost, field-deployable miniature CCD spectrometer

At present, bone marrow analysis is performed microscopically, but is time consuming and labour intensive. No automated methods have been successfully applied to classification of bone marrows cells because automated blood cell analysers have been incapable of identifying erythroblasts. The present study was designed to evaluate automated analysis of bone marrow aspirates with the CELL-DYN 4000 (CD4000) haematology analyser, which enables automated determination of erythroblast counts in both the normal mode (haemolytic time; 11.5s) and the resistant RBC mode (34.0s). The percentages of subpopulations including lymphocytes, neutrophils and erythroblasts were obtained with the CD4000, and as a reference, differential counts by microscopic observation of May&#8211;Gr&#252;nwald&#8211;Giesa-stained films of bone marrow aspirates were performed (n=98). Significant correlations (P &#60; 0.01) between the results obtained with the two methods were observed for total nucleated cell count and lymphocytes, neutrophils, erythroblasts and myeloid/erythroid (M/E) ratio. However, there were biases in the average percentages of erythroblasts, lymphocytes and M/E ratio obtained using the normal mode with the CD4000 toward values lower than those obtained with the microscopic method. Using the RBC resistant mode with the CD4000, the average percentages of erythroblasts, lymphocytes and M/E ratio approximated those obtained with the microscopic method. In conclusion, the CD4000 in resistant RBC mode is more useful for analysis of bone marrow aspirates than is the normal mode, because the former better approximates the M/E ratio than the latter.

Ryousuke  Yamamura

Kensuke  Ohta

Ki-Ryang  Koh

Izumi  Tsuda

Takayuki  Takubo

Automated bone marrow analysis using the CD4000 automated haematology analyser

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Journal of Analytical Methods in Chemistry publishes research into the methods and instrumentation used in chemical analysis, including spectroscopic, spectrometric and wet chemistry techniques, and their applications in real-world problems.

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Chief Editor, Dr. María José Trujillo-Rodríguez, is based in the Chemistry Department (Analytical Chemistry Division) at Universidad de La Laguna, Spain.

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Journal of Analytical Methods in Chemistry - Table of contents 2000

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Journal of Analytical Methods in Chemistry publishes research into the methods and instrumentation used in chemical analysis, including spectroscopic, spectrometric and wet chemistry techniques, and their applications in real-world problems.

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Journal of Analytical Methods in Chemistry

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Journal of Analytical Methods in Chemistry