ISSN 1660-5276
Tecan Journal Edition 2/2006
Four new software packages from Tecan page 4
Cover shows Dr Gerlinde Zerza-Schnitzhofer, Application Specialist/Marketing, Tecan Austria Ges.m.b.H, Austria
All Tecan’s manufacturing sites achieve ISO accreditation page 7
Unlimited throughput with Tecan and Illumina’s combined technologies page 8
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W E LCO M E
thank you
A big to you, our customers
I would like to take this opportunity to personally thank you for the trust you have placed in Tecan, a trust that allowed us to achieve a milestone year of growth in 2005. This year we will build on that trust, starting with the creation of a simplified and more effective organizational structure and the simultaneous strengthening of our service organizations in all markets. Together, these efforts reflect our commitment to providing you with the best possible levels of customer service and support.
This edition of the Tecan Journal features a number of exciting projects using our sample storage, liquid handling and detection solutions for diverse applications, ranging from genotyping for the diagnostic market to the compilation and maintenance of RNAi and compound libraries in the biopharma sector. You can also find out about our powerful new software packages, and learn about Tecan’s recent ventures to standardize manufacturing facilities and improve customer services in the US.
We will, of course, continue to concentrate on targeted product innovation. Outstanding new solutions will be launched in the coming year, giving you the speed, reliability and modularity that you have come to expect from Tecan, and ensuring that you achieve the best possible performance from your Tecan investment.
We hope you enjoy this issue of the Tecan Journal and look forward to supporting you for many more years to come.
Thomas Bachmann Chief Executive Officer (CEO)
Tecan Journal 2/2006
CO N T E N T S
Welcome and Contents Thomas Bachmann, CEO, thanks you, our customers pages 2-3
Unlimited throughput with Tecan and Illumina’s combined technologies A powerful new toolset for understanding the genetics of complex disease pages 8-9
High throughput microsomal stability assays for analyzing candidate drugs Drug development is faster with Tecan’s automated systems pages 16-17
Software focus: four new packages from Tecan enhance the connectivity of Tecan’s modules for seamless automation and data control page 4
Freedom EVOware® on Freedom EVO® Clinical IVD-D compliance opens doors to clinical applications page 5
Production of a genome-wide RNA interference library for functional gene characterization in cultured human cells Tecan’s Freedom EVO® handles esiRNA libraries for automated cell transfection pages 10-12
Building a world-class compound management system Amphora uses REMP systems to manage its compound library pages 20-21
In conversation with Craig Williamson, Director of Customer Support for Tecan US pages 6
High performance fluorescence scanning of tryptophan with Tecan’s Safire2™ microplate reader Automation helps with the design of tailor made reporter genes pages 13-15
All Tecan's manufacturing sites achieve ISO accreditation Günter Weisshaar, Executive Vice President Global Quality and Regulatory Compliance, explains
The importance of high throughput SNP genotyping for complex disease research in Spain Tecan’s Freedom EVO® workstation is used for reliable, low cost genotyping pages 18-19
Analyzing cholinesterase in organophosphate poisoning The German army automates the determination of AChE activity in whole blood pages 22-23
pages 7
Events for 2006 Meet Tecan at these events page 24 Tecan Journal 2/2006
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LGAT L OEBST A L PNR EOW DU S CTS
Software focus: four new packages from Tecan that unite to integrate your data At Tecan we realize that communication between your instruments is key, so we have released a range of new software packages that enhance connectivity, for simple integration of your plate readers with Tecan’s Freedom EVO® workstations and seamless combination of your data. The four new software packages are Freedom EVOware® Sample Tracking, Freedom EVOware 1.2, i-control™ and Magellan™ 6.0. Our Freedom EVOware Sample Tracking makes it easy to electronically record the status of all your samples throughout your entire application. Sample Tracking can register scanned labware then integrate and process all data acquired for each sample, including pipetting and plate reader data. These are stored and then used to generate and export reports containing the results. Sample Tracking is compatible with an extensive range of Tecan products, simplifying the logistics of handling large numbers of samples and improving your quality controls. It also integrates data from Freedom EVOware 1.2 and Magellan 6.0 for simple combination of plate readers and pipetting platforms. Sample Tracking is very much central to the interactions of all the new software (see figure), since it tracks movement of all plates (as controlled by Freedom EVOware) and all pipetting actions. Data and results from plate readings are fed to Sample Tracking for the generation of reports at the end of the process and these can also be accessed remotely.
Freedom EVOware 1.2 integrates the new i-control software (see below), for straightforward combination of the Infinite™ 200 multimode microplate reader with your Freedom EVO, and is also compatible with the new Magellan 6.0, allowing integration of other multimode plate readers with the workstation. The integration means that the Infinite reader, for example, will appear in Freedom EVOware’s control bar and script editor in the same way that other supported devices appear and are called up, for easy process definition. Freedom EVOware’s functionality can be extended with add-ons such as Sample Tracking, Normalization and Freedom EVOware Sample Oriented software. It supports a variety of hardware, both from Tecan and from third parties, bringing greater flexibility to your laboratory.
Get plate from Carousel
Freedom EVOware 1.2 has many other new, user friendly features to help your applications run more smoothly. These include a start-up wizard, which provides simple access to common daily routines, helping you to get your processes up and running quickly and easily, and the intuitive graphical user interface, which makes new assay protocols easy to create. There are also wizards to help you create advanced pipetting procedures, including relatively complex routines, and robotic moves are graphically defined, helping you to visualize processes. Sequences can be simulated and viewed, making it quicker and easier to develop new protocols as well as allowing remote monitoring. The package also comes with an expanded library of device drives and its modular architecture makes it easy to extend the software as new devices become available.
Pipette to reaction plate
Read plate
Freedom EVOware Freedom EVOware Sample Tracking Sample Tracking DB
Access data remotely
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Generate reports
L AT E ST P R O D U C T S
Freedom EVOware supports a wide range of Tecan modules, including the LiHa arm and dual LiHa arm, RoMa arms, Pick and Place (PnP), Te-MO™, Te-MagS™, incubators, hotels, PosID™ 2 and PosID 3. The new Freedom EVOware 1.2 additionally supports Infinite (through i-control), Sample Tracking, Sample Oriented. Freedom EVOware 1.2 is available in two versions, Freedom EVOware Standard and Freedom EVOware Plus, which has extra benefits including a fully integrated pipetting scheduler, which can scale up your application for multiple plate runs without requiring new software. There is a dynamic scheduler for efficient use of resources and an autocalibration of schedule feature, which uses real time to create a better sample processing schedule, allowing higher throughput. The Freedom EVOware Standard allows synchronous or asynchronous pipetting with two LiHa arms, while in Freedom EVOware Plus the two LiHa arms can either be used for one process or different LiHa arms can be used for different processes. Tecan’s new i-control software installs itself into the Freedom EVOware 1.2, so that the Infinite 200 plate reader can be fully combined with other application processes running on the Freedom EVO. Typically, when there is a plate reader integrated into a Freedom EVO platform, a RoMa arm transports the sample plate from the workstation and puts it into the reader, where the plate is read and data are generated; the RoMa arm then removes the plate. Fully integrating the reader’s control software into Freedom EVOware allows complete automation of the reader processes by your Freedom EVO and the i-control takes over from Freedom EVOware once the microplate is transferred into the reader. It then passes the control back when the reading is complete, so this compatibility between the software is a really beneficial new feature. The new i-control has the same look and feel as Freedom EVOware 1.2.
The new Magellan version 6.0 is compatible with all of Tecan’s multimode plate readers and has powerful data reduction capabilities, including being able to perform multiplate analyses, comprehensive spectra calculations and 3D scans. It supports all major detection modes, as well as the injector system, and is designed to allow customers to comply with 21 CFR part 11 regulation, for electronic records and electronic signatures. The new program has an intuitive, wizard-guided user interface
that maximizes its ease of use, allowing seamless process control, automated data import functions in variable formats, and easy and fully automated data export to LIMS. The program even comes with example files of typical applications to get you started quickly. For more information on our new software, please contact Jason Meredith, Global Product Manager Software: jason.meredith@tecan.com
Freedom EVOware® on Freedom EVO® Clinical, Tecan’s automated platform for clinical diagnostics The Freedom EVO Clinical with Freedom EVOware and its new module for sample oriented processing, which is predominantly used for clinical applications, is a powerful tool for controlling the Freedom EVO Clinical family of application-oriented open platforms for clinical diagnostics and blood bank environments. The Freedom EVO Clinical/EVOware system offers: • application-oriented, scalable precision liquid handling and robotic platform for clinical applications like EIA preparation and pooling • extended functional spectrum by integration of new options for clinical use • enhanced process control and safety features without compromising instrument flexibility • scheduler function for increased throughput • true walk-away capability Freedom EVO Clinical is not available in the USA although sample oriented Freedom EVOware is available with pooling functions disabled.
For more information on Freedom EVO Clinical please contact Christoph Beck, Product Manager Corporate Account & Clinical Diagnostics: christoph.beck@tecan.com Tecan Journal 2/2006
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C U STO M E R S U P P O RT
at your service
Craig Williamson is the Director of Customer Support for Tecan US, and has held the position since 1999. He is responsible for Expertline, the helpdesk and the entire service organization for North, Central and South America.
How do you manage to look after all your customers simultaneously? Our service structure is tailored to the needs of our customers and the US market to maximize our efficiency. We are separated into four regional sales and service regions: the north east, the south east, the mid-west and the west. In each of these regions we have a regional service manager who takes care of that territory and makes sure the customers receive the best care.
And what do you do in Customer Support? My organization ensures that our customers receive first class care and support, providing them with complete peace of mind. We run a helpdesk with eight people from 8 am to 8 pm and we answer whatever technical questions the customer has, organize package distribution and dispatch field engineers. We are responsible for the installation of instruments, for preventive maintenance to minimize downtime, for device upgrades and repairs and for training our customers in the proper use of the instruments. There is a lot of work involved in the Customer Support organization and, in Tecan US, we put 40% of Tecan’s resources into service. Tecan Journal 2/2006
How do you respond to customer queries?
What’s special about Tecan’s customer service?
We aim to provide our customers with courteous, timely and efficient service, so our policy is to respond and solve our customers’ requests quickly. For complex requests that cannot be solved instantly, we have experts with specialist knowledge at hand and, if an intervention is necessary, we dispatch one of our qualified and certified field engineers on-site to take care of the instrument. We have a clear escalation policy in place and, if necessary, customer requests are directed through the regional manager, then me and finally the respective Tecan production site.
A couple of years ago, Tecan US combined the two departments that used to be called Applications and Service, where the scientists worked in Applications and were separated from our electronic technicians. By changing this organization, we are able to provide our customers with the appropriate experts for every intervention, whether that requires specialist knowledge for liquid handling and applications or hardware. Our range of application scientists includes biologists, microbiologists and chemists, for example, and I think that this is a real asset for increasing customer satisfaction. There is no other organization that has switched to this system, as far as I know - it allows us to respond to calls within a shorter time and considerably increase our efficiency.
Our goals are to get the customer up and running within four hours on the telephone, or 48 hours if we have to dispatch engineers to the customer’s site, and 95% of the time we meet those goals.
How satisfied are your customers with the service you provide? Every service call that we answer has a survey attached, which the customer can fill in, to give their opinions regarding our service. I compile that information and send it to Tecan’s headquarters in Switzerland. I strive to get ratings of ‘good’ or ‘excellent’ on over 90% of those reports and, in the last quarter of 2005, we achieved that on about 96% of those reports.
What are the most important aspects of providing good service? The customers want competency, efficiency, communication, punctuality and, of course, recovery of their instruments to peak condition in the shortest time possible. It is also important for them to see the same field engineer with each visit.
What does the future hold for Customer Support at Tecan? Service is very important for our customers and, therefore, it is one of the most important departments in Tecan. Maintaining good customer service is also the key to keeping our customers and we are constantly working to make sure that our customer service is number one.
There is no other organization that has switched to this system, as far as I know - it allows us to respond to calls within a shorter time and considerably increase our efficiency.
Q UA L I T Y A S S U R A N C E
Quality Assurance and Regulatory Affairs Günter Weisshaar, Executive Vice President Global Quality and Regulatory Compliance, has news of ISO certification for all Tecan’s manufacturing sites.
Standardization and regulatory compliance are of critical importance for Tecan and its customers so, over the last few years, we have concentrated on implementing the newest ISO standards ISO 9001 and ISO 13485 - into all of our manufacturing sites; in Switzerland, Austria, Tecan SCC in Mainz, Germany, and in Tecan Systems in California. Although all these sites were already certified with local companies, our aim was to reach a universal global standardization with just one notified body. We have now achieved this aim in direct association with TÜV SÜD Product Service, chosen as one of the most common, global notified bodies with a particular expertise in the medical device business and a very strong reputation for high standards. Many of our customers also use TÜV as their regulatory partner and it makes perfect sense that we work to the same standards and can talk on the same level, especially since TÜV has been given authorization from the United States FDA since 2002 to perform and execute follow-up inspections on its behalf.
Our customers can expect that, with the ISO 9001 quality system regulation, Tecan has met all the elements required and has been audited and proved by a neutral company or party. ISO 13485 deals specifically with additional requirements for medical devices. At the same time, our sites in Switzerland and Austria are also certified according to CMDCAS Canadian Medical Device Conformity Assessment System – an additional national regulation to ISO 13485 required in Canada and also dealt with by TÜV. We are delighted with these new implementations and trust that they will give you, our customers, the reassurance of working together with a knowledgeable supplier that is fully compliant with the highest international regulations.
Tecan Switzerland Ltd. Seestrasse 103 CH-8708 Männedorf
Tecan Austria GmbH Untersbergstrasse 1a A-5082 Grödig/Salzburg
Tecan Software Competence Center GmbH Schmalweg 5 D-55252 Mainz-Kastel
Tecan Systems, Inc. 2450 Zanker Road USA-San Jose, CA 95131
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A P P L I C AT I O N B I O P H A R M A
Unlimited throughput with Tecan and Illumina’s combined technologies creates a powerful new toolset for understanding the genetics of complex disease Illumina, a San Diego (CA)-based company, has integrated Tecan liquid handling workstations into its revolutionary, endto-end solutions for large-scale genotyping applications. The collaboration has been crucial for the development of a number of products, including Illumina’s production-scale BeadLab systems, which were deployed by major investigators world wide as part of the International HapMap Project and, more recently, Illumina’s bench-top BeadStation. Both of these platforms offer automated liquid handling capability using Tecan’s flexible workstations. Illumina initially started talking to Tecan to automate the GoldenGate® Assay, Illumina’s custom genotyping assay that can multiplex up to 1536 targets per sample. Illumina’s microplate compatible Array Matrix processes 96 samples in BeadStation with Freedom EVO
Tecan Journal 2/2006
parallel and this scale of sample throughput was crying out for automation, so Illumina turned to Tecan for help. Tecan liquid handling workstations can be used to automate the pre-PCR steps or both the pre- and post-PCR steps involved in the assay, streamlining the workflow and reducing the potential for human error. Processing these assays with Tecan workstations combined with Illumina’s BeadArray™ Readers, allele-calling software and tied together with LIMS essentially formed the BeadLab systems.
“Many of our scientists had worked previously with Tecan’s systems in production sequencing environments,” said Bill Craumer, Director of Corporate and Marketing Communications at Illumina. “They valued the
flexibility of automated, robust pipetting into either tubes or plates with high precision, so it was natural to specify Tecan workstations for our genotyping BeadLab.” Illumina was both a principal investigator and a supplier of BeadLabs for other principle investigators in the $100 million International HapMap Project and, by extension, Tecan was a key contributor to the Project’s success. Illumina has installed BeadLabs automated with Tecan workstations at virtually every HapMap research center, including the Wellcome Trust Sanger Institute, the Broad Institute, Génome Québec and Japan’s RIKEN. The International HapMap Consortium was established in 2002 to develop a haplotype map of the human genome, with the aim of identifying common patterns of DNA sequence variation in the human genome1. Sets of nearby single nucleotide polymorphisms (SNPs) on the same chromosome are inherited in blocks and the pattern of SNPs on a block forms a haplotype. Although the haplotype may contain a large number of SNPs, only a few SNPs are needed to uniquely identify the haplotype. The HapMap Project has mapped the haplotype blocks and the specific SNPs that identify the haplotypes, which are called tag SNPs. Each institution in the consortium was assigned a certain chromosomal region to map, resulting in large amounts of data that were made publicly available from 2003 onwards2 and, in 2005, the completed haplotype map was announced3. The mapped tag SNPs can now be used instead of the ten million SNPs to examine the entire human genome for variation, such as in
A P P L I C AT I O N B I O P H A R M A
HumanHap 300
association with a particular disease or phenotype. Depending on the population studied, between 250,000 and 500,000 tag SNPs provide comprehensive genomic coverage, and these large-scale disease association studies are currently ongoing. Illumina has now deployed tag SNPs on its new HumanHap BeadChips, which can query up to 500,000 different SNP loci on a single microarray. The BeadChips rely on Illumina’s Infinium™ assay, which allows interrogation of the human genome at single base resolution without needing PCR or ligation steps. Precise liquid handling is critical to the Infinium assay so, once again, Illumina chose Tecan for automating the application. Tecan worked closely with Illumina to provide the Te-Flow™4 flow cell technology on the Freedom EVO® family of workstations to enable parallel processing of 24 BeadChips simultaneously. The Freedom EVO can be used to automate every step of the Infinium genotyping process, offering pre-optimized and ready-to-run protocols, and allowing much more efficient management of large sample numbers. Most HumanHap BeadChip users have at least one Freedom EVO with a modified robotic manipulator (RoMa) arm to help achieve higher sample throughput and allow LIMS integration for positive sample tracking.
“A key driving force behind this strategic partnership was to deepen our understanding of customers’ needs and provide the technology they really want,” explained Christoph Beck at Tecan. “Illumina was able to successfully automate the Infinium assay with BeadChips by combining our high precision,
robust liquid handling technology with the Te-Flow cell flow technology. At Tecan, we can provide customers with access to our instruments’ application programming interface, and this made it much quicker and easier for Illumina’s developers to integrate the workstations into their own software environment, and tailor the instruments’ performance to exactly meet requirements.” As a result of Illumina and Tecan’s combined technology, researchers can now routinely generate millions of genotypes per day. “Reliability and quality are critical for our customers, and the fact that Tecan’s workstations integrate so nicely into a LIMS-controlled environment for all required production capacities is really important,” said Bill. “Over the last five years, Tecan’s relationship with Illumina has evolved from that of an equipment supplier to a highly strategic collaborator, and Tecan has provided essential value to Illumina and to our customers,” added Alex Chan, Illumina’s Associate Product Manager, Systems & Software. Illumina has also started offering Tecan’s Freedom EVO workstations as a standard upgrade to bench-top BeadStations for users that want the automated, liquid handling capability for smaller scales of throughput. In the future, Illumina anticipates incorporating Tecan automation options with its gene expression applications.
Illumina’s beads-in-wells technology. The 3 μm features are the smallest in the microarray industry
Illumina’s BeadArray technology The BeadArray technology involves the random assembly of oligo-coated 3 μm beads into wells etched into one of two different substrates: 1) the 96 sample Sentrix Array Matrix (which was standard for the HapMap Project), where each array is composed of 50,000 fiber optic strands 2) the Sentrix BeadChip, designed for denser, whole genome applications. The BeadChips are available in six different configurations and can contain up to 12 million beads (or features) each, and every feature is represented on average over 30 times, providing really high confidence calls
References 1. The International HapMap Consortium (2003) The International HapMap Project Nature 426: 789-796 2. International HapMap Project http://www.hapmap.org 3. Altshuler D, Brooks LD, Chakravarti A, Collines FS, Daly MJ, Donnelly P & International HapMap Consortium (2005). A haplotype map of the human genome Nature 437: 1241-1242 4. Patented Te-Flow technology is exclusively distributed by Tecan Switzerland AG
For more information, please visit: http://www.hapmap.org http://www.illumina.com GoldenGate is a registered trademark and BeadArray and Infinium are trademarks of Illumina, Inc.
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AU TO M AT E D L I Q U I D H A N D L I N G
Production of a genome-wide RNA interference library for functional gene characterization in cultured human cells Hannes Grabner1, Anne-Kristin Heninger2, Ralf Kittler2, Annett Lohmann1, Ina Poser2, Jan Wagner1, Karol Kozak1, Frank Buchholz2 and Eberhard Krauß1 1 HT-Technology Development Studio (TDS), 2Research Group Buchholz, Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany
RNA interference (RNAi) is a mechanism that gives researchers the ability to investigate gene function quickly and efficiently by specifically silencing the expression of the individual gene in question. Although quite easy in some species, RNAi initially proved more difficult in cultured mammalian cells because the long double-stranded RNA activated the cells’ natural antiviral defence. To an extent, this has been overcome by using chemically synthesized small interfering RNAs (siRNAs) around 21 nucleotides long but this technique is still not always effective. Although there are now good algorithms available to predict sequences that will function well with a reasonable probability, only experimentation can show whether the selected siRNA actually degrades the mRNA in the cell efficiently. To address this problem, scientists at the Max Planck Institute in Dresden are using Tecan equipment to create a human genome-wide library of endoribonucleaseprepared, so-called, esiRNAs1,2 to use for a range of scientific applications. At the same time, they have developed a robust and automated transfection method that introduces the esiRNA molecules efficiently into the cell under constant conditions.
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Figure 1: Production scheme of the esiRNA library 3
The method for esiRNAs involves cleaving long double-stranded RNA molecules with Dicer or bacterial RNaseIII in vitro into overlapping siRNA molecules to introduce into the mammalian cell. The starting material (see figure 1; reproduced with permission from reference 3) is a collection of 15,500 E. coli bacterial clones containing a genome-wide cDNA library which covers, to the greatest possible extent, all known and predicted human genes. For each individual gene, the cDNA insertion fragments from the plasmids are amplified in 96-well microplates using PCR, and T7 polymerase promoters are attached on both sides. The T7 polymerase reads off mRNA on both sides from these promoters and it can then be
hybridized to the RNA double strand. After adding RNaseIII from E. coli, the double-stranded RNA is cleaved into short overlapping siRNA fragments which contain the highly active effector molecules in the “pool”, in addition to the less active or completely inactive molecules. This effector molecule mixture is purified through columns and, finally, the esiRNA concentrations for all molecule mixtures are measured and adjusted to the same concentrations in new microplates (normalization).
AU TO M AT E D L I Q U I D H A N D L I N G
Gel-based quality control steps are included at all important stages in order to verify the lengths of the fragments, their purity and concentration. All data obtained are fed into a database, and all steps of the operation monitored by means of a LIMS. So far, a library has been obtained with over 14,000 molecule mixtures available in the same concentrations in 384-well microplates, in order to switch off a corresponding number of human genes in cultured cells and to investigate the effect on biological processes. A genome-wide library has subsequently been produced for mice. The fundamental precondition for a welladjusted and usable library is high precision aliquoting and subsequent fully integrated measurement in terms of both hardware and software. For these reasons, a Freedom EVO® Workstation (figure 2) with eight-channel LiHa tips is used for the final step - the standardization of the library. An integrated 96channel Te-MO™ pipetting system rapidly and precisely aliquots for UV measurement of the esiRNA concentration using optional disposable pipette tips or a 96-channel Teflon® needle head. Teflon-coated steel needles have the advantage that practically no serious retention of negatively charged siRNA occurs as it does on the plastic surfaces of conventional interchangeable tips which usually have differing degrees of static charge.
Figure 2: The Freedom EVO system to produce and normalize the genome-wide esiRNA libraries. For UV measurements to determine the RNA concentration, the RoMa arm lifts the plates down from the work table to the GENios Plus reader, which is placed on the level below, as seen in the lower right corner.
The workstation includes a fully integrated Tecan GENios™ Plus photometer to conduct the measurements at 260 and 280 nm, so that both the concentration and the purity can be determined by means of the quotients from the two measurements. For high throughput, the samples are aliquoted into 384well microplates (Corning UV-Star) specially manufactured for this application. The best measurements are obtained when the samples are thoroughly mixed in the diluting medium, by what is known as ‘sandwich’ pipetting (14 μl of diluting medium, 2 μl of sample, 14 μl of diluting medium) and by means of a separate mixing pipetting step.
The eight tip LiHa pipetting system is used to assemble the standardized library. All eight tips can introduce the diluting medium first, independently of each other, and then add the appropriate quantities of the esiRNA mixture as a summand according to the instructions given by the LIMS. The sum results in equal final volumes of the various esiRNAs in the same concentrations. Samples are then taken from this newly assembled library on the 384-well scale, checked on a gel and measured again in the UV spectrophotometer. The standard deviations after standardization lie within a range of less than 20% if effective use is made of the sample quantities (2 μl).
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Transfection in high throughput procedures The next important step for this newly created library is automated transfection into human cancer cells. Positively charged liposomes are used to insert nucleotides of any type into the cells, compressing and “packaging” the nucleotide strands, helping to penetrate the cell membranes. The entire transfection process is standardized as far as possible, the CASY® cell counter (Schärfe System GmbH, Reutlingen, Germany) ensures a consistent way of counting, and variable distribution patterns resulting from pipette angle and other human errors are avoided by automated cell seeding (WellMate® Dispenser, MATRIX Corp., Hudson, USA).
Figure 3: Transfection workflow 3
Again, the Freedom EVO system is used to automate other stages of the transfection process (figure 3, reproduced with permission from reference 3, figure 3). The transfection unit is completely housed in a S2 safety cabinet (BDK Luft- und Reinraumtechnik, Sonnenbühl-Genkingen, Germany) to protect from external contamination (such as fungal spores, yeast or bacteria) during transfection and to allow working with genetically engineered viral vectorbased RNAi libraries at biosafety level S2. Improvements of the shown workflow allowed a throughput of up to 30,000 samples in triplicate (90,000 in total) within 12 hours of pure robot time.
This genome-wide RNA interference library places an extraordinary tool in the hands of researchers for the systematic investigation of individual genes of the human genome for their function in all elemental biological processes in the cell. In addition, we have developed transfection methods which enable our screening department to conduct gene characterization projects of this sort at a high throughput rate. CASY is a registered trademark of Schärfe System GmbH Teflon is a registered trademark of DuPont or its affiliates WellMate is a registered trademark of Matrix Technologies Corporation
References 1. Yang D, Buchholz F et al. (2002). PNAS 99: 9942-9947 2. Kittler R et al. (2004). Nature 432: 1036-1040 3. Krauß E et al. (2005). Biospektrum 11: 436-441
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DETECTION
High performance fluorescence scanning of tryptophan with Tecan’s Safire2™ microplate reader Most proteins have intrinsic UV fluorescence and this is due to the presence of aromatic amino acids in their structure, in particular the amino acids phenylalanine, tyrosine and tryptophan (Trp). Of these, Trp has the strongest fluorescence emission, although the spectrum’s wavelength and intensity can vary according to the local environment, including factors such as the pH and solvent polarity. Furthermore, the location of Trp within the protein’s structure can affect its characteristics, for example, Trp residues buried within the protein’s hydrophobic core can have fluorescence spectra that are shifted by 10 to 20 nm compared with those on the protein’s surface. Standard Trp is encoded by a single UGG triplet and, over the years, it has been shown that Trp analogues can be introduced into various proteins by genetic manipulation, serving as reporter genes for a wide range of studies on protein structure or function. In the 1970s, solution studies demonstrated that manipulating the structure of Trp itself, such as by substituting particular indole
atoms with electron donating atoms, leads to intramolecular charge transfer (figure 1) that is extremely sensitive to pH changes. For example, the presence of a nitrogen atom at different positions of the Trp indole ring can significantly alter the protein’s absorption properties, resulting in a red
wt-barstar
Figure 1: Left: Barstar - first protein whose Trp residues were substituted by 4-NH2 Trp and 5-NH2 Trp. The wt-barstar has three Trp residues, i.e. the fully or partially solvent-exposed Trp38 and Trp44 and Trp53 which is completely buried in the protein hydrophobic core. Right: Charge transfer in Trp and its amino analogues arises as a consequence of the presence of amino group as a good electron donor in the amino indole moieties. Such size increase of the indole ring feature promotes more (i.e. an additional three and four, respectively) mesomeric structures in aminotryptophans.
or blue shift in fluorescence emission. These aminotryptophans can be inserted into proteins, replacing the existing parent Trp, and so transferring the new pH-sensitive fluorescence emission properties into the proteins, serving as valuable reporter genes for a range of experimental situations. In this study, Dr Nediljko Budisa and his colleagues at the Max Planck Institute of Biochemistry in Martinsried, Germany, used a Tecan Safire2 microplate reader to rapidly and reliably detect the differences in fluorescence emission of tryptophan and its analogs, 5-aminotryptophan (5-NH2Trp) and 4-aminotryptophan (4-NH2Trp), upon pH titration. Different fluorescence behaviors of the native and substituted proteins have been well documented in previous studies, so the main aim of these measurements was to confirm that the fluorescence properties of Trp and its analogues as free amino acids in solutions are fully transmitted upon their incorporation into the proteins barstar and human annexin V. The amino acids, proteins and substituted proteins were prepared and purified as described in [1]. Amino acids and analogs were measured at a concentration of 1 mM and barstar at 0.5 mM, in a volume of 300 ml in black 96-well plates (Greiner®). Emission spectra were detected from 310 to 500 nm (bandwidth 5 nm) for the free amino acids and from 310 to 450 nm (bandwidth 5 nm) for barstar, in 1 nm steps using 285 nm (bandwidth 10 nm) for excitation. Detection flash mode was set to high sensitivity mode with an integration time of 20 ms (40 ms for barstar). The gain for each pH titration was determined from the sample with the highest emission value. For 4-NH2Trp only, two different gains were used due to the weak emission at higher pH. Z-position was set manually after measurement of five Tecan Journal 2/2006
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DETECTION
14
80000 60000 HN 40000 H 2N
COOH
20000
RF 0 310
410 360 wavelength [nm]
Figure 2: Effects of pH on fluorescence emission profiles of Trp (black), 4-NH2 Trp (blue) and 5NH2 Trp (red) as free amino acids in solution. Fluorescence intensity regularly drops down with the pH decrease but there are almost no changes in the position of the emission maximum upon pH titration. In contrast, the position of the emission maximum in both 4NH2 Trp and 5-NH2 Trp fluorescence spectra is pH-dependent. Low spectral intensities at higher pH (7.0, 9.0 and 11.0) for 4-NH2 Trp make it difficult to recognize the trend in the spectral shift, so sample measurements were performed with optimized gain. The values of fluorescence emission maximum for each amino acid derived from these measurements are summarized and plotted in figure 3.
460
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Figure 3: Plot of the variations in fluorescence emission maxima versus pH of solutions containing free amino acids Trp (black), 4-NH2 Trp (blue) and 5-NH2 Trp (red). Data for Trp are fitted using linear regression method (R = 0.9316) and aminotryptophan data were submitted to sigmoid plot (Origin®, Ver. 6.1G). Derived parameters are presented and discussed in the text.
Tecan Journal 2/2006
randomly chosen samples. Trp and 5-NH2 Trp were measured at 30 °C, 4-NH2 Trp at 31 °C and barstar at 28 °C and the number of flashes was set to 10 (Trp and 5NH2Trp) and 20 (4-NH2Trp). The spectral data presented in figure 2 clearly demonstrate a regular decrease in the fluorescence intensity of free Trp that correlates strongly with decreasing solvent pH (from 11.0 to 1.0). However, such intensity changes were not seen when the native proteins barstar and annexin V were measured. This is not surprising, because protein Trp residues are often involved in complex interactions with neighboring residues and solvent so that at lower pH, for example, a native-like molten globule state (i.e. exposure of buried Trp side chains) can be detected in the structure of many proteins. In both native proteins and free L-Trp in solution, the fluorescence emission maxima were almost unchanged throughout the measured pH range (1-11), as shown in figure 2. However, the fluorescence emission spectra of 4-NH2 Trp and 5-NH2 Trp generally decreased with increasing pH of the solvent, reaching a plateau level at pH > 9.0. Dramatic changes were also seen in the intensity of the anionic form of 5-NH2 Trp’s emission spectrum, compared with that of the cationic forms at pH 1.0 and 2.0 (figure 2). This trend was also observed to a certain extent in 4-NH2 Trp and in the fluorescence measurements of substituted barstar residues.
DETECTION
The most interesting and important feature that emerged from these measurements was the change in the emission maximum of aminotryptophans upon pH titration: the two analogs showed opposite trends in their spectral shift (figure 3). Both spectral shifts occurred at higher pH (7 and above) and were characterized by reduced fluorescence intensity. For 4-NH2 Trp, a blue-shifted cooperative titration curve was obtained and curve fitting revealed a transition midpoint between pH 5.2. Conversely, in 5-NH2 Trp, this cooperative transition of the emission maximum was red-shifted with a transition point at pH 5.95 (derived from sigmoid curve fitting; figure 3). Both transitions were found in the substituted annexin V and barstar, where transition midpoints were estimated to be in the pH region between 5.0 and 6.0 (precise determination was not possible since both proteins have a pI-value around pH 5.0). These large differences in spectra behaviors between 4-NH2 Trp and 5-NH2 Trp and related protein variants can be explained by the increased polarity in one of the electronic states, whose stability depends upon the nature, orientation and position of the substitute in the parent indole molecule in a particular solvent.
Conclusion The Safire2 rapidly identified and measured changes in fluorescence emission maxima and intensity in Trp analogs during pH alterations, as a result of the changes in intramolecular charge that originated from cation-to-anion transitions of the amino indoles in the pH range 5.0 to 6.0. The conversion of proteins such as barstar or annexin V from a pH-insensitive native form into a pH-sensitive protein in fluorescence is primarily the result of the intrinsic properties of the aminotryptophan analogs, 4-NH2 Trp and 5-NH2 Trp, which were integrated into the structures of related proteins. Future research will provide a further increase in the number of Trp-like amino acids that are available for redesign of native Trp residues and will enable novel strategies to be developed for generating proteins, peptides and their derivatives with tailored spectral properties. This process can be extremely laborious and time consuming without automation, but the Safire2 offers a range of high speed fluorescence techniques that help to increase throughput and reduce these bottlenecks.
[1] High performance fluorescence scanning of tryptophan and its pH sensitive analogs 4-aminotryptophan and 5-aminotryptophan spectra with the Tecan Safire2 microplate reader. Acknowledgement Tecan wishes to thank Sandra Lepthien and Dr Nediljko Budisa for performing the experiments and providing these data. Dr Budisa’s research group is based at the Max-Planck-Institute of Biochemistry in Martinsried (Germany), and focuses on reprogramming the protein translation machinery of living cells. Greiner is a registered trademark of Greiner Labortechnik GmbH
e-mail: budisa@biochem.mpg.de http://www.biochem.mpg.de/budisa/
Tecan Journal 2/2006
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A P P L I C AT I O N B I O P H A R M A
High throughput microsomal stability assays for analyzing candidate drugs Drug discovery researchers from the Istituto di Ricerche di Biologia Molecolare (IRBM) “P. Angeletti”, the Italian site of Merck Research Laboratories, have developed automated methods for performing high throughput microsomal stability assays using a Tecan Genesis RWS™ 200 Workstation. Automating these assays allows the researchers to predict the pharmacokinetics of multiple candidate drugs in just two days, instead of four, and vastly increases the throughput. Pharmacokinetic analysis of candidate drugs is a critical part of the drug development process, and helps to eliminate compounds with inappropriate bioavailability, toxicity, effective concentrations or duration or persistence of action. Such compounds would fail at later stages in drug development, so eliminating these earlier in the process saves a great deal of money, time and labor.
Dr Massimiliano Fonsi, DMPK Chemist at IRBM, has developed these microsomal stability assays using the Tecan workstation equipped with an eight tip liquid handling (LiHa) arm and robotic manipulator (RoMa) arm, Thermomixer and heater/cooler system. This set-up performs all the liquid handling steps, including sample pooling. The assay can be automated on other Tecan liquid handling workstations, including from the Freedom EVO® series. The protocol includes 1,200 LC/MS/MS runs that represent single incubation steps and, using the novel sample pooling method based on cassette analysis, all of these data can be acquired within 48 hours (as opposed to 96). This system has a number of advantages, as Dr Fonsi explained.
Dr Fonsi at the Istituto di Ricerche di Biologia Molecolare Tecan Journal 2/2006
A P P L I C AT I O N B I O P H A R M A
“My aim is to be able to screen as many candidate drugs as possible. LC/MS is a serial technique, so you cannot read an entire plate in one go - each well has to be analyzed with a single chromatography run. At the moment, the chromatography takes about two minutes and I generate 600 samples, which would take about 48 hours in total. Doubling the samples would take 96 hours, but the high selectivity of mass spectrometry means that it is possible to pool two (or more) samples for analysis, using the Tecan Genesis platform, and each drug can then be analyzed with a specific transition in the mass spectrometer. At the moment our analysis time has been halved; increasing the velocity of the RoMa arm will make it possible to increment the assay throughput by running multiple incubations at the same time and pooling more samples in a final plate for analysis in a single run. Pooling the samples in this way has been used previously for in vivo pharmacokinetics but, as far as I know, nobody else has set up this automated system for HTS microsomal stability in vitro screening.”
“Each of our projects focuses on a specific class of compound, but IRBM is working on different projects, with several different classes of drugs. This means that I have to customize the assay and the analysis for each specific class of compound using the Gemini™ software, and Tecan’s flexibility is fundamental in this respect. We also have to be able to customize the protocol to suit various experimental conditions, such as different concentrations of proteins, different species models and different cofactors. This is very important and, currently, other laboratories using these methods do not have so much flexibility and are only able to run the assay under fixed conditions. We developed this protocol in order to assay different cofactors within the same experiment. We have also introduced control samples that do not contain any cofactors in order to obtain further information about unexpected phenomena that could overlap with the microsomal metabolism, such as precipitation, hydrolysis or other chemical reactions.”
“The method that we have developed also allows us to measure multiple time points, which means that we can calculate the intrinsic clearance of the drug as well as measure the residual percentage. With our method, 20 different compounds can be screened in duplicate, in three different species, collecting six time points over 90 minutes’ incubation. The same basic procedure can also be modified to screen 32 compounds with cofactors but without duplicates, or 10 compounds in duplicate with two different separated cofactors (e.g. NADPH and UDGPA).”
“In the future, we are hoping to get a Freedom EVO 200 Workstation with a Te-MO™ 96 head, which will allow us to increase our throughput even further as well as provide better prediction of a drug’s pharmacokinetics.”
Tecan Journal 2/2006
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A P P L I C AT I O N B I O P H A R M A
The importance of high throughput SNP genotyping for complex disease research in Spain Dr Chris Phillips, Platform Manager at the Santiago de Compostela division of the Centro Nacional de Genotipado (National Genotyping Centre)(CeGen) in Spain
The technique of SNP analysis has become more widely used over the last four to five years because the high density maps it provides make it easier to locate small but potentially significant changes in the genome. It is useful for a wide range of applications – from biomedical investigations in hospitals and academic institutes to drug development studies for biotech or pharmaceutical companies. Three laboratories in Barcelona, Madrid and Santiago de Compostela, collectively the Centro Nacional de Genotipado (National Genotyping Centre)(CeGen), were established in 2003/4 to provide a flexible and high throughput facility for medium to large scale SNP genotyping research projects throughout Spain and beyond.
Tecan Journal 2/2006
The Santiago de Compostela division of CeGen, based at the university, has concentrated on establishing reliable and fast systems to meet its role as a highly efficient contract services laboratory. Two automated liquid handling workstations from Tecan were chosen to achieve a high throughput and prevent bottlenecks in certain parts of the process; a Freedom EVO® 150 platform deals with pre-PCR procedures and an Aquarius™ multichannel pipettor with a 96-channel head handles post-PCR manipulations, both systems running alongside two genotyping platforms, an Applied Biosystems SNPlex™ Genotyping System and a Sequenom MassArray® DNA analysis platform. The Freedom EVO is equipped with a number of additional features, including a Te-MO™ multichannel pipetting option with a 96 channel head, an eight channel liquid
handling (LiHa) arm and a robotic manipulator (RoMa) arm. The complete system automates extraction of genomic DNA from sets of 96 fresh or frozen blood samples with a NucleoMag 96 Blood kit (Macherey-Nagel®). This magnetic beadbased technology yields 1-4 μg of high purity DNA from 100 μl blood. Following extraction, the DNA concentration is determined using PicoGreen® (Molecular Probes), a fluorescent dye that binds the DNA and can be quantified using the laboratory’s Tecan GENios™ plate reader. Measurements are obtained for two serial dilutions for each sample and this highly sensitive technique allows the detection of up to 25 pg/ml of DNA. Five 96-well plates are processed per day by the GENios reader, equating to 480 samples. Using SNPlex, the DNA can then be genotyped on a medium to large scale at low cost and, using 384-well plates, up to 48 SNPs can be simultaneously genotyped in one sample, which equates at present to 70,656 genotypes carried out per week.
A P P L I C AT I O N B I O P H A R M A
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Dr Chris Phillips and his team at the Santiago de Compostela division of the Centro Nacional de Genotipado (National Genotyping Centre)(CeGen) in Spain
Throughput is no doubt a major consideration for the laboratory and the current operation has a potential capacity of 45,000 genotypes per week. The Tecan workstations have achieved the high workflow with reliability and, very importantly for a contract services laboratory such as this, with flexibility. Flexibility and ease of programming is hugely important because often the laboratory has to tailor its processes to suit a project’s needs, for example, researchers might send samples in unusual plates or formats or, even more likely, will need the optimization of an entirely new set of assays. SNP analysis can be very straightforward when you are only dealing with, for example, six markers that remain constant; you know what causes problems and what works well. For the laboratory in Santiago, however, there is normally a completely new set of markers every few weeks and, each time this happens the assays and techniques need to be re-optimized which takes a significant amount of time. On occasion, the set of markers may only be for a small number of samples, say 400, and the analyses might be complete within just a couple of days but the optimization may have taken a week or more. This is a challenge compared to a diagnostic set-up, for example, where the markers are known and similar samples are regularly received.
Under these circumstances, flexibility and easy programming is very important and is undoubtedly met by Tecan’s instruments, especially the Freedom EVO workstation. A new workflow for handling different volumes; a five-tip head for cherry picking and the 96-tip head working well together; features like this are important advantages of the Freedom EVO that have helped the Santiago laboratory to be prepared for whatever project comes round the corner.
SNPlex is a trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries MassArray is a registered trademark of SEQUENOM, Inc. Macherey-Nagel is a registered trademark of Macherey-Nagel PicoGreen is a registered trademark of Molecular Probes, Inc.
SNP Tecan Journal 2/2006
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SAMPLE MANAGEMENT
Building a world-class compound The fully integrated drug discovery and development company Amphora, based in North Carolina’s Research Triangle Park in the USA, has chosen REMP automated storage and retrieval systems to take control of its compound library. Over the last five years, Amphora’s compound management laboratory has been building a library of over 130,000 compounds to serve the company’s research projects that use chemogenomic, system biology and microfluidic technologies to develop targeted therapies for oncology, inflammation, Alzheimer’s disease and diabetes. Managing such a library and keeping up with all the internal and external customers’ demands is no easy task, as Ioana Popa-Burke, Associate Director at Amphora, explained: “Although we are a relatively small biotech company, our compound management still needs to be first class. Tracking all the samples manually is logistically extremely complicated so we evaluated all the major compound store manufacturers prior to purchasing the REMP system. Many of us have previous experience of The compound management lab at Amphora
Tecan Journal 2/2006
compound management from bigger pharmaceutical companies and we wanted to apply those same management principles to build a world-class management system on a smaller scale.” “We were really attracted to REMP for several reasons; firstly, the company has an extremely good reputation and every single independent user we talked to spoke very highly of it; secondly, by acquiring just the core of REMP’s SmallSize Store™ (SSS) and Mid-Size Store™ (MSS) we are able to stay within budget and grow the system as we need to.” A high quality sample storage system such as REMP’s is essential for Amphora as the company puts significant effort into ensuring that all of its compounds are of the highest quality. All of the compounds in the library are purified by LC/MS and are all more than 95% pure. Using analytical chemistry techniques,
the compounds are constantly monitored for stability and solubility and, therefore, the reliable storage conditions and tracking advantages of REMP’s systems are critical for Amphora’s standards. This also provides further advantages for Amphora’s screening processes, as Ioana explained. “We depend on very high quality screening to be able to find highly selective, target-specific compounds. We screen kinases, proteases, phosphatases and some ion channels and we can pick compounds with any selectivity profile we want. If our compounds were not of sufficiently high quality then all our screening efforts would be effectively useless. This also has ramifications for all of our disease research areas. For one of our inflammation projects, for example, we have worked on a p38α inhibitor. Using our industrialized drug discovery
SAMPLE MANAGEMENT
management system
platform, of which the REMP system is an integral part, we have been able to pick a p38α selective compound while most people are working on p38α/p38β dual inhibitors! Overall, we have been extremely pleased with the system, it has made a big impact on our daily routine as well as on the quality of the compounds and data tracking.” Amphora’s REMP system includes the Tube Punching Module™ (TPM) and associated devices, the Reatrix™ DataMatrix Scanner for reading the storage tube rack bar codes and 2D barcoded REMP 96 Tube Technology™ consumables. The TPM is a desktop device that allows PC-controlled selection of REMP 96 (or 384) tubes, which are directly transferred from storage tube racks into delivery tube racks for collecting and reformatting. The TPM performs the transfer through a single axis movement, reducing handling error
Tube Punching Module
Small-Size Store Automated Capper/Decapper
True scalability with REMP’s Tube Technology Reatrix
Storage Tube Rack with 2D Code
rates, and is faster and more reliable than conventional pick and place methods. It allows tubes to be cherry picked at any time, leaving the destination tube racks within the storage environment until instructed otherwise, and is extremely useful for aiding sample tracking. Brian Hardy, the Lead Scientist at Amphora, who runs the REMP system on a daily basis has found its ease of use and
reliability particularly important: “The REMP system has been essential for us to set up our tube-based library. It has considerably improved our process, in terms of both speed and data quality, and we have had no problems at all. The TPM and Reatrix Scanner work seamlessly and are very reliable; they have drastically reduced our potential for error. We can now cherry pick compounds as and when we like, we can use multiple formats, and the technology has helped us a great deal already to meet customer and collaborator requirements. I operate the whole system daily and have never had to use REMP’s helpline since the system has been up and running!”
REMP’s sample processing and storage concepts were originally adopted by large facilities, but are equally important for smaller scale laboratories to be able to access high quality sample management. The REMP Tube Technology consumables provide this all important entry level step, allowing you to set up your compound library in the tubes, which are supported by devices such as the Tube Punching Module (TPM), automated Capper/ Decapper™ or Tube Sealer and the Reatrix 2D scanner. The TPM is easy to integrate into automated systems, so once your library grows it becomes easy to move the tubes into a fully automated REMP store, such as the SSS.
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A P P L I C AT I O N B I O P H A R M A
Analyzing cholinesterase in organophosphate poisoning The German army automates the determination of AChE activity in whole blood Bodo Pfeiffer, Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany Agrochemicals belonging to the organophosphate group are used extensively around the world, due to their efficacy and comparatively low environmental persistence. They are, however, highly toxic and present a considerable risk to humans, as demonstrated by the estimated 3 million acute pesticide poisonings and 200,000 fatalities globally every year. A few particularly poisonous organophosphates, including soman, sarin, tabun and VX (known as nerve agents), were developed as chemical weapons and have even been used recently, notably in the Iran-Iraq war and the Tokyo underground in 1995, despite being outlawed around the world. Organophosphates inhibit serine esterases, particularly acetylcholinesterase (AChE), by covalently binding to the enzyme and disturbing its physiological function – i.e. preventing cleavage of the neurotransmitter, acetylcholine (ACh), by AChE. This leads to an accumulation of ACh in cholinergic synapses of both the central and peripheral nervous systems and at the neuromuscular junction, resulting in hyperexcitation and subsequent paralysis of the target organs and, eventually, death due to central and peripheral respiratory paralysis1,2. Organophosphate poisoning can be treated by immediate administration of atropine and AChE reactivators (oximes; Obidoxime in Germany, pralidoxime in the UK and USA) but it is necessary throughout treatment to measure and Tecan Journal 2/2006
monitor levels of the affected enzyme within the body. Muscular AChE is not easily accessed in patients, so erythrocyte AChE and plasma cholinesterase (butyrylcholinesterase; BChE) are analyzed instead. The Bundeswehr Institute of Pharmacology and Toxicology co-operates with the South Asian Clinical Toxicology Research Collaboration Centre for Tropical Medicine, University of Oxford, England3 in a prospective cohort clinical trial in Sri Lanka. Blood samples from patients are analyzed in the Institute for AChE activity, BChE activity and hemoglobin. Until recently, the parameters have been analyzed manually by measuring a 3 ml sample using a standard commercial spectrophotometer with a temperaturecontrolled cuvette holder and water bath at 37ºC. However, this manual procedure quickly reached its limits when analyzing large numbers of samples and, to solve this problem, an automated microplate method was developed to process multiple samples in parallel. Tecan’s Genesis 150 workstation was introduced, with automated pipetting and a robotic arm as well as an integrated spectrophotometer, a 37°C incubator, temperaturecontrolled microplates racks and cooled sample preparation (figure 1a). Adapting the protocol for automation was relatively straightforward because the
parameters used previously could almost all be translated and known incubation times, wavelengths and measuring times were not altered. This application can be automated on other Tecan liquid handling workstations, including from the Freedom EVO® series. Cholinesterase activity is measured in a whole blood sample collected from the patient and immediately diluted and cooled to slow down any ex vivo reactions that can rapidly take place between AChE, organophosphates and oximes. Cholinesterases convert specific substrates and, in the conversion process, thiocholine is formed as a product (figure 1b). This product reacts with DTNB (5-5'dithio bis-(2-nitrobenzoic acid)) present in the sample to give a yellow dye that can be measured with a spectrophotometer; the color development over time serves as a direct measure of the enzyme’s activity. The hemoglobin levels in the dilutions are also measured, using Zijlstra’s method4, to allow correction of any diluting errors in preparation of the samples; the AChE activity can be correlated with the hemoglobin levels5,6. To validate this set-up, extensive comparative measurements were made using both the automated and manual methods. AChE activity was analyzed in aliquots of native donor whole blood that
A P P L I C AT I O N B I O P H A R M A
The determination of cholinesterase status is primarily intended for analyzing patients poisoned with organophosphates (accidental or suicidal poisoning), but it can also be applied after the use of nerve agents in military conflicts or terrorist attacks, as well as in the context of occupational medical examinations.
OR:
Butyrylcholinesterase Butyrylthiocholine + H2O thiocholine + butyrate
→
Thiocholine + 5,5´dithio-bis(2-nitrobenzoate) 5-mercapto-2-nitrobenzoate + 5-mercaptothiocholine nitrobenzoate [yellow] (TNB)
Figure 1a: Tecan GENESIS 150 workstation. The workstation is an open source system and can also be used now for kinetic samples. It has an integrated Sunrise™ spectrophotometer and uses TOPS™ 4.0 with Magellan
Figure 1b: Formation of DTNB produces a yellow dye that serves as a direct measure of cholinesterase activity
Quinalphos poisoning
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Figure 2: Example of Quinalphos poisoning. The reactivation capability shows the maximum achievable AChE activity. The per cent inhibition shows that no residual inhibitor remains after 4 hours
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Once validated, the automated method was used to analyze and monitor AChE and BChE activity in samples from the Sri Lanka patients who had attempted suicide by oral ingestion of pesticides (quinalphos or fenthion), (figures 2 and 3). Whole blood samples were collected from the patients (t = 0) and oxime therapy was started with pralidoxime (bolus dose and infusion therapy); further samples were collected within a predefined time window. Quinalphos results in diethylphosphorylation of AChE, this enzyme species can be readily reactivated (figure 2) and so, four hours after admission to the hospital, no residual inhibitor remained in those affected patients. Fenthion results in a dimethylphosphorylated enzyme that ages quickly and cannot be reactivated (figure 3), meaning that in patients who had ingested this pesticide, no significant increase could be recorded in AChE activity, and BChE activity remained completely inhibited.
Acetylcholinesterase Acetylthiocholine + H2O thiocholine + acetate
AChE activity (mU/ml)
were inhibited with 100 nM soman; BChE activity in plasma harvested from somaninhibited blood samples was measured and hemoglobin levels were determined in dilutions of whole blood samples with different proportions of erythrocytes and added plasma. In addition, the reactivation capability of whole blood inhibited with either 100 nM soman, which does not allow AChE reactivation, or 200 nM paraoxon-ethyl (PxE), which readily allows AChE reactivation, was analyzed and the residual inhibitor was also measured in donor plasma samples inhibited with five different concentrations of paraoxon-ethyl. The results obtained with both methods correlated very strongly in all measurements.
2500 2000 1500 1000 500 0u uu 0
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Figure 3: Example of Fenthion poisoning. Due to ageing, reactivation is almost impossible (top). In spite of a decline in residual inhibitor (middle) no significant increase in AChE activity is recorded (top). BChE activity is completely inhibited (bottom)
References 1. Wiener SW, Hoffman RS. (2004) Nerve agents: a comprehensive review. J. Intensive Care Med. 19, 22-37 2. Koelle GB. (1992) Pharmacology and toxicology of organophosphates. In: B. Ballantyne and T. C. Marrs (Eds.), Clinical and experimental toxicology of organophosphates and carbamates. Butterworth & Heinemann, Oxford, pp. 35-39 3. Eddleston et al. (2005) Differences between organophosphorous insecticides in human selfpoisoning: a prospective cohort study. Lancet 366: 1452-1459
4. van Kampen EJ, Zijlstra WG (1961) Standardization of hemoglobinometry, II. The hemoglobincyanide method. Clin Chim Acta 6: 538-544 5. Thiermann H, Szinicz L, Eyer F, Worek F, Eyer P, Felgenhauer N, Zilker T. (1999) Modern strategies in therapy of organophosphate poisoning. Toxicol. Lett. 107, 233-239 6. Worek et al. (1999) Improved determination of acetylcholinesterase in human whole blood. Clin Chim Acta 288: 73-90
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EVENTS 2006
Meet Tecan at these events China 4th Transfusion Congress of Chinese Society of Blood Transfusion
Hangzhou
May 11-13 2006
Tecan/Eastwin Biopharma: Forensic workshop
Tianjin
May 18-19 2006
Tecan/Eastwin Biopharma: Drug screening workshop
Shanghai
June 13-15 2006
5 Youth Conference on Clinical Diagnostic Medicine
Guiyang
June 21-23 2006
CCLab 2006
Qingdao
Sept 15-19 2006
Analytica
Munich
April 25-28 2006
Safe and Flexible Biotechnology
Munich
May 04-05 2006
38th European Human Genetics Conference
Amsterdam
May 06-09 2006
MipTec 2006
Basel
May 08-11 2006
Compound Management, Integrity & QC
London
May 23-24 2006
10th European Workshop of Molecular Cytogenetics in Human Solid Tumours
La Grande Motte
June 08-11 2006
XXX Nordic Congress in Clinical Chemistry
Copenhagen
June 14-17 2006
50˚ Siga Annual Congress
Ischia
Sept 10-14 2006
IFR 128 Journees Screening
Strasbourg
Sept 11-18 2006
DGTI and ISCT Europe
Frankfurt
Sept 19-22 2006
Biotech Forum and Scanlab
Copenhagen
Sept 26-28 2006
th
Europe
Japan International Bio Expo
Tokyo
May 17-19 2006
International Congress of Biochemistry and Molecular Biology
Kyoto
June 19-23 2006
Orlando, FL
May 22-24 2006
AACC
Chicago, IL
July 23-27 2006
Drug Discovery
Boston, MA
Aug 7-10 2006
SBS
Seattle, WA
Sept 17-21 2006
ISHI
Nashville, TN
Oct 9-12 2006
ASHG
New Orleans, LA
Oct 9-13 2006
Neuroscience
Atlanta, GA
Oct 14-18 2006
AABB
Miami Beach, FL
Oct 21-24 2006
ASCB
San Diego, CA
USA ASM
Tecan Journal, Customer Magazine of Tecan Trading AG., ISSN 1660-5276 Design: OTM/London www.otmcreate.com Photography: Marc Wetli/Zürich www.wetli.com, Günter Bolzern/Zürich www.bolzern.net, Susanne Völlm/Zürich www.susannevoellm.ch Editor: kdm/UK www.kdm-communications.com Print: DAZ Druckerei Albisrieden AG/Zurich www.daz.ch Address: Tecan Switzerland AG, Marketing Communications, Seestrasse 103, CH-8708 Männedorf, Switzerland, journal@tecan.com,www.tecan.com Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, however, it is possible that omissions or errors might have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this
Dec 9-13 2006
publication can be made at any time without notice. All mentioned trademarks are protected by law. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. This brochure may contain reference to applications and products which are not available in all markets. Please check with your local sales representative. Aquarius, Gemini, Genesis RWS, GENios, i-control, Infinite, Magellan, PosID, Sample Tracking, Safire2, Te-Flow, Te-MagS, Te-MO and TOPS are trademarks and Freedom EVO, Freedom EVOlyzer and Freedom EVOware are registered trademarks of Tecan Group Ltd., Männedorf, Switzerland. Tecan is in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland. Automated Capper/Decapper, Mid-Size Store, Reatrix, Small-Size Store,Tube Punching Module and Tube Technology are trademarks of REMP AG, Oberdiessbach, Switzerland © 2006 Tecan Trading AG, Switzerland, all rights reserved.
Headquarters: Tecan Group Ltd., Seestrasse 103, CH-8708 Männedorf, Switzerland T +41 44 922 88 88 F +41 44 922 88 89 info@tecan.com www.tecan.com Asia (Pte) Ltd +65 644 41 886 Tecan Sales Austria GmbH +43 62 46 89 33 Tecan Sales International GmbH +43 62 46 89 33 Tecan Benelux B.V.B.A. +32 15 42 13 19 Tecan Benelux B.V.B.A. +31 18 34 48 17 4 Tecan Group Ltd., Beijing Rep. Office +86 10 586 95 936 Tecan Deutschland GmbH +49 79 51 94 170 Tecan France S.A.S. +33 4 72 76 04 80 Tecan Italia S.r.l. +39 02 215 21 28 Tecan Sales International GmbH +43 62 46 89 33 Tecan Japan Co. Ltd +81 44 556 73 11 Tecan Nordic AB +46 31 75 44 000 Tecan Nordic AB, Rep. Office Denmark +45 70 234 450 Tecan Portugal +351 21 000 82 16 Tecan Sales Switzerland AG +41 44 922 89 22 Tecan Iberica Instr. S.L. +34 93 490 01 74 Tecan UK Ltd. +44 11 89 300 300 Tecan US Inc. +1 919 361 5200 REMP AG (Switzerland) +41 31 770 70 70
Tecan Journal 2/2006
REMP (USA) Inc. +1 508 429 2200
REMP Deutschland GmbH +49 6126 5831 0
REMP Nippon AG +81 3 3539 1771
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