tecan journal edition 02/2006

Tecan JournalEdition 2/2006

ISSN 1660-5276

Four new software packages from Tecanpage 4

All Tecan’s manufacturing sites achieve ISO accreditationpage 7

Unlimited throughput with Tecan andIllumina’s combined technologiespage 8

Cover shows Dr Gerlinde Zerza-Schnitzhofer,Application Specialist/Marketing,Tecan Austria Ges.m.b.H, Austria

2 W E LCO M E

Tecan Journal 2/2006

A big thank youto you, our customers

I would like to take this opportunity topersonally thank you for the trust youhave placed in Tecan, a trust that allowedus to achieve a milestone year of growthin 2005. This year we will build on thattrust, starting with the creation of asimplified and more effective organi-zational structure and the simultaneousstrengthening of our service organi-zations in all markets. Together, theseefforts reflect our commitment toproviding you with the best possiblelevels of customer service and support.

We will, of course, continue toconcentrate on targeted productinnovation. Outstanding new solutionswill be launched in the coming year,giving you the speed, reliability andmodularity that you have come to expectfrom Tecan, and ensuring that youachieve the best possible performancefrom your Tecan investment.

Thomas BachmannChief Executive Officer (CEO)

This edition of the Tecan Journal featuresa number of exciting projects using oursample storage, liquid handling anddetection solutions for diverse appli-cations, ranging from genotyping for thediagnostic market to the compilation andmaintenance of RNAi and compoundlibraries in the biopharma sector. You canalso find out about our powerful newsoftware packages, and learn aboutTecan’s recent ventures to standardizemanufacturing facilities and improvecustomer services in the US.

We hope you enjoy this issue of the TecanJournal and look forward to supportingyou for many more years to come.

Welcome and ContentsThomas Bachmann, CEO, thanks you,our customerspages 2-3

Software focus: four new packages from Tecan enhance the connectivity of Tecan’smodules for seamless automation anddata controlpage 4

Freedom EVOware® on Freedom EVO®ClinicalIVD-D compliance opens doors to clinicalapplicationspage 5

In conversation with Craig Williamson,Director of Customer Support for Tecan USpages 6

All Tecan's manufacturing sites achieveISO accreditation Günter Weisshaar, Executive Vice President Global Quality and RegulatoryCompliance, explainspages 7

Unlimited throughput with Tecan andIllumina’s combined technologies A powerful new toolset for understandingthe genetics of complex diseasepages 8-9

Production of a genome-wide RNAinterference library for functional genecharacterization in cultured human cellsTecan’s Freedom EVO® handles esiRNAlibraries for automated cell transfectionpages 10-12

High performance fluorescence scanningof tryptophan with Tecan’s Safire2™microplate readerAutomation helps with the design of tailormade reporter genespages 13-15

High throughput microsomal stabilityassays for analyzing candidate drugs Drug development is faster with Tecan’sautomated systemspages 16-17

The importance of high throughput SNPgenotyping for complex disease researchin SpainTecan’s Freedom EVO® workstation is usedfor reliable, low cost genotypingpages 18-19

Building a world-class compoundmanagement systemAmphora uses REMP systems to manageits compound librarypages 20-21

Analyzing cholinesterase inorganophosphate poisoningThe German army automates thedetermination of AChE activity in whole bloodpages 22-23

Events for 2006Meet Tecan at these events page 24


3CO N T E N T S

4 G LO B A L N E W S4 L AT E ST P R O D U C TS

Software focus: four new packages fromTecan that unite to integrate your data


Our Freedom EVOware Sample Trackingmakes it easy to electronically record thestatus of all your samples throughoutyour entire application. Sample Trackingcan register scanned labware thenintegrate and process all data acquiredfor each sample, including pipetting andplate reader data. These are stored andthen used to generate and export reportscontaining the results. Sample Tracking iscompatible with an extensive range ofTecan products, simplifying the logisticsof handling large numbers of samplesand improving your quality controls.It also integrates data from FreedomEVOware 1.2 and Magellan 6.0 for simplecombination of plate readers andpipetting platforms. Sample Tracking isvery much central to the interactions ofall the new software (see figure), since ittracks movement of all plates (as con-trolled by Freedom EVOware) and allpipetting actions. Data and results fromplate readings are fed to Sample Trackingfor the generation of reports at the endof the process and these can also beaccessed remotely.

Freedom EVOware 1.2 integrates the newi-control software (see below), forstraightforward combination of theInfinite™ 200 multimode microplatereader with your Freedom EVO, and is alsocompatible with the new Magellan 6.0,allowing integration of other multimodeplate readers with the workstation.The integration means that the Infinitereader, for example, will appear inFreedom EVOware’s control bar and scripteditor in the same way that othersupported devices appear and are calledup, for easy process definition. FreedomEVOware’s functionality can be extendedwith add-ons such as Sample Tracking,Normalization and Freedom EVOwareSample Oriented software. It supports avariety of hardware, both from Tecan andfrom third parties, bringing greaterflexibility to your laboratory.

Freedom EVOware 1.2 has many othernew, user friendly features to help yourapplications run more smoothly. Theseinclude a start-up wizard, which providessimple access to common daily routines,helping you to get your processes up andrunning quickly and easily, and theintuitive graphical user interface, whichmakes new assay protocols easy tocreate. There are also wizards to help youcreate advanced pipetting procedures,including relatively complex routines, androbotic moves are graphically defined,helping you to visualize processes.Sequences can be simulated and viewed,making it quicker and easier to developnew protocols as well as allowing remotemonitoring. The package also comes withan expanded library of device drives andits modular architecture makes it easy toextend the software as new devicesbecome available.

At Tecan we realize that communication between your instruments is key, so we havereleased a range of new software packages that enhance connectivity, for simpleintegration of your plate readers with Tecan’s Freedom EVO® workstations andseamless combination of your data. The four new software packages are FreedomEVOware® Sample Tracking, Freedom EVOware 1.2, i-control™ and Magellan™ 6.0.

Get platefromCarousel

Pipette toreactionplate

Read plate

Generatereports

Access dataremotely

Freedom EVOware

Freedom EVOware Sample Tracking

SampleTracking DB

5L AT E ST P R O D U C TS

The Freedom EVO Clinical with FreedomEVOware and its new module for sampleoriented processing, which is predominantlyused for clinical applications, is a powerfultool for controlling the Freedom EVO Clinicalfamily of application-oriented openplatforms for clinical diagnostics and bloodbank environments.

The Freedom EVO Clinical/EVOware systemoffers:

• application-oriented, scalable precisionliquid handling and robotic platform forclinical applications like EIA preparationand pooling

• extended functional spectrum byintegration of new options for clinical use

• enhanced process control and safety features without compromisinginstrument flexibility

• scheduler function for increasedthroughput

• true walk-away capability

Freedom EVOware® on FreedomEVO® Clinical, Tecan’s automatedplatform for clinical diagnostics


Freedom EVOware supports a wide rangeof Tecan modules, including the LiHa armand dual LiHa arm, RoMa arms, Pick andPlace (PnP), Te-MO™, Te-MagS™,incubators, hotels, PosID™ 2 and PosID 3.The new Freedom EVOware 1.2additionally supports Infinite (through i-control), Sample Tracking, SampleOriented.

Freedom EVOware 1.2 is available in twoversions, Freedom EVOware Standard andFreedom EVOware Plus, which has extrabenefits including a fully integratedpipetting scheduler, which can scale upyour application for multiple plate runswithout requiring new software. There isa dynamic scheduler for efficient use ofresources and an autocalibration ofschedule feature, which uses real time tocreate a better sample processingschedule, allowing higher throughput.The Freedom EVOware Standard allowssynchronous or asynchronous pipettingwith two LiHa arms, while in FreedomEVOware Plus the two LiHa arms caneither be used for one process or differentLiHa arms can be used for differentprocesses.

Tecan’s new i-control software installsitself into the Freedom EVOware 1.2, sothat the Infinite 200 plate reader can befully combined with other applicationprocesses running on the Freedom EVO.Typically, when there is a plate readerintegrated into a Freedom EVO platform,a RoMa arm transports the sample platefrom the workstation and puts it into thereader, where the plate is read and dataare generated; the RoMa arm thenremoves the plate. Fully integrating thereader’s control software into FreedomEVOware allows complete automation ofthe reader processes by your FreedomEVO and the i-control takes over fromFreedom EVOware once the microplate istransferred into the reader. It then passesthe control back when the reading iscomplete, so this compatibility betweenthe software is a really beneficial newfeature. The new i-control has the samelook and feel as Freedom EVOware 1.2.

The new Magellan version 6.0 iscompatible with all of Tecan’s multimodeplate readers and has powerful datareduction capabilities, including beingable to perform multiplate analyses,comprehensive spectra calculations and3D scans. It supports all major detectionmodes, as well as the injector system, andis designed to allow customers to complywith 21 CFR part 11 regulation, forelectronic records and electronicsignatures. The new program has anintuitive, wizard-guided user interface

For more information on our newsoftware, please contact Jason Meredith,Global Product Manager Software:[email protected]

For more information on Freedom EVOClinical please contact Christoph Beck,Product Manager Corporate Account &Clinical Diagnostics:[email protected]

Freedom EVO Clinical is not available in theUSA although sample oriented FreedomEVOware is available with pooling functionsdisabled.

that maximizes its ease of use, allowingseamless process control, automateddata import functions in variableformats, and easy and fully automateddata export to LIMS. The program evencomes with example files of typicalapplications to get you started quickly.

6 C U STO M E R S U P P O RT


Craig Williamson is the Director ofCustomer Support for Tecan US, and hasheld the position since 1999. He isresponsible for Expertline, the helpdeskand the entire service organization forNorth, Central and South America.

How do you manage to look after allyour customers simultaneously?

Our service structure is tailored to theneeds of our customers and the USmarket to maximize our efficiency. We areseparated into four regional sales andservice regions: the north east, the southeast, the mid-west and the west. In eachof these regions we have a regionalservice manager who takes care of thatterritory and makes sure the customersreceive the best care.

And what do you do in CustomerSupport?

My organization ensures that ourcustomers receive first class care andsupport, providing them with completepeace of mind. We run a helpdesk witheight people from 8 am to 8 pm and weanswer whatever technical questions thecustomer has, organize package distri-bution and dispatch field engineers. Weare responsible for the installation ofinstruments, for preventive maintenanceto minimize downtime, for deviceupgrades and repairs and for training our customers in the proper use of theinstruments. There is a lot of workinvolved in the Customer Supportorganization and, in Tecan US, we put40% of Tecan’s resources into service.

How do you respond to customerqueries?

We aim to provide our customers withcourteous, timely and efficient service, soour policy is to respond and solve ourcustomers’ requests quickly. For complexrequests that cannot be solved instantly,we have experts with specialist knowledgeat hand and, if an intervention is neces-sary, we dispatch one of our qualified and certified field engineers on-site totake 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 Tecanproduction site.

Our goals are to get the customer up and running within four hours on thetelephone, or 48 hours if we have todispatch engineers to the customer’s site, and 95% of the time we meetthose goals.

How satisfied are your customerswith the service you provide?

Every service call that we answer has asurvey attached, which the customer canfill in, to give their opinions regarding ourservice. I compile that information andsend it to Tecan’s headquarters inSwitzerland. I strive to get ratings of‘good’ or ‘excellent’ on over 90% of thosereports and, in the last quarter of 2005,we achieved that on about 96% of thosereports.

What are the most importantaspects of providing good service?

The customers want competency,efficiency, communication, punctualityand, of course, recovery of theirinstruments to peak condition in theshortest time possible. It is alsoimportant for them to see the same field engineer with each visit.

What’s special about Tecan’scustomer service?

A couple of years ago, Tecan US combinedthe two departments that used to becalled Applications and Service, wherethe scientists worked in Applications andwere separated from our electronictechnicians. By changing this organi-zation, we are able to provide ourcustomers with the appropriate expertsfor every intervention, whether thatrequires specialist knowledge for liquidhandling and applications or hardware.Our range of application scientistsincludes biologists, microbiologists andchemists, for example, and I think thatthis is a real asset for increasingcustomer satisfaction. There is no otherorganization that has switched to thissystem, as far as I know - it allows us torespond to calls within a shorter timeand considerably increase our efficiency.

What does the future hold forCustomer Support at Tecan?

Service is very important for ourcustomers and, therefore, it is one of themost important departments in Tecan.Maintaining good customer service isalso the key to keeping our customersand we are constantly working to makesure that our customer service is number one.

at your service

There is no otherorganization thathas switched to thissystem, as far as Iknow - it allows usto respond to calls within ashorter time andconsiderablyincrease ourefficiency.


7Q UA L I T Y A S S U R A N C E

Standardization and regulatorycompliance are of critical importance forTecan and its customers so, over the lastfew years, we have concentrated onimplementing the newest ISO standards -ISO 9001 and ISO 13485 - into all of ourmanufacturing sites; in Switzerland,Austria, Tecan SCC in Mainz, Germany,and in Tecan Systems in California.Although all these sites were alreadycertified with local companies, our aimwas to reach a universal globalstandardization with just one notifiedbody. We have now achieved this aim indirect association with TÜV SÜD ProductService, chosen as one of the mostcommon, global notified bodies with aparticular expertise in the medical devicebusiness and a very strong reputation forhigh standards.

Many of our customers also use TÜV astheir regulatory partner and it makesperfect sense that we work to the samestandards and can talk on the same level,especially since TÜV has been givenauthorization from the United States FDAsince 2002 to perform and executefollow-up inspections on its behalf.

Our customers can expect that, with theISO 9001 quality system regulation, Tecanhas met all the elements required andhas been audited and proved by a neutralcompany or party. ISO 13485 dealsspecifically with additional requirementsfor medical devices. At the same time, oursites in Switzerland and Austria are alsocertified according to CMDCAS -Canadian Medical Device ConformityAssessment System – an additionalnational regulation to ISO 13485 requiredin Canada and also dealt with by TÜV.

We are delighted with these newimplementations and trust that they willgive you, our customers, the reassuranceof working together with a knowledge-able supplier that is fully compliant withthe highest international regulations.

Quality Assurance and Regulatory Affairs

� Tecan Switzerland Ltd.Seestrasse 103CH-8708 Männedorf

� Tecan Austria GmbHUntersbergstrasse 1aA-5082 Grödig/Salzburg

� Tecan Software Competence Center GmbHSchmalweg 5D-55252 Mainz-Kastel

� Tecan Systems, Inc.2450 Zanker RoadUSA-San Jose, CA 95131

Günter Weisshaar, Executive Vice President Global Quality and RegulatoryCompliance, has news of ISO certification for all Tecan’s manufacturing sites.

8 A P P L I C AT I O N B I O P H A R M A

parallel and this scale of sample through-put was crying out for automation, soIllumina turned to Tecan for help. Tecanliquid handling workstations can be usedto automate the pre-PCR steps or boththe pre- and post-PCR steps involved inthe assay, streamlining the workflow andreducing the potential for human error.Processing these assays with Tecanworkstations combined with Illumina’sBeadArray™ Readers, allele-callingsoftware and tied together with LIMSessentially formed the BeadLab systems.

“Many of our scientists hadworked previously with Tecan’ssystems in production sequencingenvironments,” said Bill Craumer,Director of Corporate andMarketing Communications at Illumina. “They valued the

flexibility of automated, robustpipetting into either tubes orplates with high precision,so it was natural to specify Tecan workstations for ourgenotyping BeadLab.”

Illumina was both a principal investigatorand a supplier of BeadLabs for otherprinciple investigators in the $100 millionInternational HapMap Project and, byextension, Tecan was a key contributor tothe Project’s success. Illumina hasinstalled BeadLabs automated with Tecanworkstations at virtually every HapMapresearch center, including the WellcomeTrust Sanger Institute, the Broad Institute,Génome Québec and Japan’s RIKEN.The International HapMap Consortiumwas established in 2002 to develop ahaplotype map of the human genome,with the aim of identifying commonpatterns of DNA sequence variation in thehuman genome1. Sets of nearby singlenucleotide polymorphisms (SNPs) on thesame chromosome are inherited in blocksand the pattern of SNPs on a block formsa haplotype. Although the haplotype maycontain a large number of SNPs, only afew SNPs are needed to uniquely identifythe haplotype. The HapMap Project hasmapped the haplotype blocks and thespecific SNPs that identify the haplotypes,which are called tag SNPs. Each institutionin the consortium was assigned a certainchromosomal region to map, resulting inlarge amounts of data that were madepublicly available from 2003 onwards2

and, in 2005, the completed haplotypemap was announced3. The mapped tagSNPs can now be used instead of the tenmillion SNPs to examine the entirehuman genome for variation, such as in

Unlimited throughput with Tecan andIllumina’s combined technologies creates a powerful new toolset for understandingthe genetics of complex disease


BeadStation with Freedom EVO

Illumina, a San Diego (CA)-based company,has integrated Tecan liquid handlingworkstations into its revolutionary, end-to-end solutions for large-scale genotypingapplications. The collaboration has beencrucial for the development of a numberof products, including Illumina’sproduction-scale BeadLab systems, whichwere deployed by major investigatorsworld wide as part of the InternationalHapMap Project and, more recently,Illumina’s bench-top BeadStation. Both ofthese platforms offer automated liquidhandling capability using Tecan’s flexibleworkstations.

Illumina initially started talking to Tecanto automate the GoldenGate® Assay,Illumina’s custom genotyping assay thatcan multiplex up to 1536 targets persample. Illumina’s microplate compatibleArray Matrix processes 96 samples in


9A P P L I C AT I O N B I O P H A R M A

HumanHap 300

Illumina’s beads-in-wells technology. The 3 μmfeatures are the smallest in the microarrayindustry

association with a particular disease orphenotype. Depending on the populationstudied, between 250,000 and 500,000tag SNPs provide comprehensive genomiccoverage, and these large-scale diseaseassociation studies are currently ongoing.

Illumina has now deployed tag SNPs onits new HumanHap BeadChips, which canquery up to 500,000 different SNP loci ona single microarray. The BeadChips rely onIllumina’s Infinium™ assay, which allowsinterrogation of the human genome atsingle base resolution without needingPCR or ligation steps. Precise liquidhandling is critical to the Infinium assayso, once again, Illumina chose Tecan forautomating the application. Tecan workedclosely with Illumina to provide the Te-Flow™4 flow cell technology on theFreedom EVO® family of workstations toenable parallel processing of 24BeadChips simultaneously. The FreedomEVO can be used to automate every stepof the Infinium genotyping process,offering pre-optimized and ready-to-runprotocols, and allowing much moreefficient management of large samplenumbers. Most HumanHap BeadChipusers have at least one Freedom EVO witha modified robotic manipulator (RoMa)arm to help achieve higher samplethroughput and allow LIMS integrationfor positive sample tracking.

“A key driving force behind thisstrategic partnership was todeepen our understanding ofcustomers’ needs and provide the technology they really want,”explained Christoph Beck atTecan. “Illumina was able tosuccessfully automate theInfinium assay with BeadChips by combining our high precision,

robust liquid handling technologywith the Te-Flow cell flowtechnology. At Tecan, we canprovide customers with access to our instruments’ applicationprogramming interface, and thismade it much quicker and easierfor Illumina’s developers tointegrate the workstations intotheir own software environment,and tailor the instruments’performance to exactly meetrequirements.”

As a result of Illumina and Tecan’scombined technology, researchers cannow routinely generate millions ofgenotypes per day. “Reliability and qualityare critical for our customers, and the factthat Tecan’s workstations integrate sonicely into a LIMS-controlled environmentfor all required production capacities isreally important,” said Bill.

“Over the last five years, Tecan’srelationship with Illumina has evolvedfrom that of an equipment supplier to ahighly strategic collaborator, and Tecanhas provided essential value to Illuminaand to our customers,” added Alex Chan,Illumina’s Associate Product Manager,Systems & Software.

Illumina has also started offering Tecan’sFreedom EVO workstations as a standardupgrade to bench-top BeadStations forusers that want the automated, liquidhandling capability for smaller scales ofthroughput. In the future, Illuminaanticipates incorporating Tecanautomation options with its geneexpression applications.

Illumina’s BeadArray technology

The BeadArray technology involves the randomassembly of oligo-coated 3 μm beads into wells etchedinto one of two different substrates:

1) the 96 sample Sentrix Array Matrix (which wasstandard for the HapMap Project), where each array is composed of 50,000 fiber optic strands

2) the Sentrix BeadChip, designed for denser, wholegenome applications. The BeadChips are available insix different configurations and can contain up to 12 million beads (or features) each, and every feature is represented on average over 30 times, providingreally high confidence calls

References

1. The International HapMap Consortium (2003) The International HapMap ProjectNature 426: 789-796

2. International HapMap Projecthttp://www.hapmap.org

3. Altshuler D, Brooks LD, Chakravarti A, Collines FS, DalyMJ, Donnelly P & International HapMap Consortium(2005). A haplotype map of the human genomeNature 437: 1241-1242

4. Patented Te-Flow technology is exclusivelydistributed 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.

10 AUTO M AT E D L I Q U I D H A N D L I N G

Production of a genome-wide RNAinterference library for functional genecharacterization in cultured human cells

RNA interference (RNAi) is a mechanismthat gives researchers the ability toinvestigate gene function quickly andefficiently by specifically silencing theexpression of the individual gene inquestion. Although quite easy in somespecies, RNAi initially proved moredifficult in cultured mammalian cellsbecause the long double-stranded RNAactivated the cells’ natural antiviraldefence. To an extent, this has beenovercome by using chemically synthe-sized small interfering RNAs (siRNAs)around 21 nucleotides long but thistechnique is still not always effective.Although there are now good algorithmsavailable to predict sequences that willfunction well with a reasonable proba-bility, only experimentation can showwhether the selected siRNA actuallydegrades the mRNA in the cell efficiently.

To address this problem, scientists at theMax Planck Institute in Dresden are usingTecan equipment to create a humangenome-wide library of endoribonuclease-prepared, so-called, esiRNAs1,2 to use for arange of scientific applications. At thesame time, they have developed a robustand automated transfection method thatintroduces the esiRNA moleculesefficiently into the cell under constantconditions.

The method for esiRNAs involves cleavinglong double-stranded RNA moleculeswith Dicer or bacterial RNaseIII in vitrointo overlapping siRNA molecules tointroduce into the mammalian cell.The starting material (see figure 1;reproduced with permission fromreference 3) is a collection of 15,500 E. coli bacterial clones containing agenome-wide cDNA library which covers,to the greatest possible extent, all knownand predicted human genes. For eachindividual gene, the cDNA insertionfragments from the plasmids areamplified in 96-well microplates usingPCR, and T7 polymerase promoters areattached on both sides. The T7 poly-merase reads off mRNA on both sidesfrom these promoters and it can then be

hybridized to the RNA double strand.After adding RNaseIII from E. coli, thedouble-stranded RNA is cleaved intoshort overlapping siRNA fragments whichcontain the highly active effectormolecules in the “pool”, in addition to theless active or completely inactivemolecules. This effector molecule mixtureis purified through columns and, finally,the esiRNA concentrations for allmolecule mixtures are measured andadjusted to the same concentrations innew microplates (normalization).

Hannes Grabner1, Anne-Kristin Heninger2, Ralf Kittler2, Annett Lohmann1,Ina Poser2, Jan Wagner1, Karol Kozak1, Frank Buchholz2 and Eberhard Krauß1

1HT-Technology Development Studio (TDS), 2Research Group Buchholz, Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany


Figure 1: Production scheme of the esiRNA library3

11AUTO M AT E D L I Q U I D H A N D L I N G

Gel-based quality control steps areincluded at all important stages in orderto verify the lengths of the fragments,their purity and concentration. All dataobtained are fed into a database, and all steps of the operation monitored bymeans of a LIMS. So far, a library has beenobtained with over 14,000 moleculemixtures available in the sameconcentrations in 384-well microplates,in order to switch off a correspondingnumber of human genes in cultured cellsand to investigate the effect on biologicalprocesses. A genome-wide library hassubsequently been produced for mice.

The fundamental precondition for a well-adjusted and usable library is highprecision aliquoting and subsequent fullyintegrated measurement in terms of bothhardware and software. For thesereasons, a Freedom EVO® Workstation(figure 2) with eight-channel LiHa tips isused for the final step - the standard-ization of the library. An integrated 96-channel Te-MO™ pipetting system rapidly and precisely aliquots for UVmeasurement of the esiRNA concen-tration using optional disposable pipettetips or a 96-channel Teflon® needle head.Teflon-coated steel needles have theadvantage that practically no seriousretention of negatively charged siRNAoccurs as it does on the plastic surfaces ofconventional interchangeable tips whichusually have differing degrees of staticcharge.

The workstation includes a fully integratedTecan GENios™ Plus photometer toconduct the measurements at 260 and280 nm, so that both the concentrationand the purity can be determined bymeans of the quotients from the twomeasurements. For high throughput,the samples are aliquoted into 384-well microplates (Corning UV-Star)specially manufactured for this appli-cation. The best measurements areobtained when the samples arethoroughly mixed in the dilutingmedium, by what is known as ‘sandwich’pipetting (14 μl of diluting medium, 2 μlof 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 standardizedlibrary. All eight tips can introduce thediluting medium first, independently ofeach other, and then add the appropriatequantities of the esiRNA mixture as asummand according to the instructionsgiven by the LIMS. The sum results inequal final volumes of the variousesiRNAs in the same concentrations.Samples are then taken from this newlyassembled library on the 384-well scale,checked on a gel and measured again inthe UV spectrophotometer. The standarddeviations after standardization liewithin a range of less than 20% ifeffective use is made of the samplequantities (2 μl).


Figure 2: The Freedom EVO system to produce and normalize the genome-wide esiRNA libraries. ForUV measurements to determine the RNA concentration, the RoMa arm lifts the plates down fromthe work table to the GENios Plus reader, which is placed on the level below, as seen in the lowerright corner.

12 AUTO M AT E D L I Q U I D H A N D L I N G


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

Transfection in high throughputprocedures

The next important step for this newlycreated library is automated transfectioninto human cancer cells. Positivelycharged liposomes are used to insertnucleotides of any type into the cells,compressing and “packaging” thenucleotide strands, helping to penetratethe cell membranes. The entiretransfection process is standardized as far as possible, the CASY® cell counter(Schärfe System GmbH, Reutlingen,Germany) ensures a consistent way of counting, and variable distributionpatterns resulting from pipette angle and other human errors are avoided byautomated cell seeding (WellMate®Dispenser, MATRIX Corp., Hudson, USA).

Again, the Freedom EVO system is used to automate other stages of thetransfection process (figure 3, reproducedwith permission from reference 3,figure 3). The transfection unit iscompletely housed in a S2 safety cabinet(BDK Luft- und Reinraumtechnik,Sonnenbühl-Genkingen, Germany) toprotect from external contamination(such as fungal spores, yeast or bacteria)during transfection and to allow workingwith genetically engineered viral vector-based RNAi libraries at biosafety level S2.Improvements of the shown workflowallowed a throughput of up to 30,000samples in triplicate (90,000 in total)within 12 hours of pure robot time.

This genome-wide RNA interferencelibrary places an extraordinary tool in thehands of researchers for the systematicinvestigation of individual genes of thehuman genome for their function in allelemental biological processes in the cell.In addition, we have developed trans-fection methods which enable ourscreening department to conduct genecharacterization projects of this sort ata high throughput rate.

CASY is a registered trademark of Schärfe System GmbH

Teflon is a registered trademark of DuPontor its affiliates

WellMate is a registered trademark of MatrixTechnologies Corporation

Figure 3: Transfection workflow3

13D E T E C T I O N

High performance fluorescence scanning of tryptophan with Tecan’s Safire2™ microplate readerMost proteins have intrinsic UV fluorescence and this is due to thepresence of aromatic amino acids in their structure, in particular theamino acids phenylalanine, tyrosine and tryptophan (Trp). Of these,Trp has the strongest fluorescence emission, although the spectrum’swavelength and intensity can vary according to the local environ-ment, including factors such as the pH and solvent polarity. Furthe-rmore, the location of Trp within the protein’s structure can affect itscharacteristics, for example, Trp residues buried within the protein’shydrophobic core can have fluorescence spectra that are shifted by 10to 20 nm compared with those on the protein’s surface.

atoms with electron donating atoms,leads to intramolecular charge transfer(figure 1) that is extremely sensitive topH changes. For example, the presenceof a nitrogen atom at differentpositions of the Trp indole ring cansignificantly alter the protein’sabsorption properties, resulting in a red


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 andTrp53 which is completely buried in the protein hydrophobic core. Right: Charge transfer in Trp andits amino analogues arises as a consequence of the presence of amino group as a good electrondonor 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 insertedinto proteins, replacing the existingparent Trp, and so transferring the newpH-sensitive fluorescence emissionproperties into the proteins, serving asvaluable reporter genes for a range ofexperimental situations.

In this study, Dr Nediljko Budisa and hiscolleagues at the Max Planck Institute of Biochemistry in Martinsried, Germany,used a Tecan Safire2 microplate reader torapidly and reliably detect the differencesin fluorescence emission of tryptophanand its analogs, 5-aminotryptophan (5-NH2Trp) and 4-aminotryptophan (4-NH2Trp), upon pH titration. Differentfluorescence behaviors of the native andsubstituted proteins have been welldocumented in previous studies, so themain aim of these measurements was toconfirm that the fluorescence propertiesof Trp and its analogues as free aminoacids in solutions are fully transmittedupon their incorporation into the proteinsbarstar and human annexin V.

The amino acids, proteins and substitutedproteins were prepared and purified asdescribed in [1]. Amino acids and analogswere measured at a concentration of 1 mM and barstar at 0.5 mM, in a volumeof 300 ml in black 96-well plates(Greiner®). Emission spectra weredetected from 310 to 500 nm (bandwidth5 nm) for the free amino acids and from310 to 450 nm (bandwidth 5 nm) forbarstar, in 1 nm steps using 285 nm(bandwidth 10 nm) for excitation.Detection flash mode was set to highsensitivity mode with an integration timeof 20 ms (40 ms for barstar). The gain foreach pH titration was determined fromthe sample with the highest emissionvalue. For 4-NH2Trp only, two differentgains were used due to the weakemission at higher pH. Z-position was setmanually after measurement of five

Standard Trp is encoded by a single UGGtriplet and, over the years, it has beenshown that Trp analogues can beintroduced into various proteins bygenetic manipulation, serving as reportergenes for a wide range of studies onprotein structure or function. In the1970s, solution studies demonstrated thatmanipulating the structure of Trp itself,such as by substituting particular indole

wt-barstar

14 D E T E C T I O N


randomly chosen samples. Trp and 5-NH2Trp were measured at 30 °C, 4-NH2 Trp at31 °C and barstar at 28 °C and the numberof flashes was set to 10 (Trp and 5-NH2Trp) and 20 (4-NH2Trp).

The spectral data presented in figure 2clearly demonstrate a regular decrease inthe fluorescence intensity of free Trp thatcorrelates strongly with decreasingsolvent pH (from 11.0 to 1.0). However,such intensity changes were not seenwhen the native proteins barstar andannexin V were measured. This is notsurprising, because protein Trp residuesare often involved in complex inter-actions with neighboring residues andsolvent 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 manyproteins.

In both native proteins and free L-Trp insolution, the fluorescence emissionmaxima were almost unchangedthroughout the measured pH range (1-11),as shown in figure 2. However, thefluorescence emission spectra of 4-NH2Trp and 5-NH2 Trp generally decreasedwith increasing pH of the solvent,reaching a plateau level at pH > 9.0.Dramatic changes were also seen in theintensity of the anionic form of 5-NH2Trp’s emission spectrum, compared withthat of the cationic forms at pH 1.0 and2.0 (figure 2). This trend was alsoobserved to a certain extent in 4-NH2 Trpand in the fluorescence measurements ofsubstituted barstar residues.

� �

�

�

��

��

� �

��

� �

�

� � �

HN2

HN2

R

360

380

340

320

0 2

pH-value

Fluo

resc

ence

Em

issi

on

Max

imum

[nm

]

4 60

8 10 12

HN

R

HN

R

HN

80000

60000

40000

20000

310 360

wavelength [nm]

RF

410 460

H2N COOH

0

HN

48000

36000

24000

12000

310 360

wavelength [nm]

RF410 460

H2N

HN2

COOH

0

HN

60000

45000

30000

15000

310 360

wavelength [nm]

pH 1.0pH 3.0

pH 5.0pH 7.0

pH 9.0pH 11.0

RF410 460

H2N

HN2

COOH

0

HN

Figure 2: Effects of pH on fluorescence emissionprofiles of Trp (black), 4-NH2 Trp (blue) and 5-NH2 Trp (red) as free amino acids in solution.Fluorescence intensity regularly drops downwith the pH decrease but there are almost nochanges in the position of the emissionmaximum upon pH titration. In contrast, theposition of the emission maximum in both 4-NH2 Trp and 5-NH2 Trp fluorescence spectra ispH-dependent. Low spectral intensities at higherpH (7.0, 9.0 and 11.0) for 4-NH2 Trp make itdifficult to recognize the trend in the spectralshift, so sample measurements were performedwith optimized gain. The values of fluorescenceemission maximum for each amino acid derivedfrom these measurements are summarized andplotted in figure 3.

Figure 3: Plot of the variations in fluorescence emission maxima versus pH of solutions containingfree amino acids Trp (black), 4-NH2 Trp (blue) and 5-NH2 Trp (red). Data for Trp are fitted using linearregression 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.


15D E T E C T I O N

The most interesting and importantfeature that emerged from thesemeasurements was the change in theemission maximum of aminotryptophansupon pH titration: the two analogs showedopposite trends in their spectral shift(figure 3). Both spectral shifts occurred at higher pH (7 and above) and werecharacterized by reduced fluorescenceintensity. For 4-NH2 Trp, a blue-shiftedcooperative titration curve was obtainedand curve fitting revealed a transitionmidpoint between pH 5.2. Conversely, in5-NH2 Trp, this cooperative transition ofthe emission maximum was red-shiftedwith a transition point at pH 5.95 (derivedfrom sigmoid curve fitting; figure 3). Bothtransitions were found in the substitutedannexin V and barstar, where transitionmidpoints were estimated to be in the pHregion between 5.0 and 6.0 (precisedetermination was not possible sinceboth proteins have a pI-value around pH5.0). These large differences in spectrabehaviors between 4-NH2 Trp and 5-NH2Trp and related protein variants can beexplained by the increased polarity in oneof the electronic states, whose stabilitydepends upon the nature, orientation andposition of the substitute in the parentindole molecule in a particular solvent.

Conclusion

The Safire2 rapidly identified andmeasured changes in fluorescenceemission maxima and intensity in Trpanalogs during pH alterations, as a resultof the changes in intramolecular chargethat originated from cation-to-aniontransitions of the amino indoles in the pH range 5.0 to 6.0. The conversion of proteins such as barstar or annexin Vfrom a pH-insensitive native form into apH-sensitive protein in fluorescence isprimarily the result of the intrinsicproperties of the aminotryptophananalogs, 4-NH2 Trp and 5-NH2 Trp, whichwere integrated into the structures ofrelated proteins. Future research willprovide a further increase in the numberof Trp-like amino acids that are availablefor redesign of native Trp residues andwill enable novel strategies to bedeveloped for generating proteins,peptides and their derivatives withtailored spectral properties. This processcan be extremely laborious and timeconsuming without automation, but theSafire2 offers a range of high speedfluorescence techniques that help toincrease throughput and reduce thesebottlenecks.

[1] High performance fluorescencescanning of tryptophan and its pHsensitive analogs 4-aminotryptophan and 5-aminotryptophan spectra with the Tecan Safire2 microplate reader.

Acknowledgement

Tecan wishes to thank Sandra Lepthienand Dr Nediljko Budisa for performingthe experiments and providing thesedata. Dr Budisa’s research group is basedat the Max-Planck-Institute ofBiochemistry in Martinsried (Germany),and focuses on reprogramming theprotein translation machinery of livingcells.

Greiner is a registered trademark of GreinerLabortechnik GmbH

e-mail: [email protected]

http://www.biochem.mpg.de/budisa/


High throughput microsomalstability assays for analyzingcandidate drugsDrug discovery researchers from the Istituto di Ricerche di Biologia Molecolare(IRBM) “P. Angeletti”, the Italian site of Merck Research Laboratories, have developedautomated methods for performing high throughput microsomal stability assaysusing a Tecan Genesis RWS™ 200 Workstation. Automating these assays allows theresearchers to predict the pharmacokinetics of multiple candidate drugs in just twodays, instead of four, and vastly increases the throughput. Pharmacokinetic analysisof candidate drugs is a critical part of the drug development process, and helps toeliminate compounds with inappropriate bioavailability, toxicity, effectiveconcentrations 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 Fonsi at the Istituto di Ricerche di Biologia Molecolare

Dr Massimiliano Fonsi, DMPK Chemist atIRBM, has developed these microsomalstability assays using the Tecanworkstation equipped with an eight tipliquid handling (LiHa) arm and roboticmanipulator (RoMa) arm, Thermomixerand heater/cooler system. This set-upperforms all the liquid handling steps,including sample pooling. The assay canbe automated on other Tecan liquidhandling workstations, including fromthe Freedom EVO® series. The protocolincludes 1,200 LC/MS/MS runs thatrepresent single incubation steps and,using the novel sample pooling methodbased on cassette analysis, all of thesedata can be acquired within 48 hours (as opposed to 96). This system has anumber of advantages, as Dr Fonsiexplained.




“My aim is to be able to screen as manycandidate drugs as possible. LC/MS is aserial technique, so you cannot read anentire plate in one go - each well has tobe analyzed with a single chromatographyrun. At the moment, the chromatographytakes about two minutes and I generate600 samples, which would take about 48hours in total. Doubling the sampleswould take 96 hours, but the highselectivity of mass spectrometry meansthat it is possible to pool two (or more)samples for analysis, using the TecanGenesis platform, and each drug can thenbe analyzed with a specific transition inthe mass spectrometer. At the momentour analysis time has been halved;increasing the velocity of the RoMa armwill make it possible to increment theassay throughput by running multipleincubations at the same time andpooling more samples in a final plate foranalysis in a single run. Pooling thesamples in this way has been usedpreviously for in vivo pharmacokineticsbut, as far as I know, nobody else has setup this automated system for HTSmicrosomal stability in vitro screening.”

“Each of our projects focuses on a specificclass of compound, but IRBM is workingon different projects, with severaldifferent classes of drugs. This meansthat I have to customize the assay andthe analysis for each specific class ofcompound using the Gemini™ software,and Tecan’s flexibility is fundamental inthis respect. We also have to be able tocustomize the protocol to suit variousexperimental conditions, such as differentconcentrations of proteins, differentspecies models and different cofactors.This is very important and, currently,other laboratories using these methodsdo not have so much flexibility and areonly able to run the assay under fixedconditions. We developed this protocol inorder to assay different cofactors withinthe same experiment. We have alsointroduced control samples that do notcontain any cofactors in order to obtainfurther information about unexpectedphenomena that could overlap with themicrosomal metabolism, such asprecipitation, hydrolysis or other chemicalreactions.”

“The method that we have developedalso allows us to measure multiple timepoints, which means that we cancalculate the intrinsic clearance of thedrug as well as measure the residualpercentage. With our method, 20different compounds can be screened induplicate, in three different species,collecting six time points over 90minutes’ incubation. The same basicprocedure can also be modified to screen32 compounds with cofactors butwithout duplicates, or 10 compounds induplicate with two different separatedcofactors (e.g. NADPH and UDGPA).”

“In the future, we are hoping to get a FreedomEVO 200 Workstation with a Te-MO™ 96 head,which will allow us to increase our throughputeven further as well as provide better predictionof a drug’s pharmacokinetics.”



The importance of highthroughput SNP genotypingfor complex diseaseresearch in Spain

The technique of SNP analysis hasbecome more widely used over the lastfour to five years because the highdensity maps it provides make it easier tolocate small but potentially significantchanges in the genome. It is useful for awide range of applications – frombiomedical investigations in hospitalsand academic institutes to drugdevelopment studies for biotech orpharmaceutical companies. Threelaboratories in Barcelona, Madrid andSantiago de Compostela, collectively theCentro Nacional de Genotipado (NationalGenotyping Centre)(CeGen), wereestablished in 2003/4 to provide a flexibleand high throughput facility for mediumto large scale SNP genotyping researchprojects throughout Spain and beyond.

Dr Chris Phillips, Platform Manager at the Santiago de Compostela division of theCentro Nacional de Genotipado (National Genotyping Centre)(CeGen) in Spain

The Santiago de Compostela division ofCeGen, based at the university, hasconcentrated on establishing reliable andfast systems to meet its role as a highlyefficient contract services laboratory.Two automated liquid handlingworkstations from Tecan were chosen toachieve a high throughput and preventbottlenecks in certain parts of theprocess; a Freedom EVO® 150 platformdeals with pre-PCR procedures and anAquarius™ multichannel pipettor with a96-channel head handles post-PCRmanipulations, both systems runningalongside two genotyping platforms, anApplied Biosystems SNPlex™ GenotypingSystem and a Sequenom MassArray®DNA analysis platform. The Freedom EVOis equipped with a number of additionalfeatures, including a Te-MO™multichannel pipetting option with a 96channel head, an eight channel liquid

handling (LiHa) arm and a roboticmanipulator (RoMa) arm. The completesystem automates extraction of genomicDNA from sets of 96 fresh or frozen bloodsamples with a NucleoMag 96 Blood kit(Macherey-Nagel®). This magnetic bead-based technology yields 1-4 μg of highpurity DNA from 100 μl blood. Followingextraction, the DNA concentration isdetermined using PicoGreen® (MolecularProbes), a fluorescent dye that binds theDNA and can be quantified using thelaboratory’s Tecan GENios™ plate reader.Measurements are obtained for twoserial dilutions for each sample and thishighly sensitive technique allows thedetection of up to 25 pg/ml of DNA. Five96-well plates are processed per day bythe GENios reader, equating to 480samples. Using SNPlex, the DNA can thenbe genotyped on a medium to large scaleat low cost and, using 384-well plates, upto 48 SNPs can be simultaneouslygenotyped in one sample, which equatesat present to 70,656 genotypes carriedout per week.

SNP Tecan Journal 2/2006


Throughput is no doubt a majorconsideration for the laboratory and thecurrent operation has a potential capacityof 45,000 genotypes per week. The Tecanworkstations have achieved the highworkflow with reliability and, veryimportantly for a contract serviceslaboratory such as this, with flexibility.Flexibility and ease of programming ishugely important because often thelaboratory has to tailor its processes tosuit a project’s needs, for example,researchers might send samples inunusual plates or formats or, even morelikely, will need the optimization of anentirely new set of assays. SNP analysiscan be very straightforward when you areonly dealing with, for example, sixmarkers that remain constant; you knowwhat causes problems and what workswell. For the laboratory in Santiago,however, there is normally a completelynew set of markers every few weeks and,each time this happens the assays andtechniques need to be re-optimizedwhich takes a significant amount of time.On occasion, the set of markers may onlybe for a small number of samples, say400, and the analyses might be completewithin just a couple of days but theoptimization may have taken a week ormore. This is a challenge compared to adiagnostic set-up, for example, where themarkers are known and similar samplesare regularly received.

Under these circumstances, flexibility andeasy programming is very important andis undoubtedly met by Tecan’s instruments,especially the Freedom EVO workstation.A new workflow for handling differentvolumes; a five-tip head for cherry pickingand the 96-tip head working welltogether; features like this are importantadvantages of the Freedom EVO thathave helped the Santiago laboratory tobe prepared for whatever project comesround the corner.

Dr Chris Phillips and his team atthe Santiago de Composteladivision of the Centro Nacional deGenotipado (National GenotypingCentre)(CeGen) in Spain

SNPlex is a trademark of Applera Corporation or itssubsidiaries in the US and/or certain other countries

MassArray is a registered trademark of SEQUENOM, Inc.

Macherey-Nagel is a registered trademark ofMacherey-Nagel

PicoGreen is a registered trademark of MolecularProbes, Inc.

20 S A M P L E M A N AG E M E N T

Building a world-class compound

Over the last five years, Amphora’scompound management laboratory hasbeen building a library of over 130,000compounds to serve the company’sresearch projects that use chemogenomic,system biology and microfluidictechnologies to develop targetedtherapies for oncology, inflammation,Alzheimer’s disease and diabetes.

Managing such a library and keeping up with all the internal and externalcustomers’ demands is no easy task, asIoana Popa-Burke, Associate Director atAmphora, explained: “Although we are a relatively small biotech company,our compound management still needsto be first class. Tracking all the samplesmanually is logistically extremelycomplicated so we evaluated all themajor compound store manufacturersprior to purchasing the REMP system.Many of us have previous experience of

compound management from biggerpharmaceutical companies and wewanted to apply those same managementprinciples to build a world-classmanagement system on a smaller scale.”

“We were really attracted to REMP forseveral reasons; firstly, the company hasan extremely good reputation and everysingle independent user we talked tospoke very highly of it; secondly, byacquiring just the core of REMP’s Small-Size Store™ (SSS) and Mid-Size Store™(MSS) we are able to stay within budgetand grow the system as we need to.”

A high quality sample storage systemsuch as REMP’s is essential for Amphoraas the company puts significant effortinto ensuring that all of its compoundsare of the highest quality. All of thecompounds in the library are purified byLC/MS and are all more than 95% pure.Using analytical chemistry techniques,

the compounds are constantly monitoredfor stability and solubility and, therefore,the reliable storage conditions andtracking advantages of REMP’s systemsare critical for Amphora’s standards.This also provides further advantages forAmphora’s screening processes, as Ioanaexplained.

“We depend on very high qualityscreening to be able to find highlyselective, target-specific compounds.We screen kinases, proteases,phosphatases and some ion channels andwe can pick compounds with anyselectivity profile we want. If ourcompounds were not of sufficiently highquality then all our screening effortswould be effectively useless. This also hasramifications for all of our diseaseresearch areas. For one of ourinflammation projects, for example, wehave worked on a p38α inhibitor. Usingour industrialized drug discovery


The fully integrated drug discovery and development company Amphora, based inNorth Carolina’s Research Triangle Park in the USA, has chosen REMP automatedstorage and retrieval systems to take control of its compound library.

The compound management lab at Amphora


21S A M P L E M A N AG E M E N T

management system

platform, of which the REMP system is anintegral part, we have been able to pick ap38α selective compound while mostpeople are working on p38α/p38β dualinhibitors! Overall, we have beenextremely pleased with the system, it hasmade a big impact on our daily routine aswell as on the quality of the compoundsand data tracking.”

Amphora’s REMP system includes theTube Punching Module™ (TPM) andassociated devices, the Reatrix™DataMatrix Scanner for reading thestorage tube rack bar codes and 2Dbarcoded REMP 96 Tube Technology™consumables. The TPM is a desktop devicethat allows PC-controlled selection ofREMP 96 (or 384) tubes, which aredirectly transferred from storage tuberacks into delivery tube racks forcollecting and reformatting. The TPMperforms the transfer through a singleaxis movement, reducing handling error

reliability particularly important: “TheREMP system has been essential for us toset up our tube-based library. It hasconsiderably improved our process, interms of both speed and data quality, andwe have had no problems at all. The TPMand Reatrix Scanner work seamlessly andare very reliable; they have drasticallyreduced our potential for error. We cannow cherry pick compounds as and whenwe like, we can use multiple formats, andthe technology has helped us a great dealalready to meet customer andcollaborator requirements. I operate thewhole system daily and have never hadto use REMP’s helpline since the systemhas been up and running!”

True scalability with REMP’sTube TechnologyREMP’s sample processing and storageconcepts were originally adopted bylarge facilities, but are equally impor-tant for smaller scale laboratories tobe able to access high quality samplemanagement. The REMP TubeTechnology consumables provide thisall important entry level step, allowingyou to set up your compound library inthe tubes, which are supported bydevices such as the Tube PunchingModule (TPM), automated Capper/Decapper™ or Tube Sealer and theReatrix 2D scanner. The TPM is easy tointegrate into automated systems,so once your library grows it becomeseasy to move the tubes into a fullyautomated REMP store, such as the SSS.

rates, and is faster and more reliable thanconventional pick and place methods.It allows tubes to be cherry picked at anytime, leaving the destination tube rackswithin the storage environment untilinstructed otherwise, and is extremelyuseful for aiding sample tracking.

Brian Hardy, the Lead Scientist atAmphora, who runs the REMP system ona daily basis has found its ease of use and

Tube Punching Module

Storage Tube Rack with 2D Code

Reatrix

Small-Size Store

Automated Capper/Decapper



Analyzing cholinesterasein organophosphate poisoning

The German armyautomates thedetermination of AChEactivity in whole blood Bodo Pfeiffer, BundeswehrInstitute of Pharmacology andToxicology, Munich, Germany

Agrochemicals belonging to theorganophosphate group are usedextensively around the world, due to theirefficacy and comparatively lowenvironmental persistence. They are,however, highly toxic and present aconsiderable risk to humans, asdemonstrated by the estimated 3 millionacute pesticide poisonings and 200,000fatalities globally every year. A fewparticularly poisonous organophosphates,including soman, sarin, tabun and VX(known as nerve agents), were developedas chemical weapons and have even beenused recently, notably in the Iran-Iraq warand the Tokyo underground in 1995,despite being outlawed around theworld.

Organophosphates inhibit serineesterases, particularly acetylcholinesterase(AChE), by covalently binding to theenzyme and disturbing its physiologicalfunction – i.e. preventing cleavage of theneurotransmitter, acetylcholine (ACh), byAChE. This leads to an accumulation ofACh in cholinergic synapses of both thecentral and peripheral nervous systemsand at the neuromuscular junction,resulting in hyperexcitation and sub-sequent paralysis of the target organsand, eventually, death due to central andperipheral respiratory paralysis1,2.

Organophosphate poisoning can betreated by immediate administration ofatropine and AChE reactivators (oximes;Obidoxime in Germany, pralidoxime inthe UK and USA) but it is necessarythroughout treatment to measure and

monitor levels of the affected enzymewithin the body. Muscular AChE is noteasily accessed in patients, so erythrocyteAChE and plasma cholinesterase(butyrylcholinesterase; BChE) areanalyzed instead.

The Bundeswehr Institute of Pharmacologyand Toxicology co-operates with theSouth Asian Clinical Toxicology ResearchCollaboration Centre for TropicalMedicine, University of Oxford, England3

in a prospective cohort clinical trial in SriLanka. Blood samples from patients areanalyzed in the Institute for AChE activity,BChE activity and hemoglobin. Untilrecently, the parameters have beenanalyzed manually by measuring a 3 mlsample using a standard commercialspectrophotometer with a temperature-controlled cuvette holder and water bathat 37ºC. However, this manual procedurequickly reached its limits when analyzinglarge numbers of samples and, to solvethis problem, an automated microplatemethod was developed to processmultiple samples in parallel. Tecan’sGenesis 150 workstation was introduced,with automated pipetting and a roboticarm as well as an integrated spectro-photometer, a 37°C incubator, temperature-controlled microplates racks and cooledsample preparation (figure 1a). Adaptingthe protocol for automation wasrelatively straightforward because the

parameters used previously could almostall be translated and known incubationtimes, wavelengths and measuring timeswere not altered. This application can beautomated on other Tecan liquidhandling workstations, including fromthe Freedom EVO® series.

Cholinesterase activity is measured in awhole blood sample collected from thepatient and immediately diluted andcooled to slow down any ex vivo reactionsthat can rapidly take place betweenAChE, organophosphates and oximes.Cholinesterases convert specific substratesand, in the conversion process, thiocholineis formed as a product (figure 1b).This product reacts with DTNB (5-5'dithiobis-(2-nitrobenzoic acid)) present in thesample to give a yellow dye that can bemeasured with a spectrophotometer; thecolor development over time serves as adirect measure of the enzyme’s activity.The hemoglobin levels in the dilutionsare also measured, using Zijlstra’smethod4, to allow correction of anydiluting errors in preparation of thesamples; the AChE activity can becorrelated with the hemoglobin levels5,6.

To validate this set-up, extensivecomparative measurements were madeusing both the automated and manualmethods. AChE activity was analyzed inaliquots of native donor whole blood that



were inhibited with 100 nM soman; BChEactivity in plasma harvested from soman-inhibited blood samples was measuredand hemoglobin levels were determinedin dilutions of whole blood samples withdifferent proportions of erythrocytes and added plasma. In addition, thereactivation capability of whole bloodinhibited with either 100 nM soman,which does not allow AChE reactivation,or 200 nM paraoxon-ethyl (PxE), whichreadily allows AChE reactivation, wasanalyzed and the residual inhibitor wasalso measured in donor plasma samplesinhibited with five different concen-trations of paraoxon-ethyl. The resultsobtained with both methods correlatedvery strongly in all measurements.

Once validated, the automated methodwas used to analyze and monitor AChEand BChE activity in samples from the SriLanka patients who had attemptedsuicide by oral ingestion of pesticides(quinalphos or fenthion), (figures 2 and 3).Whole blood samples were collectedfrom the patients (t = 0) and oximetherapy was started with pralidoxime(bolus dose and infusion therapy); furthersamples were collected within apredefined time window. Quinalphosresults in diethylphosphorylation ofAChE, this enzyme species can be readilyreactivated (figure 2) and so, four hoursafter admission to the hospital, noresidual inhibitor remained in thoseaffected patients. Fenthion results in adimethylphosphorylated enzyme thatages quickly and cannot be reactivated(figure 3), meaning that in patients whohad ingested this pesticide, no significantincrease could be recorded in AChEactivity, and BChE activity remainedcompletely inhibited.

The determination of cholinesterasestatus is primarily intended for analyzingpatients poisoned with organophosphates(accidental or suicidal poisoning), but itcan also be applied after the use of nerveagents in military conflicts or terroristattacks, as well as in the context ofoccupational medical examinations.

Figure 1a: Tecan GENESIS 150 workstation.The workstation is an open source systemand can also be used now for kineticsamples. It has an integrated Sunrise™spectrophotometer and uses TOPS™ 4.0 with Magellan

Figure 1b: Formation of DTNB produces a yellowdye that serves as a direct measure ofcholinesterase activity

Acetylcholinesterase

Acetylthiocholine + H2O thiocholine + acetate OR:

Butyrylcholinesterase

Butyrylthiocholine + H2O thiocholine + butyrate

Thiocholine + 5,5´dithio-bis(2-nitrobenzoate) →5-mercapto-2-nitrobenzoate + 5-mercaptothiocholine nitrobenzoate [yellow]

(TNB)

Figure 2: Example of Quinalphos poisoning.The reactivation capability shows themaximum achievable AChE activity. The percent inhibition shows that no residualinhibitor remains after 4 hours

Figure 3: Example of Fenthion poisoning.Due to ageing, reactivation is almostimpossible (top). In spite of a decline inresidual inhibitor (middle) no significantincrease in AChE activity is recorded (top).BChE activity is completely inhibited (bottom)

600

Quinalphos poisoning

AC

hE a

ctiv

ity (

mU

/µm

olH

b) 500

400

300

t=h

200

100

00 24 48 72

AChEReac

n

u

u u

u uu

nn nn

n


% In

hib

ition

100

75

50

25

0

t=h

0 24 48 72uu

uu u


AC

hE a

ctiv

ity (

mU

/ml)

uu u u u

3000

2500

2000

1500

1000

500

0

t=h

0 24 48 72

nnn n n n n

600

Fenthion poisoning

AC

hE a

ctiv

ity (

mU

/µm

olH

b) 500

400

300

t=h

200

100

00 4824 72 96

AChEReac

uu

u u u u

u

n

Fenthion poisoning

% In

hib

ition

100

75

50

25

0

t=h

0 24 7248 96

uu

uu

uu u

Fenthion poisoning

AC

hE a

ctiv

ity (

mU

/ml)

u u u uuuu

3000

2500

2000

1500

1000

500

0

t=h

0 24 48 72 96

References

1. Wiener SW, Hoffman RS. (2004) Nerve agents: acomprehensive review. J. Intensive Care Med. 19, 22-37

2. Koelle GB. (1992) Pharmacology and toxicology oforganophosphates. In: B. Ballantyne and T. C. Marrs(Eds.), Clinical and experimental toxicology oforganophosphates and carbamates. Butterworth &Heinemann, Oxford, pp. 35-39

3. Eddleston et al. (2005) Differences betweenorganophosphorous insecticides in human self-poisoning: a prospective cohort study. Lancet 366:1452-1459

4. van Kampen EJ, Zijlstra WG (1961) Standardization ofhemoglobinometry, 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 intherapy of organophosphate poisoning. Toxicol. Lett.107, 233-239

6. Worek et al. (1999) Improved determination ofacetylcholinesterase in human whole blood. Clin ChimActa 288: 73-90


24 E V E N TS 2 0 0 6

Tecan Journal 2/2006 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 19Tecan 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 170Tecan 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 11Tecan Nordic AB +46 31 75 44 000 Tecan Nordic AB, Rep. Office Denmark +45 70 234 450 Tecan Portugal +351 21 000 82 16Tecan 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 REMP (USA) Inc. +1 508 429 2200 REMP Deutschland GmbH +49 6126 5831 0 REMP Nippon AG +81 3 3539 1771

Meet Tecan at these events

Tecan Journal, Customer Magazine of Tecan Trading AG., ISSN 1660-5276Design: OTM/London www.otmcreate.comPhotography: Marc Wetli/Zürich www.wetli.com, Günter Bolzern/Zürich www.bolzern.net,Susanne Völlm/Zürich www.susannevoellm.chEditor: kdm/UK www.kdm-communications.comPrint: DAZ Druckerei Albisrieden AG/Zurich www.daz.chAddress: Tecan Switzerland AG, Marketing Communications, Seestrasse 103, CH-8708Männedorf, Switzerland, [email protected],www.tecan.comTecan Group Ltd. makes every effort to include accurate and up-to-date information within thispublication, however, it is possible that omissions or errors might have occurred. Tecan GroupLtd. cannot, therefore, make any representations or warranties, expressed or implied, as to theaccuracy or completeness of the information provided in this publication. Changes in this

publication can be made at any time without notice. All mentioned trademarks are protected bylaw. For technical details and detailed procedures of the specifications provided in this documentplease contact your Tecan representative.This brochure may contain reference to applications and products which are not available in allmarkets. 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 FreedomEVOware 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 Moduleand 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, SwitzerlandT +41 44 922 88 88 F +41 44 922 88 89 [email protected] www.tecan.com

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

5th Youth Conference on Clinical Diagnostic Medicine Guiyang June 21-23 2006

CCLab 2006 Qingdao Sept 15-19 2006

Europe

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

Japan

International Bio Expo Tokyo May 17-19 2006

International Congress of Biochemistry and Molecular Biology Kyoto June 19-23 2006

USA

ASM 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 Dec 9-13 2006

tecan journal edition 02/2006

Documents

tecan investment

tecan uspages

expectfrom tecan

tecan journaledition

tecan austria ges

new packages

unlimited throughput

new software packages