Tecan Journal Edition 02/2007

Tecan Journal Edition 2 / 2007

From bluetongue to bird flu, detecting infectious diseases with the Freedom EVO® page 7

High throughput nucleofection with amaxa technology pages 9 – 11

New tools for predicting the recurrence of breast cancer pages 12 – 13

C E O WE LCOME

Welcome...

...to this, the second issue of the Tecan Journal for 2007.

Tecan Journal 2/2007

Our recently published financial results confirm that 2006 was a year of strong growth for our company and I would like to personally thank you all for your continued confidence and support.

Our successful year can, of course, be attributed to our innovative products, but also to our ongoing efforts to improve and streamline our organization. Our companies around the world share the common goal of perfecting their quality management systems for all manufacturing, sales and service activities, and continually strive to listen more closely and respond more effectively to feedback from you, our customers.

There are updates from customers working in a range of diverse applications, from avian influenza and nucleic acid testing to forensics and cell biology, who count on our systems to produce the consistently reliable results they need every day. We are also delighted to share with you details of exciting new product developments which demonstrate our commitment to continually improving and expanding the breadth of products we offer.

In this issue, we bring you news of several carefully-chosen collaborations with other life science companies. Many of these partnerships add additional capability to Tecan’s liquid handling workstations and increase their range of applications, providing you with even more application-specific automated laboratory solutions.

Thomas Bachmann, CEO

I hope you enjoy this issue of the Tecan Journal, and look forward to another successful year.

CONTE N TS CEO welcome Thomas Bachmann, CEO, welcomes you to this issue of the Tecan Journal. page 2

Automated virus testing in animals From bluetongue to bird flu, detecting infectious diseases with the Freedom EVO®. page 7

Corporate news Find out about some of Tecan’s recent collaborative projects. page 4

Working for ISO accreditation around the world Implementing a new quality management system through all Tecan’s sales and service departments. page 8 High throughput nucleofection with the Freedom EVO workstation and amaxa 96-well Shuttle® Fully automated, high throughput transfection of primary cells and difficult-to-transfect cell lines. pages 9-11

Product news: Painting a picture of 20,000 genes in the mouse brain Tecan’s GenePaint™ systems are used to create a groundbreaking new gene expression atlas. page 5 7 detection modes Tecan’s highly sensitive and extremely fast Safire2™ and Infinite™ F500 microplate readers offer seven different detection modes. page 5

The target of personalized medicine moves ever closer Genomic Health in California develops tools to predict breast cancer recurrence using Tecan workstations. pages 12-13 Genetic fingerprinting of crime scene samples Forensic scientists in Stuttgart, Germany, have commissioned a semi-automated sample processing system from Tecan. pages 14-15

Engineering new biomaterials for human implants Optimizing automated tissue culture methods with the Freedom EVO liquid handling workstation. pages 18-19 Plasma technology for fast, effective tip cleaning Tecan teams up with Cerionx to combine ‘cold’ plasma tip cleaning systems with the Freedom EVO. pages 20-21

Building a cattle DNA database in Japan Automated DNA extraction for analyzing cattle parentage with the Freedom EVO 200. pages 22-23 Managing worldwide compound delivery with REMP technology Boehringer Ingelheim’s Screening Support group has a number of REMP systems to manage its library of more than 800,000 compounds. pages 24-25

Cellerity™ is now available with the MultiChannel Arm™ The MCA brings increased speed and throughput to automated cell culture. page 5

HS Pro: miRNA profiling and Exiqon LNA™ arrays Profiling the expression patterns of hundreds of microRNAs with the HS 4800™ Pro Hybridization Station. pages 16-17

Freedom EVOlyzer® received with great enthusiasm in Spain The automated ELISA platform satisfies a wide range of clinical diagnostics laboratories. page 6

Te-PoolSafe™ boosts pipetting accuracy for testing donor blood The German Red Cross Institute screens thousands of donated blood samples every day and has validated Tecan’s liquid arrival check system. pages 26-27 Events 2007 Meet Tecan at a number of events around the world over the next six months. page 28 Tecan Journal 2/2007

CO R PO R AT E N EWS

Tecan teams up with high profile business partners in a number of new developments In recent months, even more collaborations with industry partners have come to fruition, bringing application-specific, automated laboratory systems to researchers working in a range of disciplines.

Tecan and Applied Biosystems have jointly developed the HID EVOlution™ System for the automated analysis of forensic DNA casework samples.

This new standardized system provides fully automated sample quantitation, normalization and short tandem repeat (STR)-PCR set-up, by integrating Tecan’s Freedom EVO® 150 liquid handling workstation with the Applied Biosystems 7500 Real-Time PCR System, 3130xl Genetic Analyzer, and Quantifiler® and AmpFℓ STR® DNA testing kits. The HID EVOlution is expected to reduce the amount of time required to process forensic DNA samples and help laboratories to increase their sample throughputs.

Tecan and amaxa, Inc. have successfully integrated amaxa’s Nucleofector® 96-well Shuttle® System with the Tecan Freedom EVO liquid handling workstation, allowing fully automated, high throughput transfection of Tecan Journal 2/2007

primary cells and difficult-to-transfect cell lines, including non-dividing neurons and T cells, with siRNA, DNA and other substrates. The development is ideal for large-scale studies that involve high throughput transfection stages, such as RNAi-based screening for drug development. The 96-well Shuttle® relies on unique electroporation technology for transfection of primary cells with high efficiency and excellent survival rates.

Integrating this system with the Freedom EVO workstation entirely removes all manual steps for cell preparation and transfection, allowing 96-well microplates to be completely processed in just minutes, depending on the protocol used. You can read more about the collaboration on pages 9-11.

Tecan and VWR International, Inc. have announced a strategic alliance to unite the strengths of each company and together provide Tecan’s cutting edge

microarray instruments and microplate readers to the global research market.

The collaboration takes advantage of VWR’s BioSciences program, which aims to provide researchers with the highest quality services and products available, whatever their application, and a comprehensive sales network – including more than 120 sales representatives worldwide and 15 BioSciences managers in Europe alone. Tecan’s range of technically excellent microarray products and microplate readers offers perfect solutions for customers of the BioSciences program working in all areas of drug discovery and life science.

Tecan and Cerionx, Inc. have made an exclusive distribution agreement for the supply and marketing of the Cerionx 8-channel TipCharger™ System to the worldwide biopharmaceutical market. Addition of the TipCharger™ System as an integrated and supported component to Tecan’s automated liquid handling and robotics workstations offers uniquely superior pipette tip cleaning and sterilization. The TipCharger™ System uses room temperature (‘cold’) atmospheric pressure plasma to clean and sterilize pipette tips by removing contaminants at the molecular level without using hazardous solvents. This revolutionary plasma cleaning technology reduces the potential for contamination at the sample and consumable levels, reduces run time and improves assay productivity. Find out more about the Cerionx TipCharger™ System on pages 20-21.

Quantifiler and AmpFℓ STR are registered trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.

The Nucleofector 96-well Shuttle System is covered by patent and/or patent-pending rights owned by amaxa.

TipCharger is a trademark of Cerionx, Inc.

PRODuCT N EWS

Painting a picture of 20,000 genes in the mouse brain Tecan’s GenePaint™ systems for automated, high throughput in situ hybridization have been used to develop a groundbreaking, genome-wide atlas of gene expression in the adult mouse brain.

The comprehensive digital atlas details the expression patterns of approximately 20,000 genes, providing an invaluable source of information for scientists studying brain organization, gene functions and behavior. Watch out for a special feature on the project in the next issue of the Tecan Journal!

7 detection modes Tecan’s highly sensitive and extremely fast Safire2™ and Infinite™ F500 microplate readers offer seven different detection modes, saving valuable time for a wide range of high throughput applications in the biotech, pharmaceutical, research and cosmetics industries.

These two modular and upgradeable systems provide very reliable performance with top quality results and are easily integrated into Tecan’s Freedom EVO® series of liquid handling workstations.

Relax your reader is a Tecan www.tecan.com/relax

Cellerity™ is now available with the MultiChannel Arm™ The Cellerity™, Tecan’s fully automated cell culture system, is now available with the MultiChannel Arm™ (MCA) module, increasing the speed and throughput of plating cells into microplates for assays.

The MCA module has a 96-channel pipetting head and can be operated with washable and disposable tips. The new option is compatible with lidded and insert plates, and all standard Cellerity options, delivering a greater level of flexibility to your cell culture platform.

Tecan Journal 2/2007

5

C L I N I CA L D I AGNOSTICS

Freedom EVOlyzer® received with great enthusiasm in Spain The Freedom EVOlyzer, Tecan’s new generation of automated ELISA systems, has proved to be increasingly popular with laboratories throughout Spain since being introduced to the country in July 2006 by Inverness Medical Iberica (IMI), one of Tecan’s principle diagnostic distributors for the area. IMI, formerly part of Innogenetics Diagnostics, has a long and proven track record as a distributor for professional diagnostic products in Spain, with a strong focus on enzymelinked immunosorbent assays (ELISAs) for infectious diseases, diagnostics and autoimmunity. IMI’s customers range from large hospital laboratories performing routine testing, to reference laboratories and testing laboratories for small- to mediumsized hospitals.

Tecan’s cooperation with IMI started in the early 1990s and, during the last decade, IMI has placed a broad

The Freedom EVOlyzer for automated ELISAs

Tecan Journal 2/2007

range of Tecan instruments in Spain, including Genesis RMP™ systems, Minilysers, Profiblot™ analyzers, Sunrise™ plate readers and Columbus™ plate washers. IMI is a distributor for nine international reagent manufacturers and the company’s combined kit portfolio consists of more than 300 different tests, which IMI has successfully automated using Tecan’s open ELISA platforms such as the Minilyser and the Genesis RMP. The addition of the Freedom EVOlyzer has significantly strengthened IMI’s IVD-D compliant portfolio.

“We chose to take full advantage of the large range of customization and configuration possibilities that the Freedom EVOlyzer offers, such as being able to freely combine stainless steel tips and disposable tips on the same instrument,” explained Mr Sergio Gordillo, Automation Product Manager at IMI. “The instrument’s flexibility for both hardware and software options means

we are able to satisfy almost all kinds of ELISA customer with just one single platform, which has been a key factor in the Freedom EVOlyzer’s success. We always deliver these instruments with a touchscreen monitor, because the Freedom EVOlyzer software has a touchscreen compatible interface available in both Spanish and Catalan, and this has proven to be a real hit among our local customers.”

“We have been able to transfer 100% of our kits to the Freedom EVOlyzer with excellent results,” continued Mr Gordillo.

“This new system is a more than worthy successor of Tecan’s Genesis RMP and its function scope shows a clear improvement in terms of both throughput and flexibility.”

“We have relied on Tecan instrumentation for a good number of years now and it has proven to be the right choice. With the Tecan instruments and our broad range of assays we have managed to gain a significant market share of the Spanish market and, with more than 80 ELISA platforms placed in Spain, everybody knows that we are a force to be reckoned with!” claimed Mr Gordillo. “With the Freedom EVOlyzer we can now offer superior kits, automated with superior instruments supported by superior service. What customer would ever say no to that?”

CLI N ICAL DIAGNOST I C S

Automated virus testing in animals Scientists at the Staatliches Veterinäruntersuchungsamt (National Veterinary Laboratory) Arnsberg in Germany, responsible for veterinary diagnoses, have chosen Tecan’s Freedom EVO® workstation for the rapid detection of viruses which cause potentially devastating diseases. The workstation is already being used to detect the virus responsible for a recent outbreak of bluetongue disease in cattle, and will soon be used for the detection of the avian influenza virus in local bird populations.

The Staatliches Veterinäruntersuchungsamt is a government institution in the administrative district of Arnsberg, North Rhine-Westphalia, with departments for pathology, serology, virology and molecular biology for the diagnosis of animal diseases. It also investigates food and feed quality, and performs the detection of pathogens in foodstuff. Within the organization, the molecular biology department exists as a service laboratory to the other departments by performing molecular testing for various diseases.

Dr Jochen Kilwinski of the molecular biology department explained: “Since September we have been detecting bluetongue virus RNA in samples from cattle using Tecan instrumentation, working in parallel with the serology department which is detecting antibodies to bluetongue virus in the same samples. Bluetongue is an insectborne disease, primarily of sheep, and there is currently no effective treatment.

It is normally rare in cattle, however, an outbreak in the Netherlands in August 2006, then spread to the North RhineWestphalia region in autumn. Very interestingly, the virus detected in this outbreak belongs to serotype 8, which normally only occurs in the south of the Sahara, India, Pakistan, or in central and South America. Furthermore, serotype 8 is transmitted by biting midge species of the genus Culicoides, which are not found in our latitudes.”

“We are using a Freedom EVO 150 to isolate and purify RNA from blood samples, to detect the specific RNA

From left to right: Yvonne Kullman, Beatrix Hanstein, Daniela Reckling and Jochen Kilwinski

segment of the bluetongue virus serotype 8 by reverse transcription and real-time PCR. We obtained the detection protocol from the Friedrich-Loeffler Institute in Insel Riems, the National Reference Laboratory, which is also equipped with a Tecan instrument. Our Freedom EVO is working with the Te-VacS™ vacuum manifold, with a 96-well format to handle a large throughput of samples.”

Dr Kilwinski is delighted with the results he has been getting from the Freedom EVO. “During initial testing, there was 100% agreement between the preparations made by the Freedom EVO and our manual preparations.

Furthermore, the automated preparation was shown to be in good accordance with the handmade preparations while participating in a national bluetongue virus ring trial. We have a lot of experience with manual handling, so this gave us complete confidence in Tecan’s automated system. The performance of the Freedom EVO is excellent with no reliability problems, and we would

not have been able to handle our current throughput manually. We are very happy to have this instrument.

I strongly feel that other institutions should also have the opportunity to carry out their investigations with a workstation like this.”

With the flexibility of the Freedom EVO, Dr Kilwinski’s workstation is not restricted to bluetongue virus detection.

“We are also responsible for the detection of avian influenza in wild bird populations within the administrative district of Arnsberg. The protocol for the detection of the avian influenza virus has already been implemented on the Freedom EVO, and it is very easy to switch from testing for bluetongue virus to avian flu. We are now waiting for spring, when the migration of birds starts again, and we are ready to use the workstation to test for avian flu in local bird populations, should the need arise.”

Tecan Journal 2/2007

Q ualit y A ss u rance

Working for

ISO accreditation around the world

Following the successful ISO certification of our manufacturing sites, work is already under way to implement similar high standards in all of Tecan’s organizations around the world. The first sales organization, Tecan France, has now been certified according to ISO 13485, an international standard intended for medical device companies.

Implementation of this quality management system means that all sales and service activities throughout our sales organizations will be structured in a similar way, and customers everywhere will receive the same consistently high standard of service and support from Tecan. As with our manufacturing facilities, we have chosen to work with TÜV Product Service, a well-known global notified body with a particular expertise in the medical device business and a strong reputation for high standards.

Günter Weisshaar, Tecan’s Executive Vice President Global Quality and Regulatory Compliance, is leading the implementation at a global level, and explained: “Implementing the right structure and procedures into our market units will help us to become more customer oriented and to respond in a more timely and efficient manner.

An important benefit will be a change in the way we handle information from our customers; whether in the form of a complaint or a product suggestion, we will be able to handle this information in a far better way.”

Tecan Journal 2/2007

Erika Hildebrandt, Quality Manager for Tecan Europe, led the project in France in close association with Denis Coulet, Business Manager for Tecan France. She explained: “Implementing ISO 13485 helped us to document successful processes already in place, and to improve other procedures. The main focus of all the changes is to serve our customers in a better and more professional way and, only a few months on from the implementation in France, we can already see the advantages. The everyday running of Tecan France has become more precise and our customers are already feeling the benefits. Installations, maintenance and overall customer service have all improved as we are able to record and track customers’ requests more easily and respond more quickly. For the long term, too, we have refined our methods of collecting customer information and feeding this back to our manufacturing and R&D sites so that it can be fed into new product designs where appropriate.”

The quality system now established in France will be systematically implemented into all Tecan’s sales organizations and we look forward to a time very soon when our customers around the world will be able to enjoy the same advantages as our customers in France.

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

High throughput nucleofection® with the Freedom EVO® workstation and amaxa 96-well Shuttle® Tecan and amaxa GmbH have successfully integrated the amaxa Nucleofector® 96-well Shuttle® System with the Tecan Freedom EVO® liquid handling workstation, allowing fully automated, high throughput transfection of primary cells and difficult-to-transfect cell lines, including non-dividing neurons and T cells, with siRNA, DNA and other substrates. The collaboration was originally instigated for Dr Claudia Merz at Bayer Schering Pharma AG, Germany, who needed fully automated, high throughput transfection of immune system cells for RNAi-based screening experiments.

Scientists routinely use transfection techniques to insert DNA or RNA into cells and modify the expression levels of a certain gene or genes. This approach is often used for functional studies of genes, such as RNA interference (RNAi) to silence gene expression; for cell-based manufacturing of therapeutic products (eg. antibodies, hormones or vaccines); and for structural studies of proteins and amplification of DNA or RNA in cells for purification. Many of these applications require high throughput transfection in combination with primary cells or nondividing cell lines that are notoriously difficult to transfect with standard lipofection approaches.

The Nucleofector® technology from amaxa, a German biotechnology company, has been specifically designed for difficult-to-transfect cell lines and primary cells, including stem cells, T lymphocytes and non-dividing neurons.

The technology uses electroporation instead of lipofection, where electrical currents cause the gene of interest to be transferred directly into the cell and even the cell’s nucleus. This quick and simple approach is the only non-viral method to achieve very high transfection efficiencies while maintaining high survival rates in such cells, with up to 80% efficiencies in human T cells, for example, compared with 1-3% or fewer usually transfected by lipofection. As nucleofection causes the DNA to directly enter the nucleus, this

Tecan Journal 2/2007

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

The entire nucleofection process is controlled by the Freedom EVOware® software, using an interface provided by amaxa. Nucleofection conditions for the 96-well Shuttle® are defined in the amaxa software and the resulting parameter files are uploaded from Freedom EVOware and executed on the 96-well Shuttle®.

method does not require cell division, and expression of the transfected gene can be analyzed shortly after nucleofection. For some cell types this can be within as little as two hours, rather than the standard 24 to 48 hours.

Integrated cooling and heating elements.

Transportation of amaxa’s Nucleocuvette™ plate to the 96well Shuttle.

Tecan Journal 2/2007

Tecan and amaxa have teamed up to integrate amaxa’s Nucleofector® 96-well Shuttle® System with Tecan’s Freedom EVO® liquid handling workstation. All necessary steps for transfection can be automated, including cell harvesting, diluting and plating; DNA/RNA normalization; preparation of reagent mixes; resuspension of cells and substrates in Nucleofector® solution; the nucleofection process; and analysis of transfection results. Nucleofection occurs in disposable, 96-well Nucleocuvette™ modules, which consist of an innovative conductive polymer electrode material that ensures no metal ions are released into the cell suspension during transfection. The system uses identical transfection parameters for any nucleic acid substrate, so that DNA vectors, such as expression plasmids, and RNAi vectors, including short hairpin (sh)RNA vectors or short interfering (si)RNA duplexes, can be transfected using the same protocol.

“Many easily-transfected cell lines are quite different from primary cells of the human body so are not applicable for most research and drug development studies,” explained Andreas Schroers, Product Manager at amaxa. “The integration enables, for the first time, transfection of medically relevant primary cells in a fully automated environment, providing a valuable tool for research and drug discovery studies.”

The collaboration originally began in response to a request from Dr Claudia Merz, scientist in Target Discovery at Bayer Schering Pharma AG in Berlin, Germany. Dr Merz had been performing lipofection using a Freedom EVO workstation for some time, and needed to expand the set-up for large scale, automated nucleofection of human T lymphocytes and the Jurkat lymphocyte cell line using focused siRNA libraries.

These small libraries have between 200 and 800 siRNAs that are applied in triplicate, requiring multiple 96well Nucleocuvette™ plates to be completed each day. Dr Merz’s integrated system automates the complete plate preparation, nucleofection and replating of nucleofected cells for one 96-well plate in about 40 minutes, and twelve 96well plates can be processed in one day, equating to 1,152 samples. A Freedom EVO 200 workstation is used for all additional steps of the RNAi studies, such as mRNA knockdown analysis and analysis of phenotypic changes of the knocked down cells.

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

Come and see the amaxa integration on show on Tecan’s booth 311 at the Society for Biomolecular Screening exhibition in Montreal, Canada, April 2007.

96-well Nucleofection Lipid-based reagent L

100 % transfection efficiency 90 80 70 60 50 40 30 20 10 0 HL-60

Jurkat

K562

U937

Nucleofection versus lipofection of suspension cells Cells were transfected with (pmaxGFP™). The efficiency was measured on a BD FACSCalibur™ 24h post transfection.

% transfection efficiency

“We are very excited and pleased that the final installation of the integrated system into our laboratory was achieved so smoothly, including setting up the software protocols to run the 96-well transfections,” said Dr Merz. “Our protocols for manual transfection of primary human T cells or Jurkat cells, for example, have been fully adapted for high throughput nucleofection on the Freedom EVO workstation. We can now run RNAi screens using difficult-to-transfect cells, primary cells or suspension cells in an automated fashion, which will enhance and facilitate our target discovery efforts in dermatology or immunology, for example.”

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100 90 80 70 60 50 40 30 20 10 0 Column 1

Column 2

Column 3

Column 4

Reproducible intra-plate transfection efficiency in nucleofected Jurkat E6-1 cells (ATCC® TIB-152™) Analysis was performed on a BD FACSCalibur™ 24h post Nucleofection. The transfection efficiency of each well is shown per column of a 96-well Nucleocuvette Module. Column 4 contained two control samples (no pulse, no plasmid). (Data kindly provided

by C. Merz. Bayer Schering Pharma AG, Berlin)

100 relative expression (% pulse only, sample C) 50 Not for clinical use or blood and plasma screening.

The Nucleofector 96-well Shuttle System and the 96-well Nucleocuvette plates and modules are covered by patent and/or patentpending rights owned by amaxa. PmaxGFP is a trademark of amaxa.

BD FACSCalibur is a trademark of BD Biosciences.

ATCC is a registered trademark and TIB-152 is a trademark of American Type Culture Collection.

40 30 20 10 0

1

3

5

7

9

11

13

15

C

siRNA-mediated depletion of vimentin in human T-cells Knockdown on mRNA level measured by qRT-PCR. 15 samples compared to control (C) set to 100% (Data kindly provided by C. Merz, Bayer Schering Pharma AG, Berlin)

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L IQ U I D H A N DLI NG AN D ROB OTICS

The target of personalized medicine moves ever closer Researchers at Genomic Health Inc., California, are using several Freedom EVO® 200 workstations and a Genesis platform to automate the development and routine screening of gene panels for predicting the risk of recurrence and response to treatment of breast cancer.

Scientists now firmly believe that an individual’s genetic makeup has a strong influence on susceptibility to disease onset or recurrence and response to different treatments, and, as a consequence, understanding these genetic variations is seen as central to creating personalized drugs, tailored to the individual, with greater efficacy, safety and fewer adverse side-effects.

Screening genomes for these variations is logistically awesome as there are potentially many candidate genes that are likely to be involved. The focus of research and development at life science and clinical diagnostics company Genomic Health Inc. is to do just that

Tecan Journal 2/2007

and current efforts are concentrated on developing a gene panel which serves as an initial screen of many genes.

In 2004, Genomic Health launched the Oncotype DX™, the first diagnostic multi-gene expression test commercially available that has clinical evidence validating its ability to quantify the likelihood of breast cancer recurrence in women with newly diagnosed, early stage, node-negative, estrogen receptorpositive breast cancer. The Oncotype DX™ assay analyzes the expression of a panel of 21 genes from a tumor specimen using RT-PCR, and the results are presented as a Recurrence Score™ that estimates the risk of cancer recurrence within ten years, on

a scale from 0 to 100. These scores are more accurate and reproducible than the variables that clinicians currently rely on, like tumor size and grade, and patients’ ages.

The assay has five steps, starting with manual RNA extraction from formalinfixed, paraffin-embedded tumor tissue (FPET) samples. All the subsequent steps are automated, from RNA quantification by fluorescence assay, to a quantitative PCR assay that checks for DNA contamination, and reverse transcription (RT) which produces complementary DNA from the mRNA. The final step is quantitative TaqMan® RT-PCR using the complementary DNA as

LIQU I D HAN DLI NG AN D ROB OT I C S

the template in 384-well plates, with the expression of each of 16 genes measured in triplicate and then normalized relative to a set of five reference genes.

RT-PCR is highly reproducible as it is sensitive, specific, unaffected by inherent variability in the original tumor blocks, and amenable to high throughput and real-time quantification, with successful amplification even from degraded starting RNA. The work to develop the Oncotype DX™ assay down to a much more manageable 21 genes has relied on three independent clinical studies and the assay has been validated in a large, independent multicenter clinical trial (NSABP Study B-14) and in a large population-based case-control study in breast cancer patients at the Northern California Kaiser Permanente. The magnitude of these and potentially more studies in the future made the use of automation in the development work essential, as Jay Snable, Director of Process Automation, explained:

“We needed automated systems for our ongoing assay development and, although we chose Tecan for several reasons, the primary reason was the highly flexible technology, offering so many accessories that we can basically do whatever we need to. As a clinical reference laboratory, we receive the patient samples in tubes, and Tecan provided the best solutions for handling all the different formats we need, from single tubes through to 384-well microplates, in a highly integrated system with quality controls all the way.

It incorporates integral barcode scanning and identification, enabling tracking of individual samples through all the assay steps.”

“A big labor-saving feature of the platform comes from the barcode information that the system generates during assembly of samples into assay plates. We can put samples on in any order; they are scanned and information is sent to our LIMS where the tube and plate barcodes are married, eliminating manual batching steps. This was just one of the benefits coming from the successful integration of the equipment with our LIMS. For example, we also use the LIMS for normalization. Tubes are placed on the robot and scanned. The resulting file is exported to the LIMS and a Gemini™ script for adjusting sample volumes and concentrations is generated and sent back to the platform. The samples are then normalized for use in some of the intermediate steps of the Oncotype DX™ assay, the genomic DNA assay and reverse transcription.”

Most of the Tecan workstations at Genomic Health are configured with Tecan’s Multichannel Pipettor, Te-MO™.

The PCRs are set up on the Freedom EVO workstations using Te-MO 3/5 multipipettors with 96-channel pipetting heads (Te-MO 96). These workstations are also equipped with a PosID™ System for automated barcode reading. Applied Biosystems’ 384-well thermocyclers (7900HT Fast Real-Time PCR systems) are used for the RT-PCR and resulting quantification. The data are reduced and analyzed using custom software developed in-house and, after passing a series of quality control steps, a Recurrence Score™ is calculated.

A report is generated, reviewed by clinical staff, and released to doctors who advise the patients.

Jay concluded: “In 2005, we ran about 7,000 RNA samples in the clinical laboratory with a targeted turnaround time of 10 to 14 days per sample, including review and quality control processes. We continued to scale up through 2006 and processed approximately 13,500 patient samples. Genomic Health is also now scaling up the development process. For example, we are now looking at 761 genes in an initial screen to create a gene profile for an assay to test for risk of recurrence of another cancer type, and this is where the Tecan technology will prove especially useful for the high throughput we need to achieve.”

From left to right: Jay Snable, Darrin Chan, Keith Volk, Kenneth Hoyt, Anhthu Nguyen and Ian Storer from Genomic Health.

Not all options discussed in this article have been cleared for use in all markets. Contact your local sales office for specific information.

Oncotype DX and Recurrence Score are trademarks of Genomic Health, Inc. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Tecan Journal 2/2007

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FO R E N S I C S

Genetic fingerprinting of crime scene samples Forensic scientists at the Landeskriminalamt police office in Stuttgart, Germany, have commissioned a semi-automated sample processing system from Tecan to handle about 20,000 crime scene samples it receives annually for genetic fingerprinting. The specially designed system includes one MiniPrep™ and two Tecan Freedom EVO® liquid handling workstations. In addition, the system includes an integrated thermal cycler, real-time PCR and capillary electrophoresis systems from Applied Biosystems.

The Landeskriminalamt (LKA) is the central criminal police office for the federal state of Baden-Württemberg (BW), in South-West Germany, and its forensic services division is responsible for investigating most of the biological crime scene samples received from the deferring police departments in the state. Approximately 20,000 samples per year come from crimes of all types, including murders, thefts, sex crimes and vandalism. Forensic scientists need to be able to process the complete range of sample materials. For each sample, genomic DNA is extracted from the cells, purified and quantified prior to amplification of short tandem repeats (STRs) by PCR. Sequencers are then used for fragment analysis of STRs, using capillary electrophoresis, and a genetic fingerprint is formed.

In 2005, Dr Kai Vollack and his colleagues Dr Barbara Haak and Dr Ralph Schwenzer, the automation integration team at the LKA-BW, started looking for a semiautomated method for processing their samples in order to cope with the demanding workload. The laboratory had a number of stringent requirements that the automated system needed to meet. It needed to be able to handle a wide range of biological sample types and volumes; to provide the maximum DNA yield and highest analysis sensitivities; to ensure minimal risk of DNA contamination; to incorporate barcode-based sample identification; and to integrate real-time PCR using human-specific primers and performing PCR of STR DNA sequences.

Tecan Journal 2/2007

The members of the automation integration team of the LKA-BW (from left to right: Dr Barbara Haak, Dr Werner Pflug, Anja Gruber, Angelika Jelinski, Dr Ralph Schwenzer, Jessica Loidl, Marga Kugler and Dr Kai Vollack).

Another requirement of the LKA was based on the pioneering work, started in the early nineties by Dr Werner Pflug, the head of the DNA division, using evidence from perpetrators’ skin cells for DNA profiling. “When a person touches an object, he leaves single particles from his skin on that object,” explained Dr Vollack. “It was originally thought that these particles were dead cells with totally degraded DNA but we now know that this is not the case and we often have enough DNA in these particles to create a complete genetic fingerprint.”

For this reason, it was particularly important that the set-up could separate epithelial cell samples that do not contain any DNA from those that do contain DNA at an early stage within the processing workflow. No available, off-the-shelf system could meet these requirements so the project went to public tender and Tecan was chosen for a number of reasons.

“The main advantage of choosing Tecan was the flexibility of the whole system,” explained Dr Vollack. “We

needed a platform that was open to new developments and compatible with devices and reagent kits from different companies. We had seen a Tecan system in action at Professor Scheithauer’s laboratory at the department of legal medicine, Medizinische Universität Innsbruck, and we appreciated its outstanding performance, particularly with the sample identification provided by the integrated barcode reader and the automated data exchange with the LIMS. We were also invited by Tecan to see the automated genetic sample processing system that was recently created for the South African Police Service and we were very impressed with this.”

The forensic science division at the LKABW now has its own fully operational system provided by Tecan which has semi-automated sample analysis. The first step of the process involves manual separation of the cell lysate from the stain carrier, which could be a skin particle, piece of tissue or swab, for example. The lysate is transferred to a Freedom EVO 150 workstation, which is equipped with

FOR E N S I C S Manual separation of the cell lysates from the stain carriers and preparation of the crime scene samples for the ChargeSwitch® Technology magnetic bead extraction of genomic DNA.

an 8-channel liquid handling (LiHa) arm, two Te-MagS™ magnetic bead separation modules and a PosID™ barcode sample identification device. Samples undergo magnetic bead-based DNA extraction and purification using the ChargeSwitch® Technology from Invitrogen and eluted DNA is pipetted into 96-well PCR plates.

These plates are transferred to the second Freedom EVO 150 workstation for the PCR set-up for DNA quantification using the Quantifiler® Human DNA Quantification Kits from Applied Biosystems. Samples undergo real-time PCR using an integrated Applied Biosystems 7500 RealTime PCR System and data are exported to the LIMS and imported into the second Freedom EVO workstation for STR PCR set-up of samples containing sufficient DNA, then sent to an Applied Biosystems 9600 Thermal Cycler. The samples are prepared for capillary electrophoresis by the third Tecan liquid handling system, a single-channel MiniPrep 75, and finally subjected to capillary electrophoresis with a 16-capillary Applied Biosystems 3130xl Genetic Analyzer.

The system includes a number of extra safety precautions in the workflow, including several that are thought to avoid cross-contamination of samples.

For example, the liquid handling arms do not travel across open wells or tubes other than their targets, and using the Te-MagS module instead of a shaking device for mixing prevents the formation of potentially contaminating aerosols.

The forensics division at LKA-BW has a ‘four-eye’ policy, where a second member of staff must confirm every sample

transfer, which is labor-intensive as well as time-consuming, but incorporating Tecan’s automated sample identification processes within the workflow has helped to free up personnel for other tasks.

“We are really happy with the system, it is very sensitive and we have already tested a wide range of possible samples, including the more difficult skin particle samples,” said Dr Vollack. “The automated processing also reduces operational error and, therefore, improves the quality of our data.”

Preparation of single epithelial particles from the butt of a pistol used as a murder weapon.

ChargeSwitch is a registered trademark of Invitrogen.

Quantifiler is a registered trademark of Applera Corporation or its subsidiaries in the US and/or certain other countries.

The Tecan Freedom EVO 150 liquid handling workstation with two Te-MagS magnetic bead separation modules for

DNA extraction of crime scene samples using the ChargeSwitch® Technology from Invitrogen.

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M I C ROA R R AyS

Profiling the expression patterns of hundreds of microRNAs with the HS 800™ Pro Hybridization Station Exiqon is a Danish biotechnology company that provides products and services for the life sciences, research and drug discovery industries all over the world. It specializes in microRNA (miRNA) analysis and produces unique, locked nucleic acid (LNA™)-based high affinity arrays for miRNA profiling. Tecan HS 800™ Pro Hybridization Stations were used during the development of these arrays and Exiqon now relies on the HS 800 Pro systems to provide miRNA profiling and analysis services.

MicroRNAs have recently generated a great deal of interest and are now recognized as important posttranscriptional regulators of gene expression in a variety of biological functions (reviewed in 1- ). However, the actual functions of miRNAs and the mechanisms by which they are controlled remain unclear. Many organisms express hundreds of miRNAs that vary in abundance across cell and tissue types.

The total number of miRNAs in vertebrate genomes is currently unknown, but hundreds have already been isolated and the final numbers may reach over 1,000. A great deal of research remains to be carried out into these molecules, including their possible roles in human diseases and potential for novel therapeutic tools.

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Exiqon, a biotechnology company based in Copenhagen, Denmark and Woburn, USA, produces the miRCURY™ LNA microarrays, which were specifically developed for miRNA expression profiling. Using a high affinity nucleotide analogue, locked nucleic acid (LNA™), Exiqon creates oligonucleotide probes with greatly increased affinity for their complementary targets, due to the increased melting temperature (Tm). This has proved to be particularly useful for detecting short nucleic acid targets such as miRNAs, as standard oligonucleotide technologies have insufficient specificity and affinity for these molecules. The miRCURY™ LNA arrays have almost 1,500 LNA™-based capture probes, making it possible for researchers to profile all miRNAs from vertebrates, invertebrates,

M I C R OA R R AyS plants and viruses (as registered in the miRBase 8.1 database at The Wellcome Trust Sanger Institute, UK) and to crossprofile between different species. The microarrays also contain a number of capture probes (miRPlus™ probes) for detection of miRNAs that are not included in miRBase. The microarrays have low sample usage requirements, being able to work with as little as 1 µg total RNA, and do not require miRNA enrichment.

Exiqon’s miRCURY™ LNA arrays were developed using a Tecan HS 800 Pro Hybridization Station, which offers fully automated, highly sensitive microarray processing with high reproducibility and flexibility. “We started using the HS 800 Pro during the development of our array products because we needed an automated system for hybridizing and washing the slides,” explained Ina Dahlsveen, Product Manager for Services at Exiqon. “It is very important to have as little variability between slides as possible, and using the HS 800 Pro improves our reproducibility and minimizes technical issues that can arise during manual hybridization. Although our slides are compatible with the manual systems available, these are less efficient for processing large numbers of slides.”

In May 2006, Exiqon started providing miRCURY™ LNA array profiling services, as Peter Roberts, Brand Manager at Exiqon, explained: “The services were actually set up in response to specific demand.

After we launched our microarrays, we were getting at least one or two enquiries every week asking if we could run an analysis service, despite the fact that we never advertised one. So when we set the services up, it was a natural progression for us to incorporate the Tecan hybridization stations with them.”

samples from plants, zebrafish, C. elegans, domestic cow and so on – there are customers out there working on all kinds of wonderful organisms. Our turnaround time is variable depending on the size of the project; at the moment, an average project involves about 10 to 12 slides and takes about two weeks from when we receive the samples until we deliver the results. Our services include data analysis, depending on the customer’s requirements. Every project is different so we try to be as flexible as possible and customize our services for each customer and their samples.”

“The arrays work very well with the Tecan hybridization stations, there is very good compatibility between them,” added Peter. “We have had a few instances where customers that are using our arrays for the first time come across some technical problems but, by taking a Tecan hybridization station on-site and running the arrays with the customer, the problems have been solved.”

The compatibility is so convincing that Exiqon’s US office in Woburn, Massachusetts, routinely sends Tecan hybridization stations to its new miRCURY™ LNA customers to aid them in setting up new array procedures. Mikkel Nørholm, Manager of Technical Sales Support at Exiqon in the US, explained: “When a laboratory starts using a new technology, it normally takes some time

to get everything working perfectly.

During that process it’s really helpful to have a gold standard, something that you know works. Combining a Tecan hybridization station with our predeveloped miRCURY™ LNA protocol means that even for inexperienced users, the procedures work straight away.”

The program for Exiqon’s automated microarray hybridization protocol was originally developed in collaboration with Erasmus MC, based in Holland.

“The reproducibility you can get with the hybridization stations, even across different laboratories, is so high that we could simply install the program exchanged by e-mail and it worked instantly!” said Mikkel. “This is also a major benefit for our customers as they can start on their experiments much more quickly after receiving the program from Exiqon electronically.”

References: 1. Bartel DP (200 ). MicroRNAs: genomics, biogenesis, mechanism, and function.

Cell 116(2): 281-297 2. Shivdasani RA (2006). MicroRNAs: regulators of gene expression and cell differentiation. Blood 108: 6 6- 65 3. Wang Y et al. (2007). MicroRNA: past and present. Frontiers in Bioscience 12: 2 16-2 29 LNA, miRCURY and miRPlus are trademarks of Exiqon A/S, Vedbaek, Denmark.

“Obviously with services you also need high throughput, as well as good reproducibility,” Ina continued. “We soon acquired a second HS 800 Pro system, and we will get a third station in the near future. Most of the samples we receive are human, but we also get many samples from mouse and rat, and our newest array contains capture probes for miRNA from all types of organisms. We have received

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LSIQ D HRAEN DLI NG AN D ROB OTICS O FUTIWA

Engineering new

biomaterials for human implants Scientists at the University of Applied Sciences in Zurich, Switzerland, are collaborating with Tecan to optimize automated liquid handling systems for cell and tissue culture. The Cell Biology Division at the University’s Institute of Chemistry and Biotechnology (ICB) specializes in developing cell culture technologies and tissue engineering, including the in vitro development and investigation of biomaterials for bone, cartilage, intervertebral discs, teeth and other connective tissue implants.

It is essential that these materials are tested to high standards of quality with minimal variation, so the group has been developing automated tissue culture methods using a Tecan Freedom EVO® liquid handling workstation as part of their research activities.

Professor Dr Ursula Graf-Hausner, Group Leader of the Cell Biology Division, said: “We are collaborating with industrial partners who develop biomaterials, such as titanium-based products, as potential foreign implants. We begin by carrying out in vitro studies of these biomaterials and, if the results of our preliminary studies are successful, then the materials proceed to animal model studies and, eventually, to clinical applications. Some of the technologies we use are still in the early stages of development but I believe an essential factor for the success of any method is that it can be automated.

Reproducibility is an extremely important

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part of reaching our clinical goals when working with these materials, and we have started a very good collaboration with Tecan to develop these methods.”

The Cell Biology group has been evaluating the capabilities of the Freedom EVO workstation to perform fully automated cell culture, including cell harvesting and plating, for several different cell lines. The Freedom EVO is equipped with a FlaskFlipper™ module for handling the automation-friendly Corning® RoboFlask™ vessels and an Infinite™ F200 luminescence reader for analyzing the cells. It is also configured with a modified, 8-channel liquid handling arm equipped with 5 ml syringes that is capable of both rapid bulk media dispensing and precise low-volume pipetting for reagents, as well as a robotic manipulator arm, and a Te-Shake™ robotic shaker and stirrer.

The group uses a variety of mammalian cell lines and human primary cells for its research and has been testing the Freedom EVO’s ability to handle cell types with different adhesion and aggregation characteristics, including mouse fibroblasts (moderate adherence and aggregation), human osteogenic sarcoma (SaOS-2) cells (strong culture flask attachment and aggregation) and human breast adenocarcinoma (MCF-7) cells (weak attachment to flasks, moderate aggregation). The harvesting efficiencies

of the workstation and the viabilities of the cultured cells were compared to manually obtained results, and it was found that the automated methods achieved comparable or better reproducibility and viability of cells than manual methods1 (see Figure 1).

Dr Stephanie Mathes, research scientist in the Cell Biology group, has been running the Freedom EVO workstation, and said: “The flexibility of the Freedom EVO is really positive and specific design features can easily be added to suit a customer’s application. I particularly like the Freedom EVOware® software; it is very easy to operate and you can simply drag and drop icons to create a whole new automated procedure. We have plans for more projects now that we’ve seen the true flexibility of Tecan’s workstation and all its possibilities.”

“The collaboration we have with Tecan is very close and we have really been able to share our knowledge and experiences,” Professor Graf-Hausner added. “For example, in some instances we have achieved better data by using slightly different volumes of reagents and have passed this on to Tecan for optimizing the system.” The team has recently developed a range of complete, automated cell harvesting and plating protocols that will accommodate different operating conditions, such as the time required for the cells to detach from culture flasks,

LIQU I D HAN DLI NG AN D ROB OT I C S

100

8 7

99

6

Cell viability (%)

“The automated system saves us a lot of time in preparing cells, not least because we can leave it to run unattended over the weekends and during holiday periods,” Professor Graf-Hausner continued. “This is a huge advantage for members of our team, including several students, who would normally be manually pipetting the cells, as they can now spend their time doing much more interesting and sophisticated tasks. The collaboration as a whole has given them an excellent insight into the early steps of process development, and the system itself shows what cell culture technology and tissue engineering research might involve in the future and how new technology will really make our lives much easier in the laboratory.”

From left to right: Lorenz Wohlwend, Stephanie Mathes and Ursula Graf-Hausner.

Cell number/RoboFlask (mio)

to suit a variety of cell types. The results generated are easily transferable to other Tecan cell culture equipment such as the Cellerity™.

5 4 3 2

Cell number Robot

Reference: 1. Graf-Hausner U, Mathes S, Baumgartner S, Mueller U (2007). Automated processing of cell cultures on the Tecan Freedom EVO robotic workstation. Poster presented at ALA 2007.

Further details of the study are available at www.tecan.com Corning is a registered trademark and RoboFlask is a trademark of Corning, Inc. Not all applications discussed in this article have been cleared for use in all markets. Contact your local sales office for specific information.

97 96

1 0

98

95

Cell viability

Manual

Figure 1 SaOS-2 cells were harvested manually and automatically by a Freedom EVO workstation equipped with a FlaskFlipper and Corning® RoboFlask™ vessels.

Cell number and viability were compared. Repeatability of the process and viability of the harvested cells were higher for the automated process.

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A P P L I CAT I O N B IOPHARM A

Plasma technology for fast, effective tip cleaning Tecan has teamed up with Cerionx Inc., the sole provider of automated pipette tip cleaning systems using ‘cold’ plasma, in an exclusive global distribution agreement for the supply and marketing of the Cerionx 8-channel TipCharger™ System.

It is estimated that over 99% of all matter in the known universe is in the form of plasma – lightning and flames are the only two naturally occurring forms on earth. However, through advancements in physics and chemistry as well as the harnessing of alternating currents, the breadth of man-made plasmas continues to widen. Pennsylvania-based company Cerionx uses a man-made, atmospheric pressure ‘cold’ plasma in its unique TipCharger™ System to rapidly and thoroughly clean pipette tips more efficiently than traditional solvent-based methods.

Atmospheric plasma begins with the capturing of free electrons to charged dielectric plates. Through changes in polarity, these electrons are forced to mobilize between a set of plates resulting

Figure 1: Generation of atmospheric plasma and the subsequent ionization/activation of room air.

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in an electron avalanche (Figure 1).

By drawing air in between these plates and through the electron field, the components of air are bombarded with electrons, causing the fracturing and/or activation of CO2, H2O, O2, and N2. The result is a ‘cold’ or ‘non-thermal’ plasma field where most of the input energy is channeled to the electron component of the plasma while the resulting ions and neutral components remain at or near room temperature. In addition, highly energetic metastable atomic species are generated. These species have the appropriate number of electrons, but are displaced to higher shells and exhibit high reactivity and some selectivity in their reaction capabilities. The plasma ions and metastable atomic species react rapidly with biologicals and organic solvents to facilitate their breakdown and eventual removal (Figures 2 and 3).

The patented cold plasma technology developed by Cerionx is quite unique and, although plasma has been used in other cleaning applications, the TipCharger™ is the first to apply it to pipette tip cleaning in the laboratory environment.

In contrast to the conventional solventbased cleaning methods by dilution, TipCharger™ generates no solid or liquid waste, eliminates the use of bleach washes and generally speeds up current wash protocols.

The collaboration between Tecan and Cerionx began in the summer of 2005, when Mr Paul Hensley, founder of Cerionx, visited the Tecan facilities with an early model of the TipCharger™ system. Tecan’s R&D laboratory tested the unit thoroughly to fully assess its potential with Freedom EVO® liquid handling workstations, and an exclusive global distribution agreement for Tecan to market the Cerionx 8-channel TipCharger™ System was finalized in December 2006.

Ms Tia Smallwood, Director of Marketing at Cerionx, elaborated: “We knew that TipCharger™ would provide a manufacturer of liquid handling workstations with significant added value and help differentiation in a crowded market. We approached Tecan as the worldwide leader recognizing they could position TipCharger™ to maximum advantage. We really believe that the sum of these two products is greater than each of the parts.”

APPLICATION B IOPHA RNMTS A COMPONA

Figure 3: Artist’s impression of plasma cleaning action of pipette tips in the TipCharger™ cleaning station.

Even before the agreement was signed, Tecan and Cerionx worked closely together to provide several customers with the first systems to combine the TipCharger™ with Tecan instruments.

One of the early users was Transnetyx Inc., a genotyping service provider in Memphis, Tennessee. Mr Bryan Rushing, Automation Engineer at Transnetyx, explained: “We had been using a bleach solution rinse protocol on our six Tecan Freedom EVO and Genesis RWS™ workstations but, by switching to the TipCharger™, we found that our protocol achieved a 40% reduction in processing time. Processing speed is critical to our business. We guarantee delivery of our results in 24 or 72 hours, achieving 99.973% accuracy.”

In the future, Cerionx is planning to validate the TipCharger™ for clinical environments, making the technology available to even more Tecan users, and new formats of the technology are also under development. Mr Robillard said: “Tecan is a great partner for us; we work in very similar ways. Both companies are aligned around the notion of proving technologies one application at a time, and are committed to development through proof of application. Tecan is a tremendous partner to work with and, at Cerionx, we are really excited to be working together. We hope and expect the collaboration to lead to a very active exchange in the future.”

TipCharger is a trademark of Cerionx Inc.

Mr Mark Robillard, Vice President of Sales and Marketing of Cerionx, added: “This early proof of concept demonstrated the good technical fit of the TipCharger™ with Tecan workstations, and the experience confirmed our belief that the genotyping, genomics and like markets offer extensive opportunity.”

Figure 2: Plasma cleaning action in and around a pipette tip.

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L IQ U I D H A N DLI NG AN D ROB OTICS

Building a cattle DNA database in Japan Over the last decade, the Livestock Improvement Association of Japan (LIAJ) has developed a DNA and beef sample database capable of parentage discrimination analysis for pedigree registration and individual identification of every head of cattle in Japan. To handle the large number of samples involved, scientists at LIAJ have chosen Tecan’s Freedom EVO® 200 workstation, with barcoded sample identification, for the automated extraction of DNA. Since its establishment in 1965, LIAJ has developed its activities, with the improvement of cattle as the primary objective. Today, the association continues to work on livestock improvement, using various methodologies to support the production of superior breeding stock and to develop efficient methods for the individual identification of animals. The Maebashi Institute of Animal Science, LIAJ’s livestock improvement technology laboratory in Gunma, Japan, was opened in 1977 to carry out blood typing and artificial fertilization research in cattle.

The institute has since expanded to handle embryo transplantation, sperm sorting, as well as all DNA-based testing such as parentage testing, testing for hereditary diseases and individual identification. In addition to the various inspection businesses, the Maebashi Institute is involved in research work, including quantitative trait loci (QTL) analysis of Japanese varieties of cattle using DNA markers, in collaboration with other research institutions.

As a technical scientist in the Division of Animal Genetics at the Maebashi Institute, Dr Kazuhito Kurogi is in charge of the individual identification and parentage discrimination analysis. Dr Kurogi explained: “In Japan, a blood typing-based cattle identification database was created in the 1970s, which LIAJ took over from the Japan Holstein Registration Association in 1978. The work to create a DNA-based database for cattle Tecan Journal 2/2007

started in 1997 and we started DNAbased parentage testing in July 2000.

By October 2004, DNA-based parentage testing completely superseded the blood typing system and the DNA database is now fully operational, with all testing carried out using DNA.” The cattle DNA database is used for various objectives like monitoring registration and parentage testing, verifying biotechnology techniques such as artificial fertilization and embryo transplantation, and for meat traceability.

Meat traceability is one of several recently implemented government initiatives to win back consumer confidence following the discovery of a bovine spongiform encephalopathy (BSE)-infected cow in Japan in 2001. All steps in the movement of beef in Japan are subject to traceability testing, from birth to slaughter, storage,

The Division of Animal Genetics at the Maebashi Institute of Animal Science, LIAJ.

and sale at supermarkets, butchers, and even steakhouses and specialist restaurants which handle cuts of beef for final preparation at the diners’ table.

The new traceability legislation has been actively enforced since December 2004.

Dr Kurogi elaborated: “In Japan, each animal is identified by a unique 10digit number. The cattle wear ear tags bearing this number, which is also used as a key for DNA testing. Each pack of dressed meat is labeled with the same 10-digit number, and anybody can find out the past history of each animal from birth to slaughter using this number.

This information is readily available to consumers through in-store computers as well as on the internet through the National Livestock Breeding Center’s homepage.”

“The Ministry of Agriculture, Forestry and Fisheries of Japan has entrusted LIAJ with the task of storing beef samples taken after slaughter, as well as individual identification testing of the stored samples and samples collected from retailers. By carrying out the DNA testing we verify that the traceability system is functioning precisely as specified. For this testing, the cattle DNA analysis technology is essential for individual identification.”

LIQU I D HAN DLI NG AN D ROB OT I C S

In response to the recent and rapid increase in the number of samples handled for registration and testing, in 2004 Dr Kurogi decided to automate the DNA extraction process using Tecan’s Freedom EVO 200 workstation with an integrated robotic manipulator (RoMa) arm, 8-channel liquid handling arm (LiHa) and a PosID™ barcode reader. Dr Kurogi uses the Freedom EVO with its integrated Te-MagS™ magnetic bead separation module to extract and purify DNA from hair root, blood and tissue samples. “We operate the Freedom EVO all day, almost every day. Our extraction program takes approximately two hours to process one plate of 96 samples, so it is possible to handle approximately four plates – 384 samples – in a working day. It is also possible to operate the Freedom EVO unattended so, when we need to process more than four plates in a day, the workstation is run overnight.

At present, we are extracting DNA from approximately 75,000 samples in a year, and the cattle DNA database is updated daily.”

“We have optimized and standardized our programs so that we can extract clean DNA from blood and hair roots using essentially the same method. It is very easy to use and all operations can be carried out by one person, simply by pressing buttons on a PC. We just line up the samples in the morning and leave the workstation to run for the whole day, only stopping briefly to make slight adjustments to the program if different sample types are being processed. We are now making efforts to shorten the process time by simplifying the program without compromising the quality of results.”

“The Freedom EVO has saved labor and time by automating the DNA extraction procedures and the complete barcode management is extremely useful for us, eliminating human error. In a testing facility such as ours, the thoroughness of sample management is critical so the PosID barcode identification is particularly important.”

The flexibility of the Freedom EVO is allowing the scientists to automate other laboratory tasks. Dr Kurogi concluded: “Currently, we are continuing to transfer the tasks which were previously performed manually to Tecan’s workstation. The possibility to expand the application in response to increased workload in the future, of course, is another important characteristic that attracted us to the Freedom EVO.”

Dr Kazuhito Kurogi, Dr Tomohito Ito and Mr Yusuke Kozono with the Freedom EVO workstation.

The Freedom EVO workstation at the Maebashi Institute of Animal Science, LIAJ.

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Managing worldwide compound delivery with

REMP technology As a result of the relentless pace of today’s drug discovery, pharmaceutical companies face ever-increasing throughput demands for compound screening and lead identification.

Many pharmaceutical companies have active screening libraries that contain hundreds of thousands or millions of compounds, and effective and reliable management of these samples is critical to drug development processes.

Boehringer Ingelheim is one of the world’s top 20 pharmaceutical companies, with headquarters in Ingelheim, Germany, and 143 affiliated companies spread around the world.

The company’s principle research site is based in Biberach, Germany, where current research focuses include diseases of the central nervous system, metabolic disorders and respiratory diseases. At Biberach, the Screening Support group relies on several REMP automated sample storage and retrieval systems to provide compound management services for Boehringer Ingelheim’s high throughput screening (HTS) laboratories and other laboratories worldwide.

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Dr Gerhard Mihm, head of the Screening Support group, explained what the services involve: “On a daily basis, we need to be able to acquire compounds, register the compounds into our corporate and dispensary databases, store them under appropriate conditions and/or provide them to our customers in various HTS and project laboratories, on time and prepared to high standards of quality. We deliver compounds in either solid or dissolved forms and sample preparation includes weighing, dissolution and plating. There are only six of us within the group providing this service, so it is essential that all of our processes are highly streamlined and very efficient, otherwise we could not possibly cope with these requirements.”

The compounds come from various sources, including archive compounds that have been previously synthesized

by Boehringer Ingelheim’s medicinal chemists, screening compounds from the combinatorial chemistry groups, and many externally acquired compounds.

The Screening Support group was established 15 years ago and its workload has dramatically increased during this period. “Over the years, the number of compounds to be managed has increased tremendously, as have the numbers of assays and additional requirements from our many different customers. In order to cope with these demands, we rely heavily on our REMP sample storage systems,” said Dr Mihm. “We now have more than 800,000 compounds in our collection so our service would not be possible without this technology.”

Dosage and Solution Preparation Station with tube sealing and capping.

The REMP Dosage and Solution Preparation station.

SA MPLE M ANAGEM E N T

The Screening Support group’s sample store.

“We first started talking to REMP back in 1996,” continued Dr Mihm, “and one of the key reasons we chose these systems was for the rapid service and maintenance the company could offer us. We were also convinced that the technological concepts of the REMP systems would meet our requirements.

In 1998 we had the first ever REMP solution store for 96-well tubes installed and, since then, we have acquired three more automated REMP systems. These include stores for our solid samples and a -20°C store for compound solutions using REMP 96 and 384 Tube Technology™ consumables and automated cherrypicking functions. Due to the constant growth of our collection, a new plate store at -20°C was implemented at the end of 2006 as well as an expansion of our vial store to accommodate another 250,000 vials.” With the REMP technology, the Screening Support group can comply with Boehringer Ingelheim’s preferred practice of using single-use only tubes and plates for the DMSO solutions, ensuring high quality, contamination-free samples for all of its laboratories.

In addition to the REMP storage systems, the group depends on a number of REMP workstations, including the REMP Automated Plate Replicator™ (APR) for copying plates and the REMP Dosage and Solution Preparation Station™ (DSP; a collaborative development between Boehringer Ingelheim and REMP) for automated weighing of solid compounds in vials with special REMP weighing caps, and for preparing stock DMSO solutions and generating 96well mother plates. Compounds that are already available in microplates or

96-well tubes are dissolved and plated with a Tecan automated compound dissolution and reformatting system (CIPDISS). This new system is based on a Freedom EVO® liquid handling workstation and includes a Te-MO™ 96 multichannel pipetting option and a CRS F3 robot. REMP devices are also integrated with the workstation, including the Automated Capper/ Decapper™ (ACD96) and the LHS heat sealer. Boehringer Ingelheim’s engineers have developed another Freedom EVO-based workstation (CIVDISS) for dissolving and plating solid compounds that cannot be weighed automatically.

“In general, we are happy with REMP technology. For us, the development of an excellent relationship with the people at REMP has been a major benefit.

Communication with them is absolutely perfect in most cases and it could be described as more like a partnership than a vendor-buyer relationship,” Dr Mihm concluded.

Automated Plate Replicator for copying plates.

Dr Hans-Peter Sattler, Project Manager, Tecan Integration Group and Nadine Hauser, Laboratory Assistant, Boehringer Ingelheim.

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Te-PoolSafe™ boosts pipetting accuracy for testing donor blood The German Red Cross Institute in Frankfurt is responsible for screening thousands of donated blood samples every day for blood banks in Germany and further afield. Scientists at the Institute have developed high throughput PCR-based testing methods to screen for a number of viruses, and samples are pooled prior to screening using three Tecan liquid handling workstations. In 2006, the Institute worked with Tecan to validate the recently launched Te-PoolSafe™ option for monitoring pipetting during pooling.

Dr Kai Hourfar at the Red Cross Institute.

As one of the first laboratories to use PCR for testing donated blood, the German Red Cross Institute in Frankfurt, Germany, was an ideal candidate for perfecting Tecan’s new pipette monitoring module in a rigorous diagnostic setting. Dr Kai Hourfar, deputy of the Blood Donor Screening Lab, said: “We demonstrated years ago that PCR testing of blood samples saved lives, because we had cases where some samples were PCRpositive for viruses, even though antibody tests of the same samples were negative.

Because we were pioneers and still have a very good reputation for this application, many institutes from other countries asked us, and continue to ask us, to perform PCR testing on their behalf. The challenge to begin with was that there was no automation for PCR testing, and the only feasible way to test the large numbers of donations was to create mini pools of the samples. We had worked with Tecan previously when developing our serological tests, and decided to choose Tecan instruments again to automate pooling for PCR.” The Institute initially acquired a Genesis RSP™ liquid handling workstation for pooling the samples, followed more recently by two Genesis Freedom® workstations. All three workstations

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are dedicated to pooling the thousands of samples that the Red Cross Institute receives each day.

“We create pools of 96 donations, which means that if we receive 1,000 samples per day then we reduce these samples to ten pools and only need to perform ten PCRs for each parameter being tested,” Dr Hourfar explained.

“We include enrichment procedures in our method and always use positive controls with our screens to ensure that our testing is sufficiently sensitive, and this approach allows us to meet the strict testing criteria defined by the Paul Ehrlich Institute.” The pooled samples are subjected to PCR and serological tests simultaneously, as platelets are only useful for a maximum of around five days, and this approach clears a unit of blood for release just one day after donation. Blood samples arrive at the Red Cross Institute at night, so pooling begins at 11:00 pm and is normally finished by 6:00 am the following day, ready for the technicians to begin subsequent procedures. Samples from first-time donors are always pooled separately, because these samples have not been previously screened. However, the incidence of positive results during testing at the Red Cross Institute is extremely low.

CLI N ICAL DIAGNOST I C S

“All the samples are barcoded and managed by our LIMS software, and we have several safety measures in place to prevent any mix-up of the sample identities,” said Dr Hourfar. “Most institutes that we test for send us a list of the samples they are sending and we compare the tubes we receive with this list to make sure none are missing or duplicated. If two samples arrive with the same barcode, for example, then both donations will have to be discarded.”

or, alternatively, may be transferred to the LIMS for automated processing.

Inaccurately dispensed samples are selectively identified and can be separately re-pipetted.

Safety precautions are vital in any diagnostics laboratory and, in 2006, Tecan launched the Te-PoolSafe option, a liquid arrival check system based on weight measurement, that allows blood banks and nucleic acid testing laboratories to monitor and evaluate the performance of their pooling application. The module ensures full sample traceability and provides documented proof of performance, leaving users confident that the appropriate amount of liquid has been pipetted from each sample into the pool. This is a very fast and sensitive balance with a 16-position tube holder and it is supported by software that measures and evaluates each dispense from primary samples to the sample pool, to confirm liquid arrival. Results are displayed to the system operator

“We have pooled well over 5,000,000 donations since we first started testing at the Red Cross Institute and any additional safety measures, such as the visual inspection of archive plates to verify automated safety features, will always be important,” Dr Hourfar explained. “It is critical that we do everything we can to ensure our pooling procedures are safe and it is becoming increasingly necessary in diagnostics laboratories to prove these safety measures are adequate.

The Te-PoolSafe provides valuable documentation to show that all blood pooling steps are accurately performed.”

Dr Hourfar and his colleagues performed a number of validation studies for the Te-PoolSafe on behalf of Tecan1, and were impressed by the module’s potential to improve monitoring and documentation during blood pooling.

The Te-PoolSafe option has not been cleared for use in all countries. Contact your local sales office for specific information.

Reference: 1. Hourfar MK, Koller M, Roth WK, Kehrli R, Seifried E, Schmidt M. (2007).

Balance module allows consistent monitoring and documentation of the pooling process for NAT-testing. Vox Sanguinis (in press).

Balancing samples with the Te-PoolSafe.

Tecan Journal 2/2007

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28

EV E N TS 2 0 07

Meet Tecan at these events in the next six months Americas American Association for Cancer Research (AACR)

Los Angeles, CA

14-18 Apr 2007

Society for Biomolecular Sciences 2007 (SBS)

Montreal

15 Apr 2007

FASEB Experimental Biology

Washington, DC

29 Apr - 1 May 2007

American Association for Clinical Chemistry (AACC)

San Diego, CA

15 Jul 2007

Drug Discovery Technology 2007 (DDT)

Boston, MA

06 Aug 2007

The 6th International BioExpo

Tokyo

20 Jun 2007

JAIMA Show 2007

Makuhari

29 Aug 2007

The Japanese Society for the Study of Xenobiotics

Sendai

09 Oct 2007

Sanguin Spring Seminars 2007

Amsterdam

19 Apr 2007

MC-GARD Molecular Profiling of the Genome

Amsterdam

02 May 2007

MipTec 2007

Basel

07 May 2007

The Swedish Microbiology Spring Meeting 2007

Stockholm

09 May 2007

AMT – Advances in Microarray Technology

Edinburgh

15 May 2007

Euromedlab 2007

Amsterdam

03 Jun 2007

European Human Genetics Conference 2007

Nice

15 Jun 2007

ESACT 2007

Dresden

17 Jun 2007

Molecular Life Sciences 2007 – Herbsttagung der GBM

Hamburg

16 Sep 2007

DGTI Jahreskongress

Friedrichshafen

18 Sep 2007

Biotech Forum & Scanlab

Stockholm

24 Sep 2007

ILMAC

Basel

25 Sep 2007

106th General Meeting

Asia and Pacific

The 28th Annual meeting of Molecular Biology Society of Japan Europe, Middle East and Africa

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, Beat Glauser/www.beatglauser.com, Tim Cook/The Day Publishing Company (New London, CT) 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 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.

Cellerity, Columbus, Gemini, Genesis RMP, Genesis RWS, GenePaint, HID EVOlution, HS 4800 Pro, Infinite, MiniPrep, MultiChannel Arm, PosID, ProfiBlot, Safire2, Sunrise, FlaskFlipper, Te-MO, Te-MagS, Te-PoolSafe, Te-Stack and Te-VacS are trademarks and Freedom EVO, Freedom EVOlyzer, Freedom EVOware and Genesis Freedom 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, Automated Plate Replicator (APR), DSP and REMP Tube Technology are trademarks of REMP AG, Oberdiessbach, Switzerland. © 2007 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 Tecan Austria +43 62 46 89 33 Belgium +32 15 42 13 19 China +86 10 586 95 936 Denmark +45 70 23 44 50 France +33 4 72 76 04 80 Germany +49 79 51 94 170 Italy +39 02 215 21 28 Japan +81 44 556 73 11 Netherlands +31 18 34 48 17 4 Portugal +351 21 000 82 16 Singapore +65 644 41 886 Spain +34 93 490 01 74 Sweden +46 31 75 44 000 Switzerland +41 44 922 89 22 UK +44 118 9300 300 USA +1 919 361 5200 ROW +43 62 46 89 33 REMP Switzerland +41 31 770 70 70 Japan +81 44 542 70 21 USA +1 508 429 2200

Tecan Journal 2/2007