Tecan Journal Edition 01/2015 by Tecan Group Ltd.

TJ Edition 1/2015

Tecan Journal

Life Sciences and Diagnostics

Ignite the productivity of your lab with the SparkTM 10M reader pages 4 - 5

A helping hand for vitamin testing pages 18 - 19

Pouring oil on troubled waters pages 20 - 21

Providing diagnostic solutions for Alzheimer's disease pages 24 - 25

Welcome to 2015 and the new Tecan Journal

This year will see a wave of exciting new products for Tecan, beginning with the introduction of two major new platforms at one of the laboratory automation industry's leading events – SLAS2015. Visitors to the exhibition – which takes place from the 8th to the 10th of February in Washington, DC – will be able to see a wide range of innovative Tecan instruments in operation, including the latest Fluent™ liquid handling solution and the new Spark™ multimode reader. Bringing the next level of innovation to automated liquid handling, the Fluent Laboratory Automation Solution for compound management builds on the success of the Fluent cell-based assay workstation launched in 2014, setting new standards for simplicity, productivity and confidence. This advanced liquid handling workstation will be complemented by Spark, an all-new multimode microplate reader which is designed to streamline the everyday activities of virtually any life sciences laboratory, from fast absorbance scanning for basic colorimetric assays to integrated cell counting and incubation capabilities for 3D cell culture analysis. We also continue to invest in the tremendously popular Freedom EVO® platform, and will be showcasing a number of application-specific, easy-to-use solutions for the new look instrument – including the Freedom EVO NGS workstation – at SLAS2015. And it's not just our Life Sciences Business that has a lot to offer in 2015. Our Partnering Business is also looking forward to a busy year, supporting our clinical diagnostics partners with their regional product launches of new instruments – developed and manufactured by Tecan – and the associated ramp up in our serial production. The latest of these – the ORTHO VISION™ Analyzer from Ortho-Clinical Diagnostics – is now offering enhanced safety for transfusion medicine laboratories in Europe, Japan and Australia. You'll find details of many of our new and existing products in this issue of the Tecan Journal, as well as ways in which our ingenious customers are using their Tecan equipment. I hope you enjoy the issue, David Martyr, CEO

CEO WELCOME TECAN JOURNAL 1/2015

pages 4 - 5

This cutting edge system means that laboratories no longer have to make a trade-off between flexibility and sensitivity pages 14 - 15

CEO Welcome

4 - 5

Ignite the productivity of your lab with the Spark 10M reader

6 New Fluent Laboratory Automation Solution for compound management 7

NGS library preparation at the touch of a button

8 HP D300 now offers aqueous liquid dispensing 8

CavroÂŽ Disposable Tips launched for OEM customers

An important piece of the HIV puzzle

10 - 11

Simplicity by design

12 - 13 Automating NGS protocols for a wide range of sample types

pages 20 - 21

pages 28 - 29

Automation offers several advantages over manual testing, in particular the reduction in hands-on time required, and improved reproducibility

14 - 15

High throughput cell separation â&#x20AC;&#x201C; a model for upscaling

16 - 17

Nanoliter liquid transfers aid search for new antibiotics

18 - 19

A helping hand for vitamin testing

20 - 21

Pouring oil on troubled waters

22 - 23

World-first automation for influenza surveillance

24 - 25

Providing diagnostic solutions for Alzheimer's disease

26 - 27 Rapid generation of dose-response curves for high throughput screening 28 - 29 Automation allows simultaneous antimicrobial susceptibility testing and MALDI-TOF sample preparation for bacterial identification 30 - 31

Seeing the light

Leading the debate

32 Events

CONTENTS TECAN JOURNAL 1/2015

Ignite the productivity of your lab with the Spark™ 10M reader The new Spark 10M multimode microplate reader is designed to offer greater flexibility and increased productivity for cell biology and genomics customers. From microbiology research and cell-based assays to rapid DNA quantification, the all-new platform delivers a combination of advanced capabilities and exceptional ease of use to simplify your daily work.

At the heart of the Spark 10M are Tecan’s unique Fusion Optics, which offer users the choice of filter- or Quad4 Monochromators™based measurements – or even a combination of both – at the touch of a button. By allowing users to independently choose between filters and monochromators for both excitation and emission, this cuttingedge system means that laboratories no longer have to make a trade-off between flexibility and sensitivity. This exceptional format flexibility is complemented by patent-pending High Speed Monochromators which allow complete absorbance scans – from 200 to 1,000 nm – in under five seconds. This option combines the speed of a diode array with the precision and flexibility of a monochromator, offering wide-ranging and detailed spectra with unsurpassed wavelength accuracy for rapid DNA and protein quantitation. The Spark 10M reader has been developed from the outset with cell-based assays in mind, and includes a host of software and hardware features – including a built-in cell counter – designed to simplify cell biology protocols. Precise regulation of environmental conditions is crucial to any cell-based assay, and the system’s fully integrated Gas Control Module (GCM™) offers simultaneous control of CO2 and O2 concentrations within the measurement

PRODUCT NEWS TECAN JOURNAL 1/2015

The Spark 10M reader is designed to improve productivity without compromising on flexibility

chamber. Combined with precise temperature regulation and plate shaking options, this provides predictable culture growth under physiological, hypoxic or anaerobic conditions, allowing long-term, walkaway studies. Productivity and reproducibility are further improved by the addition of a patent-pending humidity cassette to provide evaporation protection for any SLAS-format microplate, and automated lid handling to precisely balance gas exchange and minimize the risk of contamination for any measurement mode.

For even greater efficiency, the Spark 10M also features a bright-field cell counting module for automated, label-free cell counting and analysis. This innovative system offers accurate, reproducible cell counting and trypan blue-based cell viability analysis using disposable Cell Chip™ devices. To maximize sensitivity, even for low cell concentrations, the system allows flexible area selection and automated replicate processing, providing simple mean-value calculations, greater accuracy and more precise data.

All these measurement modes and options – along with an injector module with unique stirring and heating capabilities for rapid kinetic assays – are fully integrated and supported by the Spark 10M’s user-oriented SparkControl™ software. Accessed via a PC or the instrument’s intuitive touchscreen interface, SparkControl makes it easier than ever before to set up and run even complex assay protocols, with one-click operation and remote control for even greater efficiency and productivity.

SparkControl makes it easy to run even complex assays

To find out more about Tecan’s Spark 10M multimode reader, visit www.tecan.com/spark

PRODUCT NEWS TECAN JOURNAL 1/2015

New Fluent™ Laboratory Automation Solution for compound management Tecan will soon be launching a new Fluent Laboratory Automation Solution designed to offer greater throughput and flexibility for compound management workflows*. Following the successful launch of the Fluent cell-based assay workstation in summer 2014, this second application-oriented system will introduce a number of new hardware and software options aimed at simplifying and improving the productivity of compound management and assay plate generation activities. The compound management workstation is ideally suited to drug discovery laboratories and centralized compound library facilities, offering a host of features intended to streamline day-to-day activities such as daughter plate generation, plate reformatting and hit picking.

Liquid handling versatility has been increased with the introduction of washable fixed tips for the instrument’s Flexible Channel Arm. Complementing the existing disposable tip options to provide cost-effective reagent and compound distribution – as well as cap piercing capabilities – the new fixed tips are also available in a low volume option, offering reliable pipetting below 0.5 µl to help minimize compound consumption and enable effective serial dilutions. This increased flexibility is complemented by the ability to

use DMSO – the solvent of choice for most compound libraries – as the system liquid, allowing fast, efficient plate filling without the need for a bulk dispenser module. Labware logistics have also been enhanced with the addition of a tube handling option for the Robotic Gripper Arm (RGA), and a Stacker module. Compatible with a wide range of common tube formats, the tube gripper can be manually exchanged with the RGA’s existing microplate gripper, allowing efficient transfer of tubes and simplifying barcode scanning. The Stacker – which is compatible with SLAS-format consumables – is ideally suited to high capacity tip storage, allowing up to 40 racks of Nested LiHa disposable tips per stack for increased walkaway times. These new hardware options are fully supported by the platform’s updated FluentControl™ software, which also includes a number of new applications and pre-programmed ‘Smart Commands’ for common activities such as plate replication and reformatting. To streamline integration with upstream and downstream processes, the software will also support both worklisting and 2D barcode scanning, ensuring the Fluent Laboratory Automation Solution for compound management fits seamlessly into even the most demanding of workflows. To find out more about Tecan’s Fluent Laboratory Automation Solution, visit www.tecan.com/fluent * Will be released soon. For research use only within the USA. Not for clinical diagnostics.

The Fluent Laboratory Automation Solution offers greater throughput and flexibility for compound management workflows

PRODUCT NEWS TECAN JOURNAL 1/2015

NGS library preparation at the touch of a button Tecan has launched the Freedom EVO® NGS workstation to simplify NGS sample preparation and PCR set-up. Offering user-friendly, walkaway automation of library preparation at the touch of a button, it eliminates the need for extensive manual processing.

The Freedom EVO NGS workstation guides the user through instrument set-up

NGS is the fastest growing application in genomics, yet many sample preparation protocols can be labor intensive and time consuming. The Freedom EVO NGS workstation has been designed to offer robust and reliable automation of library preparation, quantification, qPCR set-up, normalization, pooling and capture. It includes all the modules required for precise set-up of NGS sequencing libraries and, with one-touch method selection and step-by-step user instructions via Tecan’s intuitive TouchTools™ graphical interface, it reduces hands-on time and increases productivity – even for inexperienced users.

To further streamline processing, Tecan has worked with leading industry partners to supply ready-to-run protocols for a number of popular NGS technologies. Protocols for Illumina’s TruSeq® Nano DNA, TruSeq Stranded mRNA* and Nextera® Rapid Capture Exome* chemistries have been developed in collaboration with Illumina and the High-Throughput Sequencing Facility, University of North Carolina at Chapel Hill, with thorough testing and sequencing data analysis to ensure high quality results.

preparation workflow for Ion AmpliSeq™ panels, developed and supported by Thermo Fisher Scientific, helping you to hit the ground running and simplifying day-to-day activities. To find out more about Tecan’s Freedom EVO NGS workstation, visit www.tecan.com/ngs *Illumina-qualified protocol.

In addition, the system is supplied with protocols covering the Ion Torrent™ library

PRODUCT NEWS TECAN JOURNAL 1/2015

HP D300 now offers aqueous liquid dispensing The popular HP D300 Digital Dispenser now allows customers to titrate low volumes of biomolecules in aqueous solutions, offering greater flexibility for drug discovery workflows. Users can choose between dispensing DMSO, for small molecule studies, and aqueous solutions in combination with a surfactant, for the investigation of proteins, antibodies, enzymes and nucleic acids. Newly launched T8+ and D4+ Dispenseheads, combined with updated software, allow both new and existing customers to benefit from this breakthrough, offering straightforward set-up of dose-response curves and synergy studies. New ‘fluid class’ options allow users

to optimize liquid dispensing performance to their specific buffers and surfactants, and an Enzyme Profile function enables fast and easy set-up of titrations using differing combinations of enzymes, substrates and other compounds. These innovative features, combined with the system’s proven low volume dispensing performance, make it easy to perform complex experiments – such as enzyme characterization, Km determination and exploring the effects of enzyme inhibitors – ensuring a comprehensive solution for drug discovery applications. To find out more about the updated HP D300, visit www.tecan.com/digitaltitration

The new Enzyme Profile function enables rapid setup of any combination of enzymes, substrates and compounds with just a few clicks

Cavro® Disposable Tips launched for OEM customers Tecan has created a range of Cavro Disposable Tips for use with the Company’s popular Cavro brand OEM components. Offering customers a selection of premium consumables to complement their Cavro Omni Robot and Cavro Air Displacement Pipettor (ADP) options, these high quality tips have been developed and validated to ensure optimal performance and reliable operation. All Cavro Disposable Tips are manufactured to the same high quality standards as Tecan’s broad portfolio of consumables for life sciences customers, using state-of-the-art production processes and integrated quality control procedures to ensure consistent pipetting performance and provide complete peace of mind. The conductive polypropylene tips are available in a variety of tip volumes, and can be supplied in customer-branded packaging for large volume orders, providing a complete liquid handling solution to enhance your instrument design. To find out more on Tecan’s Cavro Disposable Tips, visit www.tecan.com/cavroditi 8

PRODUCT NEWS TECAN JOURNAL 1/2015

Cavro Disposable Tips offer exceptional performance and peace of mind

An important piece of the HIV puzzle The Irkutsk Regional AIDS Center is taking advantage of the flexibility and ease of use of an Infinite® 200 PRO multimode reader to help monitor HIV patients. Combining luminescence- and absorbance-based assays with a range of other diagnostic tests, the Center is able to accurately assess the immune status of its patients. The prevention and diagnosis of HIV infection is a major public health issue in Russia, with an estimated 645,000 people living with HIV at the end of 2013. The relatively high prevalence of HIV-positive individuals – corresponding to approximately 0.45 % of the country’s overall population – includes between 60,000 and 80,000 of these cases diagnosed in the last five years. To help cope with this urgent health problem, the Russian Federation has a network of AIDS centers across every major region. Each center has responsibility for the prevention, monitoring and treatment of HIV infections within its local population, as well as providing psychological care to patients and coordinating the various services concerned with HIV. The Irkutsk Regional AIDS Center in Eastern Siberia has recently moved into a new, purpose-built facility close to the shores of Lake Baikal. The complex effect of HIV on the human immune system means that a variety of tests are required to accurately determine a patient’s immune status, and the Center’s clinical immunology laboratory is equipped with a range of modern diagnostic instruments intended to simplify its workflow. Two of the components which must be measured – the phagocytic activity of white blood cells and the concentration of circulating immune complexes (CICs) – are determined using microplate-based optical assays, and the laboratory has chosen an Infinite M200 PRO multimode microplate reader to perform these tests. The phagocytic activity of white blood cells is analyzed by luminol-dependent chemiluminescence. A suspension of the patient’s white blood

cells is mixed with opsonized zymosan or a saline control in the presence of luminol. Neutrophils and monocytes from the patient’s blood then ingest the zymosan particles and generate reactive oxygen species that oxidize the luminol. The resulting chemiluminescent signal is measured for 30 minutes using the Infinite M200 PRO in luminescence mode, and the system’s Magellan™ software automatically calculates the stimulation index. Determination of the CIC concentration is also performed on the Infinite M200 PRO reader. Diluted patient serum is mixed with a solution of polyethylene glycol, which precipitates the immune complexes. After an hour of incubation, the turbidity of the sample wells is measured directly using the instrument’s absorbance mode.

Inna N Savintseva, head of the clinical immunology laboratory, commented: “The most important thing is that the Infinite reader is easy to use. We are not a research organization, so any instrument that is too complex or unreliable is not suitable for routine screening. Our local Tecan representative was very helpful in teaching the laboratory personnel to use the instrument, which we were very grateful for, and we have not had any issues. The instrument’s high sensitivity is also very important, as the results must be reliable and tally with the results of other assays in order to provide an accurate picture of each patient’s immune status.” To find out more on Tecan’s Infinite 200 PRO, visit www.tecan.com/infinite200pro

The Irkutsk Regional AIDS Center laboratory team

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

Simplicity by design Genera Biosystems has been testing a HydroSpeed™ plate washer with its Sirocco™ multiplexed diagnostics automation platform, allowing rapid, efficient washing of its proprietary AmpaSand™ beads for high retention rates and accurate results.

"We looked at several plate washers on the market, and the HydroSpeed washer was the only system that met our specifications." Karl Poetter, Chief Scientific Officer at Genera Biosystems

Genera Biosystems in Melbourne, Australia, develops and manufactures molecular diagnostic testing kits based on its proprietary AmpaSand bead technology. Used for molecular diagnostics in clinical pathology laboratories throughout Australia, AmpaSand beads are based on silica microspheres of various diameters to offer multiplexed testing based on PCR and flow cytometry. The Company offers a number of diagnostic assay kits which take advantage of this technology’s multiplexing capabilities, including the RTIplex™ Respiratory Pathogen Panel assay, which can distinguish between 15 common infections of the upper respiratory tract, including influenzas, parainfluenzas, respiratory syncytial virus, human adenovirus, human rhinovirus, Bordetella pertussis, Chlamydophila pneumoniae and Mycoplasma pneumoniae. This broad spectrum test is designed to aid clinical decision-making by providing a fast and easy method of identifying the pathogen responsible for the patient’s symptoms, improving antimicrobial stewardship and enabling appropriate infection control measures to be implemented as soon as possible. The surface of each AmpaSand bead is chemically activated to allow covalent bonding of oligonucleotides in high concentrations, providing stable beads that can be used directly for PCR amplification and cytometric analysis. Designed for use in a 96-well microplate format, a key feature of AmpaSand beads is their high density. This ensures very fast, efficient settling during centrifugation, removing the need for magnetic separators or filtration plates. Karl Poetter, Chief Scientific Officer, explained: “Following PCR, AmpaSand plates are spun down to remove the supernatant, then washed to remove unbound reagents or PCR

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

products prior to flow cytometry analysis. Manual bead washing is tedious and error-prone – much hands-on time is needed and there is a high risk of erroneous results through disturbing the pellet and losing beads – which is far from ideal in a clinical setting. We needed to automate the post-PCR washing steps to provide the accuracy and reliability necessary for diagnostic applications, and so looked for an automated 96-channel plate washer that offered us effective removal of unincorporated primers and reagents without disturbing the pellet. We looked at several plate washers on the market, and the HydroSpeed washer from Tecan was the only system that met our specifications. It offers precise control over the wash parameters and needle positioning, allowing us to obtain very high bead retention rates and ensuring effective washing.” To further simplify diagnostic testing, Genera Biosystems has evaluated the HydroSpeed washer for integration with a multiplexed diagnostics automation platform – the Sirocco Automation System. This set-up combines bead washing using the HydroSpeed with automated sampling, high throughput flow cytometry and data analysis, driven by the Company’s proprietary QPlots software. This system greatly reduces the number of manual plate handling steps by providing automated post-PCR processing and analysis of AmpaSand beads, allowing efficient processing of completed PCR reaction plates from multiple PCR instruments. Karl continued: “The HydroSpeed system is designed with laboratory automation in mind, so was very straightforward to integrate into the Sirocco platform, and the engineers from Tecan Australia were very helpful during this process.”

Following the successful trial integration of the HydroSpeed into the Sirocco platform, the Genera Biosystems team now plans to develop a fully automated diagnostics platform that offers complete automation of the assay. “Our aim is to create a streamlined, compact and user-friendly automated system that offers walkaway automation of the entire process, simply requiring the patient sample to be loaded onto the instrument. This will lower the risk of pathogen exposure for users, and minimize the risk of erroneous results, with potential benefits for both clinical laboratories and patients around the world. The Tecan team has always been positive and supportive of our automation efforts, and we are confident that their expertise will help us to develop this next generation of our automated system,” Karl concluded. To find out more on Tecan’s HydroSpeed washer, visit www.tecan.com/hydrospeed

Precise control over wash parameters ensures high retention rates for AmpaSand beads

To read more about Genera Biosystems, go to www.generabiosystems.com

The HydroSpeed offers precise control over critical wash settings

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

Automating NGS protocols for a wide range of sample types

PTP’s Genomics Platform has automated all the steps from DNA/RNA extraction to the preparation of sequencing pools. Using three Freedom EVO® platforms, comprehensive automated workflows ensure high quality data and complete sample traceability, with a daily throughput of up to 288 genomic DNA samples. PTP’s Genomics Platform facility

Parco Technologico Padano (PTP) in Lodi, in the north of Italy, is a science park serving universities and publicly-funded research centers in Italy – as well as other private companies and institutes across Europe – in the agriculture, food, animal feed and biotechnology sectors. PTP’s Genomics Platform (PGP), headed by Valentina Gualdi, was set up in 2005 and carries out genotyping, Sanger sequencing, next generation sequencing (NGS) and high throughput genomic analyses. Established in 2012, the NGS laboratory performs a mixture of genotyping services on plant, animal and human samples for in-house research groups, dietary and fitness counseling, and several clients in the food industry, including the analysis of durum wheat, herbs, rice and tomatoes, and checking kosher foods for the presence of pork. Valentina explained: “Our three Freedom EVO platforms were installed 10 years ago, and have been reconfigured many times since as our needs have changed. We first turned to automation to manage the large numbers of samples from our collaborative Italian and

GENOMICS TECAN JOURNAL 1/2015

European projects more easily. Automation saves us money by allowing us to process smaller volume samples – as low as 5 μl – while avoiding errors and maintaining full sample traceability, which is essential to our laboratory’s European ISO 9001 and Italian Accredia quality control regulations. Having one manufacturer also makes transferring samples and protocols between the instruments much easier.” Samples received by the laboratory are aliquoted into 96-well plates by a Freedom EVO 100 platform equipped with a four-channel Liquid Handling (LiHa) Arm and disposable tips. These plates are then transferred to a Freedom EVO 150 workstation for extraction and fluorimetric quantification on an integrated GENios™ microplate reader. After quantification, this instrument is also used to set up PCR reactions, including KASP™ genotyping assays. Post-PCR processing is performed on a second Freedom EVO 150 platform configured with an eight-channel LiHa Arm using fixed tips, a

MultiChannel Arm™ (MCA) 96 and a Robotic Manipulator Arm – as well as a cooling system, a shaker and an integrated Infinite® M1000 microplate reader. The post-PCR steps include automated magnetic bead PCR and Sanger sequencing dye clean-ups, microsatellite analysis, pooling and NGS library preparation with Nextera® XT or TruSeq™ kits for DNA or RNA analysis. PTP’s NGS facility is also a Certified Service Provider for Illumina, and the Company recommends double quantification with both PicoGreen® dye labeling and quantitative PCR with KAPA kits. For PicoGreen quantification, the Freedom EVO 150 sets up the 96-well plates, builds the standard curve, makes sample replicates, and runs fluorimetric readings on the Infinite M1000 reader. Data is then exported into an Excel® file, from which the workflow template is generated, to ensure all samples are represented equally in the pooled libraries loaded onto the Illumina sequencers (HiSeq™ 2000 and MiSeq™). “For each project, we do all the DNA or RNA extractions first, which can take up to a month,” Valentina continued. “Extraction of each 96-well plate takes two to three hours, with a maximum daily output of 288 samples. Accurate quantification of DNA or RNA for library preparation is critical for good quality NGS data and so, once extraction of all the samples is complete, we perform all the DNA/RNA quantifications and quality checks on the same day. We then verify results and normalize the samples before beginning the PCR set-up. For genotyping set-ups, we can perform 10 or more PCRs per plate in one day, generating as many as 100 data points per sample – the only limiting factor is the amount of DNA/RNA we can extract. For NGS analysis with TruSeq DNA, one sample is generally loaded per lane, with up to 16 per run, whereas the Nextera XT protocol for

amplicon sequencing or metagenomics allows the pooling of up to 96 or 384 samples.” “We have always been satisfied with Tecan’s products and service. We usually develop our own scripts on the Freedom EVO platforms, but help from Tecan’s specialists is always available by telephone or email. I attended Tecan’s training course when we first had the platforms, and, in turn, trained PGP’s technicians; with experience the Freedom EVOware® software is very intuitive, and they can all write and edit scripts on the platforms. This flexibility is essential for handling the wide range of samples which arrive in the laboratory daily,” Valentina concluded. To find out more about Tecan’s genomics solutions, visit www.tecan.com/genomics To read about the PTP Genomic Platform, visit www.tecnoparco.org/index. php?option=com_content&view=article&id= 103&Itemid=244&lang=en

"Flexibility is essential for handling the wide range of samples which arrive in the laboratory daily." Valentina Gualdi, Laboratory and Quality Manager at the Genomics Platform

The NGS laboratory team

GENOMICS TECAN JOURNAL 1/2015

High throughput cell separation – a model for upscaling The Karlsruhe Institute of Technology has automated protocols for high throughput separation of cells for therapeutic applications on a Freedom EVO® 200 liquid handling platform. The process maintains cell viability and is more rapid and reproducible than manual methods, enabling processing of 96 samples in four hours.

Traditional antibody-based methods of cell separation and purification are extremely costly due to the large amounts of clinical-grade antibodies required, and the difficulty in removing the antibody labels from cell membranes for subsequent clinical applications. There is also a risk of initiating undesirable cellular reactions, and some target cells lack a known cell-specific surface marker. Researchers at the Institute of Process Engineering in Life Sciences, Section IV: Department of Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Germany, have developed alternative, automated cell separation methods based on aqueous two-phase systems (ATPS). ATPS is a label-free separation technology that uses two immiscible polymer solutions – for example PEG and dextran – to provide gentle separation of cells according to their physicochemical properties. PhD student Sarah Zimmermann explained: “ATPS offers many advantages compared to antibody-based methods of cell separation; cost-effectiveness, scalability and very mild, gentle conditions for purification of cell products. Initially, we performed ATPS manually, but this is tricky because the polymer phases are extremely viscous. Manual pipetting can give rise to large errors, and data is not very reproducible. Automation was the way forward.” The Institute, led by Professor Jürgen Hubbuch, has been using Tecan liquid handling systems for high throughput purification and formulation development of therapeutic proteins for over 10 years, and has three Freedom EVO workstations – two Freedom EVO 200s and a Freedom EVO 100. The high throughput ATPS protocols were automated on a Freedom EVO 200, using different liquid classes to optimize pipetting of extremely viscous solutions. The required concentrations of the various polymers, salts and other additives that determine the

CELL BIOLOGY TECAN JOURNAL 1/2015

pH of the aqueous two-phase systems is entered into an Excel® spreadsheet, and the volume of each reagent needed to prepare the final ATPS is calculated using MATLAB® (MathWorks), which also converts the data to a format readable by Freedom EVOware®. The workdeck is then set up with the stock solutions and cells, the run started, and the Freedom EVO left to prepare the aqueous two-phase systems and pipette the different solutions into 96-well plates. After shaking, the cells are added and the plates shaken again, before settling for 30 minutes to allow phase separation. Aliquots are removed from both phases of the ATPS for dilution and staining, and analyzed on a flow cytometer. “One of the challenges we faced during process development was the accurate pipetting of small cell suspension volumes,” Sarah explained. “Since the cells sediment, we needed to implement resuspension protocols. However, because of the small diameter of the standard fixed tips, cell viability was an issue. We overcame this problem by optimizing the pipetting speed, the number of mixing cycles, the use of air gaps and the partitioning volumes, and compared the results to manual cell handling. At the end of this study, we were able to reproducibly transfer 5 x 105 cells in a volume of 13 µl with a standard deviation of less than 3 %.” Automation has made the ATPS process faster and more reproducible, enabling systematic screening of different conditions. More samples are prepared in less time – 96 samples can be screened and analyzed in four hours – with much greater precision compared to manual methods. Volumes ranging from 10 to 990 µl are pipetted using the Freedom EVO’s Liquid Handling Arm with fixed tips, with standard deviations ranging from 1.5 % for the smallest volumes to less than 0.5 % for larger volumes, even with very viscous polymer solutions. The validated high throughput screening process has

From left to right: Jürgen Hubbuch, Sarah Gretzinger and Sarah Zimmermann in front of the Freedom EVO 200

been successfully applied to the separation of differentiated and undifferentiated cells from the HL-60 cell line, adjusting the ATPS conditions so that the cells partition differentially between the two phases. “ATPS is a very sensitive way of separating cells according to their surface properties, offering a gentle, cost-effective and scalable approach for the purification of cell products that does not significantly influence the cell viability, differentiation state or differentiation potential of HL-60 cells.

Automation of the process on a Freedom EVO workstation has enabled us to establish high throughput screening, allowing rapid directed design of purification protocols for therapeutic cells, which has proved very successful,” concluded Sarah.

“Manual pipetting can give rise to large errors, and data is not very reproducible. Automation was the way forward.”

To find out more about Tecan’s cell biology solutions, visit www.tecan.com/cellbiology To learn more about the Department of Biomolecular Separation Engineering (MAB) at KIT, visit mab.blt.kit.edu/english

CELL BIOLOGY TECAN JOURNAL 1/2015

Nanoliter liquid transfers aid search for new antibiotics Researchers at Germany's University of Konstanz have established a fluorescence-based method for screening small molecules to identify potential antimicrobial agents. Using the Freedom EVO® workstation’s MultiChannel Arm™ 384, the University’s Screening Center has developed an innovative technique allowing reliable liquid transfers down to 80 nanoliters.

Most antimicrobial agents that target the ribosome as their mode of action act on the fully assembled ribosome, preventing translation at different points of the cycle; some are bacteriostatic, others bacteriolytic. However, a team at the University of Konstanz, Germany, is taking a new approach, looking earlier in the process and trying to identify drug candidates that might inhibit assembly of the ribosome in the first place. Dr Rainer Nikolay, a post-doctoral researcher in the Department of Molecular Microbiology, explained: “I wanted a research project that had clear medical or pharmaceutical relevance, and decided to develop a fluorescence-based screening method for drug candidates that block ribosome formation, preventing protein translation and ultimately stopping bacterial growth. With the rise of antimicrobial resistance, this is a hot topic, and can also give us a better insight into how ribosomes form at the basic molecular level.” The project makes full use of the University’s Screening Center, a service facility within the Faculty of Biology that is equipped with a Freedom EVO workstation and has access to a library of over 38,000 compounds. Silke Müller, a biologist in the screening facility, has extensive experience of performing large-scale library screens using the system, and worked closely with Rainer to establish the automated screening method. Silke

DRUG DISCOVERY TECAN JOURNAL 1/2015

added: “Our Freedom EVO is equipped with a MultiChannel Arm 384 (MCA 384), two Monitored Incubator Options (MIO™) and an Infinite® F500 microplate reader, which allows us to be very flexible in the types of assays we can perform. We work with a wide range of samples – enzymes, mammalian cells, bacteria – using various assay formats including serial dilutions, time-course assays, and virtually any kind of high throughput screening assay that the groups here want to do. The results we get are very reproducible and the system itself runs very reliably.” To analyze the ribosome assembly in vivo, the team used chromosomal gene knock-in techniques to create an E. coli strain harboring large and small ribosomal subunits labeled with the fluorescent proteins EGFP

Dr Rainer Nikolay and Silke Müller

and mCherry respectively. This reporter strain showed growth properties and translation apparatus that were similar to the wild type, allowing alterations in ribosome assembly to be detected using the ratio of EGFP to mCherry fluorescence*. Rainer described the principle of the assay: “Only intact ribosomal subunits exhibit fluorescence activity so, by measuring the fluorescence intensity of both proteins, we could identify potential subunit-specific assembly defects. Based on this principle, we developed a screening assay in 384-well microplates, using 80 µl of cell suspension per well. This small reaction volume is ideal for screening applications – allowing large-scale studies to be performed cost effectively – but can require wasteful predilution of the 10 mM stock solutions from the compound library.”

“With Silke’s help, we figured out all the different parameters,” Rainer continued. “Everything now works smoothly and the system is very reliable. The two MIOs make it possible to run 12 plates daily, taking several fluorescence measurements at reasonable time intervals. We believe this is the first method to directly detect ribosome assembly defects in vivo in a high throughput compatible format and, as a result, we discovered some promising compounds that we would like to characterize further.”

To find out more about Tecan’s drug discovery solutions, visit www.tecan.com/drugdiscovery To find out more about the University of Konstanz Screening Center, visit cms.unikonstanz.de/screening-center/screenings

“This method has proved highly reliable, with CVs of ~10 % for 80 nl transfers.”

*Rainer Nikolay, Renate Schloemer, Sabine Schmidt, Silke Mueller, Anja Heubach and Elke Deuerling. Validation of a fluorescencebased screening concept to identify ribosome assembly defects in Escherichia coli. Nucl Acids Res (2014) 42 (12): e100.

Aspiration/dispense volume (µl)

To achieve a final compound concentration of 10 µM without the need for dilution of the stock solution, the team developed a novel liquid transfer technique based on the MCA 384. Silke explained: “The minimum transfer volume specified for the MCA 384 is 500 nl, but we needed to achieve reliable transfer of just 80 nl of liquid. Using fixed steel tips, I developed a technique similar in principle to a pin tool device, relying on the residual liquid volume on the inside and outside of the tips following several aspiration and dispense cycles to provide very low volume transfers. Although the exact aspiration/dispense volume and number of cycles needs to be optimized for each assay buffer, this method has proved highly reliable, with CVs of ~10 % for 80 nl transfers.”

25 20 15 10 5 0

0 50 100 150 200 Transfer volume (nl)

A calibration curve is performed for each assay buffer, demonstrating the excellent reproducibility of the liquid transfer technique

DRUG DISCOVERY TECAN JOURNAL 1/2015

A helping hand for vitamin testing A Freedom EVO® workstation is proving key to the provision of high quality vitamin testing services at the Medical Laboratories of the Reinier de Graaf Hospital. This versatile solution has enabled full automation of the majority of sample preparation workflows for LC analysis of vitamins, generating major time and cost savings, and meeting the laboratory’s increasing throughput demands.

The Medical Laboratories of the Reinier de Graaf Hospital, based in the picturesque city of Delft, are major suppliers of medical diagnostic services in the Netherlands. As well as providing routine services for the hospital – including clinical chemistry, hematology, blood banking, immunology, pharmacy and microbiology – the laboratory is a renowned Dutch reference laboratory that offers sophisticated diagnostic services to other medical laboratories, universities, the food industry and organizations such as the Netherlands Forensic Institute. The Department of Chromatography plays a key role in delivering these advanced testing services, as Clinical Chemist Dr Frans van der Horst explained: “We have an obligation to our patients, colleagues and external clients to provide fast, high quality services at the lowest cost. Liquid and gas chromatography (HPLC/GC) offer rapid, cost-effective testing for a wide range of analytes, but sample

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

clean-up prior to analysis has traditionally been performed manually, making sample preparation labor intensive, time consuming and prone to errors. As an ISO 15189 compliant laboratory, quality control and process security are very important to us, and so we looked for ways to eliminate these tedious manual processes. Automation was the obvious solution, as this would not only help to streamline our workflow, but also to improve traceability and deal with the double digit growth in sample numbers that we are seeing every year.” Yolanda van Leusden, Manager of the Department of Chromatography, took up the story: “One of the main reasons for looking into automation was to cut the costs of testing. We had already optimized our manual sample pretreatment procedures as far as was practical, and so further improvements to our existing workflow were not feasible. The repetitive nature of sample preparation was ideally suited to automation, and this would free up staff to perform other activities, reducing the cost per test. Another

benefit of automation was the potential to scale down many protocols into a 96-well microplate format, offering significant savings on reagent costs.” Frans continued: “We didn’t have any previous experience with automated liquid handling systems, and so looked at the various instruments on the market. Because the pre-analytical procedures for each assay vary significantly, we needed a very flexible platform that would be able to accommodate these individual sample preparation protocols. The Freedom EVO workstation is clearly a very versatile system, and its open architecture also provided the opportunity to integrate several devices already being used with our manual techniques onto the platform. Our Freedom EVO 150 instrument is equipped with an eight-channel Liquid Handling Arm using a combination of fixed and disposable tips, a Pick and Place Arm, a Robotic Manipulator Arm, a Te-Shake™ module and a Hettich centrifuge, as well as a custom heating module and microplate transfer racks. This flexible set-up enables

The Freedom EVO implementation team. Left to right: Louwra Koolmees, Marion Pleunes, Yolanda van Leusden and Johan Hardin

us to perform the wide range of individual sample processing steps we require. The first protocol we chose to automate was our vitamin B1/B6 workflow, as this is one of our most frequently requested assays, and is very complex to perform manually. This gave us the opportunity to assess the Freedom EVO platform’s capabilities using a method we were very familiar with, and would obviously have a bigger impact on our throughput once it was brought into routine use.”

scripts, we put a lot of effort into validating every step – paying particular attention to the reproducibility of pipetting for the various reagents and biological fluids – to ensure they met our high quality standards. The results clearly demonstrated that the automated protocols were better than our traditional manual procedures.”

preparation protocols. Our next goal is to develop liquid-liquid extraction protocols, a technique which, although very cheap and robust, is technically quite complex to automate. If we can accomplish that, then we will have a Freedom EVO platform covering the full sample preparation range which, to me, appears to be a big paradigm shift for medical laboratories.”

Following the successful implementation of the fully automated vitamin B1/B6 protocol, the laboratory has now transferred preparation of the majority of its vitamin assays to the Freedom EVO. Frans concluded: “The instrument is set up to perform protein precipitation, centrifugation, incubation and solid phase extraction – as well as aliquotting and pipetting, of course – giving us access to a wide range of techniques to meet the needs of individual assays. As far as we can see, there are no technical limitations to automating all of our vitamin sample

Louwra Koolmees, a member of the team responsible for developing the automated protocols, commented: “The system’s user-friendly Freedom EVOware® software meant that we were able to transfer our manual procedures onto the platform quite easily, with very little support from Tecan required.” Her colleague Marion Pleunes added: “Once we had set up the basic

To learn more about Tecan’s clinical diagnostics solutions, visit www.tecan.com/ clinicaldiagnostics To find out more on the Reinier de Graaf Hospital, go to www.reinierdegraaf.nl

Preparation Pipetting of samples and reagents Centrifugation Pipetting Incubation Automated

Manual

Pipetting of stabilization reagent

Centrifugation 10

Sample pipetting Vial capping/plate sealing

100

120

140

160

180

Time (minutes) Comparison of time required to perform vitamin B extraction using manual and automated methods. The automated method takes just 120 minutes to extract 96 samples, compared with 170 minutes using manual techniques, representing a 30 % reduction in total extraction time. In addition, automation reduces hands-on time from 120 to just 30 minutes, freeing up the user to carry out other work. Steps marked with indicate walkaway times

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

Pouring oil on troubled waters

Tecan’s Infinite® M1000 PRO multimode reader has helped Aqsens to develop a portable testing platform for on-site quantitative monitoring of polymeric scale inhibitors in oil field-produced waters.

Scientists at the Aqsens R&D site in Turku, Finland, are focused on developing assay chemistries and portable readers for environmental monitoring, clean technology and life sciences applications, with an initial emphasis on the oil industry. Using its expertise in liquid fingerprinting, the Company has established the AQSENS™ Q-technology platform, offering quantitative and qualitative analysis of liquids or dissolved solids. This versatile technology is ideal for industries requiring rapid, accurate on-site analysis, and forms the basis of the recently launched KemSens™ SI – developed in collaboration with Kemira Oyj – a rapid, portable testing platform for monitoring residual scale inhibitors in oil field-produced waters. The oil industry depends on chemical inhibition to minimize scale deposition in

Joonas Siivonen and Satu Tiittanen using the Infinite M1000 PRO

PETROCHEMICAL INDUSTRY TECAN JOURNAL 1/2015

“We knew straight away that the Infinite M1000 PRO would meet our needs.” oil field-produced waters, and must actively perform monitoring of residual scale inhibitors. Analysis is traditionally a lengthy procedure, requiring complex equipment, which is performed off-site. Inevitably, the time taken to transport samples to the laboratory causes a delay in reporting the results. The AQSENS QS system is designed to overcome these issues, and Tecan’s Infinite M1000 PRO reader played a key role in its development, as Joonas Siivonen, Application Scientist at Aqsens, explained: “During the oil extraction process, a large volume of water is produced. Depending on the composition of this water, there can be a build-up of scale in the piping, increasing the pressure and potentially creating a blockage. Polymeric scale inhibitors are used to prevent this happening, and the residual levels in the oil field-produced waters must be monitored to ensure treatment is effective, enabling safe operation of the oil platform. However, oil fields are usually in difficult to reach locations; it can be up to two weeks before samples reach the laboratory, and a further couple of weeks before the results are received. Data quality can also be compromised, as the people taking the samples are not performing the analysis, increasing the likelihood of samples being mixed up. In addition, traditional laboratory

methods struggle to achieve the sensitivity needed; scale inhibitors remain active well below current detection limits.” Joonas continued: “We developed the portable AQSENS QS platform to help overcome these issues, using a patented detection technology combining timeresolved fluorescence (TRF) with chemical modulators. In the early development stages, we relied on a rented plate reader. However, this did not have enough sensitivity for our assay – we could not perform testing at the low concentration ranges necessary for monitoring residual scale inhibitors – and the cost of renting was also relatively high. We decided to purchase our own reader and, initially, looked at filter-based readers, assuming that monochromator-based readers would

not be sufficiently sensitive. However, after speaking to the Tecan distributor in Finland, our opinion changed. The technical knowledge of the Finnish sales team was excellent; in just one phone call, they described the capabilities of the Infinite M1000 PRO to us in perfect detail. We knew straight away that it would meet our needs, helping us to develop both the assay chemistry and a TRF reader for use in the field.” “The Infinite M1000 PRO has proved more sensitive for our applications than most filter-based instruments, and also gives us additional flexibility. The ability to control the gain setting is a really big bonus, allowing us to adjust the instrument’s sensitivity and detect TRF signals at a much lower level than was previously possible. The Infinite M1000 PRO

has also enabled us to determine the optimum wavelength and measurement settings for our chemistries, establish manufacturing specifications for our TRF reader, and validate the assay protocols. The final product, using the AQSENS QS platform, offers oil producers a fast, accurate means of monitoring residual scale inhibitors on site. Sample transfer is eliminated, reducing the likelihood of a mix-up, and the results are available in around 15 minutes, which is a huge difference compared to off-site laboratory testing. Sensitivity is improved too, and residual scale inhibitor concentrations as low as 1 ppm can now be detected,” concluded Joonas. To find out more about Tecan’s Infinite M1000 PRO reader, visit www.tecan.com/infinitem1000 To find out more about Aqsens, visit www.aqsens.com

The AQSENS QS system offers rapid, portable testing within 15 minutes

The system combines TRF with specific chemical modulators

PETROCHEMICAL INDUSTRY TECAN JOURNAL 1/2015

World-first automation for influenza surveillance Researchers at the Victorian Infectious Diseases Reference Laboratory have completely automated an influenza hemagglutination inhibition assay on a Freedom EVO® workstation using the newly developed FluHema™ module from SciRobotics. It is the first laboratory in the world to achieve this, now running and analyzing up to 128 microplates daily with far better flexibility, reproducibility and overall assay performance.

The WHO Collaborating Centre for Reference and Research on Influenza – part of the Victorian Infectious Diseases Reference Laboratory (VIDRL) in Melbourne, Australia – is one of five centers around the world that oversee the surveillance of human influenza viruses in the global population. As part of the World Health Organization’s Global Influenza Surveillance and Response System (GISRS), information from the Center is used by the WHO to make recommendations on the appropriate viruses to be included in the annual seasonal human influenza vaccines for the northern and southern hemispheres. Dr Ian Barr, Deputy Director, explained: “Our laboratory performs molecular and serological assays on influenza viruses isolated from samples gathered around the region. After growing the viruses in cell

culture, we look for any significant changes in the circulating human influenza viral population which might require an update to the biannual recommendations for the influenza vaccine composition.” “Despite sample processing and culture growth being predominantly manual activities, we automated our analytical workflow some years ago to standardize the processes as much as possible, as well as to ensure high quality data. More recently, we have introduced a Tecan Freedom EVO workstation to increase flexibility and improve the reproducibility of some of our assays. The first assay we automated on this platform was our routine neuraminidase inhibitor resistance testing. This fairly straightforward assay involves a series of

“The platform has given us a very flexible, reproducible and precise system.”

Rob Shaw, Iwona Buettner and Ian Barr with the automated workstation

VIROLOGY TECAN JOURNAL 1/2015

pipetting steps followed by fluorimetric analysis – which is fully automated using the system’s integrated microplate reader – but transferring it to the Freedom EVO has certainly improved performance in terms of assay reproducibility and lower CVs. However, our main reason for purchasing the platform was the automation of our hemagglutination inhibition (HI) assay.” Developed over 70 years ago, the HI assay can be used to detect even small changes in the viral serotype using specific antisera, and relies on the influenza virus’ ability to agglutinate red blood cells. Ian continued: “By using a series of very specific, postinfection antisera from ferrets, we are able to selectively block this agglutination process. The ferret sera are serially diluted to determine the point at which the serum no longer blocks agglutination, allowing us to profile the characteristics of each viral sample, comparing them to a reference panel to identify new variants. Until now, identifying the point at which agglutination is no longer inhibited has always been performed visually by a trained technician. Using a Freedom EVO workstation, we have been able to develop a fully automated system that can not only do all of the liquid handling steps, but can also ‘read’ the plates, and this breakthrough was made possible through Tecan’s connection with SciRobotics. By combining our knowledge of this assay with the liquid handling capabilities of the Freedom EVO 200 platform and SciRobotics’

expertise in imaging and analysis, we can now automatically process and analyze the HI assay plates.”

To view a short video of the fully automated HI system in action, go to www.youtube.com/watch?v=fvZ034BDSqQ

The VIDRL worked closely with SciRobotics to develop a new automated imaging system – the FluHema module – which photographs the plates and analyzes images from individual wells and columns. Depending on the red blood cell type, the 96-well plates can be tilted at angles of up to 65 degrees, allowing compact cell ‘buttons’ shaped like inverted teardrops to form, offering more precise endpoints and easier interpretation. The module’s software uses specially developed algorithms to determine the settling patterns, recognizing where changes in shape and size occur across a dilution series. Data is then exported to an Excel® spreadsheet, which shows the final results along with photos of each well.

To learn more about Tecan’s clinical solutions, visit www.tecan.com/clinicaldiagnostics To find out more about the WHO Collaborating Centre for Reference and Research on Influenza, visit www.influenzacentre.org

“Overall, our collaboration with Tecan and SciRobotics has been very successful. Reliable automated interpretation of the HI assay is now possible, enabling a typical run of around 64 plates to be performed in just a few hours and doubling our previous daily throughput. Our technical expert at Tecan Australia, Luke Danielewski, played a significant part in this, and his knowledge and expertise were invaluable. The platform has given us a very flexible, reproducible and precise system, freeing up staff time for other activities,” Ian concluded.

VIROLOGY TECAN JOURNAL 1/2015

Providing diagnostic solutions for Alzheimer’s disease IBL International, the latest addition to the Tecan Group, offers a range of microplate-based immunoassays to help diagnose Alzheimer’s disease, a devastating condition that significantly impacts the lives of both sufferers and their families. Early diagnosis is essential for implementation of appropriate treatment and support mechanisms, enabling patients to live as independently as possible. Alzheimer’s disease (AD) is the most

therapeutics for Alzheimer’s. Biomarker

picture, and the total level of Aβ should

commonly encountered form of dementia,

analysis has the potential to change this

be studied to account for people who are

affecting not only the elderly but, in

situation. We know that amyloid plaques are

naturally low or high Aβ 1-42 producers.

some cases, much younger people. IBL

a characteristic of AD, and that the precursor

This could give rise to a false positive

International has been marketing assays

amyloid-beta proteins that form these

diagnosis and so, to prevent this happening,

for research purposes for more than

deposits are present in the cerebrospinal

the Aβ 1-42 result is normalized to the level

15 years and, since early 2014, has offered

fluid (CSF); measurement of these precursor

of Aβ 1-40. Because Aβ 1-40 is consistently

an in vitro diagnostic (IVD) assay for

proteins therefore provides an indication of

around 60 % of the total Aβ produced,

amyloid-beta (Aβ) 1-42* – a known biomarker

a patient’s disease status.”

this presents a clearer picture, and clinical

of Alzheimer’s – as well as the reference

studies have shown the superiority of using

protein Aβ 1-40. International Product

Oliver continued: “Formed by enzyme-mediated

the Aβ 1-42 to Aβ 1-40 ratio rather than

Manager Oliver Schmidt discussed the

cleavage of the amyloid-beta precursor

measurement of Aβ 1-42 alone.”

pathology of the disease and the Company’s

protein (APP), amyloid-beta peptides

diagnostic solutions: “Alzheimer's disease is

can exist in several isoforms, but the

Generally, subjective clinical measures such

characterized by cognitive impairment, but

one that is pathologically important in

as the ‘clock drawing’ and ‘three-word delay’

it is only during post-mortem examination

Alzheimer’s disease is Aβ 1-42. This is the

exercises are used for the initial assessment

of the brain that the hallmark protein

main constituent of the brain plaques,

of cognitive impairment, supported by the

deposits – amyloid plaques and tau fibrils

and AD patients typically have a lower

analysis of biomarkers – such as amyloid

– and brain shrinkage can be definitively

than usual concentration of Aβ 1-42 as the

and tau – using immunoassays, helping

identified. However, AD will have begun to

protein oligomerizes to form plaques, with

to confirm the diagnosis. “We already

take hold many years earlier, long before

a corresponding decrease in monomer

had good manual amyloid-beta assays for

cognitive deficiencies were visible, and this

concentration. Although Aβ 1-42 is the main

research purposes, and optimized these

is one of the main limitations in developing

isoform, this alone does not give a complete

for IVD use* with CSF,” said Oliver. “These assays offer several advantages. Low variance is essential, and our kits typically have CVs of far less than 10 %, comparing well with other available assays. Another benefit is that they are sensitive enough to allow predilution of the sample. The amyloid assays, for example, include a 1:20 predilution step, eliminating matrix effects and enabling amyloid-beta measurement in the low picomolar range, as well as allowing both Aβ biomarkers to be determined in a single dilution of a small volume of CSF. Assay times are also short, enabling results to be generated in just three and a

Normal brain

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

Alzheimer's-affected brain

half hours. The Aβ assays have now been adapted for fully automated processing

on the Freedom EVOlyzer®, and we are

results, explained: “We process about

also in the process of implementing a tau

150 samples a month for each of the AD

assay on this platform. With automation,

biomarkers. At present, we use the Aβ 1-42

we simply prepare the workdeck, program

ELISA for routine testing – the kit includes

the workstation and start the assay. The

three sets of standards and controls, which

reproducibility is excellent.”

is a perfect match for our schedule of two runs a week – and, now that the evaluation

“We chose IBL assays for their wide linear range and good reproducibility, due to low inter-lot variation.”

IBL assays have already been implemented

is complete, will be implementing the

at MVZ Volkmann in Karlsruhe, Germany.

Aβ 1-40 assay in early 2015. We chose IBL

Chemist Oliver Bauer, who is responsible

assays for their wide linear range and good

The kit design is another benefit; ready to

for technical validation of the laboratory’s

reproducibility, due to low inter-lot variation.

use standards and controls, plus matched protocols and reagents for the different

1,800 1,600

[Aβ 1-42] (pg/ml) by automated processing

biomarker assays, make it easier to process

Fit line 95 % pointwise CB 95 % individual PB

the ELISAs. We really appreciate the effort IBL puts into developing products to meet its customers’ requirements.”

1,400

To find out more about IBL International, visit

1,200

www.ibl-international.com/en

1,000

To find out more about MVZ Volkmann, visit

800

www.laborvolkmann.de/analysenspektrum/ HTML/index.html

600

*CE-IVD marked for Europe, not available for clinical use in USA.

400 200 0 0

200

400

600

800

1,000

1,200

1,400

1,600

[Aβ 1-42] (pg/ml) by manual processing

Comparison of results obtained manually with automated processing on the Freedom EVOlyzer

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

Rapid generation of dose-response curves for high throughput screening The HP D300 Digital Dispenser is enabling researchers at the Columbia Genome Center’s High-Throughput Screening Facility to quickly and simply create dose-response curves for combinations of up to nine compounds, allowing rapid generation of EC50 data for faster experimental progress.

The High-Throughput Screening Facility at the JP Sulzberger Columbia Genome Center, part of the Department of Systems Biology at Columbia University Medical Center in New York City, USA, provides molecular screening services to researchers within the University and in external laboratories. The Center is also the core screening facility for NYSTEM – the New York State Stem Cell Foundation – which supports stem cell researchers across the state, and has particular expertise in developing stem cell screening assays. Dr Charles Karan, Scientific Director of the High-Throughput Screening Facility, explained: “We work with scientists engaged in long-term research projects – mainly

traditional high throughput screening (HTS) and discovery processes – to design, develop and execute their assays, and to understand the complex follow-up procedures and the implications of their experimental results. We also have a next generation sequencing (NGS) facility, and are currently working to bring these tools together.” “Over the years, our collection of automated systems has evolved in response to specific needs. We run most of our ultra-high throughput assays on a large, automated system, purchasing other instruments – such as the HP D300 Digital Dispenser – to address specific needs as they arise. We do a lot of

Left to right: Ronald Realubit, Xhensila Hyka and Charles Karan with the HP D300

DRUG DISCOVERY TECAN JOURNAL 1/2015

drug synergy experiments, simultaneously studying the effect of two compounds on cells, and the traditional HTS method of creating master plates and then running a dilution series is extremely laborious and material intensive. There is also an element of risk in using plastics with small molecules. We needed a rapid, flexible platform for more cost-effective drug synergy experiments, and chose the HP D300 rather than the alternative acoustic technology because it allows us to generate highly reproducible dose-response curves economically.” Charles continued: “The HP D300 fills a particular niche in our department, allowing us to react rapidly, set up and run an experiment. Another big advantage for drug synergy studies is the excellent reproducibility of individual EC50 curves. With this level of confidence in our results, we can run our chosen experiments knowing that the results are consistent from one run to the next. The robust liquid handling capabilities of the HP D300 let us focus on the drug concentrations we are really interested in and, as we don’t need to look at the initial curves and then create a different dilution series, assay series progress faster; we can generally hone in quite rapidly on an observed result for in-depth investigation. The HP D300 is invaluable in this respect.” “In the beginning, we carried out small-scale experiments with a single combination of compounds. Today, using the latest HP D300 software, we run larger, 30-plate assays, looking at replicate combinations of eight or nine drugs across different time points. We prepare stem cell plates on our large automation system, then simply transfer the

plates to the HP D300 to dispense screening compounds into the plate wells, using the instrument’s plate randomization function to ensure robust data that is free from edge effects. For small molecule screening in DMSO – a solvent that can affect subsequent NGS assays – best practice is to normalize DMSO concentrations, and the large D4 cassette is perfect for this, allowing us to use far fewer cassettes per plate.”

“We can generally hone in quite rapidly on an observed result for in-depth investigation. The HP D300 is invaluable in this respect.”

“The system is highly intuitive, and has proved very popular. We are increasingly finding that people from outside the facility are coming in to use the HP D300 for their own projects. It provides a simple method of generating dose-response curves that eliminates serial dilution, and is ideal for our drug combination studies. The instrument does exactly what we want it to do, fulfilling our need for an instrument that staff can just walk up to and use straight away for small molecule studies, with very little set-up time required. I am really happy with the HP D300,” concluded Charles. To find out more about the HP D300 Digital Dispenser, visit www.tecan.com/digitaltitration To find out more about the High-Throughput Screening Facility, visit systemsbiology. columbia.edu/genome-center/highthroughput-screening-automation

Charles Karan setting up an experiment on the HP D300

DRUG DISCOVERY TECAN JOURNAL 1/2015

Automation allows simultaneous antimicrobial susceptibility testing and MALDI-TOF sample preparation for bacterial identification A Freedom EVO®-based platform for automated antimicrobial susceptibility testing and MALDI-TOF sample preparation is proving beneficial to the Medical Laboratory Bremen, offering enhanced reproducibility compared to manual processes and greater accuracy of results.

The Medical Laboratory Bremen in Germany is a private laboratory offering specialist analytical services across all areas of laboratory medicine and medical microbiology to clients throughout the country, as well as the rest of Europe. The laboratory is renowned for employing the most advanced analytical technology available, and has worked extensively with microbiological diagnostic products manufacturer MERLIN Diagnostika to tailor the Freedom EVO-based MICRONAUT ASTroID workstation to meet its particular needs. This automated platform combines sample preparation for microbial identification by MALDI-TOF

(matrix-assisted laser desorption ionization - time of flight) mass spectrometry (MS) with antimicrobial susceptibility testing (AST) by microbroth dilution. Dr JohannWolfgang Wittke, the laboratory’s specialist in microbiology and infectious disease epidemiology, explained: “Typically, we receive around 450 medical microbiology analysis requests a day from the state of Bremen and neighboring Niedersachsen, performing measurements to determine the antimicrobial resistance of E. coli and other enterobacteria, Pseudomonas and other non-fermenters, oxidase-positive fermenters, many different gram-positive rods and cocci, as well as some yeasts. We needed to

“Automation offers several advantages over manual testing, in particular the reduction in handson time required and improved reproducibility.” Mariola Richter and Dr Johann-Wolfgang Wittke perform automated antimicrobial susceptibility testing and MALDI-TOF sample preparation on the Freedom EVO-based MICRONAUT ASTroID workstation

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

improved reproducibility, making the process much less labor intensive and time consuming than manual pipetting of numerous microplates. Hands-on time is reduced by 30 to 40 % and, because there is a significant reduction in variation, reliability and precision are much better; far fewer tests need to be repeated. When the analysis is performed manually, around 10 to 15 % of tests are likely to be repeated. Using automation, this falls to 3 % or less. In addition, we find that while this method may take slightly longer than the commonly used agar diffusion technique, greater accuracy is achieved for beta-lactamases (including extended-spectrum), plasmid-encoded AmpC and carbapenemases, enabling us to provide more reliable results, allowing the patient to receive the most appropriate treatment earlier.”

automate this process; it was impractical to continue using manual procedures for AST and bacterial identification sample preparation. We acquired our current ASTroID system in January 2011, and this was the start of a collaboration with MERLIN to optimize the platform and implement it in our daily routine testing procedures.” While agar dilution is considered the gold standard for AST of fast-growing bacteria, it places considerable demands on laboratory resources and, for a routine testing laboratory, the microdilution technique employed by the ASTroID system offers a cost-effective alternative, delivering accurate, precise results with minimal hands-on time. The ASTroID workstation is based on a Freedom EVO platform equipped with a four-channel Liquid Handling Arm, a PosID™ barcode scanner, a temperature-controlled adapter for MALDI-TOF target plates, plus carriers for up to nine MICRONAUT AST plates and one 96-well dilution plate. After barcode identification of the microplates and sample tubes, the ASTroID takes aliquots of each sample for preparation of the AST inoculum, or spotting onto a MALDI-TOF target plate. The inoculation, incubation, measurement and evaluation of the AST plates are performed on the workstation, according to the MICRONAUT standard operating procedure. For MALDI-TOF MS, the system adds a matrix solution to the dried sample on the target plate, which is then transferred to the mass spectrometer. After MALDI-TOF analysis, the identification result is transferred to the MICRONAUT 6 software via direct online connection and linked to the corresponding AST pattern. The integrated expert system checks the plausibility of the results, determining resistance mechanisms

and providing clinical interpretation. Dr Wittke continued: “We perform antimicrobial resistance testing in 96-well microplates, mainly using the breakpoint method to determine whether an organism is sensitive, intermediate or resistant, and always include a growth control. Depending on how many wells are needed for each organism, we can analyze as many as four micro-organisms per plate. In special circumstances, for example multi-resistant organisms, determination of minimum inhibitory concentration is required; this uses an entire plate for one bacterium.” “Automation offers several advantages over manual testing, in particular the reduction in hands-on time required and

“Our collaboration with MERLIN has been very successful. The Freedom EVO-based ASTroID workstation is fully validated and has been in routine operation in our laboratory for around three years, giving us the capability to simultaneously perform resistance testing and MALDI-TOF sample preparation. The system is now wellestablished, and works very reliably, providing precise, accurate results,” concluded Dr Wittke. To find out more on Tecan’s clinical diagnostics solutions, visit www.tecan.com/clinicaldiagnostics To find out more about the Medical Laboratory Bremen, visit www.mlhb.de To find out more about MERLIN Diagnostika, visit www.merlin-diagnostika.de

CLINICAL DIAGNOSTICS TECAN JOURNAL 1/2015

Seeing the light An Infinite® M200 PRO reader is helping Californian company Sevident to establish rapid, sensitive immunofluorescence and colorimetric assays based on its proprietary NETs technology.

Sevident, based in Hayward, USA, has developed an advanced rapid capture platform (NETs) that allows the isolation and detection of multiple targets of interest in small sample volumes. This proprietary technology uses a unique three-dimensional molecular scaffold to maximize specific binding capacity and minimize non-specific binding. NETs can be applied to single- or multi-target evaluation of virtually any molecule of interest, including cells. The technology can also be applied to pre-existing assays to help enhance assay sensitivity, achieving detection levels as low as femtomoles per ml (fmol/ml). Emily Stein, President and Chief Scientific Officer of Sevident, explained: “Sevident was founded when I began experimenting in my kitchen in 2008! Initially, my aim was to resolve some of the issues I experienced in my work

Emily Stein (front) and the Sevident team

MULTIPLEX ASSAYS TECAN JOURNAL 1/2015

analyzing patient samples. I began with some colorimetric experiments, using a borrowed plate reader. We then progressed to a small laboratory in Palo Alto, where we spent a year honing the technology and developing it to allow simultaneous protein and nucleic acid detection, before moving to our current ‘proper’ facility in Hayward.” “Our technology is multifaceted, focusing on multi-chemistry multiplex capture. NETs can bind and extract a wide variety of molecules, including proteins, nucleic acids, carbohydrates, lipids, cells and small molecules. This allows targets of interest to be captured and isolated, simplifying removal of contaminants and producing extremely high purity samples. We can tailor this technology for a plethora of applications – testing, screening, monitoring, over-the-counter products and point-of-care diagnostics, as well as research and a broad range of environmental, industrial and manufacturing

applications – working with commercial partners to achieve our joint objectives.” Emily continued: “We moved from simple colorimetric tests to fluorescence-based assays, as we found the multiplex capabilities very exciting. Multiple colors can be read simultaneously in the same well or on the same beads, which we thought was pretty intriguing. However, our original plate reader was quite old and cumbersome to use, and so we decided to invest in a modern, up-to-date instrument more suitable for these multiplex assays, with the flexibility to perform colorimetric, luminescence- and fluorescence-based assays as our project requirements changed. After testing all the major brands, we set our sights on the Infinite M200 PRO because of its ease of use, sensitivity and precision. It also allows us to change between different types of assay, accommodating our own fluorescence-based development programs and the colorimetric and luminescence-based programs of some of our partners. At that time, we had just entered a partnership with a government laboratory to develop a rapid immunoassay for surveillance of dengue virus infection. Reproducibility between the two laboratories was essential – we needed to know that if the same samples were run in both laboratories, the results would be similar – and so it made sense for us both to use a Tecan reader. We knew that we would have greater confidence in the comparability of the results if the laboratories were using the same equipment.” “Intuitive software was another key requirement. We needed a reader with software that could be picked up quickly, as we cannot afford long delays in our development cycles. I really like the Infinite M200 PRO’s software, and find i-control™ very nice to use.

Achim von Leoprechting, Head of Partnering Business, Executive Vice President

Leading the debate

Emily Stein loading samples onto the Infinite M200 PRO

Magellan™ is user-friendly too, and we are gradually implementing this as we need it. The flexibility of the reader’s software and the analytical prowess of Magellan was a significant factor in the decision to buy the Infinite M200 PRO.” “At this point in time, the Infinite M200 PRO is one of the most widely used pieces of equipment in our R&D program, enabling us to use our technology to help our partners enhance the sensitivity of their assays. The reader has proved ideal for our purposes, and we are easily achieving sensitivities in the fmol/ml range. The local Tecan staff have been excellent too; the sales team is very knowledgeable, and the technical support has been really good. Everything has worked well, and I am very happy with the reader,” concluded Emily. To find out more on Tecan’s Infinite M200 PRO, visit www.tecan.com/infinite200pro

Tecan’s Partnering Business has a long history as an OEM manufacturer, working closely with its customers to meet the demands of new and emerging technologies, reduce costs and develop solutions to cope with a greater need for data connectivity, financial pressures in the healthcare sector and changing disease patterns. As a company, we are in a privileged position, benefitting from early exposure to new trends and enabling technologies – such as next generation sequencing – as they are introduced into research laboratories and test environments. As these technologies cross over into mainstream diagnostics, the knowledge we have acquired allows us to help our partners develop highly efficient and cost-effective hardware and software solutions, leading to faster market entry. This transition is a big change, but also a great opportunity to use the knowledge that we have accumulated. But this is just one of the challenges facing the diagnostics industry; there is also an intense focus on de-risking procedures, eliminating the likelihood of manual errors as far as possible. Combining our expertise as an OEM manufacturer with the know-how of our partner companies, we can help to establish very powerful, yet easy-to-use, software solutions, ensuring that even complex diagnostics systems provide user-friendly operation plus extreme control and traceability of results. In addition, there is great interest in developing highly functional and robust point-of-care diagnostic devices, avoiding logistic and sample deterioration issues, and this is another field in which Tecan is actively engaged. Wherever the future takes us, the Partnering Business will continue to be at the forefront. To let us know your thoughts, contact us at hello@tecan.com Find out more at www.tecan.com/linkedin

For more information about Sevident, visit www.sevident.com TALK TO TECAN TECAN JOURNAL 1/2015

TJ Meet Tecan at these events Americas SLAS Society for Laboratory Automation & Screening 2015

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Shenzhen, China

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Tecan are pioneers in automated liquid handling and innovative life science solutions. For over 30 years we continue to enable and support our customers to make the world a healthier and safer place.

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Tecan Journal, Customer Magazine of Tecan Trading AG., ISSN 1660-5276 Design: OTM/London www.otmcreate.com Photography: Günter Bolzern/Zürich www.bolzern.tv Editor in Chief: Tecan Trading AG, Cornelia Kegele Project Lead: Tecan Trading AG, Cornelia Kegele/Antonietta Allocca Editor: kdm/UK www.kdm-communications.com Print: DAZ Druckerei Albisrieden AG/Zurich www.daz.ch Address: Tecan Trading AG, Marketing Communications, Seestrasse 103, CH-8708 Männedorf, Switzerland, journal@tecan.com, www.tecan.com To register for the Tecan Journal please go to www.tecan.com/journal © 2015 Tecan Trading AG, Switzerland, all rights reserved.

EVENTS TECAN JOURNAL 1/2015

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. In general, the trademarks and designs referenced herein are trademarks, or registered trademarks, of Tecan Group Ltd., Mannedorf, Switzerland. A complete list may be found at www.tecan.com/trademarks. Product names and company names that are not contained in the list but are noted herein may be the trademarks of their respective owners. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. This journal may contain reference to applications and products which are not available in all markets. Please check with your local sales representative: www.tecan.com/contact