Focus on Liquid Handling and 3-D Imaging at SLAS 2013

Most commonly, laboratory automation involves a few hundred wells run for a specific purpose. SLAS 2013, the meeting of the Society for Laboratory Automation and Screening, was held at the Gaylord Hotel in Orlando, FL, from January 12–16. The 3800 specialists who attended convened to talk about systems built around automated liquid handlers and their enabled applications. I asked one vendor if he had counted the different liquid handling systems on the floor. He had not, but offered the estimate of “over 70.” This is a well-served market indeed. Imaging, particularly label-free imaging, was a hot topic, as was 3-D imaging of cells, all with many new products.

Liquid handling equipment

Liquid handlers are used in most screening experiments. AndrewTM from Andrew Alliance (Geneva, Switzerland) was the most unusual. It is a dual-arm robotic tower that provides hands-free pipetting using adjustable handheld pipets from several major vendors. The workspace is made from interlocking dominos. Thus, programming requires locating only one well and the positions of the remaining plates, or the computer calculates the coordinates for each tip with a program called Andrew AssistantTM. The main advantage appears to be the ability to use an existing inventory of manual pipets. Andrew was recognized as one of the three most significant new products at the meeting.

The Biomek® 4000 from Beckman Coulter (Brea, CA) was previewed last year. The 4000 features an eight-position deck that can be expanded to 12 positions, plus two off deck for tool racks and waste. Plates range from 24 to 96 wells. Pipetting tools are compatible with assay volumes in the range of 200–1000 μL. Unique software control is intuitive and flexible. Software modules support routine techniques such as formatting and serial dilutions, PCR setup, and cell staining. The 4000 is manufactured in the U.S.A.

Agilent (Santa Clara, CA) introduced the Encore Multispan System for liquid handling. This is a large (56 × 32 × 29 in.) workstation with room for 24 pipetting and 32 gripping stations. Plus, the robotic arm has a reach 21 in. outside the box to pick up plates and tools, etc. The robot is fast, with an average time for pick and place of 4 sec. Two integrated pipetting stations aspirate and dispense over the range of 300 nL to 1 mL with a CV of ±2.5% for volumes 1 μL and larger. It is a nice piece of engineering.

Moving down the scale in volume size to the mid-nanoliter range, IMTEK (Institute for Microsystems Technology) (University of Freiberg, Germany) introduced a contactless liquid handler for dispensing liquid volumes in the range of 250 nL to 10 μL. A piezoelectric piston squeezes a polymer nozzle to displace small drops. A microcapacitance sensor monitors the tip performance. At 1 μL volume, accuracy is better than 3% and precision better than 5%. The liquid handler is designed for plates with low sample volume as well as microdevices, including chips. A poster also reported use of the device for calibration of other nanoliter-scale dispensers.

Volume calibration on the picoliter scale

Noncontact dispensing at the picoliter/nanoliter/microliter scale was the topic of a poster from Dr. Dong Liang, et al. (HSG-IMIT, Freiburg, Germany). Poster MP018 described a calibration system for liquid dispensing in the size range of 200 pL to 100 nL. Drops are formed and immediately imaged with fully automated software.

The easyPunch STARlet liquid handler from Hamilton Robotics (Reno, NV) automates sample processing of punch cards. The cards are identified, punched, and passed to the sample well for extraction. Next it is on to the analyzer, be it an LC, MS, or hybrid. Hamilton developed the easyPunch in collaboration with GE Healthcare Life Sciences (Piscataway, NJ).

3-D imaging

The meeting title includes the key word, “screening,” but up until two years ago—when the Society for Laboratory Automation and Screening was formed by the merger of the Society for Laboratory Automation and the Society for Biological Screening (SBS)—“biological” was the focus. It still is; “bio” is just not in the meeting title. After liquid handlers, imagers and enabled bioapplications were the focus, particularly 3-D label-free imaging.

One of the two tracks at HCA 2013 focused on 3-D cells. At SLAS 2013, a poster from Markus Ryman and colleagues at Zurich University of Applied Sciences (Switzerland) described hydrogels, which provide the 3-D environment that some cells seem to prefer. They used a Tecan Freedom EVO® liquid handling robot (Tecan, Männedorf, Switzerland) to prepare three different hydrogels for evaluation. HCT-116 cells were cultured for 2, 5, and 8 days. Dose-response analysis of treatment of the cells with taxol provided results similar to the reference (manual) protocol.

At SLAS, numerous products were offered for 3-D cell culture with scaffolds, etc. More information on 3-D cell culture systems can be found at http://www.biocompare.com/Editorial-Articles/117891- 3D-Cell-Culture/.

Flexible fluorescence plate reader

Berthold Technologies (Wildbad, Germany) introduced a premium-performance plate reader for fluorescence and luminescence called Mithras2 LB 943. It features dual monochromators, one for excitation and the other for emission. Light sources include a halogen or xenon flash lamp, or 680-nm laser. Two low-noise photomultipliers operate in the single photon mode. The dynamic range is more than 106. A photodiode covers the range of 200–1000 nm. Stray light is very low, which enables luminescence detection of as little as 3 zmol of firefly luciferase per well. The 943 is compatible with all plate formats.

Internal reflection ellipsometry

Maven Biotechnologies (Pasadena, CA) introduced a novel reader for measuring the interaction of unlabeled cells with extracellular matrixes. The Polaron ReaderTM is built on Label Free Internal Reflection Ellipsometry (LFIRETM). Using an optical diagram similar to surface plasmon resonance (SPR), a sample cell is illuminated from the bottom with polarized light that is phase modulated. Adhesion of the sample to the surface produces Angstrom-scale changes in density, which show up as differential phase delays of the two polarizations. These are converted to an image with a megapixel charge coupled device (CCD). Detection sensitivity is better than 1 pg/mm2, or sub-ng/mL for typical antibody–antigen interactions. Speed is faster than 2 sec; a 96-well plate is processed in less than 4 min. One application compares the adhesion of cells to a surface 25 min after dosing with a drug candidate. The time course can be followed indefinitely. This is superior to typical “endpoint” assays, which require fixing and staining.

Two-dimensional imaging plus SPR

Sierra Sensors GmbH (Greenville, RI) introduced an imager for two-dimensional imaging with SPR to study molecular interactions. No labeling is required. Samples are isolated by hydrodynamic isolation facilitated with a custom-designed microfluidic plate. Each plate contains eight dual-sensor flow paths for sample and reference. Detection cell volume is 30 nL. Sample volume is 1–300 μL. Minimum sample concentration is 50 pM. Output is association and dissociation rate.

Light scattering

Protein interactions are the basis of communication in biosystems, for good or bad. Nano Discovery, Inc. (Orlando, FL) introduced NanoDLSayTM, a system based on light scattering to study label-free interactions. Recall that light scattering is very sensitive to the largest size particles in the detection volume. A typical assay involves selecting and injecting antibody-labeled gold nanoparticles (AuNP) followed by the protein of interest. The antibody captures the protein, which increases the size of the AuNP. The increase in size from light scattering should be about twice the size of the protein. If this is the case, then the protein is exploring possible interactions with other proteins and antibodies. Appropriate solutions are added and the change in size is measured. No size change, no interaction. However, if the size of the AuNP is much larger than predicted, this indicates the presence of a complex. If it is very large, it is probably a homoaggregate incorporating several gold particles. The nature of the complex can be probed by adding antibodies to suspected members of the complex and observing the change in size. The selectivity of the AuNP can be changed by adding ligands to the surface such as citrate.

Optical trapping and digital holographic microscopy

Current protocols for identification of rapists use amplified DNA profiles. However, in about 25% of the samples, the high concentration of DNA of the victims overwhelms the signal from the rapist. This was addressed by Dr. Dan Mueth of Arryx (A Haemonetics Company). Current protocols involve multiple extractions that suffer from excessive carryover from the victim. Arryx developed a fluorescent video microscope that uses holographic optical trapping (HOT) to select sperm cells. These are captured by the HOT device and moved to a collection well for digestion and DNA amplification. This greatly reduces carryover from the victim and reduces uncertainty in the identification based upon DNA matches.

A related poster described digital holographic microscopy (DHM) in which interferometry is used to image transparent cells without labeling in real-time. To demonstrate the utility, a team at École Polytechnique Fédérale (Lausannne, Switzerland) used DHM to monitor real-time cellular modulation in response to chemical and environmental challenges. The authors recommend DHM for screening and mode of action studies.

Compound libraries

Screens for lead development need a fresh supply of new compounds. Initially, the goal is to expand diversity to find novel leads. Once identified, the challenge is to make a focused library for use in structure–activity relationships and ultimately select the candidate for clinicals. Most compounds to date have been prepared using conventional batch synthesis mixing reagents in flasks, followed by chromatographic workup. Prof. Timothy Jamison of MIT (Cambridge, MA) described multistep synthesis using continuous flow. For many reactions this is faster and improves yields due to rapid mixing and improved temperature control. Ideal candidates are reactions that are exothermic and fast (<1 min), such as those involving azides.

Next, Kevin Woller described the High Throughput Chemistry System developed at AbbVie Inc. (North Chicago, IL, formerly Abbott) to support the need for 20,000 new compounds/year. It features on-line reversed-phase liquid chromatography (RPLC) purification. The most common problem is products with low solubility. However, the major upgrade is improved management of the reaction planning, registration, curation of supporting information including physical properties, and results from use in screens. A similar system was described by Dr. Alex Godfrey (Eli Lilly, Indianapolis, IN).

Automated tube labeling

Computype (St. Paul, MN) introduced the Flex TVF, a novel tube and vial feeder as a front end for the company’s tube labeler. This automates the boring, manual task of positioning vials for the labeler. Throughput is up to 40 pieces per min. All vials are used. The TVF is also quiet. It even processes vials with rubber rings or gaskets. No lip is required. It will detect vials that are missing caps. The TVF should make biobanking faster and more reliable.

Circulating tumor cells

Dr. Mehmet Toner, Founding Director of the NIH BioMEMS (Micro Electro Mechanical Systems) Resource Center (Charlestown, MA) opened the formal meeting with a keynote lecture on circulating tumor cells (CTCs). He noted that CTCs are sloughed off by the primary tumor and then colonize new locations in the host. Patient outcome is inversely related to the metastatic spread of the primary cancer. Metastasis is responsible for most mortalities.

CTCs are very rare; there are typically less than 10 in a 7-mL blood draw. For diagnostics, staining and counting are useful in patient stratification and measuring therapeutic efficacy. For research, harvesting the cells for further characterization is desired. Toner described development of a novel CTC capture plate. First editions were an array of posts micromachined out of silicon. The posts were covalently covered with antibodies to tumor cells. These were useful but too expensive. Second-generation plates were molded plastic with microvortexes that selectively trap the CTCs. These are stained and counted. The second half of the keynote described chips for studying the transition of epithelial cells to mesenchymal stem cells, which metastasize with fatal results if untreated.

Micro cell culture dishes

What laboratory supply is older than the Petri dish? Changhuei Yang (California Institute of Technology, Pasadena) described an improved design for automated imaging of small-format Petri dishes for automated processing after incubation. The new dishes are designed to present the surface so that it is always in focus. The parts are carefully selected to be high volume and low cost. For example, the imager is designed for cell phones. It can image a 5 × 5 mm area with high resolution since the optics use computer-aided resolution improvement.

ICP-MS for proteomics

Quantitative proteomics often involves stable isotope tags. However, the obvious candidates, including isotopes of hydrogen, carbon, and nitrogen, are really quite limited. Prof. Gary Nolan (Stanford University, CA) presented a major extension of stable isotope labeling technology by covalently labeling proteins with strings of chelating groups, loading them with lanthanides, and feeding the mixture into an inductively coupled plasma-mass spectrometer (ICP-MS). Specificity is provided by the particular ion in the label and the number of chelating sites in the chelator. Even with a single cell, the response is 104 times better than electrospray ionization (ESI) detection. Throughput is very high, with a plate in less 6 min. Prof. Nolan calls the process Single-Cell Mass Cytometry. Results so far indicate that in acute myeloid leukemia (AML), the cells have more order than previously expected. This is attributed to adulterated differentiation cascade or a pseudo-hierarchy in differentiation. In addition to novel instrumentation, Nolan showed a unique false color clustering extension of informatics that helps group cells along relevant pathway branches. The color and organization inherent in this display provide superior display to red/green heat maps.

Bromodomains

Several lectures and posters touched on bromodomains since they are implicated in epigenetics. Bromodomains are a group of proteins exhibiting four alpha helices that recognize acetylated lysine residues, which are often found on N-terminal tails of histones. Bromodomains are a potential druggable target; hence there is a need for screening. Dr. Ellen Berg of DiscoveRx’s BioSeek Division (Fremont, CA) described a platform that facilitates screening of more than 30% of the human bromodomains. This was part of a satellite meeting held at the Sanford Burnham Medical Research Institute (La Jolla, CA).

Capillary electrophoresis (CE)

DNA separations are a common application of HPCE. In contrast, CE of proteins is less popular since the run times are longer. QIAGEN (Valencia, CA) reasoned that productivity could be improved if 12 runs were made in parallel, hence the QIAxcel® 12-channel system for proteins in the range of 10–200 kDa. Throughput is 96 samples in 2 hr, with no operator attention required. Lifetime of the 12-channel gel cartridge is expected to be 100 runs, which is 1200 samples. The fluorescence detection reagent labels lysine residues. The detection limit is 2.5 ng/μL with light emitting diode (LED)-induced fluorescence. Initial applications include cell lysates, antibody fragments, and recombinant proteins.

Genotyper

GenCell Biosystems, Ltd. (Limerick, Ireland) introduced the automated GT-Series Genotyper, which utilizes micromachined technology for small sample volumes. This instrument was selected as one of the three most significant new products at SLAS 2013.

Genetic sequencing as a service

Beckman-Coulter has a reputation as a vendor of tests and instruments for clinical diagnostics, including genetic sequencing with capillary electrophoresis. Recently, the company announced Clinical Laboratory Improvement Amendments (CLIA) licensure for clinical sequencing and formation of Beckman Coulter Genomics. The business also obtained a license from the State of Massachusetts. Beckman plans to begin accepting samples for detection of BRAF exon 11 (codons 439–477) and exon 15 (codons 581–620) for mutations. This is the first clinical molecular diagnostic assay developed by the company. In the future, Beckman plans to use next-generation sequencing for a range of assays in oncology and infectious diseases.

Industry outlook

The Laboratory Products Association (Fairfax, VA) hosted a breakfast that featured a report by K.C. Warawa on the results of a survey of 1000 laboratory managers in the U.S.A. regarding staffing and spending plans for 2013. This has been an ongoing series since 2002. These managers forecast that 2013 will be a repeat of 2012, which had growth in the range of 2–5%—not great, but much better than a decline.

Future venues for SLAS

SLAS 2014 will return to San Diego, CA, January 18–22. The 2015 meeting is scheduled to be held in Washington, DC. This sets up an intended long-term rotation between coasts with even years in San Diego and odd years in The District. Please see www.slas.org for details.

Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: rlsteven@yahoo.com.

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