SLAS2015 was the fourth annual meeting of the Society for Laboratory Automation and Screening. SLAS reflects a merger of the Society for Laboratory Automation with the Society for Biological Screening. Before the merger, laboratory automation was maturing rapidly, while bio screening was just starting to get exciting, with assays involving live zebrafish and synthetic functioning human tissue. The merger seems to be working. The meeting drew almost 5000 scientists to the Walter E. Washington Convention Center in Washington, D.C. from February 8 to 12, 2015.
Some highlights of the meeting are described below.
Organs on a chip
Until about 10 years ago, animal testing for toxicity and efficacy of candidate therapeutics was standard operating procedure, despite many problems, including that the data from rodents did not correlate well with subsequent human trials. About five years ago, there were reports that it was possible to use human cells in screens instead. Next, the 2D array of cells was extended to 3D clumps, aggregates, spheroids, etc. A year later, there were reports of testing involving beating human cardiomyocytes.
Fast forward to SLAS 2015, where Prof. Don Ingber of the Wyss Institute at Harvard (Cambridge, Mass.) presented a keynote lecture describing construction and evaluation of live human organ synthesized microchips. He described the lung chip in detail. A scaffold membrane is deposited between two flexible plastic lines mounted on a glass surface. The lines are pneumatically actuated to provide rhythmic motion of the membrane, since motion is required for lung function. The bottom of the membrane was inoculated with endothelial cells, which expand to form an epithelial layer as in the blood supply. The top of the membrane is inoculated with human alveolar cells. These grow to form a lung surface that produces alveolar fluid. The lung on a chip can be used to support respiration.
Prof. Ingber went on to show that lung chips can be combined with other chips such as heart to form simple systems that mimic living systems in animals, including humans. This is a work in progress, but it is very exciting since it may lead to the creation of more valid in vitro models for testing efficacy and safety in human systems.
Later in the day, Prof. Joo Hun Kang, also of the Wyss Institute, lectured on "Bioinspired Spleen-on-a-Chip for Sepsis Therapy.” Sepsis affects about 750,000 in the U.S. each year and is a frequent cause of morbidity. The spleen has many functions, including removing pathogens and toxins from the blood. In acute infections, sepsis occurs when the spleen is overwhelmed by the toxic load, leading to toxic shock, organ failure and death.
Kang reported that blood from infected rats is drawn from an extracorporeal shunt and mixed with magnetic nano beads coated with mannose binding lectin (MBL). MBL has a general avidity for a variety of pathogens and toxins. After mixing, blood and particles flow to a magnetic separator where the pathogen-loaded particles are removed and passed to a parallel saline channel and on to waste. The cleansed blood is returned to the host. The spleen chip removed more than 90% of the pathogens in the challenge. Throughput is 1.25 L/hr. Multiple units can be connected in parallel for larger animals. The spleen chip may find use in research, therapeutic and diagnostic applications.
Spheroids
Spheroids have been developed as in vitro models of the initial avascular stage of solid tumors. The 3D structure of spheroids is often a better assay model than conventional monolayers. Dr. Pauline Menager of CYTOO (Beverly, Mass.) described an easy method to attach and grow nine spheroids per well in the bottom of a multiple-well plate. This facilitates screening with high-speed plate readers. She claims that CYTOO’s spheroids improve throughput, reliability and convenience compared to other formats.
Rapid bioassays
For several years there has been a real effort to increase the speed of bioassays, often with multiplexing. However, Dr. Daniel Chelsky of Caprion Proteomics (Montreal, Canada) reported that targeted mass spectrometry using multiple reaction monitoring (MRM) facilitates rapid quantitation of hundreds of proteins from only 5 μL of plasma. First the proteins are proteolytically digested to peptides. In the research phase for lung cancer, peptides from lung nodules are quantitatively compared to those in the surrounding tissue to find possible markers that are up- or down-regulated in malignant tumors. Retrospective assays further differentiate between metastatic and benign lung nodules. From this, a set of 388 peptides is selected for use in a laboratory-developed test (LDT) using n MS assay of 5 μL of digested plasma. A retrospective study of patients with suspected lung nodules showed that the MS assay of plasma provides a negative predictive value of better than 90%, thus saving many from surgery. The assay has been commercialized by Integrated Diagnostics (Seattle, Wash.).
IntelliCyt (Albuquerque, N.M.) demonstrated the iQue Screener HD, which scans cells in 1536 multiple-well plates in less than 1 hour. The software is intuitive and interactive. Only 1 μL is aspirated from each well. The cytometer processes 10,000 cells, beads, microbes, etc., per second with up to six readouts. Supported MultiCyt Kits for apoptosis and cytokines complete the package.
The MultiCyt cell proliferation dye panel utilizes two spectrally distinct, proprietary dyes that are multiplexed to monitor two subpopulations within a well and with other MultiCyt reagents. The dyes are cell-permeable fluorescent reporters. When the dye enters a cell, it binds selectively to intracellular targets. Cell proliferation is detected and quantified based on the twofold dilution of the dye after each round of cellular division. MultiCyt cellular assay reagents include those for monitoring cell health and for multiplexing using a cell encoding system. Cell health screens can monitor a variety of endpoints including proliferation, membrane integrity, apoptosis and genotoxicity.
Data quality
Concerns about data quality and reproducibility permeated the meeting. In the post-lecture discussion, Dr. Francis Collins, Director of the NIH, responded to a question on assay reproducibility. He said that he was not sensitive to the issue of irreproducibility of results until asked by a senator while testifying. Dr. Collins has since elevated data quality and reproducibility as an issue for the NIH.
Dr. Tim Errington, founder of the Center for Open Science (Charlottesville, Va.), addressed reports that about 75% of the time scientists at two corporations could not reproduce the results appearing in the scientific literature. The publications were from academic institutions, where staff turnover is rapid and record retention is often weak. The implication is that the studies, conclusions and claims are suspect.
To remedy the situation, Dr. Errington recommended that the experimental protocols be described in much more detail than is common today. The Center for Open Science is planning to offer a validation service that would try to bridge the gap by trying to independently verify selected studies.
Clearly there is some discrepancy. Perhaps experimental protocols can be reduced to a brief description as opposed to listing all the details. Complex analytical methods in particular, such as those published by ASTM and USP, are difficult to reproduce. Transferring a method from one laboratory to another can uncover previously unappreciated details. Differences in water purity, temperature, freezing and thawing protocols, light, etc., can have major effects. Even within the same enterprise, keeping analytical methods under control at multiple production sites can require interlaboratory visits by analysts and special protocols for training and maintaining proficiency.
Liquid handling
With the Artel (Westbrook, Maine) VMS (volume management system), which will be distributed in North America, Australia, Germany and Switzerland, analysts can verify that all dispenses and aspirations are performed as planned. This verification should improve the quality of data and aid in detecting the cause of an out-of-specification result.
Artel sponsored a vendor workshop in which Dr. Catherine Quintero of Merck (Boston, Mass.) described how her division has retained Artel to perform scheduled maintenance and calibration of about 10 automated liquid handlers. This is cost-effective for Merck, since it avoids adding full-time staff employees to perform noncore functions. When problems with out-of-specification results were encountered, the Artel team was effective in diagnosis and remediation of the problem instrument. Once diagnosed, the Artel team found imminent failures in several liquid handlers of the same model. Dr. Quintero reported that the verification service has quickly paid off, since data quality has improved and assay failures cost over $10,000 per event.
STRATEC Biomedical AG (Birkenfeld, Germany) introduced the Tholos technology for pneumatic measuring of remaining well volume in 96- and 384-well plates. The well is sealed and a precise aliquot of gas is released into the well. The gas expands and the pressure increases. Boyle’s Law (P1V1) = (P2V2) can quickly provide the well volume (V2) with an accuracy of 1%. The measurement is not sensitive to shape, color or contents of the well. This is a convenient check on the contents of each well.
Sample preparation
Glygen Inc. (Columbia, Md.) has a reputation for introducing novel products for sample preparation. Additions to Glygen’s Lab-in-a-Tip include NuTip, with embedded chromatographic material, TopTip and PrickTip. The latter is a stainless steel needle to prick septum vials or plate closing mat. The needle is attached to the polypropylene pipet tip without glue so that inertness is reserved.
Additions to Glygen’s Lab-in-a-Plate line include crash plates for protein precipitation, Slit Plate for fritless solid-phase extraction and Embedded Plate for conventional SPE. The latter two use Glygen’s 1-μm slit design, which retains the solid sorbent without frits, facilitating handling liquid volumes as small as 1 μL.
Fast protein liquid chromatography
Fast protein liquid chromatography (FPLC) was a contentious topic in the 1980s when LKB and Pharmacia (now with GE, Piscataway, N.J.) competed for liquid chromatographs optimized for proteins. Practichem (Research Triangle Park, N.C.) introduced the Arista Slice, a compact, modular liquid chromatograph with biocompatible liquid flow path and integrated fraction collector. The pump is rated at 25 mPa, or 3525 psi. Detection is with dual-wavelength UV absorbance and conductivity.
Summary and credits
The staff of SLAS deserves special recognition for organizing an outstanding scientific program and supporting exhibition. SLAS2016 is scheduled for January 23‒27, 2016 in San Diego, Calif. Please visit www.slas.org for updates.
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected].