Monday, June 25, 2012

Increasing Throughput with Automated Liquid Handling

Steady advances in automated liquid handling at the µL to nL level are enabling more scientists around the world to reap the benefits of increased throughput, decreased costs, and more efficient use of reagents. Many of the latest techniques were discussed at the recent “European Lab Automation” conference. For example, Hugues Ryckelynck, scientific associate II, oncology disease area, biochemistry unit, NIBR (Novartis Institute of Biomedical Research), described how to quickly implement automatic liquid handlers to dispense accurately and rapidly in the nL and μL ranges. He noted that “automated liquid-handling systems represent a great opportunity to increase experimental throughput and reduce reagents costs through assay miniaturization.” However, he emphasized that setting up an automatic liquid dispenser to operate optimally in the nL to low μL ranges requires careful observations at the bench, or the results are likely to be highly variable and of low quality. Ryckelynck went on to describe simple operations to be done by hand with air and positive-displacement pipettes in order to anticipate problems such as high liquid viscosity, surface bonding, foaming, and surface tension that will be faced during the development of assays on nL and μL automatic liquid dispensers. He further stressed that “when pipetting nanovolumes, an important source of variation is also the carryover from the source and from well to well.” Ryckelynck gave practical examples of procedures developed by his group to optimize liquid handling on air/positive displacement (pipettes) and injector (pressure-based) systems that can be applied when working at submicromolar and subnanomolar concentrations of target proteins or reagents. He explained that the choice of robotic dispenser, i.e., dispensing technique (injectors vs. pipette) and speed of dispensing, and the experimental setup in general, should be made based on the biophysical properties of compounds and solutions. The usual programming for complex solutions would be to pipette in a single pipetting mode (take once, dispense once, and change the tips). A faster and more efficient technique is serial dispensing (take once, dispense many), which allows the user to save on reagents and consumables and reduces experimental variability when optimally set (changing tips is like getting a new tool with its own new variability). By performing sequential dummy runs, Ryckelynck showed high variability at the beginning and at the end of serial dispenses with all the dispensing systems used. This variability can be compound/reagent-related, a carryover effect, a result of forward pipetting, or inherent to the dispensers. Ryckelynck developed simple dispensing procedures in order to avoid these problems. These procedures involve: (i) working in the optimal dispensing series of the instruments and getting rid of carryover by performing prime and post-dispenses, and (ii) saving time by minimizing well-to-well contamination flaws by performing dispenses in logical series. He gave practical examples of how compound dilution protocols that avoid contamination and time loss in the nL range can be carried out quickly, and be easily implemented on a positive-displacement system. He explained his “reverse-half-Log” dilution technique, which provides the double advantage of an internal control on all dilutions and data best suited to Log presentations. In conclusion, Ryckelynck presented a flexible laboratory setup for an optimal use of nL and µL liquid handlers that is used in his unit. Key features of this setup include the use of standalone instruments working on modules of experiments running in parallel: (i) bulk dispense of microvolumes for limited experimental conditions is performed with pressure-based dispensers, (ii) filling from complex sources is performed using an air-displacement liquid handler for microvolumes and nanoliter positive dispenser for nanovolumes, (iii) source and plate (re)organization is done using an air-displacement liquid handler while (iv) compound dilutions and nL transfer to limit solvent impact on the experiments is performed with a nanoliter positive dispenser. Cycloolefin Microplates Rainer Heller, Ph.D., is a leader in Greiner Bio-One’s high-throughput screening (HTS) group, where he is responsible for launching many new products dealing with HTS. Greiner Bio-One boasts newly designed microplates made from cycloolefins. Due to their excellent optical, chemical resistance, and physical properties, cycloolefin microplates have become increasingly popular in research and high-throughput applications, Dr. Heller said. A variety of different cycloolefin microplates are now, or will soon be, available from Greiner Bio-One for different purposes, including cell-based assays, compound storage, liquid handling, and biochemical assays. The new 1,536-well SCREENSTAR Microplate, for example, is a cycloolefin microplate designed for microscopic applications, high-content screening, and high-throughput screening. The SCREENSTAR Microplate was co-developed by Greiner Bio-One and GNF Systems and features a black pigmented frame with a 190 µm ultra-clear film bottom for ideal compatibility with instrument optics. Well bottoms display excellent optical properties for the highest optical transparency, with reduced autofluorescence in the lower UV range, low birefringence, and a refractive index of 1.53, the same as glass. Recessed microplate wells enable complete periphery access for high-magnification objectives. Cell culture treatment and sterility assure exceptional performance, he said, for high-content screening, especially with fluorescence microscopy in the lower UV range. A smooth microplate top, absent of alphanumeric coding, facilitates flush lid mounting for use with ultra-high-throughput screening systems. A similarly constructed 96-well cycloolefin microplate will soon be available from Greiner Bio-One, and the company is currently developing a similar 384-well cycloolefin microplate. Another application of cycloolefin microplates is for compound storage, as cycloolefins exhibit low water absorption, low impurities, high transparency, and resistance to polar solvents, particularly DMSO, which is commonly used to preserve biological samples. In order to make the latest technical and design innovations available for HTS, Greiner Bio-One will soon be introducing a new 1,536-well cycloolefin microplate for compound storage, liquid handling (including acoustic systems and pin tools), and transmission measurements in biochemical assays. This new 1,536-well microplate will follow the most relevant ANSI recommendations and feature a smooth microplate top, also absent of alphanumeric coding to facilitate flush lid mounting for use with the GNF ultra-high-throughput screening system. The wells are more tapered than in classic 1,536-well microplates, reducing the dead volume in different liquid-handling applications. Regulated Bioanalysis Joseph A. Tweed, a bioanalytical scientist working in the pharmacokinetics, dynamics, and metabolism department at Pfizer, described the development of an internal, regulated, automated sample-preparation and extraction platform for use on the Hamilton MICROLAB® STAR liquid-handling workstation. Tweed noted that the sample-preparation and extraction techniques used for regulated preclinical and clinical bioanalysis of serum, plasma, urine, and cerebrospinal fluid are often very repetitive and tedious tasks that can greatly benefit from automation. His group chose the Hamilton STAR because of its air-displacement pipetting technology and its ability to pipet microliter (µL) volumes with reliable precision and accuracy. In addition, the Hamilton STAR offers flexible and customizable deck platforms with robotic manipulation arm(s) and integrated one-dimensional (1-D) and two-dimensional (2-D) bar code scanners, he said. Tweed and his group developed a graphical user interface to couple with the Hamilton STAR liquid-handling method. This approach allows the bioanalytical scientist increased flexibility and customization of study- and assay-specific parameters for any given bioanalytical sample-preparation technique selected. Tweed said the platform incorporates the ability to batch process study samples among five automated extraction techniques: protein precipitation, solid-phase extraction, liquid-liquid extraction, plate-based protein precipitation, and supported liquid extraction. Additional features include the ability to prosecute routine sample batches via an ordered laboratory information management system sequence or randomized 1-D or 2-D barcodes. Tweed said that the software package and the modular method design provide a flexible and versatile approach for routine bioanalytical sample preparation. The advantages provided by this technology are that it offers increased throughput, improved chain-of-custody for study sample analysis, and a streamlined approach for routine bioanalytical sample preparation. Tweed noted that after sample preparation has been accomplished, specimens are analyzed via liquid chromatographic tandem mass spectrometric analysis.

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