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Feature Articles : Jun 15, 2011 (Vol. 31, No. 12)

Dispensing with Manual Pipetting

Automated Systems Trump the More Labor-Intensive Methods in Almost Every Situation
  • Vicki Glaser

Laboratories performing a diverse range of applications, including sample preparation for next-gen sequencing, high throughput, low-volume compound screening or PCR reactions, enzyme-based assays, or maintenance and storage of large compound or cell collections, for example, are looking to automate processes previously done manually and, in particular, liquid transfer and dispensing.

When faced with the many automated liquid-handling systems and robotic platforms available today, labs need to consider the range of protocols they might like to automate to meet not only their current but also their future needs. They can then compare the breadth of automation capabilities and the potential flexibility of the different commercial instruments to find the best match and, ideally, avoid having to purchase multiple instruments over time, each dedicated to a particular application and not amenable to broader-scale integration into the lab’s overall workflow.

A variety of automated liquid-handling solutions are available. Their range of features and capabilities vary as do the types of applications for which they are most suitable and beneficial.

The service group at Tecan uses Artel’s MVS® Multichannel Verification System to confirm that Freedom EVO liquid-handling systems are performing to manufacturer’s specification for installations or following maintenance on an existing system.

MVS uses standardized reagents that are dispensed by the liquid handler being evaluated, and the volume of liquid in each well is measured using a dual-dye, dual-wavelength ratiometric method. When dispensing into a 96-well microplate the MVS specification for inaccuracy is less than 3% and less than 1.5% for imprecision, and less than 10% and less than 3%, respectively, for a 384-well plate.

Using the components of the MVS ensures that true volume performance is being measured, explains Debbie Bowers, director of sales for liquid-handling robotics at Tecan. “The Artel technology allows laboratories to compare standardized results across labs, whether in the same building, state, or country.”

The partners report better results for a PCR assay performed using a manual protocol that called for the transfer of a 15 µL volume of liquid than for the same assay performed with an automated liquid-handling instrument. Whereas, the initial assumption was that the problem was with the robot, application of the Artel method showed that the manual pipettor was actually dispensing 15.67 µL of liquid. When the robot was programmed to dispense 15.67 µL, the results of the PCR assay performed using the two methods were similar.

The overriding message, emphasizes Bowers, is that selection, utilization, and validation of an automated liquid-handling system should be matched to the user’s specific needs and applications. When comparing liquid handlers, the overall specifications of an instrument should be secondary to the specifications that are relevant to a particular application.

“Tailor the features of a pipetting channel to the application needs,” Bowen says, taking into consideration, for example, liquid volumes, properties of the liquids to be dispensed, types and sizes of tips, plates, and tubing to be used, and the specific protocols to be automated.

With this in mind, she feels that flexibility and scalability are key advantages of the Freedom EVO systems, with their selection of 2- to 16-channel liquid-handling arms or the MultiChannel Arm™ 96 or 384 dispensing heads and the ability to transfer liquid volumes ranging from 100 nL to 5 mL, which can be extended to upwards of 50 mL with the DynamicFill™ Technology. Additional features include liquid-level sensing during pipetting and the Pressure Monitored Pipetting (PMP™) option, which detects pipetting errors by comparing recorded and real-time-simulated pipetting pressure signals.

According to TTP LabTech, its mosquito® HTS nanoliter liquid handler is useful for performing serial dilutions and plate replication and reformatting. For low-volume serial dilutions and sample transfer to assay-ready plates, the capability to pipette volumes from 1.2 µL down to 25 nL makes it possible to go directly from a 96-well to a 1,536-well plate format.

A recent case study presented customer data from an 11-point serial dilution that compared manual pipetting to automated pipetting of samples in DMSO. The two methods yielded virtually identical levels of a fluorescent label at each dilution point, demonstrating at least equivalent automated pipetting accuracy.

Joby Jenkins, product manager for the mosquito, attributes the liquid handler’s accuracy to two main features of the technology: positive displacement-based liquid transfer and precise Z positioning. With positive displacement, the pistons come in direct contact with the liquid in the pipette tip, pushing the liquid out of the tip. This allows for high-accuracy, low-volume pipetting of liquids with a broad range of “surface tensions and viscosities with no set-up changes” or recalibration, including dispensing of plasma or cell preparations at nanoliter scale.

Low-Volume Pipetting

TTP designed the mosquito with a dispensing head that moves only in a vertical plane. The head is lowered to perform contact pipetting into plates that move across the deck of the instrument. This design improves positioning accuracy and avoids errors that might be introduced when trying to align a dispensing head with a plate and using air-displacement dispensing to propel the liquid into the wells of the plate.

He emphasizes the importance of minimal dead volume for low-volume liquid transfer applications. The <500 nL dead volume of the mosquito allows users to access virtually all of the material in the source plate. “You can use even a 1 µL mother plate volume and then stamp out to low-volume assay-ready plates.”

In the comparative case study, the source plate was a 384-well PicoTube storage system, composed of microtubes in a 384-well plate footprint. Each microtube has an individual foil seal that must be pierced before the contents can be accessed. The 0.8 mm (external diameter), needle-like dispensing tips on the mosquito pierce the foil seals as the liquid is transferred, without the need for a separate piercing step.

TTP is developing a larger-volume pipette tip for the mosquito, with a dispensing range of 500 nL to 5 µL, and plans to launch the mosquito HV (high volume) later this year.

Manual pipetting is a practice so commonplace in laboratories that handing the task over to an automated instrument may require a real leap of faith. Thermo Fisher Scientific hopes to make that transition a bit easier with its new automated pipetting system, the Versette™. The modular device is designed to be “versatile and scalable from an instrument, pricing, software, and consumables perspective,” says Kiara Biagioni, product manager, automated liquid-handling equipment.

It targets a variety of automated liquid-handling applications including serial dilutions, cherry-picking, plate replication, assay development, compound library screening, and genomics.

Users can start with a single-channel pipetting head and a two-position stage configuration and add on to the instrument as their needs evolve, selecting from 19 interchangeable pipetting heads with 1 to 384 channels, air-displacement disposable tip or a fixed-tip format dispensing technology, a dispensing range of 0.1–1,250 µL, and a two- or six-position deck. The 96- and 384-channel pipetting heads feature Thermo’s D.A.R.T.s tips, with a surface sealing design that provides consistent tip height and alignment, “which is critical for low-volume dispensing procedures.”

Versette Clip Tips attach the tips to the single-, 8-, and 12-channel pipetting heads, with a sealing interface that prevents tips from falling off and reduces wear and tear on the instrument. All of Versette’s pipetting heads include RFID tags that identify and track usage. The ControlMate software interface includes a preset library of liquid classes, such as percent DMSO, which allows the instrument to optimize accuracy for different liquid properties.

Maximizing Productivity

Reed Kelso, co-founder and application scientist at Bionex Solutions, and his colleagues identified “a gaping hole in lab automation” in that available devices do not do any work while consumables are being loaded or unloaded and product is recovered. Bionex scientists call this “idle time.”

In contrast, automated systems operating in modern industrial settings typically do not stop working except for maintenance. Every minute a tool is not doing work, the company is losing productivity. Kelso’s group set out to apply the concepts underlying Lean Six Sigma material handling to automated liquid-handling processes in life sciences labs, which led to the development of the Hive™ automation platform.

The Hive is built around a Lazy Susan design, providing simultaneous-access plate storage that enables continuous operation. While one side of the carousel is accessible to the robot arm, the other is accessible to a technician for loading and unloading. The only downtime, explains Kelso, is the 2–3 seconds it takes for the carousel to rotate from one side to the other. Transfer of source and destination plates takes place outside of the pipetting space, and pipetting is not interrupted as plates are moved around on the deck.

Kelso reports that a customer using the Hive has documented its ability to do the work previously done by six machines, and to complete it three times faster, resulting in an 18-fold increase in throughput. This application involves transferring randomly distributed hits from 96-well collection plates to a 384-well plate format for additional screening.

Early adopters of the technology are expected to be looking for a system that can perform as many as 50,000 picks in an 18-hour day—laboratories that are doing very high-throughput applications such as cell culture screening, monoclonal antibody development, screening of synthetic molecules for drug or biofuels discovery, massively parallel PCR reactions involving a large selection of primers, or SNP analysis, for example. At this scale, hit recovery/cherry-picking becomes the bottleneck and cannot keep pace with screening technology.

The ability to reduce sample transfer time by as little as 0.1 seconds over a process that requires 40,000 picks will allow laboratories to increase the scale of screening by as much as eightfold, contends Kelso.

Each of the Hive’s pipetting channels moves independently, and the architecture of the system is designed to maximize the number of channels that can access a source plate at one time, with a minimum of two channels being able to access any combination of wells on a plate in parallel. A high-speed barcode reader and tip-exchange system, and a plate rotator that can change the plate orientation from landscape to portrait as needed contribute to the speed and flexibility of plate-processing operations.

Automating Workflows

For large-scale genomics research, Promega  offers a workflow for isolating genomic DNA (gDNA) from large volumes of whole blood using its ReliaPrep™ HT gDNA isolation system integrated on Hamilton’s MicroLab® StarPlus liquid-handling workstation.

To avoid the inherent limitations of centrifugation-based methods for gDNA preparation—mainly the need to balance sample numbers and weights, and additional restrictions related to centrifuge capacity and operation—Promega developed ReliaPrep chemistry, explains Cristopher Cowan, manager, integrated solutions and engineering.

It requires no centrifugation step to remove red blood cells or other plasma components, allowing ReliaPrep to capture all of the nucleic acid in a whole-blood sample. Storage and transport of blood samples prior to processing may expose the samples to freeze/thaw cycles or other conditions that can compromise the sample, lysing white blood cells.

“You may have already lost much of your DNA yield before you even get to the precipitation step,” says Cowan. Rapid processing of 32 3–10 mL samples at a time takes place on the ReliaPrep’s HSM 32 LV device, which performs heating, shaking, and magnetization functions in one location. Automated liquid-level sensing allows the control software to scale the chemistry in each processing tube based on the volume of individual samples.

In a poster presentation at “LabAuto”, Promega illustrated the efficiency of DNA recovery on ReliaPrep compared to a precipitation-based method using 10 mL samples of both fresh blood and blood samples exposed to two freeze/thaw cycles. The results show improved yield and purity based on UV absorbance spectroscopy data measured after elution of the gDNA or resuspension of the DNA pellets.

According to Cowan, the ReliaPrep system uses a minimal amount of plasticware for gDNA isolation. Each blood sample is processed in a single 50 mL conical tube using two pipette tips—one for the elution and one for the removal of waste.

The Hamilton MicroLab StarPlus can integrate with three 32-sample ReliaPrep HSM devices. The Promega/Hamilton method can process 96 samples in less than 8 hours, with an average yield of about 300 µg of DNA per 10 mL of blood.

The concept of a benchtop robotic workstation designed to maximize the number and types of devices with which it can integrate, while maintaining a relatively compact perimeter to preserve workspace, was the central theme of a poster presented at “LabAuto” by Agilent Technologies.

Seeing a gap not only in its own product line—between the small-scale BenchCel Microplate Handler and the Direct Drive Robot (DDR)—but also in the broader market for robotic liquid-handling systems, Agilent envisioned an instrument that would be applicable across multiple workflows, including genomics and sample preparation for next-gen sequencing, cell-based assays, maintenance of cell stores, sample management for high-throughput applications, and ELISA-based assays.

Agilent’s BenchBot, introduced at “LabAuto” and set to begin shipment to customers this month, offers customers a midsize robotic system from which they can build a customized workstation and is sized to operate inside a standard fume hood. It can accommodate up to 10 devices at a time, has a vertical reach of 20–420 mm, a radial reach of 590 mm, a 270° work space, five degrees of freedom (z-axis, shoulder, elbow, wrist, and gripper), and an average pick-and-place transfer time of five seconds.

The goal, says Marc Valer, product manager, integrated systems, is to enable customers to “create larger workstations in a smaller footprint, encompassing a whole workflow with one system.”

The software includes a menu of operations for more than 140 different devices, allows for remote monitoring of robot status, and includes the One Touch Teaching function, with which users can manually place a plate at a desired position, press a button, and the software will record the information needed to instruct the robot to dispense at that position.

The Benchbot’s gripper can grasp microplates in either portrait or landscape orientation, and the robotic arm can access various types of labware housed in a plate hotel or plate stack storage device or a unit that enables random access.

Catering to the NGS Market

Recent improvements in throughput and in the multiplexing capabilities of second- and third-generation sequencing technologies have created a need to automate the up-front sample-preparation workflows that allow researchers to realize the full potential of these next-generation sequencing (NGS) platforms. Caliper Life Sciences just launched the Sciclone NGS Workstation to address these demands.

According to Jeremy Lambert, automation applications and genomics marketing manager at Caliper, the Sciclone NGS Workstation is a fully featured automated liquid handler specifically designed to accommodate the rapidly changing protocol requirements of the NGS market, including modifications for single-molecule sequencing workflows.

The Sciclone NGS Workstation can process a range of NGS methods including TruSeq protocols from Ilumina, RNA-Seq protocols from NuGEN, and multiplexed sequence capture methods from Roche NimbleGen. The Sciclone NGS Workstation also supports workflows for the Life Technologies Solid and Ion Torrent sequencing platforms as well as the Pacific Biosciences RS system.