Handheld air-displacement pipets are used in virtually every laboratory around the world. The technique and skills of individual pipet operators are critical to the accuracy of results of liquid delivery.
Artel (Westbrook, ME) helps laboratories address liquid delivery accuracy and precision needs with regular publication of “best practices” for pipet operators. The company recently conducted a study to demonstrate how various pipetting parameters affect accuracy. The lab analyst can control these parameters by adjusting pipetting technique and by choosing the right pipet and tip for the job.
While Artel has thousands of customers in a range of industries throughout the world, Shire, a specialty biopharmaceutical company headquartered in Dublin, Ireland, stands out because of its unique approach to improving the accuracy of handheld pipetting. Shire focuses on neuroscience; rare diseases; and gastrointestinal, internal, and regenerative medicine, marketing therapeutics in 50 countries around the world.
Addressing the guesswork in analyzing failed assays
The staff at Shire’s quality control facility in Lexington, MA, is involved in the product release testing of enzyme replacement therapies, and quality is a key focus. At any given time, dozens of assays are running. As in many quality control facilities, when an assay fails or atypical results are obtained, analysts’ pipetting skills are reviewed as a potential root cause or contributing factor during the subsequent investigations. According to Patrick Deitemeyer, Senior Development Specialist at Shire, “When an assay fails it’s important to determine an assignable cause, but when it came to human errors this was difficult. We have lots of documentation on various parts of the assay, but we don’t have a videotape that would allow us to go back and review the manual pipetting process.”
Assay investigations can cause data delivery delays and loss of analyst time for repeat runs. Regularly performed assays at Shire can take anywhere from 30 min to a full week, and it is particularly challenging when one of the longer assays fails to meet system suitability requirements. The company believed that if they could quantify and manage how much human error was contributing to the assays, they could reduce variability and improve their efficiency and assay success rate.
In one particular investigation, pipetting was identified to have had a significant impact on the atypical result. “The aberrant result for this one assay brought the problem to a head,” explained Deitemeyer. “We needed to know if our samples were really out of specification or if it was the execution of the assay itself.” These challenges compelled the company to look for an “out-of-the-box” solution to identify and improve human pipetting errors that contributed to assay failures. “Our overall goal was to reduce the invalid assay rate and the time requirements for failed assay investigations,” said Deitemeyer. Based on these goals, Shire decided to develop a specialized program to individually qualify the pipetting performance of the facility’s approximately 70 analysts.
Transferring assays from bench to automation
In addition to improving the accuracy of individual assays, the company wanted to better facilitate the transfer of assays from the bench to automation. “This is always a challenge because every person doing manual pipetting on the bench is different,” explained Deitemeyer. “We needed to verify that they could perform to the same standards as our robots and vice versa. We can’t afford to deal with liquid transfer variability.”
In other parts of the facility, the Artel MVS® Multichannel Verification System was already being used to verify the performance of Shire’s robotic liquid handlers. The system is based on proprietary ratiometric photometry technology, which uses dual-dye, dual-wavelength absorbance to determine the volume of liquid dispensed to levels as low as 10 nL. The MVS technology offers significant advantages over traditional volume measurement methods and within minutes can determine the accuracy and precision of each channel of an automated liquid handler. Based on these measurements, the liquid handler methods can be immediately optimized to ensure that dispensed volumes closely match the targets of the particular assay. The system’s software includes password protection and the ability to create user log-ins, group users by pipetting ability, and therefore manage work assignments. The software also includes electronic signatures and audit trail to facilitate compliance with 21 CFR Part 11 electronic records requirements.
“Our development area had been very happy with the MVS in qualifying their robots. We knew the system was working well and it was much easier than gravimetric methods. We also knew the MVS was easy to work with, so we went to Artel and suggested that we do our personnel qualification with it,” explained Deitemeyer. “Even though this is not what the MVS is generally used for, Artel helped us to tailor the software to meet our needs. Their applications team was excited about helping us with this innovative application.”
According to Keith Albert, Ph.D., Senior Applications Scientist at Artel, “Our applications team was happy to work with Shire to help them maximize the functionality inherent to the MVS, not only for verifying the performance of their liquid handlers but also to qualify their technicians and standardize liquid dispensing accuracy and precision throughout their facility.”
Qualifying the analysts in pipetting
Shire now successfully uses the MVS for its analyst qualification program, as well as to more regularly and accurately calibrate its robotic liquid handling systems. Its portability makes it easy to take the MVS to where the analyst is working, eliminating atmospheric variability and enabling Shire to test and qualify the analysts in the exact conditions in which they work. This allows the company to control temperature, humidity, and barometric pressure while administering the test. All three of these factors can affect pipetting, particularly when working with small volumes.
Initial testing of the pipetting skills of all QC analysts was performed on staff using handheld single- and multichannel pipets; the process took almost three months. This program also includes training of all new hires and annual requalification of the pipetting performance of all analysts. Figures 1 and 2 show pre- and post-training skills assessment (data obtained from an anonymous laboratory).
Figure 1 – Representative pipetting skills assessment of 53 QC technicians prior to receiving pipetting technique training, dispensing 10-μL aliquots of sample (n = 5).
Figure 2 – Representative post-training data for the same technicians performing the same skills assessment protocol with the same pipet, as described in Figure 1.Tools for pipetting training
Figure 3 – Analyst using PCS Pipette Calibration System.Shire uses Artel’s other flagship product, the PCS® Pipette Calibration System, to train new hires and any analysts who need a pipetting skill refresher (see Figure 3). Measurements with the PCS are also based on ratiometric photometry, and the portable system can be set up anywhere, including sterile environments, eliminating the need to move people and pipets in and out of the area.
Artel studies have shown that few experienced pipet operators can achieve the highest level of accuracy and precision without some training. For example, factors and techniques that can influence pipet captive air volume must be taken into consideration, including: 1) minimizing handling of the pipet, which heats and expands the air; 2) prewetting of the tip to minimize evaporation of the sample; and 3) ensuring temperature equilibration of pipet and sample to control both evaporation and air volume.
The pipetting mode used—forward or reverse—also affects accuracy, depending on the assay circumstances. Reverse mode is better for viscous or volatile samples, while forward mode is best to prevent overdelivery of aqueous solutions. The proper positioning and immersion depth of the tip and consistent speed in depressing and releasing the plunger further ensure precise and accurate results. A brief 1-sec pause while the tip is still immersed in the source liquid allows the sample to settle in the tip to ensure precision. The common practice of wiping the tip after aspiration, which can lead to errors by “wicking” sample from the tip, should be done carefully and only if the sample is forming droplets on the outside of the tip.
Artel offers several pipetting training courses. With practice and some tips for better pipetting, analysts can use the PCS to measure their own improvement progress; thus it is an effective training tool.
Deitemeyer commented, “We were impressed with how well our analysts responded to our in-house training program with the PCS at Shire. No one blamed the test for his or her pipetting issues.” Some of our analysts were really surprised at how they were able to improve the precision and accuracy of their skills using the PCS.”
Time savings in troubleshooting
“The biggest benefit we have derived from the use of the Artel systems is time savings in our total assay process. In the event we get an atypical test result, we can immediately verify that the people and the robots were pipetting correctly, so we then can move the investigation quickly in the right direction. Moving away from gravimetric pipet calibration delivered additional significant time savings.” The company has also significantly improved the overall accuracy and precision of liquid transfers both by analysts and robots, and plans to continue the training and qualification program.
Tanya Knaide is Product Manager, Artel, 25 Bradley Dr., Westbrook, ME 04092-2013, U.S.A.; tel.: 888-406-3463/207-854-0860; fax: 207-854-0867; e-mail: [email protected]; www.artel-usa.com