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Extreme pipetting

By Dr Matt Wilkinson , 30-Mar-2007

Artel is sending three of their employees to the most extreme environments in the US to demonstrate the impact of environmental conditions on pipetted volumes.

The expeditions are designed to highlight the impact that environmental factors such as temperature, pressure and humidity can have on the accuracy and precision of pipettes.


Inaccurate pipetting can lead to inaccurate data generation, expensive quality control (QC) issues and problems with regulatory bodies if the errors remain undetected until after products have been released.


Many laboratories work in 'extreme' conditions, whether pipetting hot or cold liquids, or while working inside a glovebox or walk-in incubator.


For accurate quantities of liquid to be delivered under these conditions, it is critical to account for these environmental variations, and calibrate equipment accordingly.


"The objective of Artel's Extreme Pipetting Expedition is to increase awareness of how environmental conditions affect pipetted volume so that laboratories can eliminate a potential source of error and strengthen quality assurance," said Kirby Pilcher, president of Artel.


The first expedition saw Artel's Aaron Davis and Doreen Rumery trek to the Mount Washington Weather Observatory, which is situated at an altitude of 6,288 feet. The facility's barometric pressure was measured at 805 mbars, well below the sea-level average of 1,013 mbars.



The pressure at this altitude is so low that water boils at a reduced temperature of 93°C, and a three minute egg takes six minutes to cook, with the result being nowhere near palatable.


The low pressure was also responsible for a repeatable pipetting error - approximately 3 per cent for a 10µl pipette. This increased to 9 percent when using a 1µl pipette.


These variations can be compensated for in the laboratory by adjusting the internal mechanism of the pipette, as long as the accuracy of the pipettes is known in the first place.


Laboratory workers often assume that their liquid handling instruments such as pipettes and automated liquid handlers consistently operate to specification. However, research by Artel shows that up to thirty percent of liquid delivery devices are operating out of specification at any given moment.


Assuming accurate volume dispensing is a risky assumption for operators to make because the consequences of adding the wrong amount of solution can be serious: from wasting a batch of product after a failed QC inspection, to releasing an out of specification product which can damage not only a company's reputation and revenues, but also land them in trouble with legislative bodies such as the US Food and Drug Administration (FDA).


Artel has designed two instruments to help avoid accuracy problems, the PCS (Pipette Calibration System) and the MVS (Multichannel Verification System), to calibrate the liquid handling devices and provide traceable documented results in less than 10 minutes.


The MVS allows the calibration of liquid handlers with up to 384 tips, over a range from 0.03µl to 200µl.


Artel has recently enhanced the MVS to provide laboratories with the first standardised technology to verify the accuracy of dilution ratios in serial dilution protocols.


This type of dilution experiment frequently occurs in drug discovery response screening to determine the optimal concentration of active molecule. If the dilution ratio is inaccurate the compound will be too strongly or weakly concentrated resulting in either a dangerous or ineffective pharmaceutical product.


The PCS and MVS instruments use a ratiometric photometry approach to measure the amount of liquid dispensed, rather than the more laborious gravimetric weighing techniques.


According to Dr George Rodrigues, Artel's senior scientific manager, ratiometric photometry works by adding a known concentration of a red azo-dye from the pipette into a known volume and concentration of blue dye.


The two dyes absorb light at distinct wavelengths with no overlap and show a linear photometric response across a wide concentration range, allowing the accurate calibration of a 1µl pipette to a resolution of one-tenth of a nanoliter. .


The accuracy of the results gained using the dyes is traceable back to the US National Institute of Standards and Technology (NIST), providing measurement assurance and equivalent results anywhere in the world.


An effective approach to good manufacturing practice (GMP) and good laboratory practice (GLP) often involves ensuring that equipment is calibrated using an established procedure, with every step being documented.


While uncalibrated pipettes are known to add error, Artel has also shown that user error tends to introduce more inaccuracies than the equipment. Although automated systems can underperform, errors tend to derive from a lack of calibration and bad programming.


Rodrigues believes that laboratory practices are coming under increased scrutiny as the need for data integrity grows. The FDA's Process Analytical Technology (PAT) initiative for the pharmaceutical industry aims to improve process efficiency and product quality by providing for nearly continuous analytical scrutiny.


At the same time, the FDA's Quality System Inspection Technique (QSIT) has led to much closer scrutiny and focus on the importance of quality system processes such as Corrective And Preventive Action (CAPA).


"Using fully traceable QC procedures to ensure data accuracy throughout the entire drug discovery and development process is certainly the safest way to go," said Rodrigues.


FDA quality system requirements have always called for personnel to be adequately qualified with respect to education, training and experience, but a new emphasis on demonstrated competency is emerging.


"The technology that Artel has invented allows fast and accurate testing of a pipette operator's human technique," said Pilcher.


The PCS gives a laboratory pipette user the chance to quantify their accuracy (or lack thereof) and improve technique and build confidence.


"People are amazed at how much they can improve the accuracy of their technique in just half an hour," said Pilcher.


The question of competency is highlighted further in ISO 17025 (General requirements for the competence of testing and calibration laboratories) which dictates that management must ensure the competence of all who operate specific equipment.


Yet another standard, ISO 15189 (Medical laboratories - particular requirements for quality and competence) calls for laboratory management to maintain records of personnel competency evaluations - putting the pressure of compliance firmly with the laboratory manager.


To ensure user competency Artel provides a Pipetting Proficiency Certification Program with two training levels offered. The company has also initiated a Liquid Handler Performance Verification service, which provides on-site third party assessment of accuracy and precision for automated liquid handlers.


The company is currently looking for suggestions on where in the US to send their team for the next 'extreme pipetting' mission . "Of the suggestions received so far, Badwater in Death Valley National Park is certainly one of the most extreme," said Rodrigues.


Not only is Badwater 86m below sea level, but the temperatures in mid-July can reach as high as 52oC.


"While a lab will never be as hot and dry as Death Valley, a lab will always be approaching an extreme from the norm," said Pilcher.

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