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Quality Digest


Extreme Pipetting Results

Published: Wednesday, September 12, 2007 - 21:00

(ARTEL: Westbrook, Maine) -- Data taken at Yellowstone National Park during Mission No. 2 of the ARTEL Extreme Pipetting Expedition show that pipettes deliver volumes inaccurate by up to 37 percent when handling fluids at temperatures different from the pipettes. Called thermal disequilibrium (TD), this source of error is prevalent in today’s laboratories, as many assays and tests require the addition of liquids which are warmer or colder than the laboratory environment and the actual pipettes. Yellowstone was selected for Mission No. 2 of the expedition because it’s emblematic of thermal variation and disequilibrium. The extreme pipetting expedition is a multi-phase, year-long scientific study to investigate and illustrate the impact of laboratory conditions on data integrity.

“From our testing at Yellowstone, it is clear that pipettes show a bias in volume delivery when dispensing fluids at different temperatures, and the error is especially significant when handling small liquid quantities,” says George Rodrigues, Ph.D., senior scientific manager at ARTEL and expedition leader. “Laboratories must account for this variation to ensure good accuracy and precision in test results.”

Data taken at Yellowstone show that error caused by TD was most significant when using smaller volume pipettes at their minimum volumes. For example, a two-microliter variable-volume pipette set to deliver 0.2 microliters over-delivered cold liquid (3°C) by 37 percent and under-delivered warm liquid (45°C) by 24 percent, compared with liquid at room temperature (21°C). When set to deliver its maximum volume (two microliters), the pipette over-delivered cold liquid by one percent and under-delivered warm liquid by seven percent.

As expected, error was present, but smaller, when working with larger liquid volumes. The 20-microliter pipette set to deliver two microliters over-delivered cold liquid by four percent. When the pipette was set to deliver its maximum volume of 20 microliters, the over-delivery amounted to 0.6 percent.

“There are not many laboratory managers who would knowingly accept error of more than a few percent, let alone error of 37 percent,” says Doreen Rumery, quality control manager at ARTEL and Extreme Pipetting Expedition member. “Yet many technicians frequently pipette warm and cold liquids in critical testing and assay work without accounting for volume variation.”

The aim of the Extreme Pipetting Expedition is to raise awareness about common laboratory extremes that can affect data quality, including the TD phenomenon. Restriction enzymes used in nucleic acid work, for example, are frequently handled at ice temperature (0°C), and higher temperatures are encountered when handling mammalian cell cultures (37°C) or polymerase chain reaction (PCR) solutions (60°C or higher).

The resulting volume differences can be explained by the air displacement operation of pipettes. The dead air volume in pipettes acts to pull liquid into the pipette tip. When inserting the pipette tip into warm fluid, the air inside the tip is at ambient temperature. During aspiration, the pipette tip heats up, causing the air to expand and push liquid out of the tip. This causes less liquid to be dispensed, leading to variation between the target and delivered volumes. The opposite occurs when pipetting cold liquids.

“Since the magnitude of error is dependent on a number of protocol-specific details, such as pipetting speed and type of sample container, a one-size-fits-all correction factor does not currently exist,” says Rodrigues. “ARTEL will continue to study TD to help laboratories develop strategies to minimize this source of error.”

The naturally unstable conditions at Yellowstone exemplified the challenges laboratories face with sometimes unpredictable or uncharacterized changes in the testing environment. For example, humidity and ambient air temperature greatly fluctuated through stages of the testing, leading to volume variation and, in some cases, inability to isolate fluid temperature as the source of error. Affected data were not reported. To reduce environmental variation, ARTEL repeated the testing in a controlled laboratory and summarized the results in a poster entitled “Errors Associated with Pipetting Warm and Cold Liquids,” which is available for download at www.artel-usa.com/extreme.

Volumes were measured using the ARTEL PCS (Pipette Calibration System), a portable, robust measurement technology based on ratiometric photometry. The system is unaffected by the environment and is highly accurate and precise, even at small volumes.

For more information, visit www.artel-usa.com/extreme/results2.htm


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