Thermal disequilibrium is prevalent in today's laboratories, as many assays and tests require the addition of liquids that are warmer or colder than the laboratory environment and the actual pipettes
Data taken at Yellowstone National Park during mission 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.
Yellowstone was selected for mission 2 of the expedition because it is 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.
Artel released these results at the 2007 AACC annual meeting and clinical lab expo, San Diego, 17-19 July 2007.
"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, 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 thermal disequilibrium was most significant when using smaller volume pipettes at their minimum volumes.
For example, a two-microlitre variable-volume pipette set to deliver 0.2 microlitres over-delivered cold liquid (3C) by 37 percent and under-delivered warm liquid (45C) by 24 percent, compared with liquid at room temperature (21C).
When set to deliver its maximum volume (two microlitres), 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-microlitre pipette set to deliver two microlitres over-delivered cold liquid by four percent.
When the pipette was set to deliver its maximum volume of 20 microlitres, 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 thermal disequilibrium phenomenon.
Restriction enzymes used in nucleic acid work, for example, are frequently handled at ice temperature (0C), and higher temperatures are encountered when handling mammalian cell cultures (37C) or polymerase chain reaction (PCR) solutions (60C 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 thermal disequilibrium to help laboratories develop strategies to minimise this source of error".
The naturally unstable conditions at Yellowstone exemplified the challenges laboratories face with sometimes unpredictable or uncharacterised 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.
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.
