Extreme Pipetting

You may not be planning to carry out your next assay on a mountain or in the desert, but the ARTEL extreme pipetting expedition have done just that to find out how changes in environmental conditions could have a profound affect on your data.

While pipetting does not normally conjure images of sweltering temperatures, exploding geysers, mountainous heights or rainforests, laboratory technicians do often work in or with extreme conditions. Pipetting hot and cold liquids, working in walk-in incubators and glove boxes, laboratory humidity, and outsourcing pipette calibration to laboratories at different elevations are common in today’s life science community. All of these tasks can expose pipettes to harsh environments and put data at risk.

Due to the mechanical function of air-displacement pipettes, environmental conditions are a major source of volume variation and, consequently, laboratory error. Given the small volumes used in today’s laboratories, a volume inaccuracy of just 1μl may significantly alter results. For data accuracy and precision, laboratories must account for the error caused by laboratory conditions.

Helping laboratory technicians understand and quantify how environmental conditions affect pipetted volumes was the goal of the ARTEL extreme pipetting expedition (www.artel-usa.com/extreme). The year-long scientific study took ARTEL scentists to extreme locations to test pipette performance using the ARTEL PCS (Pipette Calibration System). Portable and unaffected by the environment, this system is used at each mission to measure dispensed volumes. The field results are compared to data collected at ARTEL’s controlled laboratory and the resulting error is calculated. This study can be used by pipette users to more fully understand how their instruments will perform in various laboratory environments so that they can eliminate a potential source of error and strengthen quality assurance. 

This article summarises data acquired during the four missions of the ARTEL extreme pipetting expedition.

Mission 1 – Barometric pressure (Mount Washington)
To study the effect of barometric pressure on pipetted volumes, the ARTEL extreme pipetting expedition visited Mount Washington (6,288ft), the highest peak in the White Mountains of New Hampshire. Although there are not many laboratories operating at over 6,000ft, not all laboratories operate at zero elevation. Understanding how barometric pressure affects data is therefore essential.

At Mount Washington, ARTEL found that variable-volume air-displacement pipettes under-delivered by up to 33% compared to data collected in the controlled laboratory at sea-level. Error was greatest when pipetting smaller volumes and when using pipettes at the lower end of their volume range.

It was found that at the high altitude of Mount Washington, a 20μl pipette under-delivered by approximately 1μl when set to deliver its maximum volume, and under-delivered by 3% when set to deliver its minimum volume. The 2μl pipette under-delivered by 4% when set to its maximum volume but under-delivered by 32% when set to its minimum volume.

Although altitude is a constant condition within a laboratory, consider laboratories above sea level that send pipettes to a calibration service at sea-level. Even newly calibrated, the pipettes are likely to under-deliver once returned to the laboratory and produce inaccurate data. The Mount Washington data show the importance of regularly verifying pipettes in the environment in which they are used.

The volume variation found at Mount Washington is explained by the lower air density found at higher altitudes. Air-displacement pipettes contain air gaps between the piston and the liquid that is being pipetted, and these gaps act as a cushion. When an operator aspirates, presses his/her thumb down on the plunger at the top of the pipette, and inserts the pipette into the liquid, air is trapped in the pipette tip. This is called the captive air volume. When the plunger is released, the trapped air acts as a spring that connects the piston to the liquid and pulls the liquid up into the tip. If air is less dense, less liquid is pulled into the pipette tip and subsequently dispensed, resulting in under-delivery and data inaccuracy.

The magnitude of volume variation caused by altitude for each pipette is relatively constant, which facilitates correction within the laboratory. For example, the 10 μl pipette under-delivered by 2.6% and 2.8% in replicate tests. To compensate for this repeatable volume variation, laboratories have two options. The first option is to adjust the internal mechanism of the pipette for the specific environmental conditions. The second option is to adjust the delivery setting. In this case, because the pipette under-delivered by an average of 2.7%, setting the pipette to deliver 10.27μl would deliver an actual volume of 10μl.

Mission 2 – Temperature disequilibrium (Yellowstone National Park)
After collecting data to quantify the effect of barometric pressure on pipetted volumes, the expedition set out for Yellowstone National Park to study error caused by thermal disequilibrium. Laboratory assays and protocols commonly require handling liquids at temperatures different than the pipettes used to deliver them. For example, restriction enzymes used in nucleic acid work 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 team found that pipettes over-delivered cold liquids and under-delivered warm liquids, and inaccuracies as great as 37% were recorded. Data collected at Yellowstone also showed that error caused by thermal disequilibrium was most significant when using smaller volume pipettes at their minimum volume settings, similar to the findings at Mount Washington. Pipettes handling small liquid volumes were also more significantly affected than pipettes handling larger liquid volumes.

A 2μl variable-volume pipette set at its minimum volume over-delivered cold liquid by 37% and under-delivered warm liquid by 23%, compared with liquid at ambient temperature. When set to deliver its maximum volume, the same pipette over-delivered cold liquid by 1% and under-delivered warm liquid by 7%. 
 
Error was present, but smaller, when working with larger liquid volumes. When using a 20μl pipette to dispense cold liquid at its minimum volume, error of 4% was recorded, as compared with the 37% error found when using the 2μl pipette at its minimum volume. Using the same 20μl pipette at its maximum volume produced error of only 1%. This error was lower than when using a 2μl pipette at its maximum volume, which resulted in 1%. However, using the 20μl pipette to dispense its minimum volume caused greater error than when using it at its maximum volume.

As with barometric pressure, the volume differences caused by thermal disequilibrium are also the result of the air-displacement operation of pipettes. When inserting the pipette tip into warm fluid, the air inside the tip is at ambient temperature. After insertion in the vial holding the warm liquid, the pipette tip is now in a warm microenvironment. During the time taken to aspirate, the pipette tip heats up, causing the captive air to expand and push liquid out of the tip. This causes less liquid to be aspirated and dispensed, leading to variation between the target and delivered volumes.

The opposite effect happens when pipetting cold liquids. When the pipette is inserted into a cold microenvironment, the captive air volume shrinks, which causes more liquid to be aspirated and dispensed.

Unlike altitude, thermal disequilibrium is a dynamic phenomenon. While pipettes did consistently under-deliver warm fluid and over-deliver cold fluid, the magnitude of volume variation varied. This is partly because the rate at which fluids equilibrate to their microenvironment is time-dependent. 

The longer a pipette is in a warm environment, the warmer the captive air volume becomes. This leads to greater air expansion and a greater impact on volume.  Therefore, the magnitude of error caused by thermal disequilibrium depends on several protocol-specific details, such as pipetting speed, type of sample container, volume of captive air in the pipette tip, etc. A straightforward, blanket solution does not exist. 

However, actions can be taken to minimise risk of volume variation including equilibrating fluids to the temperature of the environment and liquid handling device, minimising captive air volume by pipetting as close to the maximum volume as possible, and minimising exposure of the pipette tip to the warm or cold liquid and microenvironment.

Mission 3 – Dry heat (Death Valley National Park)
Another common laboratory condition is dry heat, which can be caused by devices using high power or open flames, such as analytical instruments, ovens, incubators and freezers, as well as by heating and air-conditioning systems. To test dry heat’s effect on pipette performance, the expedition visited Death Valley National Park and conducted two experiments – with and without pre-wetting the pipette tips. Although laboratories do not commonly operate in environments as extreme as Death Valley, laboratory conditions may cause humidity to be as low as 15%.

Compared to typical laboratory conditions, pipettes significantly under-delivered in the dry heat of Death Valley. While delivery errors were partially reduced by following pre-wetting steps, under-delivery persisted. Like previous missions, the data from Mission 3 show that pipettes exhibited greater errors when set to their minimum volumes than when set to their maximum volumes, and smaller-volume pipettes were more prone to error.

The largest error recorded at Death Valley occurred when using a two-microliter pipette at its minimum volume without pre-wetting, with error of 35 percent. In this case, pre-wetting reduced error to 31 percent. At its maximum volume, the 2μl pipette exhibited error of seven percent without pre-wetting and 5% with pre-wetting.

When working with larger liquid volumes, errors were still induced by the hot and dry conditions but on a smaller magnitude. Without pre-wetting, a 20μl pipette at its minimum volume under-delivered by 17%. This was a significantly lower inaccuracy than exhibited by a non-pre-wet two-microliter pipette at its minimum volume (35% error). 

When operating at its maximum volume, the 20μl pipette, like the 2μl pipette, exhibited smaller volume delivery inaccuracies than when operating at its minimum volume, totaling 6% without pre-wetting and 1% with pre-wetting.

Pipetting error in dry heat is largely due to evaporation. In dry heat, evaporation of the fluid occurs within a pipette tip during the aspiration process. This evaporation increases the total volume of the gas phase, and therefore increases the air cushion in the pipette barrel, preventing the pipette from aspirating the full target volume.

Humidity and temperatures within a laboratory can change throughout the course of a year, can vary significantly between individual laboratories within the same building, and can even differ in various sections of the same laboratory. It is also important to account for variations in temperature and humidity when quality control, research, and manufacturing projects are outsourced to contracting laboratories or transferred to another location.

Although costly, the ideal solution is for laboratories to purchase a system to control humidity and temperature.  Alternatively, laboratory technicians can consistently monitor the humidity and heat within their facilities, determine the potential for evaporation, and adjust pipettes accordingly. Another solution is to coordinate pipette calibration frequency with humidity cycles. Lastly, pre-wetting pipette tips prior to use is recommended. In the extreme conditions at Death Valley, while pre-wetting did not fully compensate for pipetting inaccuracy, it consistently reduced the magnitude of error.

Mission 4 – Humidity (Olympic National Park)
To study pipette performance in a humid environment, ARTEL visited the temperate rainforests of Olympic National Park in Washington State. According to regulatory standards, pipette calibration laboratories must control their relative humidity at greater than 50%, while working laboratories are often significantly drier. By visiting Olympic National Park, ARTEL sought to determine how pipettes behave in a humid environment.

The team tested in several locations throughout Olympic National Park including Rialto Beach and Olympic Inn. The team found that pipettes performed extremely well in the very humid environment at Rialto Beach, 14oC and 74% relative humidity. The tested pipettes experienced average inaccuracy of only -0.35%, with -1.55% the largest inaccuracy recorded. 

The team also tested pipettes at Olympic Inn, where the relative humidity measured exactly 60% with a temperature of 20oC – a common specification for pipette calibration laboratories. Pipettes also performed well at this location, with average inaccuracy of -0.11% and an average imprecision ratio of 0.68. 

While ARTEL seems to have found the perfect environment for accurate and precise pipette performance, laboratories do not typically have humidity as high as at Olympic National Park.  The relative humidity of a common laboratory is 15-40%.

Several steps can be taken to reduce the risk of pipetting error caused by humidity changes.  First, verifying the performance of liquid handling instruments in the environment in which they are used can help to eliminate humidity as a source of error. This practice will provide information about how pipettes perform in actual conditions and allow laboratories to adjust their pipetting processes accordingly.

Second, as discussed above, ARTEL strongly recommends that laboratory technicians pre-wet pipette tips prior to use.  Pre-wetting the tips will humidify pipettes before dispensing to improve overall performance, reduce risk of evaporation, and produce more consistent volume dispenses.

Environmental conditions can have drastic effects on pipetted volumes and laboratory data integrity. To account for volume variation caused by environmental conditions, which fluctuate between laboratories and can even fluctuate within a laboratory, regular, on-site verification of liquid handling instrumentation is essential.

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