For Linco Diagnostic Services (www.lincodiagnostics.com), quality assurance is critical. Performing functions such as bioanalytical services and clinical trials for pharmaceutical and biotechnology companies, Linco is under intense scrutiny by its clients, as well as the FDA. Given the integral role of pipetting in its research activities and the fact that pipettes are a significant source of error, Linco must maintain a stringent liquid-delivery quality-assurance program and perform pipette calibration regularly. This is an issue experienced by most life science laboratories.
Linco previously relied on gravimetry for pipette calibration checks, verifying volume using analytical balances and manually calculating and recording the results. However, this procedure was lengthy, tedious, and prone to human error. It was also highly volatile, requiring a controlled environment absent of static, vibration, and temperature variations for proper function.
Given the severity of errors in its liquid-handling operations, such as delivering incorrect amounts of reagents to an assay and altering the results that it presents to customers, Linco sought a new solution in the PCS®, a photometric Pipette Calibration System from Artel (www.artel-usa.com). Using a patented dual-dye approach to pipette calibration called Ratiometric Photometry, this device allows Linco’s laboratory technicians to accurately and precisely verify liquid delivery performance in minutes, even at low volumes. The result is greater confidence in assay results, increased efficiency, and a deepening of client trust.
As the use of high-value reagents grows and the trend toward low-volume assays intensifies, it becomes increasingly important to accurately and precisely measure small volumes. When working with fewer than 200 microliters, gravimetric methods are plagued by evaporation and other environmental issues, and this impacts calibration certainty. The PCS, however, relies on a robust, dual-dye ratiometric approach to calibration to combat such inaccuracy and imprecision. This technique measures light absorption at two wavelengths by two specially formulated dye solutions, part of the PCS, to verify volumes as low as 0.1 µL.
To illustrate the greater accuracy and precision at low volumes provided by the PCS versus gravimetry, two experiments were conducted. First, to compare precision results, a pipette was checked using gravimetry. Ten data points were obtained using the same pipette tip, and the CV of these results was calculated. The same pipette with a new tip was then checked using the PCS to obtain ten data points, and the CV of these results was calculated. This process was repeated several times to assure repeatability. Representative data for six different pipette volumes with three runs for each of the two methods are shown in Figure 1. The data show that the two methods produced essentially the same results at high volumes, but the PCS produced better precision results at the lowest volumes tested (2 µL and 5 µL).
Comparing accuracy results between the two methods requires several additional steps. This is because in making repeated runs using one method (either one), the value of the means tends to drift with time and as the pipette tips are changed. Causes of this discrepancy may include warming of the pipette by the hand over time, variability between tips, and the wetting of the tips by the solution, which sometimes varies visibly from one tip to the next. Variations from one mean to another have been observed as large as 0.7%, even when the imprecision of the individual runs is much lower.
To obtain an accuracy comparison not compromised by these factors, several steps were taken. First, at the start of the PCS experiment, the unused blank vial into which solution would be pipetted was fitted with an anti-evaporation cap and accurately weighed using gravimetry. The pipette was then checked using the PCS with 10 sample additions. The vial was removed and weighed again to determine the amount of sample solution added to the vial.
After correction for evaporation and sample solution density, the gravimetric value of the mean volume added can be determined and compared to the mean value reported by the PCS. This comparison is valid regardless of the actual volume delivered by the pipette, and thus, does not use the pipette as a transfer standard. The results of this accuracy comparison for six different pipettes are shown in Figure 2. Again, the leading cause of error in this comparison is the variability of evaporation in the gravimetric method, especially evident at low volumes.