Ratiometric Photometry for Pipette Calibration
By Rodrigues, G. | Publication
Method Critical for Low-volume Measurement
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.
A key factor in the higher degree of accuracy and precision afforded by the PCS versus gravimetry is its automatic calculation and documentation features. Automatic calibration eliminates transcription errors and rules out the possibility of deliberately skewed results. With the PCS, liquid delivery instrumentation performance is instantaneously verified, and supporting documentation can be printed with the touch of a button. This documentation is necessary to prepare for client audits and FDA inspections.
Speed Enhances Data Integrity
In today’s laboratories, where resources are often stretched thin, it is important that pipette calibration methods not only produce accurate and precise results but also verify volumes quickly. The PCS’ ease of use and operation speed allow Linco to calibrate its pipettes in half the time that its gravimetric method required, and this enables more frequent calibration. Out-of-specification pipettes can be quickly and easily identified, as can the affected assays.
Figure 2: This accuracy comparison of mean values between the PCS and gravimetric calibration methods shows greater variation at lower volumes
To check the performance of pipettes using the PCS, laboratory technicians use the pipette in question to dispense the system’s reagents into a vial situated in the PCS. (Linco’s particular SOPs dictate that each month four data points be taken at the pipette’s maximum volume and four data points at its minimum volume.) The photometer then measures the light absorbance of the formulated dye solutions and uses this data to calculate the dispensed volume.
The PCS evaluates these results against Linco’s customized tolerance for error, programmed into the system’s software. Pipettes functioning outside Linco’s approved range are identified and flagged for correction.
Benchtop Training Tool
The immediacy and objectivity of the PCS’ data output allow Linco to use it as a training tool. For example, the pipetting technique of new hires can be tested and improved immediately with minimal disruption to the laboratory. The monthly calibration checks also help current employees verify and maintain their liquid-dispensing performance. This enables prompt identification and correction of pipetting technique problems, and this can produce a dramatic improvement in pipetting quality, data integrity, and efficiency.
The improvements in accuracy and precision resulting from training are evident when comparing Figures 3a and 3b, which show the pipetting skill of 50 laboratory technicians before and after training. The test subjects were actual working laboratorians from four different pharmaceutical quality control laboratories.
Before training, each operator’s skill was tested using a properly functioning pipette to make a series of liquid deliveries, all at a volume setting of 5 µL. The percent inaccuracy and imprecision (%CV) was calculated for each operator and plotted on the graph (Figure 3a). This figure shows that both CV and inaccuracy can be large, even among professional technicians.
Figure 3A: The variation in pipetting accuracy and precision displayed in (A) was notably reduced after operators received training on proper pipetting technique with (B) clearly indicating the improvement
Each operator was consequently trained in proper technique, and given the opportunity to practice these skills using the PCS. The skills test was repeated and the results plotted in Figure 3b. Each operator with poor initial skills was able to make dramatic improvement once trained on proper pipetting technique.
It is critical that life science laboratories maintain a regular pipette calibration program using proven technology that delivers accurate and precise results. Artel’s PCS, based on ratiometric photometry, can more accurately and precisely verify pipette performance at low volumes than can gravimetric methods. At Linco, this has resulted in greater confidence in data and enhanced quality assurance.