July 1, 2013 (Vol. 33, No. 13)
Keith J. Albert, Ph.D. Artel
Regular Instrument Performance Checks Help Ensure Accurate Reagent Volume Transfer
Over the past 20 years, laboratories around the world have been turning to liquid handling robots to increase the throughput and reproducibility of a wide range of assays. This widespread implementation of automation has contributed to significant advances in our understanding of science and medicine. Speed and reproducibility, however, do not guarantee accurate assay results. Assay results are dependent on reagent concentrations, which are volume-dependent. If a liquid handler is inaccurate in the volumes it dispenses, actual reagent concentrations can vary dramatically from those intended.
Most liquid handlers can be highly precise, but some can also be inaccurate when default methods and settings are employed. These performance variations may go unnoticed if volume transfer accuracy is not checked. As with pipettes, liquid handlers can also introduce errors into an assay process. Volume accuracy is especially important in any assay when a critical or limiting reagent is dispensed and when a quantitative result is expected, such as in drug discovery and molecular diagnostics. Additionally, monitoring volume transfer performance helps ensure that laboratories are meeting regulatory and/or compliance guidelines.
Gravimetric analysis with an analytical balance is often used to measure the weight of liquid dispensed and therefore, by inference, the liquid volume; however, environmental factors that cause evaporation and balance drift can negatively affect a pipettor’s performance in dispensing the low microliter and nanoliter quantities typically required of robotic instruments. While the average accuracy across multiple dispensing channels can be determined using gravimetric analysis, it is difficult and often prohibitively time-consuming to calculate the accuracy and precision of each channel of the robot.
In contrast, ratiometric photometry provides a simple and effective method for determining the volume dispensed by each channel into the individual wells of a microplate. This technology, incorporated in the Artel MVS® Multichannel Verification System, uses a dual-dye, dual-wavelength absorbance method to determine the volume of liquid dispensed, to levels as low as 10 nL.
This technology enables the accuracy and precision of each of the robot’s channels to be determined within minutes. If inaccuracy is found, in many cases it is possible to optimize the liquid handler method to ensure dispensed volumes closely match desired theoretical volumes required for the kit, application, or assay.
Application of Technology
To demonstrate the application of this technology, two different liquid handlers were used to transfer specific volumes, and by using information produced by the MVS, the volume transfer accuracies were subsequently optimized. It should be noted that while the optimization procedures vary from instrument to instrument, the inaccuracies described are fairly typical of scenarios, involving all brands of liquid handlers, found by the Artel field service teams.
Beckman Coulter Biomek NX
A Beckman Coulter Biomek NX liquid handler can be optimized for pipetting accuracy by adjusting the scaling factor (slope, m) and the offset (y-intercept, b) in the Biomek software’s Calibration tab within the Technique Editor. The calibration is based on y = mx + b. The scaling factor and offset values are meaningless, however, without a way to measure volume transfer accuracy.
Adjusting pipetting accuracy is a simple and effective process: (1) measure volume transfer performance (three or more target volumes are recommended); (2) plot measured (x) versus theoretically displaced volume (y); (3) determine the new slope and offset values; and (4) enter values and retest. A default preoptimized universal technique was employed to dispense 2, 5, and 8 µL. The preoptimized performance was (linearly) inaccurate: -27.85%, -14.68%, and -9.6%, respectively.
New scaling and offset factors were calculated and employed, and the relative inaccuracies improved to -0.05%, -1.14%, and 1.05%, respectively. The example above is represented graphically using the mean volumes measured for preoptimized and optimized trials (Figure 1).
One can also optimize volumes transferred with the Qiagen QIAgility by adjusting the p-value, which changes the amount of liquid pipetted by approximately 0.04 µL per step. The process for optimizing accuracy is simple: (1) perform initial testing; (2) determine the volume difference for measured versus desired; (3) convert the volume difference to p-value steps; (4) add or subtract steps to or from current p-value; (5) retest with new p-value.
In the data presented here, the preoptimized mean volume was 0.168 µL higher than the desired target of 2 µL (Figure 2). The p-value was decreased by 4.2 steps to 71.16 ([0.168 µL] / [0.04 µL/step] = 4.2) and the 2 µL target was retested and optimized in one simple experiment.
Compound concentration values can vary by more than 30% if one transfer is inaccurate and by more than 50% if two successive transfer steps are inaccurate (Table). The data above can be used to show how reagent concentrations are affected if two successive volume transfer steps are inaccurate, such as when a critical compound is diluted with buffer. For example, the actual 2 µL measurement data for the Biomek and QIAgility liquid handlers have been incorporated into two calculation models where the concentration of the critical compound is 10 mM.
The critical compound is further diluted with 25 µL from a bulk dispenser to create a final reagent concentration of 0.74 mM within the total volume of 27 µL. Additionally, a second calculation model assumes a ±10% difference in buffer volume, where the table shows the worst-case scenarios, i.e., the lower target volume was paired with a higher buffer volume to determine percent difference and vice versa.
In the examples provided above, the cumulative concentration after a simple two-dispense protocol was shown to deviate from the desired concentration by as much as 50%. Because reagent concentration deviations can multiply rapidly during pipetting steps, it’s important for researchers to include regular instrument performance checks, and potential optimization steps, in their assay processes.
While automated liquid handlers have improved laboratory efficiency and productivity, the promised increases in efficiency will only be realized when the volumes they dispense match the requirements of the application(s). Liquid handlers can be inaccurate, but with tools and know-how, a simple adjustment might ensure volume transfer accuracy, and therefore, confidence in assay results.