Accurate volumetric measurements and transfers are essential to all laboratories concerned with maintaining high levels of quality and productivity. The performance of pipettes varies substantially, and their accuracy cannot be taken for granted.
Many assays and experiments typically start in individual tubes and single channel pipettes are the tool of choice when it comes to pipetting at that stage. Once an assay has been developed (or established) using single channel pipettes, it can then be scaled up and transitioned from tubes to plates to process more samples at once and increase the throughput. At that point, using single channel pipettes is error prone, time consuming, repetitious and increases the risk of ergonomic issues.
For example, if you are using a single channel pipette to fill a 96-well plate in a typical assay, if you need to add 5 reagents to each well, you will need 480 individual pipetting steps (5 times 96 wells)! Keep reading to find out how you can reduce your chance for errors, as well as save time and effort by using multichannel pipettes.
Multichannel pipettes are often an obvious and easy choice for scaling up and can immediately decrease the amount of time spent for an increased number of samples. Multichannel pipettes are most frequently available in 8- or 12-channel versions spaced to work in 96- or even 384-well microplates, but larger multichannel configurations and adjustable spacing versions are available as well.
Using a multichannel pipette in the scenario above, which requires 480 pipetting steps would only require 40 steps (5 times 8 rows) with a 12-channel pipette and 60 steps (5 times 12 columns) with an 8-channel pipette. Using these devices not only decreases the time needed to fill the plate, it also helps reduce the risk of repetitive strain injury (RSI), and even mental and physical fatigue. Pipetting errors by a compromised analyst can have negative effects on the accuracy and precision of pipetting, potentially leading to false or inconclusive results. Repeating the assay is then necessary, and doesn’t just mean more pipetting, but also wasting valuable reagents and samples while diminishing productivity.
Multichannel pipettes come with their own set of benefits, as well as challenges. One challenge involves the pipette tips themselves. They can be difficult to load, and oftentimes load unevenly.
Problems with tip attachment, high tip ejection forces, and increased aspiration forces have furthered the difficulty in using them correctly. There have been some manufacturing breakthroughs in multichannel pipette technology that help to overcome these issues, such as electronic pipettes that have an electronic piston and/or electronic tip ejection mechanisms. Advances in tip attachment have made attaching tips to multichannel pipettes more consistent, which in turn leads to greater accuracy and precision. However even with these advanced features, there is no substitute for a properly trained operator who is using a multichannel pipette with each channel calibrated.
Proper technique with multichannel pipettes is critical and operator variability can cause errors that compound quickly. Below are some techniques to keep in mind when using these devices:
While multichannel pipettes do offer the user a time savings over single channel pipette use in assays and experiments, the time to calibrate multichannel pipettes can be significantly longer than single channels. It’s important to treat each of the channels as an individual instrument and verify the performance of each channel. All channels of a multichannel pipette can be calibrated simultaneously. This will not only save time, but serve as a better model of how the pipette is actually used and behaving in an assay. Evaluating the frequency of calibration needed and the calibration method available will help a lab determine the next best steps for scaling up.
As technology gets better and the levels of detection in common assays (such as PCR, immunology assays and cell-based potency assays) decrease, volume requirements also decrease. With low to very low volumes, it becomes increasingly difficult to pipette with accuracy and precision, which makes operator proficiency even more pertinent. At the same time, laboratories are more frequently calling for increased productivity. Improper use of pipettes leads to errors that are magnified when using multichannel pipettes.
For pipettes already in service, calibrations are to be performed at least once every 12 months 1, but quarterly calibration is encouraged.2 Adjustable volume pipettes should be tested at 3 volumes; nominal (the nominal value is the highest volume setting; e.g., a 10-100 μL pipette has a nominal volume of 100 μL) volume, 50% of nominal volume and 10% of nominal volume for the minimum setting. 3
Each channel of a multichannel pipette needs to be tested like a single-channel pipette, and each channel of the multichannel pipette must pass for the overall device to pass.3, 4, 5, 6 Calibrations require at least 10 replicate measurements per test volume.2, 3, 4, 5, 7, 8
Routine tests (quick checks) should be performed at least every 3 months.2, 4 Routine tests are intended to ensure the continuous fit for purpose of the pipette between calibrations, and do not require an estimation of the expanded uncertainty of the measurement. Routine tests require at least 4 replicates, which are needed for the calculation of the standard deviation (random error).3, 7, 8
Yes, each channel should be verified. ISO 8655-2022 is an international standard that provides recommendations for Piston Operated Volumetric Apparatus (pipettes). ISO 8655-6 outlines the specifica-tions and testing methods for multichannel pipette calibration and validating, “For the purposes of the volumetric performance test, each channel shall be regarded as a single-channel and tested and reported as such.”4 To comply with the standard, multichannel pipette testing should be performed on every single channel.
A typical calibration that follows the standards specified by ASTM7, ISO1, and CLSI2 calls for 10 replicates at each specified volume. Breaking that down into individual steps, in a low, mid, and high volume calibration, there will be 30 pipetting steps (3 volumes x 10 replicates).
Gravimetry takes approximately 1 minute per sample measurement and a single channel calibration like the one previously described will take 30 minutes to complete, depending on the volume of the pipette.
Following the standards, when the need to calibrate an 8- or a 12-channel pipette arises, you can multiply the number of steps by 8 and 12, respectively resulting in 240 pipetting steps for an 8-channel and 360 steps for a 12-channel pipette. The 4 hours (240 minutes) needed to calibrate an 8-channel pipette and the 6 hours (360 minutes) needed to calibrate a 12-channel pipette, may be a real resource burden on a laboratory. It’s very expensive and impractical. The ability to save the time in between pipetting steps by measuring 96 wells at once will result in a significant time savings for any laboratory.
Fulfilling the standard using a single channel gravimetric balance can be extremely time consuming. Even though multichannel pipettes are meant to save time, you may need to spend more time calibrating. However, time savings can be realized by delivering the test volume of all channels simultaneously into a microplate utilizing a ratiometric method or hybrid gravimetric/photometric approach.4,5