The use of automated liquid handling equipment to rapidly test and reproducibly screen thousands of compounds has become an essential component to life science laboratories on a global scale.Along with an increase in use of automated liquid handlers, transferred volumes have become increasingly small, and yet, there are more demands on the accuracy and precision of such transfers, which include aspirating, diluting, dispensing, mixing and washing. Automated liquid handlers are generally used to increase the productivity and repeatability of volume transfers, but as discussed below, they are still prone to error. It is therefore important to understand how some errors can be recognized and avoided to maintain liquid handling quality assurance, especially when transferring critical reagents. Because concentrations of biological and chemical species are volume dependent, the accuracy and precision of individual, or step-wise, volume transfers directly impact the amount of critical reagents transferred to or from the assay. Inaccurate or imprecise delivery could easily result in the loss of experiment integrity. Therefore, knowing the exact volume in each step of an assay and the component concentrations is critical to interpreting the results and allows data and process integrity to be maintained.
Automated liquid handlers can take the human variable (the largest source of error Artel has identified in manual pipetting) out of pipetting and therefore can offer more repeatability from one event to the next. These systems, however, are still subject to multiple types of error as they are much more complex and have many internal actions which all must work within specification. The very selling point of many systems – their flexibility and control over many variables in the automated pipetting process – means inherently that there are more opportunities for error to creep into the system. In this article, the economic consequences of error in automated liquid handling will be discussed, as well as some of the most common areas for error propagation in using automated liquid handlers for liquid transfers. Lastly, the need to implement a robust volume verification method to minimize errors before they occur will be outlined.
If automated liquid handlers are not dispensing the desired amount of critical reagent(s), then it is likely that unseen error will increasingly propagate as a process continues. Even slight discrepancies in the amount of transferred reagent can compromise results, leading to poor quality, useless data and downstream costs associated with remedial actions. The economic impact of allocating resources for a continued liquid handling process that is based on potentially false results may be severe. Moreover, if the liquid delivery systems are over-delivering target volumes of expensive and/or rare reagents, then there will be a significant economic impact due to the loss of precious materials.
A typical high-throughput screening laboratory might test 1–1.5 million wells per screen, with an average screening frequency of about 20–25 times per year. With an approximate cost of $0.10 per well, the cost for reagents is approximately $3.75 million per year (1.5 million wells x 25 screenings x $0.10/well). If liquid handlers continuously over-dispense critical reagents, this can easily lead to an average cost per well of say, $0.12 per well (a 20 percent increase). The resulting additional annual cost would be $750,000. The company would risk depletion of those rare compounds and may not even have enough of the compound to conduct a full retesting program.
Furthermore, and depending on the type of screening effort, over dispensing critical reagent in each assay could cause more false positives, and those compounds will probably be used in subsequent screenings. These false positives are not fatal to the process, but they are detrimental and will cost the laboratory time, resources, and materials to continue screening with false performers, until they are tested out of the applicant pool. On the other hand, under delivering critical reagent in each assay, may lead to an increase in false negatives, which can be severely detrimental to the integrity of the entire screening process. To the screener, a false negative is no different than a ‘non-performer’ and these compounds would not be used in subsequent screenings. The underlying point is that by under delivering critical reagent, the next blockbuster drug may go unnoticed and potentially cost the company billions in future revenues.
The types of tips employed on the liquid handler are critical to the accuracy and precision of each volume transfer. Some liquid handlers employ fixed, or permanent, tips (including pin tools), which avoid the recurring consumable cost required for disposable tips. When using fixed tips, however, there must be rigorous and effective tip washing protocols in place. Otherwise, unwanted residual reagent may be carried-over and contaminate subsequent transfer steps. Ineffective tip washing can therefore cause liquid handling error and it is recommended that users who employ tip washing methods have validation protocols to prove the efficiency of the washing steps to ensure that tips are clean and the entire sample plug is removed.
When using disposable tips, the tip types are very important to the integrity of the volume transfer. Vendor-approved tips, as opposed to the cheaper ‘bag of tips’ option, should always be employed to minimize volume transfer error and optimize liquid delivery. Tip performance has been found to be directly related to quality because tip material, shape, properties, fit and wet-ability are all important factors for repeatability. The cheaper, bulk tips may not be manufactured with the highest precision manufacturing and may have variable characteristics that affect delivery, such as differences in upper diameter, virgin plastic content and presence of ‘flash’, which is residual plastic residue inside the tip. These tips also might not fit well on the liquid handler and they may have variable wetting/delivery properties. Without using approved tip types, accuracy and precision may be at risk, and in some cases, the liquid handlers may be incorrectly blamed for variable performance when the tips are the root cause of error.
Another source of error that can occur when using automated equipment is contamination. For instance, the liquid handler gantry/head moves across the deck, aspirates reagent, moves to a pre-determined deck location, dispenses reagent or aspirates another reagent, moves to a different location, dispenses, ejects or washes tips, etc. Contamination can occur while the head is moving across the workspace where droplets can fall from the tips onto the deck workspace, especially when slippery or organic type reagents are employed. Users should evaluate their systems and tips to ensure that droplets are not remaining after a sample is dispensed. Some users address this possibility by adding a trailing air gap following a reagent aspiration to minimize the chances of liquid slipping out of the tip. Users should also carefully plan when and where disposable tips are ejected, to ensure that contamination is not caused by random reagent splatter onto the deck workspace.
In some liquid handling protocols, a relatively large volume of reagent is aspirated and then sequentially, or systematically, dispensed across a microtiter plate. Though this method can save time, there are sometimes errors associated with variable accuracy. Users must ensure that, upon dispensing, the tips are not touching any liquid in the wells to avoid contamination or dilution. It is usually recommended that this protocol be a dry dispense (dispensed into a dry well) or alternatively, dispensed in a non-contact fashion above the buffer-filled wells. If an automation method employs a sequential transfer, users should validate that the same volume is dispensed in each successive transfer as it is common for the first and/or last dispense to transfer a slightly different volume.
Many laboratories perform some type of dilution testing to determine various characteristics associated with their specific assays, such as dose response, toxicity, detection limits, percent inhibition, drug efficacy, etc. A serial dilution is a systematic assay or test process where an important reagent is sequentially reduced in concentration. The assays are predominantly carried out in a microtiter plate where the different rows (or columns) contain lowering amounts of critical reagent across the plate. In many applications of a serial dilution assay, a neat or diluted target reagent will be transferred to a column of wells containing a pre-determined volume of assay buffer.
As a specific example, 100 µL of neat target reagent could be transferred to a column of wells in a 96-well plate that already contain 100 µL of assay buffer. The 200 µL total volume is then mixed with aspirate/dispense cycles or via on-board shaking before 100 µL of the 50% less concentrated target reagent is aspirated and transferred to the next column of wells, which also houses 100 µL of buffer. This specific example is a 1:2 dilution and may occur with up to twelve steps in the 96-well plate to dilute the starting material to a final concentration of 1/212, or 1/4096, of the starting concentration.
Automated liquid handlers are routinely employed to perform serial dilution protocols and users need to verify that the volume transfer is accurate and that each well is efficiently mixed before the next transfer takes place. If the reagents in the wells are not well-mixed and therefore not homogeneous before the transfer, the concentration of critical reagent will be very different compared to the assumed theoretical concentration levels across the plate. The experimental results will be flawed and users may have no indication that inefficient mixing is to blame.
One of the first steps in minimizing error in automated liquid handling is to choose the right pipetting technique, such as forward-mode or reverse-mode pipetting. Forward mode is the most common technique where the entire aspirated reagent in the tip is discharged. Forward mode is suitable for aqueous reagents with or without small amounts of proteins or surfactants. Reverse mode is a pipetting technique where more reagent is aspirated into the tip than dispensed, i.e., if 5 µL of serum is required, the pipettor might be programmed to aspirate 8 µL serum and then dispense the 5 µL (the theoretical 3 µL remaining is dispensed back to a reagent reservoir or to waste). This method is suitable for viscous or foaming liquids.
Along with the choice of pipetting technique, automated liquid handling errors may occur when variables within the user interface (software) are incorrectly defined. For instance, the user should ensure that procedural variables (aspirate/dispense rates and heights, requested volumes, pauses, liquid class settings, etc.), deck layouts (position and location of consumables and hardware) and consumable types (microplate types/footprints, reagent reservoir size(s), etc.) are properly defined for each assay under test.
It is also important to maintain a tip depth of about 2–3 mm below the surface of the reagent when aspirating liquid. Pipetting errors may occur if the reagent continues to be removed from the reservoir and the tip heights are not being compensated for that difference. In some instances, a liquid handler might use conductive, or liquid sensing, tips which are used to indicate the surface depth of the liquid. There can be errors in aspirating reagent if liquid sensing tips are lowered into bubbly or frothy reagents where the system falsely identifies liquid being present.
To reduce liquid handling error, laboratories must implement regular calibration programs and verification checks for volume transfer accuracy and precision and to quickly identify those systems that are failing. The evaluation method should be standardized, fast, easy to implement, and minimize instrument downtime and required resources. Currently, there is only one commercially-available standardized platform²-6 that meets these requirements.
Volume transfer for critical target screening should be compared for all devices within a process, especially for liquid handlers that are performing similar or identical tasks. If liquid handlers in San Diego and Boston are performing the same tasks for the same assay or assay-type, those systems should be evaluated with a standardized procedure on a tip-by-tip accuracy and precision basis. Such a volume verification method should also offer the opportunity to understand liquid handler device behavior for quality control purposes, trending patterns, diagnostic troubleshooting, method transfer, factory and site acceptance testing and employee training.
To maintain analytical integrity by reducing error and associated downstream economic loss, it is recommended that a volume verification method of performance evaluation be continuously implemented to understand if critical volumes are being accurately and precisely dispensed. As process control within a laboratory continues to be emphasized, a robust volume verification method should be implemented so that liquid handler behavior is known, optimized, and verified to deliver the desired target volumes for all levels of assay development. The volume verification method should serve as an essential tool in all laboratories utilizing liquid handling methodology because it is hard to manage and minimize error if there is no means to identify error in the first place. The more frequently liquid handler checks are performed, the sooner malfunctioning liquid handlers will be detected, fixed and brought back on-line.
Keith J. Albert, PhD, is an expert in liquid handling performance management, providing customized services across a wide range of platforms, applications, liquid volumes, and liquid classes. As Senior Applications Specialist, Keith combines his extensive knowledge and experience with rigorous science in his work in liquid handling system performance research, customer education, and assay/method validation.