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   Home » Consumables & Supplies » Articles » Sources of Error: Automated Liquid Handlers
 
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Sources of Error: Automated Liquid Handlers
Sources of Error: Automated Liquid Handlers
10/23/2010

 A robust volume verification method can minimize the common errors of automated liquid handling and their associated economical consequences.

by Keith J. Albert, Ph.D., Technical Marketing Manager, ARTEL 
 

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The very selling points of automated liquid handlers provide more opportunities for error.

Using automated liquid handling equipment to test rapidly and screen thousands of compounds reproducibly has become an essential component to life science laboratories on a global scale. In addition, transferred volumes have become smaller, while the demands on the accuracy and precision of such transfers—aspirating, diluting, dispensing, mixing and washing—have increased. 

Automated liquid handlers are generally used to increase the productivity and repeatability of volume transfers, but they are still prone to error. Therefore, understanding how some errors can be recognized and avoided is necessary in order 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. Thus, knowing the exact volume in each step of an assay and the component concentrations is critical to interpreting results and maintaining data and process integrity. 

Economic impact 

Automated liquid handlers can take the human variable (the largest source of error) out of manual pipetting and offer more repeatability from one event to the next. These complex systems, however, are subject to multiple types of error due to many internal actions that must all work within specification. The very selling points of the systems—their flexibility and control over variables in the automated pipetting process—provide more opportunities for error. 

If automated liquid handlers are not dispensing the desired amount of critical reagents, then that unseen error most likely 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 based on potentially false results may be severe. Moreover, if the liquid delivery systems are over-delivering target volumes of expensive and rare reagents, then the loss of precious materials creates a significant economic impact as well. 

A typical high-throughput screening laboratory might test 1 to 1.5 million wells per screen, with an average screening frequency of about 20 to 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 $0.12 per well (a 20% increase). The resulting additional annual cost would be $750,000. A company would risk depletion of those rare compounds and may not have enough of the compound to conduct a full retesting program. 

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 cost laboratories time, resources, and materials to continue screening with them 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 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 a company billions in future revenues. 

Tip types and contamination 

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, rigorous and effective tip washing protocols must be in place. Otherwise unwanted residual reagent may be carried over and contaminate subsequent transfer steps. Ineffective tip washing can thus cause liquid handling error; users who employ tip washing methods should have validation protocols to prove the efficiency of the washing steps to ensure tips are clean and the entire sample plug is removed. 

When using disposable tips, the tip types are 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 have variable wetting/delivery properties. Without using approved tip types, accuracy and precision may be at risk, and the liquid handlers may be incorrectly blamed for variable performance when the tips are the root cause of error. 

"Users should evaluate their systems and tips to ensure droplets are not remaining after a sample is dispensed."

Another source of error 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 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 contamination is not caused by random reagent splattering onto the deck workspace. 

Sequential dispensing inaccuracies 

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, sometimes errors are 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. 

This protocol is usually recommended to 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; the first and last dispense commonly transfer a slightly different volume. 

Serial dilution transfers 

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. 

For 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 12 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 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. 

Methods and parameters 

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. This mode is suitable for aqueous reagents with or without small amounts of proteins or surfactants. 

With the reverse mode pipetting technique, more reagent is aspirated into the tip than dispensed: i.e. if 5 µL of serum is required, then the pipettor might be programmed to aspirate 8-µL serum and then dispense the 5 µL. The theoretical 3 µL remaining is dispensed back into a reagent reservoir or as waste. This method is suitable for viscous or foaming liquids. 

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.

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 sizes, etc.) are properly defined for each assay under test. 

Maintaining a tip depth of about 2 to 3 mm below the surface of the reagent is also important 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. 

Risk reduction 

To reduce liquid handling error, labs must implement regular calibration programs and verification checks for volume transfer accuracy and precision and to identify quickly those systems that are failing. The evaluation method should be standardized, fast, easy to implement, and minimize instrument downtime and required resources. 

Volume transfer for critical target screening should be compared for all devices within a process, especially for liquid handlers 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. 

Conclusion 

To maintain analytical integrity by reducing error and associated downstream economic loss, a volume verification method of performance evaluation should be continuously implemented to understand if critical volumes are being accurately and precisely dispensed. 

As process control within a lab 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. 

For more information, contact Keith Albert at kalbert@artel-usa.com or 207-854-0860 or visit www.artel-usa.com

 
 
 
 
 
 
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