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No Room or Time for Error in High Throughput Screening Laboratories

Laboratories seeking to create novel therapies for debilitating and life-threatening diseases are challenged by seemingly conflicting goals to maximize productivity and quality.

by Katrina M. Gugenberger
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The Need to Standardize Performance Verification of Automated Liquid Handlers

Laboratories seeking to create novel therapies for debilitating and life-threatening diseases are challenged by seemingly conflicting goals – to maximize productivity and quality. Facing a race against the clock, scientists strive to beat fierce competition to market and provide treatments to patients who simply do not have the luxury of time. Yet quality programs must be implemented to ensure that laboratory processes generate solid, reproducible data in their developmental efforts.

Early in the drug discovery process, targets pass through a High Throughput Screening (HTS) laboratory. Looking for “hits,” interactions between these targets and compounds that could indicate a potential blockbuster drug, HTS laboratories conduct a high volume of tests. Screening campaigns often use a number of automated liquid handlers to dispense a variety of solutions, such as aqueous-based phosphate buffered saline solutions to organic-based dimethyl sulfoxide (DMSO) based solutions. For accurate and precise results, these laboratories must verify that all liquid handlers are dispensing uniformly, and equipment validation is thereby critical.

To bring efficiency and standardization to its HTS laboratory’s liquid handling operations, a biopharmaceutical company involved in molecular and cellular research recently implemented a standardized volume verification system based on ratiometric photometry. The rapid and robust system enables the laboratory to evaluate and optimize the volume transfer performance of its automated liquid handlers and streamline calibration and documentation processes. Since implementing this system to enhance its liquid handling quality assurance, the laboratory has experienced stronger and quicker assurance of data integrity.

The Need for Liquid Handling Quality Assurance
Inconsistency is undesirable in life science laboratories, and steps must be taken to control sources of uncertainty. At drug discovery laboratories, where high-throughput screening campaigns typically cost tens of thousands of dollars, the financial justification for minimizing variability is apparent.

Given the large role that liquid handling plays in almost every stage of the screening process, the impact of liquid handling device uncertainty can be significant. However, the implementation of an effective performance verification program can reduce downstream costs and errors associated with troubleshooting, equipment downtime, and over-use of valuable materials and resources.

To understand the prevalence and importance of automated liquid handling devices in HTS laboratories, let’s explore a typical screening process. Once a target is identified, for example a cancer target, an assay is developed to test that target under varied conditions. A typical screen will be conducted, during which an automated liquid handler supplies 384-well plates with compound stocks from the compound management group. The device then transfers these compounds into 384-well “granddaughter” plates, where the compound is diluted to an acceptable concentration. The automated liquid handler spots the granddaughter plates into a 384-well plate containing the specific assay under evaluation. In more general terms, the assay is executed and the plates are read on a plate reader to determine the assay results. If any hits are discovered, the plate is moved to hit validation for Method of Action studies (MOA).

If an automated liquid handler is malfunctioning during any stage of an assay screening process, either by not fully transferring the target volume or by over-delivering diluent, the concentration of the compounds is altered. This unseen liquid transfer can subsequently lead to downstream false-positive or false-negative results.

Laboratories are highly concerned about false-negatives because these are potential hits that are missed. Missed hits can lead to the failure to identify the next breakthrough drug, and this translates into an almost immeasurable loss of profits.

False-positives are also detrimental, causing inefficiency and misuse of compounds. After a hit is discovered and the compound moves to hit validation, dose response assays are run to confirm the potential drug’s responsiveness. If a hit is not again found, a false-positive has occurred and malfunctioning liquid handling equipment could be one of the root causes, i.e., inaccuracies in pipetted volume to the assay could be a reason for a false-positive.

Previous Home-Brewed System
Prior to implementing a standardized volume verification system, the HTS laboratory at the biopharmaceutical company referenced above was using a home-brewed calibration system based on tartrazine dye for performance verification of its automated liquid handlers.

The tartrazine calibration method entailed a multi-step process requiring hours from a skilled technician for manufacturing the dye solution, creating dilutions of that dye, and developing a standard curve before the liquid handlers could even be evaluated. The first step required serial dilution of the tartrazine dye, which was then hand pipetted to produce a standard curve for determining and documenting the concentration of the dye. Then, the tartrazine solution was dispensed using the automated liquid handler under test. In the laboratory’s method, by comparing the liquid handler dispensing results against the generated standard curve, information about the accuracy of the volumes delivered by the automated liquid handler could be evaluated.

The main drawback of the tartrazine method is the amount of preparatory work required prior to each screening test. This process was very tedious and time consuming for the lab, requiring the production of standard curves before each validation. In addition, data was stored on the individual computers of the technicians performing the calibration. Because technicians had unique methods of preparing the dye solutions and calculating and documenting the results, inconsistent, non-reproducible data resulted. In essence, this laboratory did not have a standardized method to validate the volume transfer performance of its many liquid handlers that are used by many different technicians.

The laboratory switched to a standardized liquid handling quality control platform. The platform measures the absorbance of light by two standardized dyes to simultaneously determine the accuracy and precision of liquid transfers for all tips/channels of an automated liquid handler. This system removes the burden of manufacturing home-brew dye solutions and there is no need for producing standard curves as encountered with the tartrazine method. Instead, the concentration of the standardized dyes are controlled by the manufacturer by providing pre-made solutions with barcodes, which are automatically inputted into its software component via barcode scanning, facilitating automatic calculation and reporting, and producing standardized results.

As opposed to the time-consuming tartrazine method, requiring about two hours per liquid handler verification, the new standardized volume verification system performs measurements in less than ten minutes in 96- and 384-well plates. Additionally, the technician-to-technician variability observed with the tartrazine method is a not observed with the standardized volume verification system because results are the same regardless of operator. This rapid volume verification facilitates frequent calibration of automated liquid handlers without diminishing productivity. If any issues are uncovered, the automated liquid handler can be adjusted before and during the screening process to ensure consistent data integrity and efficiency.

Because HTS laboratories use automated liquid handling devices from a number of manufacturers, not only must each instrument’s variability be controlled, but variability between instruments must be as well.

The laboratory needs to verify that the same amount of liquid is being transferred and that its results are comparable with each liquid handler manufacturer’s system, so that one microliter from one liquid handler is the same as one microliter from another liquid handler, even when those two systems are measured in different labs or on different days.

Using the standardized volume verification system has helped the HTS laboratory discover that what technicians thought were the same target volumes on multiple systems were actually different. In HTS laboratories, inaccuracies by fractions of microliters can significantly alter assay results.

With a robust liquid handling quality assurance program, laboratories can continue to reap the speed and efficiency-enhancing benefits of their automated liquid handlers without second-guessing quality.

Katrina M. Gugenberger, Marketing Public Relations, ABI, 29 Broadway
New York, NY 10006,