Automation in Multiplexing

Multiplexing allows researchers to generate more data per sample compared to the established practice of measuring one analyte at a time. And combining multiplexing with automation increases confidence in results. However, to successfully automate a multiplexed assay workflow, choosing the right automation is key. This article comments on the advantages of both automation and multiplexing before suggesting some factors to consider when bringing the two together. It also explains how automation extends beyond sample handling to include data acquisition and analysis.

Automation and multiplexing complement one another

“Although multiplexing and automation offer similar benefits—savings in terms of time, reagents, and sample material—they provide these in different ways,” reports Kevin W.P. Miller, Ph.D., Senior Market Segment Leader and Scientific Content Manager at Hamilton Company. “Multiplexing does so by using a single chemistry to measure multiple analytes simultaneously, whereas automation increases sample throughput and improves experimental consistency, minimizing the need for assays to be repeated. Yet, the overlap in benefits means multiplexing and automation naturally complement one another; where combined, they enable researchers to glean more data—and have more confidence in those data—than ever before.”

Nick Pattinson, Head of Laboratory Automation at Automata, notes that advantages specific to automation are that it mitigates the risk of experimental cross-contamination, ensures the safety of staff working with infectious material, prevents repetitive strain injuries, and improves sample traceability. Automation also facilitates assay miniaturization, further conserving reagents and samples. “Importantly, by making labor-intensive and error-prone repetitive processes such as pipetting and sample prep faster and more efficient, automation frees up researchers’ time to focus on the high-value tasks that best utilize their skillset and training,” he says.

“The value of multiplexing to scientific research is evidenced by the number of publications citing xMAP^® Technology—a well-known bead-based technology for measuring as many as 500 analytes in parallel—which totals over 54,000 to date,” comments Chris Haake, Senior Marketing Manager, Licensed Technologies Group, at Luminex Corporation. “Not only does multiplexing deliver a more complete picture of each sample by monitoring a range of selected targets simultaneously, but it also eliminates confounding variables like different buffer systems, time points, or operators for a more accurate comparison of analyte expression within the same assay environment.”

And, while xMAP Technology is used for analyzing soluble targets, multiplexing is also applicable to the study of solid tissues. “Multiplexing is required whenever the molecular relationships among different cell populations are investigated,” comments Alexander Barang, International Sales and Business Development at TissueGnostics. “This is of particular importance in precision cancer diagnostics, where distinct cell types and tissue entities—such as malignant tissue, adjacent normal tissue, and the patient immune response—must be analyzed interdependently to understand the specific molecular mechanisms causing a disease.”

Image: Automated glomeruli detection and spatial phenotyping using TissueFAXS for data acquisition and StrataQuest for analysis. Image provided by TissueGnostics.

According to Miller, irrespective of the assay format, any lab that is measuring multiple analytes stands to benefit from an automated multiplex workflow. “Next-generation sequencing is a classic example,” he says. “A typical multiplex NGS workflow takes up to two days to complete, and it is unfeasible for technicians to pipette with the same degree of repeatability and reliability over that timeframe, week in and week out. By introducing automated liquid-handling systems to take over manual tasks, person-to-person and day-to-day variability is eliminated and the risk of error is reduced.”

Factors to consider when combining automation with multiplexing

Introducing automation into a multiplex assay requires careful planning. “The first step is to define your goals,” explains Haake. “Are you aiming to increase throughput, reduce variability, decrease hands-on time, save on reagents, or something else? You must also decide whether you wish to automate all, or part, of your process, to help determine the type of automation you need.” Available options range from single channel automated liquid dispensing systems and multichannel dispensers capable of dispensing up to 384 aliquots simultaneously, through automated plate washing systems, all the way to fully automated immunoassay platforms.

Next, Miller recommends mapping out the workflow to help identify any potential areas for improvement before they become roadblocks. “Take tip usage, for instance,” he says. “Multiplex assays are generally tip-intensive due to the number of liquid-handling steps, so you need to think about how many tips are necessary for each run, what space is available on the automated liquid handler’s deck for housing tip racks, and whether the workflow would benefit from having a storage module for such frequently used consumables. Likewise, certain items, such as semi-skirted microplates, may require changing to an automation-friendly format.” Lastly, highlighting critical process steps is key; Haake stresses the importance of taking into account elements like light-sensitive reagents or precise incubation times that absolutely must be factored into a successful automation strategy.

“When choosing automation for your multiplexed assay, there are three main factors to consider,” notes Pattinson. “Flexibility is essential; your automation requirements may change, so you need a system that can adapt to your needs. Finding a solution that is simple enough for your existing staff to adopt readily and that can be deployed within just a few weeks or months is also vital; managed services can be a good choice, especially for automation beginners. Additionally, cost will always play a part in the decision-making process. Some traditional automation systems can be incredibly expensive, making it worthwhile to shop around for more affordable alternatives; newcomers to the automation space offering open technologies can often provide innovative solutions for a lower price tag.”

Automation extends beyond sample handling and processing

Automation in multiplexing involves far more than improving the efficiency of sample handling and processing; automation is also widely applied to data acquisition and analysis. “In a tissue imaging setting, an automated process for acquiring multiplexed slides based on defined settings produces vast amounts of content-rich data, which can subsequently be mined using machine and deep learning image analysis tools,” reports Barang. “These use standardized, user-independent algorithms to automatically quantify marker expression intensity per cell and reveal the spatial distribution of target cells in relation to histological entities, providing unbiased and highly reproducible results. As the number of markers imaged per sample continues to grow, AI-based decision support systems are increasingly valuable, particularly for monitoring the complex immune and tumor microenvironment status in cancer patients or those with immune disorders.”

Hero image courtesy of TissueGnostics