Multiplex testing: Solving for efficiency and efficacy

• Multiplex testing technology can enable laboratories to get more diagnostic information from a single test, saving time and resources compared to doing multiple tests in parallel or sequentially
• Panel testing supports the detection of diseases with overlapping or asymptomatic presentations, providing comprehensive insights while ruling out non-relevant causes
• Consolidating multiple tests into one cycle reduces plastic use, reagent consumption, and waste, while potentially requiring fewer patient samples and follow-up visits

Multiplexing is an advanced diagnostic approach that enables simultaneous detection of multiple targets from a single sample or reagent or test kit. While implementation varies across technology platforms, the fundamental principle remains consistent: maximizing information yield while reducing sample requirements and maximizing resource utilization. The result is more informed clinical decision-making and less waste.

For sustainable healthcare practices, multiplex testing offers not only clinical benefit, but also environmental, social, and economic value, as it helps reduce waste, can often be done in decentralized care settings, and helps preserve valuable resources.

In molecular diagnostics, multiplex Polymerase Chain Reaction (PCR) technology enables the detection of multiple targets in a single reaction. Multiplex Point of Care testing for respiratory or sexually transmitted infections can provide rapid results for multiple analytes from a single patient sample, while the patient is still present with their healthcare provider. Each application shares the common goal of extracting maximum diagnostic value without requiring additional sample collection or unnecessary patient visits. In the diagnostic realm, to put it more succinctly, we want to increase each laboratory's ability to do more with less.

Rapid molecular Point of Care testing outside of centralized laboratory settings also provides value towards sustainable healthcare by assisting in the clinical management of respiratory or sexually transmitted infections in decentralized and community-based healthcare settings. This approach enables more informed treatment strategies and can help contain further transmission by providing timely diagnosis and eliminating the need for sample transport and its associated carbon emissions.

The cobas® liat system exemplifies sustainable Point of Care, near-patient testing through multiplexed assays for respiratory panel testing to aid in the differential diagnosis, as well as other multiplexed panels targeted for STI pathogens.  This compact system requires minimal sample volume yet delivers results for multiple pathogens in approximately 20 minutes.*

Multiplexed sexually transmitted infection (STI) panels that simultaneously detect multiple targets from a single patient sample are also now possible. This approach is highly useful as common STIs often present overlapping symptoms and can frequently be asymptomatic, making diagnosis challenging when relying solely on symptoms.²

Another example is the cobas® liat SARS-CoV-2, Influenza A/B & RSV nucleic acid test, which may be more commonly used for the general population and when those viruses are known to be circulating, yet can be harder to distinguish based on symptoms.

This consolidated approach also reduces plastic consumption, decreases biohazardous waste generation, and reduces the need for repeat sample collection, which are important contributors to a more sustainable environment.

A new technique that uses a standard PCR plate on existing cobas® 5800/6800/8800 systems, Roche scientists found a way to triple the number of targets (e.g., viruses or bacteria) that can be detected based on multiple thermo-activated dyes, temperature cycling, and relatively simple data processing. Called TAGS—short for “temperature activated generation of signal”—more information is available per single test, which can mean quicker results for patients and lower costs for healthcare professionals. And with no need for additional reagents and consumable parts, there is less waste for the landfill.

Commercially launched with the name cobas® Respiratory flex, this system is designed for flexibility - because it allows users to test only what’s needed, and to tailor how they use it, which may allow them to simplify their respiratory season logistics. Up to 12 results can be simultaneously detected and differentiated from one test using the same kit. Results can be generated for the following viral targets: influenza A virus, influenza B virus, respiratory syncytial virus (RSV), SARS-CoV-2, adenovirus (species B, C and E), human metapneumovirus, human rhinovirus/enterovirus, common human coronaviruses (229E, HKU1, NL63, OC43), parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, and parainfluenza virus 4.¹

Labs get the flexibility to tailor their testing needs using existing cobas 5800/6800/8800 systems that are ready right now without any system hardware or system software upgrade required. Respiratory testing logistics are simplified with a customizable solution from just one test kit.

Beyond PCR-based solutions, there are other modular and scalable systems, such as the cobas® eplex system uses specialized test cartridges to simultaneously detect and identify greater than 20 viral and bacterial targets from a single sample test for infectious diseases, specifically with test panels for respiratory infections and also bloodstream infections, which can lead to sepsis.³ Additional panels are in development for gastrointestinal infections, meningitis, and encephalitis.

The cobas eplex system is an easy-to-use sample-to-answer system that enables it to be used by any technician on any shift. With results in only about 90 minutes, this is especially useful for testing high-risk or hospitalized patients who require a comprehensive result rapidly. Using multiplex testing not only aids in clinical management for each affected patient, it also contributes to reducing antibiotic resistance and promoting antimicrobial stewardship efforts to help address the growing problem of antibiotic resistance.⁴

The consolidation of testing processes results in reduced per-test costs through more efficient use of reagents and materials. For instance, running a multiplexed panel potentially uses fewer resources than running each analyte test individually. The reduction in physical waste translates to decreased waste management expenses, as a direct economic benefit.

Beyond the laboratory value, for the healthcare system overall, multiplex testing may also contribute to reducing funds wasted on unnecessary or ineffective treatments, assuming that the clinical information is more timely, accurate, and actionable. With testing capability that delivers more diagnostic information from a single test and as well as requiring less transportation or repeat visits or time, resources are utilized more efficiently, especially when compared to doing multiple tests in parallel or sequentially.

The world is facing climate change challenges and environmental considerations, so it is becoming a global imperative that we find ways to protect our global environment while improving patient outcomes. Innovative diagnostic solutions, such as multiplexing, that effectively address both objectives, are worthy investments.

Increasingly, sustainability and efficiency are being prioritized, and multiplex testing technology represents a significant advancement in combining environmental responsibility with clinical excellence.

In tissue diagnostics, immunohistochemistry (IHC) uses antibodies to detect antigens in a tissue sample. Unlike next-generation sequencing, flow cytometry, and other protein expression technologies, the significance and potential for immunohistochemical (IHC) staining with multiplex protocols is to provide simultaneous detection and analysis of multiple biomarkers from a single tissue section without the loss of tissue context.⁵

Multiplexing of this process involves sequential application of antibodies and detection systems on a single tissue section, and the automation of key steps helps ensure more consistent results while minimising reagent waste and manual intervention.

Multiplexing as a holistic visualization uncovers a world of interactions, enables colocalization studies, and allows for an overall greater understanding of cell populations and protein/protein relationships”.⁵ The process involves a carefully orchestrated sequence that maintains tissue integrity while extracting multiple data points.

Currently, research approaches with the DISCOVERY ULTRA platform enable automated multiplex testing of up to five biomarkers on a single tissue section.⁶ This provides comprehensive tissue characterization while preserving valuable patient samples. The sequential staining process includes application of primary antibodies, binding of Human Receptor Protein-conjugated secondary antibodies, and enzyme-mediated deposition of fluorophores, with heat deactivation steps between rounds to prevent cross-reactivity.⁶

Furthermore, digital pathology helps reduce courier costs as digital images of stained slides can be shared electronically, further lowering the environmental footprint of diagnostic processes.