Sunday, August 06, 2023

by Aaron Medaris

Is it influenza, COVID, RSV, or some other respiratory infection? HCPs are faced with these questions every day as patients fill their exam rooms with coughs, fevers, and congestion. Your training and experience will help you in your diagnosis and subsequent treatment, but with so many similar symptoms between different infections, the need for assistance and even assurance is at an all-time high. Luckily, testing at the point of care is becoming more advanced and effective. One of the most important advancements in point of care testing is the addition of multiplex testing – the ability analyze multiple analytes or targets in a single test. Multiplex testing is nothing new but has been steadily evolving over the last several decades and is gaining a larger presence in physician offices and urgent care centers around the nation. In this article, take a closer look at multiplex testing, its development over the years, and how it’s making a difference in point of care testing. 

Understanding Multiplex Testing 

Multiplex testing, also known as multiplexing or multiplex assay, is a technique used in laboratory diagnostics to simultaneously analyze multiple analytes or targets in a single test. It allows for the efficient and cost-effective detection of multiple biomarkers or genetic variants in a single sample. 

Traditionally, diagnostic tests would focus on a single target, requiring separate tests for each analyte of interest. Multiplex testing revolutionizes this process by enabling the detection of multiple targets in a parallel and high-throughput manner. 

There are different methods of multiplex testing, depending on the specific application and technology used. Here are a few examples:

• Multiplex Polymerase Chain Reaction (PCR): PCR is a widely used technique to amplify and detect specific DNA sequences. Multiplex PCR involves designing primers that can amplify multiple target DNA sequences simultaneously. By incorporating different fluorescent dyes or molecular tags, the amplified products can be differentiated and detected in a single reaction.
• Multiplex Immunoassays: These assays are used to detect and quantify proteins or antibodies in a biological sample. Multiplex immunoassays employ different types of capture beads or microarrays, each coated with specific antibodies or antigens. By labeling the analytes with fluorescent or enzymatic tags, multiple targets can be detected and quantified simultaneously.
• Mass Spectrometry-based Multiplexing: Mass spectrometry can be used for multiplex analysis by labeling analytes with unique mass tags. By introducing stable isotopes or mass labels, multiple samples can be combined and analyzed together, allowing for the simultaneous detection of multiple targets. 

Multiplex testing offers several advantages over single-target assays, including reduced sample volume requirements, lower costs, and faster turnaround time. It has found applications in various fields, including clinical diagnostics, infectious disease testing, genetic testing, and drug discovery. 

The Evolution of Multiplex Testing 

The concept of multiplex testing has been evolving over several decades, and the specific techniques used in multiplex testing have been developed and refined over time. Here are some key milestones in the history of multiplex testing:

• 1980s: The development of enzyme-linked immunosorbent assays (ELISAs) laid the foundation for multiplex immunoassays. ELISAs allowed for the quantification of a single analyte, but researchers began exploring ways to expand the assay to measure multiple targets simultaneously.
• 1990s: Multiplex PCR techniques started to emerge during this period. Researchers began developing methods to amplify and detect multiple DNA targets using PCR, such as multiplex allele-specific PCR (AS-PCR) and multiplex ligation-dependent probe amplification (MLPA).
• Late 1990s to early 2000s: The advent of microarray technology revolutionized multiplex testing. DNA microarrays allowed for the simultaneous analysis of thousands of genetic targets. This technology enabled gene expression profiling, SNP genotyping, and other multiplex applications.
• 2000s: The field of multiplex immunoassays expanded with the introduction of microsphere-based technologies. Luminex Corporation introduced the xMAP® (Multiplexed Assays with Beads) technology, which uses color-coded beads to enable the detection of multiple analytes simultaneously.
• 2010s: Advances in next-generation sequencing (NGS) technologies facilitated high-throughput multiplex sequencing. Techniques such as targeted sequencing panels and amplicon sequencing allowed researchers to simultaneously analyze multiple genetic variants or mutations. 

These milestones illustrate the progressive development and application of multiplex testing techniques. The field continues to evolve, with ongoing advancements in technology, assay design, and data analysis methods, enabling even more comprehensive and efficient multiplex testing capabilities. 

Multiplex Testing at the Point of Care 

Multiplex testing at the point of care refers to the use of multiplex assays or devices in settings outside of a central laboratory, such as clinics, doctor's offices, or even at home. Point-of-care multiplex testing offers several advantages, including rapid results, immediate clinical decision-making, and improved patient management. Here are some examples of how multiplex testing is used at the point of care:

• Infectious Disease Diagnosis: Multiplex assays are commonly used for the simultaneous detection of multiple pathogens in infectious disease diagnostics. Point-of-care multiplex tests can rapidly identify the presence of multiple viral, bacterial, or parasitic pathogens in a single sample, enabling timely diagnosis and appropriate treatment decisions. For example, a single test may detect respiratory viruses like influenza A and B, respiratory syncytial virus (RSV), and SARS-CoV-2 (the virus that causes COVID-19).
• Syndromic Panels: Syndromic panels combine multiplex testing with a broad range of targets related to specific clinical syndromes, such as respiratory infections, gastrointestinal infections, or sexually transmitted infections. These panels can simultaneously detect multiple pathogens associated with a particular syndrome, providing a comprehensive diagnostic approach and aiding in targeted treatment.
• Pharmacogenetics: Point-of-care multiplex testing can be utilized to analyze multiple genetic variants associated with drug metabolism or treatment response. These tests can provide insights into an individual's genetic profile and help guide personalized medication selection or dosing adjustments.
• Cancer Biomarkers: Multiplex assays can be used at the point of care to detect multiple cancer biomarkers, such as specific mutations or protein markers. These tests aid in cancer diagnosis, prognosis, and treatment decision-making, allowing for a more targeted and personalized approach.
• Rapid Screening: Multiplex tests can be employed for rapid screening of various conditions or diseases. For instance, multiplex lateral flow assays are commonly used for point-of-care testing of pregnancy, infectious diseases, cardiac markers, or drug testing. These tests provide quick results and are often simple to use, making them suitable for non-laboratory settings.

Recent advancements even include CLIA Waived multiplex testing, allowing healthcare professionals to make immediate and informed decisions, leading to improved patient care and outcomes. 

As we ramp up for cold and flu season and patient begin to fill your exam room with coughs, congestion, and fevers, remember there are sound diagnostic multiplex tests available to assist in your diagnosis.