The more scientific insight and biological knowledge that accumulate during the course of a drug development process, the more potentially promising biomarkers are identified. Defining which of these may prove suitable on an individual basis is the first step. However, at an early stage, probing as many relevant mechanisms of action as possible makes sense, and the most recent efforts have led to select panels of multiple candidate biomarkers rather than a single biomarker signature. Assessing the panel in one go - a process called multiplexing - is therefore a natural move to ascertain the most effective combination in the contemplated context of use.
There are many immediate benefits that come from multiplexing. Maximising information from minimal sample volume is an obvious one when securing appropriate diseased samples is such a limiting factor of Biomarker qualification.
Making the technology affordable comes next, as the labour required to operate biomarker work is the most critical resource in a lab. Generating simultaneous data from a single run decreases the cost per reportable significantly.
Multiplexing also drives additional biomarker value by ensuring measurements are derived from the exact same aliquot, minimising technical and pre-analytical variability.
To be considered fit for purpose, the multiplex assay must be able to exhibit the same characteristics that would be expected from a single analyte measurement. This requires assay concentrations and matrices to be compatible between all of the biomarkers on a panel. More importantly, it must be demonstrated that along the selected analytical ranges, there is no cross-reactivity between the panel members - ideally less than 1% and always less than 10% to yield useful data. This is where manufacturing expertise is invaluable and the larger the panel, the more delicate the fine-tuning becomes. The panel must be large enough to retain the sample volume and economic interests that are paramount to the value of the multiplex concept, while also being small enough to permit analytical performance - this is where IVD engineering capabilities become essential to success.
Bioinformatics capabilities are also requested as the development and validation of a meaningful composite index is expected to allow optimised interpretation of the data generated from the panel.
The expertise developed in the research field could translate into further diagnostics applications, through the CDx route in the first place, as multiplex research projects become the rule.
However, why would multiplexing be limited to translational medicine when the assets of multiplexing suit the diagnostic need so well? Certainly, reducing sample volume and minimising costs while delivering high throughput and high-quality data fits the current healthcare landscape well. Adequately defined panels, and their easy combination through a user-friendly software interface, contribute to faster clinical conclusions by suppressing the need for reflex testing.
Multiplexing is about generating massive amounts of data, including technical data: calibration curves, quality-control data, duplicate matching and reflex dilutions beyond upper range limits, to name a few. Automation not only takes care of the above, but also guarantees superior execution when very small sample volumes are involved, batch sizes are variable, and results are expected in a timely and controlled manner. Full automation enables the multiplex technology to effectively perform, allowing relevant scientific decisions to be made and diagnostics testing to be carried out.
Multiplexing is already used on a large scale in technologies such as flow cytometry, LC-MS/MS and highcomplexity molecular assays - all primarily research tools, operated by highly specialised laboratories. Development and validation guidelines for ligand-binding assays are available and widely used; however, these guidelines typically exclude multiplexing, preventing the technology from being fully recognised for its diagnostics potential. Hopefully, that gap can soon be bridged so that regulation stays in sync with technological advancement.