Multiplexing Advantages

Over recent years, "biomarker" has become a buzz word and is heard in more and more clinical arenas. Whole conferences have been built around their development and use, and companies have emerged that specialize in their analysis. However, for a word that is so widely used there is often very little understanding as to what it means.

Simply put, a biomarker is a measurable biological molecule that can be used to give an indication of any biological process. This can be in the diagnosis, development, prognosis of a disease, or the safety, efficacy, or pharmacodynamics of a drug. So practically every test performed within a clinical laboratory falls under this umbrella term.

Its usage in current trials tends to refer to the specific analytes used to monitor the disease (e.g., BUN, CRE, and Ionized Ca2+ for kidney disease). Here the analytes measured are standard clinical laboratory tests that become "biomarkers" in how they are being used.

Alternatively, biomarker testing can form into a larger panel looking at biological events (e.g., inflammation and/or chemotaxis). This opens up a much wider array of analytes that are less routinely measured within the clinical laboratory, often performed as panels of 10 or 20 different analytes. The analytes of interest are frequently from the families of biological molecules referred to as cytokines and chemokines.

Impact on sample size

Due to the large range of tests to be performed for these panels, traditional clinical laboratory testing methodologies are often inappropriate. Standard immunoassays require between 20 and 500 µL of sample per test, automated platforms, also require a dead-volume. When measuring a large number of analytes, which may split across multiple platforms, sample volume requirements and sample handling requirements increase for investigators and the laboratory, introducing human error.

As such, newer multiplexing technology, involving the simultaneous measurement of multiple analytes in a single analytical run, enables the analysis of full biomarker panels with a single sample addition, often as little of 25 – 50 µL. This modest sample requirement makes biomarker multiplexing extremely attractive in clinical trials where the total blood draw is a major controlling factor in whether the trial will gain ethical approval. Multiplexing therefore provides maximum yield of results without impacting on patient safety/comfort or trial logistics and storage.

Multiplexing techniques

The numerous multiplexing systems on the market can broadly be split into two areas: in-well and bead.

In-well multiplexing works in the familiar 96-well plate format. However, the ECL (electochemiluminescence) methodology allows for up to 10 analytes per well to be determined. Being electrically stimulated and reporting by fluorimetry there is considerably less background than a standard ELISA, providing higher sensitivity. Automated processing and an optional 384-well format makes this method extremely high throughput. However, it is inherently restricted by a maximum of 10 analytes with a similar expected concentration range.

For bead multiplexing, there are two primary methods available, both of which use the principles of flow cytometry originally designed and used for the separation of blood cells.

Bead multiplexing uses bead populations with an identifiable internal fluorescence and coated with analyte specific antibodies, A common secondary antibody with a fluorescent reporter is used for the analyte quantification. The flow cytometer is able to distinguish each individual bead population, quantify the amount of reporter, and therefore the amount of analyte.

A bead multiplexing specialist is eBioscience and the FlowCytomix System, which can be used on most benchtop flow cytometers. The majority of the assay can be performed within a 96-well filter plate format, simplifying wash steps increasing throughput, and keeping sample handling as homogenous as possible. Following the labelling procedure, samples are resuspended and transferred to standard flow cytometry tubes for analysis. This procedure allows for the simultaneous quantification of up to 20 discrete analytes in 25 µL of serum. Although convenient and flexible at the bench this methodology is also limited by analyte number and sampling handling can be cumbersome.

The other leading bead multiplexing approach has come to market with the development of flow cytometer system designed specifically for multiplexed bead arrays, arguably the most well known of which is Luminex. The overall process is very similar to that of the FlowCytomix system except for two major differences allowed for by the specialization in this type of analysis.

Luminex is capable of completing the full assay within the 96-well filter plate format, removing the time consuming step of resuspending samples in a secondary tube. Luminex's dual lasers and xMAP technology enables the system to distinguish up to 100 discrete bead populations. The xMAP system is licensed to a number of major suppliers, making the range of analytes and pre-made panels quite comprehensive. Newer developments using magnetic bead populations and the FLEXMAP system allow up to 500 discrete populations. The shear number of identifiable bead types allows the inclusion of within well standards to further standardize between well measurements, and increase the robustness of the assay.

Both systems have their specific advantages depending on the desired biomarker panel, but both allow for the optimal use of sample and generation of data.

The power of biomarker testing

The advantages of multiplexing are allowing developers to perform a large number of biomarker assessments within the same panel, on a single sample aliquot. Consequently, biomarker multiplexing is very popular in pediatric trials where the total blood draw is restrictive.

The data mining potential of biomarker multiplexing has made available the inquisition of considerably more biological processes. Drug developers are either using biomarkers to assess a broader range of pharmacodynamic markers monitoring disease progress, or they are investigating potential better pharmacodynamics markers that will enable them to develop a better testing panel to shape future clinical development, as well as aid in the hunt for companion diagnostics.

As these testing procedures are a relatively new addition to clinical trial protocols, their full potential have yet to be realized. Nonetheless, biomarker multiplexing provides significant advantages to drug developers in gaining a deeper understanding of a drug and its effect on patients and disease.

Andrew Botham, PhD, Head of R&D at ACM, Aviator Court, Clifton Moorgate, York, YO30 4UZ, UK, e-mail: abothham@acmgloballab.co.uk.