– Brendan O’Farrell, DCN Diagnostics
At DCN, we regularly get requests for the development of “multiplexed” lateral flow devices. This is, in fact a growing area of application development. It brings some very specific thought processes along with it when it comes to designing, developing and commercializing products.
The term “multiplexing” means different things to different people depending on their interests. Generally, a “multiplexed” assay means one that detects or measures more than one analyte in a single device. However, the term can also refer to the act of generating multiple replicates of a single test within a single device. Additionally, it can be applied to the use of positive, negative or kinetic controls within a test coupled with the analyte-specific assay.
In actuality, the number of true multiplex lateral flow applications commercialized to date is relatively low. There are a variety of reasons for that rooted in technical, manufacturing and commercial considerations. In many instances (though not all) the technical and manufacturing issues can be less of a hurdle than the commercial. In this article, we will discuss the technical, manufacturing, and commercialization issues that impact the success of multiplexed devices.
In terms of multiplexing analytes, certain classes of assays are more amenable than others. For example, serology tests are relatively straightforward to multiplex. In these tests, the target is a single class of antibody directed against multiple analytes. On the other hand, biomarker assays can be much more complicated. This class of assays detects several different proteins from a single sample and may have multiple conjugates.
Let’s take a closer look at a simple duplex system designed to detect two protein antigens in a whole blood sample. The primary technical considerations include:
- the specificity of the available binding reagents
- potential interferences in the sample
- the availability of the analytes for detection. In other words, does the sample have to be pre-treated and does the same sample treatment work for both analytes?
- the required LOD and dynamic range for each analyte. Are they present in roughly the same concentrations so that both can be detected from the same sample?
- the stability of the binding reagents for each analyte. Is any difference in degradation rates of those binding reagents?
There has to be fundamental compatibility between the assays to allow for the multiplexing of analytes from a single sample. If that compatibility exists, we can move on to the lower order technical hurdles. Not all of these issues are deal-killers. Many can be overcome. However, the more analytes we add to the mix, the greater the complexity of answering some of these questions becomes. If we add quantification to the mix, the system becomes even more complex. In that case, we are concerned with variability in the system that can limit the accuracy of the results.
We have to work to generate highly reproducible results in each of our assays and understand the issues around degradation of reagent performance in the context of what that can do to the standard curves in the system. Again, more analytes, more complexity.
The complexity of manufacturing a multiplexed lateral flow assay is another significant consideration. The more assays we put on a device, the greater the potential risk of manufacturing failure and post-market failure of the product. Processing equipment, materials, manufacturing and QC processes may all be quite different to those used in a standard single-plex assay and the manufacturer will need deep technical capabilities to ensure ongoing quality and production yields.
From a technical perspective, it may be possible to multiplex many different assays onto a single rapid assay product. However, we have to be sure of the utility and market demand for the product. So, to state the obvious, one of the biggest questions around high level multiplexing is not “how,” but “why” we should do it. Good questions to ask include:
- What is the added regulatory complexity of the multiplex relative to single-plexed assays?
- What is the added manufacturing complexity and risk?
- What is the likely cost of the device?
- If we multiplex 2, 5, 10 or 20 assays onto a single device, can we get reimbursement for them?
- Does the market, the clinician or the end user want or need all of the data from our multiplex system? Is there clinical or diagnostic utility to generating all of this data? Is someone willing to pay for the entire multiplexed assay if they only want certain of the results?
To date, there are a relatively small number of panels that actually make sense from all of these perspectives. However, there are many assays that can benefit from the power of replicates and built-in controls that can come from a multiplexed assay.
So, why aren’t there more multiplexed lateral flow tests on the market? In short the ability to develop, manufacture and commercialize a multiplexed point of care test requires a balance between technical capability, design and market need. But there is demand in many application areas that can help to overcome the commercial issues, so developers and manufacturers need to be ready to meet the technical challenges. In part 2 of this series, we will discuss some effective multiplexed assay and product architectures and some novel approaches to overcome those challenges.
In the meantime, do not hesitate to contact us to discuss this topic further at firstname.lastname@example.org or 760-804-3886. We also invite you to join us at The Advanced Lateral Flow Course in San Diego this October where lateral flow professionals from all over the world are gathering to learn, collaborate and share ideas about the lateral flow technology and the future of the market. Learn more at www.dcnevents.com.