Lateral flow assays are the point of care testing devices that are technologically equipped. As they are technologically equipped, there is a chance of any possible problem or fault that can further confuse its end users. For the solution of the problem, and its analysis it is important to know about the troubleshooting process of the Lateral Flow Assay. Lateral Flow Assay might have some issues like false-positive results, false false results, membrane issues, and several others.
The troubleshooting guide for the lateral flow assay becomes a necessity when there is confirmation of the reported failure. In the reported failure, one must describe the failure in the lateral flow assay test so that there is adequate information. Moreover, it is important to know the frequency of the failure, the number of times it has occurred, and what likelihood ratio of this failure is to occur again.
In the troubleshooting guide, there are a number of steps which has further steps that must be followed in case the issue arises in the lateral flow assay test and device. The first step is to investigate the basics of LFA. In this step, the material history, and process variability of the LFA test must be checked. Also, if there are unusual observations noticed while doing the test, they will be also helpful in determining the problem. Now the second step is to start the test with the already finished device. By this, it is meant to test the device thoroughly before taking it for any kind of testing. You must check and inspect the strips. While inspecting the strips, there are few considerations that must be kept in mind such as checking the consistency of the assembly of the assays, contamination in the LFA, and if there is any physical damage to the device.
The next step is substituting the components that are known for working in exchange for those that are not working at that moment. For this purpose, you might need to look for the previous sets of devices if the problem found is in the device or any particular component of that LFA device. The same goes in the case of any chemical or biological elements that are thought to be the cause of the problem then they must be substituted from the previous batch of devices to overcome this problem as well.
The next step that comes in this process is to investigate the speed of the assay. For investigating the speed of the assay you can use a half-stick assay. With the help of it, it gets easy to troubleshoot the issues whether they are chemical components or biological components. By using this method, the variations from the conjugate pads, sample pads, housings, and the related assembly processes can be eliminated. And the plus point is other variables can be checked simultaneously.
Other factors that are involved in the troubleshooting guide of Lateral Flow Assay are vendors, manufacturing and QC, and the end-users of the product. By vendors, they are responsible for producing the quality with consistency and adding the specifications that are relevant to the lateral flow assay. Moreover, the manufacturing process and quality control must be checked as this is the main and foundational step to the production of good quality devices. Enhanced training must be given for the refined processes and refined quality of the product. Lastly, for the end-users, the instructions for operating must be written explicitly and clearly so that they understand using the device in a responsible manner.
While using the lateral flow assay, there is the possibility that you might see false-positive results. The false-positive result occurs when the desired analyte in the sample or matrix is not present yet the results came out positive. To solve this issue it is important to understand the different causes behind it. The reasons could be non-specific binding, heterophilic binding, or even cross-reactivity. For overcoming these issues, the optimization of the lateral flow assay or the conjugation pad must be done. The above-mentioned causes are completely different from each other in terms of affinity, antigens, and antibodies. Also, the presence of heterophilic antibodies can lead to the binding of the antibodies with the non-targeted analyte, thus resulting in false results. If in your lateral flow assay there are heterophilic antibodies you can solve the issue by purchasing the heterophilic blocking reagents or even mouse IgG conjugate can be used to solve this issue of lateral flow assay.
Another problem that can be found in the lateral flow assay is the decrease in the control and test line when the sample or matrix is put on the sample pad in the LFA device. This issue has been observed in a number of cases and the reason identified for this is the different elements in the clinical matrix or the sample. To troubleshoot this issue, the use of blocking agents in the conjugate diluent or conjugation pad can help to overcome this problem. Different blocking agents can be proteins, salt, molecules, or metabolites.
Conclusion:
Lateral Flow Assays are the most commonly and globally used devices. However, there could be issues due to the different reasons that one might face while using the device. The troubleshooting guide for the lateral flow assay helps to demonstrate what could be done for analyzing the problem and further solving it. In conclusion to all of the above-written material, it is a must to say that for the effective manufacturing of lateral flow assays and lateral flow test strips, there is a combination of biological, chemical, physical, and engineering issues. It is recommended and must-take action to consider all the minimal factors and aspects while manufacturing the product to minimize any type of variation if the device and strips could yield wrong results in the end. Also, if there is a problem with the nitrocellulose membrane of the lateral flow assay device the problem could be in the design of the device or other assembly issues.
References
https://www.emdmillipore.com/INTERSHOP/static/WFS/Merck-Site/-/Merck/en_US/Freestyle/DIV-Divisional/Events/pdfs/lateral-flow-presentations/troubleshooting-lateral-flow-tests.pdf
https://www.cytivalifesciences.com/en/us/solutions/lab-filtration/knowledge-center/lateral-flow-assay-troubleshooting-and-how-to-switch-membranes
https://nanocomposix.com/pages/lateral-flow-tips-tricks-and-frequently-asked-questions-faq#troubleshooting
