Although most contemporary immunoassays are extremely robust and display excellent sensitivity and specificity, they are still susceptible to interferences such as heterophile or human antianimal antibodies, vitamins/supplements, medications, or other unidentified interfering substances in the patient specimen. Depending on the type of immunoassay employed (competitive vs. sandwich) and mechanism of interference, results may be falsely increased or falsely decreased. Clinical laboratorians and physicians may suspect interferences when lab test results are inconsistent with the clinical presentation or with other clinical or laboratory findings. It is critical that laboratories have the ability both to detect and rule out assay interference as a source of error.
There are standard tools the laboratory can use to troubleshoot spurious results. Common approaches used to rule out an assay interference include: 1) serial dilution of the sample to verify analyte recovery upon dilution; 2) alternate testing methods to assess comparability of results between methods; and 3) commercially available kits to pretreat samples and remove possible interferences such as antianimal or heterophile antibodies or biotin.
Although these approaches are valuable, labs must consider several caveats and limitations. Strategies for developing a robust investigation protocol and avoiding red herrings, or misleading results, are described below.
Validating Dilution Protocols to Aid in Detecting Assay Interference
Assessing recovery of the analyte upon dilution of the specimen is a powerful tool for interference investigations. A common pattern observed when an interfering substance is present is that the analyte will not recover upon initial dilution of the sample. Upon subsequent dilutions, the analyte concentration, adjusted for dilution, will plateau once the interfering substance is sufficiently diluted to a concentration where it does not affect the assay. If the analyte does not recover appropriately upon serial dilution of the sample, an interfering substance should be suspected.
However, labs need to know the expected recovery in samples without interference to correctly interpret results. Some assays do not dilute in a linear fashion or are very sensitive to matrix effects introduced by the diluent. The diluent and dilution protocol must be validated in control samples to establish expected recovery.
It is best to adhere to the manufacturer’s recommendations for diluent when possible. Measuring the analyte concentration in the diluent is also necessary as some diluents may contain measurable analytes, which will yield misleading recovery results.
Under normal circumstances, sample dilution is reserved for situations where the analyte concentration is above the analytical measurement range (AMR). However, when using dilution as a tool for investigating interference, the lab may need to dilute samples with much lower analyte concentrations. Furthermore, dilutions at the low end of the AMR may exhibit poor recovery. Regardless of the diluent chosen, the lab should validate its use by diluting waste patient samples with a similar analyte concentration to that of the sample with suspected interference to establish expected recovery.
Using Alternate Testing Methods to Investigate Potential Assay Interference
The use of alternate testing methods can be useful, as assay manufacturers use different antibodies and reagents that are not usually susceptible to the same interferences. If significantly different results are obtained, an interfering substance may be present that affects one of the assays.
When interpreting results obtained from alternate methods while troubleshooting a suspected interference, labs must understand how the results would compare in patient samples with no interference present. For example, known bias between two methods needs to be accounted for. The laboratory should use method comparison data to establish expected agreement between methods and set criteria for differences that would be considered suggestive of interference. Comparable results between methods are strongly supportive evidence for ruling out interference.
Using Commercially Available Reagents and Kits to Detect Assay Interferences
Finally, samples can be treated with commercially available reagents to investigate the potential for interference by comparing results obtained pre- and post-treatment. For example, heterophile antibody blocking tubes or interference-specific blocking reagents from companies, such as Scantibodies or Veravas, can be used to remove heterophile antibodies or biotin from the patient sample.
However, the lab must demonstrate that the product does not affect the assay. Negative controls (waste patient samples) should be used to validate products either prior to or in conjunction with investigating the sample with suspected interference. Ideally, the lab would also confirm that the reagent removes the interference in a positive control. This is feasible for interferences such as biotin, which can be spiked into a sample to create a positive control. However, this may be impossible for heterophile or human antianimal antibody interference. Once the lab confirms that treating the sample does not affect measurement of the analyte in control patient samples, the commercial kit can be a useful troubleshooting tool for identifying antibody- or biotin-mediated interferences.
In collaboration with their clinical colleagues, laboratorians play a critical role in providing quality laboratory results and investigating questionable test results. While the lab’s troubleshooting toolbox includes valuable techniques, careful consideration must be given to the limitations surrounding these experiments in order to appropriately interpret results and draw conclusions about interfering substances. The ability to confidently rule out an assay interference and provide assurance of valid results is equally as important as being able to detect an assay interference.
Brooke Katzman, PhD, is an assistant professor in the Department of Laboratory Medicine and Pathology and codirector of the Hospital Clinical Laboratory and Point-of-Care Testing at the Mayo Clinic in Rochester, Minnesota. +Email: [email protected]
Nikola A. Baumann, PhD, is codirector of the Central Clinical Laboratory and Central Processing at the Mayo Clinic in Rochester, Minnesota. +Email: [email protected]