Clinical laboratories rely on accurate measurement of biomarkers to diagnose and manage various medical conditions, while interferences cause inaccuracies in test results which complicates result interpretation and potentially leads to patient harm. Although assay performance is monitored by internal and external quality assurance procedures, and common visible interferences such as hemoglobin, bilirubin, lipids are assessed at initial method validation and simultaneously with most clinical chemistry tests, endogenous protein interference is invisible and can’t be easily detected. The categories of endogenous protein interference, its causes, and the measures taken to mitigate its impact are explored in this article.

Endogenous protein interference may cross-react with the assay reagents, bind to the target protein, or affect the detection methods, thereby resulting in inaccurate measurements. There are several common endogenous protein interferences. Heterophilic antibodies are naturally occurring antibodies from previous exposures that can interference with the binding of analytes to antibodies in immunoassays, leading to false-positive or false-negative results. Rheumatoid factor is an autoantibody commonly found in patients with rheumatoid arthritis. It can cause interference in immunoassays by forming immune complexes with assay antibodies, usually leading to false-positive results. High-abundance proteins such as albumin can interfere with the detection of lower-abundance proteins.

To mitigate the adverse impact of endogenous protein interference, a number of steps can be taken. 1. Validation and quality control: Laboratories may test for potential known protein interferences and establish appropriate thresholds for detection. 2. Pre-treatment of samples: Dilution or adding blocking agents like polyethylene glycol (PEG) can reduce interference caused by high-abundance proteins or heterophilic antibodies. For example, laboratories may use PEG precipitation to remove the macroprolactin complex to yield prolactin results more reflective of the biologically active levels. Laboratories can also use the heat inactivation method or specific complement inhibitors to mitigate complement interference. 3. Alternate methodologies: In some cases, using a different antibody or detection method from another platform that is less susceptible to interference can improve accuracy of the test results. For example, thyroglobulin autoantibodies interfere with the measurement of thyroglobulin levels used in monitoring thyroid cancer recurrence. Laboratories can employ mass spectrometry-based assays that are less susceptible to autoantibody interference. In situations when the low-molecular weight lysozyme is excreted in the urine and interferes with sandwich immunoassays by bridging capturing and detection immunoglobulins, an lysozyme-resistant assay masking lysozyme by Cu2+ ions or ovalbumin is an effective method to block the linkage between lysozyme and immunoglobulins. 4. Additional analysis to identify interference: In scenarios where interference results from a detectable endogenous protein, additional analysis may be applied to identify the underlying cause. For instance, paraproteins are abnormal monoclonal immunoglobulins that particularly interfere with phosphate measurements, resulting in falsely elevated phosphate levels recognized as pseudo hyperphosphatemia. Serum protein electrophoresis can help identify the presence of paraproteins, allowing for appropriate interpretation of laboratory results.

In summary, the invisible interference from endogenous protein in clinical lab tests is a significant challenge that can affect the accuracy and reliability of test results. Understanding the causes of interference and implementing appropriate mitigation strategies are crucial for ensuring the clinical utility of lab tests. Continuous improvement in assay design, validation, quality control and in methods to remove interference is essential for minimizing the impact of endogenous protein interference.


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