A team effort to unmask the big problems: Macromolecules interference in laboratory testing

What are macromolecules? 

Laboratory testing of protein biomarkers is a critical component for patient care. However, macromolecular complexes, formed between proteins and immunoglobulins (IgG), IgM, IgA and/or IgE, can interfere with laboratory testing. These complexes do not retain the functional activity of the original protein but prolong the elimination half-life of the biomarker. While this may seem innocuous, macromolecules can interfere with immunoassays, leading to erroneous results (1). 

How do macromolecules interfere with testing and how does the interference get identified?

Immunoassays rely on the interaction between specific protein epitopes and antibodies. In the presence of a macromolecule complex assay antibodies are still able to bind to their target site if the epitope remains accessible. This interaction can typically result in falsely elevated patient results. Patients with macromolecules may exhibit persistently elevated laboratory values for certain analytes that do not align with their clinical conditions.  

Macromolecular interference has been documented for prolactin, aspartate transaminase, amylase, lactate dehydrogenase, creatine kinase, and troponin, etc (2). Identifying macromolecules in patient specimens is essential to prevent diagnostic errors.  However, laboratorians are rarely able to identify macromolecular interference by relying solely on isolated patient results. Such interferences are usually identified when clinicians contact the laboratory to inquire about a discrepancy between lab results and a patient’s presentation. Therefore, establishing collaborative relationships between care teams and the laboratory is essential for proper patient care. 

How to work up and follow up? 

Macromolecule interference may be readily identified by retesting the specimen using a different assay methodology or different set of antibodies. Antibodies binding to different epitopes or those of a different species than that initially tested may not be subject to the same interferences. Other laboratory tools available to work up macromolecule interference include polyethylene glycol (PEG) precipitation, protein A/G precipitation, and size exclusion chromatography (SEC). PEG non-specifically precipitates high-molecular weight species in a sample. It is recommended that cutoffs for identifying the presence of macromolecules are established using multiple patient control samples. Beads coated with anti-IgG/A/M/E antibodies to pull down the antibody binding to the molecule of interest may also be used. The most accessible beads in the market are protein A/G which bind to the fragment crystallizable (Fc) fragment of IgG. It is important to note that interference with IgM/A/E macromolecules will not be detected using this approach (3). Overall, the two methods mentioned above requires testing the original sample, retesting after PEG precipitation or protein A/G pull-down, and an assessment of analyte recovery. Detailed protocols have been described and are referenced here (4,5). Another less commonly utilized approach is the use of size exclusion chromatography to separate high molecule species and identify their composition. 

These approaches are often used together to determine the presence of a macro complex. However, a significant challenge is that these investigations are typically not recorded in the patients electronic medical records, underscoring the critical role of communication between laboratorians and clinicians in identifying alternative testing approaches for disease diagnosis and monitoring. 

Conclusions 

Laboratory findings from immunoassays that are inconsistent with clinical presentations should prompt further investigations, including for macromolecule interference. Early identification of macromolecule interference can prevent unnecessary and potentially harmful diagnostic workups, emphasizing the need for close collaboration between laboratorians and clinicians.  

References

1. Straseski JA, Wyness SP. When Big Complexes Cause Big Problems. ADLM CLN Article. https://myadlm.org/cln/articles/2016/september/when-big-complexes-cause-big-problems-macromolecule-interference-with-routine-measurement.
2. Moriyama T, Tamura S, Nakano K, Otsuka K, Shigemura M, Honma N. Laboratory and clinical features of abnormal macroenzymes found in human sera. Biochim Biophys Acta. 2015;1854:658–67.
3. Bularga A, Oskoui E, Fujisawa T, Jenks S, Sutherland R, Apple FS, et al. Macrotroponin Complex as a Cause for Cardiac Troponin Increase after COVID-19 Vaccination and Infection. Clinical Chemistry. 2022;68:1015–9.
4. Davidson DF, Watson DJM. Macroenzyme detection by polyethylene glycol precipitation. Ann Clin Biochem. 2003;40:514–20.
5. Wyness SP, Hunsaker JJH, La’ulu SL, Roberts WL. Reference intervals for six enzymes after polyethylene glycol precipitation and ultrafiltration. Clin Chim Acta. 2011;412:1161–2.