Resolving Heterophile Antibody Interference in Viral Serology Using Blocking Tubes

Abstract

Despite their clinical utility, immunoassays are susceptible to various types of interference. In this study, we aimed to assess the extent of interference from heterophile antibodies in routine clinical tests and evaluate the effectiveness of heterophile blocking tubes (HBT) in mitigating such interference. We collected 185 residual serum samples that tested positive or equivocal in at least one IgM assay for Epstein-Barr virus (EBV), viral capsid antigen (VCA), herpes simplex virus (HSV), varicella-zoster virus, cytomegalovirus (CMV), rubella virus, or Toxoplasma gondii. These samples were obtained from the clinical laboratory of a tertiary teaching hospital in Korea and tested between May and July 2024. For each sample, complete IgM and IgG results for all six pathogens were obtained by performing any missing assays. Each sample was then pretreated with HBT and reanalyzed, and the assay results were compared with those of untreated samples. HBT pretreatment significantly reduced both reactivity levels (e.g., EBV VCA: 32.2 ± 35.8 U/mL to 12.8 ± 15.6 U/mL; HSV: 1.4 ± 1.0 index to 0.6 ± 0.4 index) and positivity rates (EBV VCA: 38/185 [20.5%] to 5/185 [2.7%]; HSV: 92/185 [49.7%] to 5/185 [2.7%]). These changes notably altered the clinical interpretation of the EBV status, reclassifying the 46 patients previously identified as having primary EBV infection. Our findings indicate a high prevalence of heterophile antibody interference in routine IgM testing for common viruses. HBT pretreatment effectively eliminated this interference and may be valuable for resolving discrepancies in clinical laboratory results.