Persistent red fluorescence interference in anti-PLA2R indirect immunofluorescence assays associated with liposomal doxorubicin

BACKGROUND: Analytical interference can occasionally affect indirect immunofluorescence (IIF) assays and lead to unexpected fluorescence signals. CASE: We report this type of interference during anti-phospholipase A2 receptor (anti-PLA2R) testing in a patient investigated for primary membranous nephropathy (pMN). Cells incubated with the patient's sample exhibited an unexpected intense red fluorescence signal, deviating from the typical green fluorescence indicative of a positive result or the absence of fluorescence in negative cases. DISCUSSION: This unexpected signal appeared to depend on the type of fixative used for the cells and raised concerns regarding result interpretation. Subsequent analyses, including ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS), identified the source of interference as doxorubicin, a chemotherapeutic agent known for its intrinsic red fluorescence. Notably, this interference persisted for up to three weeks after the last chemotherapy administration, likely due to the prolonged circulation of its PEGylated liposomal formulation. CONCLUSIONS: This case highlights the need for heightened awareness of potential fluorescence interferences in IIF assays, particularly in patients receiving liposomal formulations of intrinsically fluorescent drugs such as doxorubicin. Clinicians and laboratory personnel should consider recent exposure to such medications when interpreting unexpected fluorescence signals in IIF assays to avoid potential misinterpretation. In these situations, alternative analytical methods such as ELISA or delayed testing may help ensure accurate assessment.