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• The performance of Liaison in detecting acute Epstein-Barr infection is optimal in pediatric patients with compatible symptoms. • The optimal cutoff points obtained for the Liaison assay differed depending on the age range and symptomatic presentation. • Some autoantibodies such as ANCA or rheumatoid factor, and IgM against viruses like VHS or Cytomegalovirus, can cause cross-reactivity. • The use of Liaison is not recommended for protocol-driven manner in oncology patients and immunocompromised individuals. • Our data exclude the possibility of Epstein-Barr IgG antibodies causing cross-reactivity. The present investigation assessed the Liaison® diagnostic performance in detecting Epstein-Barr (EBV) IgM-VCA in a large patient cohort, considering age and symptomatology. VIDAS® were employed as a benchmark for acute EBV infection. The study also probed other coexisting conditions and potential cross-reactivity for error sources. A total of 1311 samples were analyzed, with notable associations found only among paediatric (kappa=0.75) and young adult (kappa=0.58) populations with compatible symptoms. ROC analysis revealed varying optimal cutoff values based on age and symptom categorizations. Logistic regression models identified age and patients from Oncology or Infectious Disease as significant factors for false positives. Potential interferences emerged with RF, ANCA, cytomegalovirus-IgM and VHS-IgM. Notably, Liaison® couldn´t distinguish EBV patients from Oncology, Haemathology or Internal Medicine. This study provides valuable insights, such as implementing ageand symptom-specific thresholds or reviewing test requests, for optimizing EBV serology in microbiology laboratories, leading to faster and more reliable responses. [ABSTRACT FROM AUTHOR]