Identification of Epstein-Barr Virus (EBV)-Infected Lymphocyte Subtypes by Flow Cytometric In Situ Hybridization in EBV-Associated Lymphoproliferative Diseases

To diagnose Epstein-Barr virus (EBV)-associated diseases and to explore the pathogenesis of EBV infection, not only must the EBV load be measured, but EBV-infected cells must also be identified. We established a novel flow cytometric in situ hybridization assay to detect EBV(+) suspension cells using a peptide nucleic acid probe specific for EBV-encoded small RNA (EBER). By enhancing fluorescence and photostability, we successfully stained EBER and surface antigens on the same cells. In 3 patients with hydroa vacciniforme- like lymphoproliferative disease, we demonstrated that 1.7%-25.9% of peripheral lymphocytes were infected with EBV and specifically identified these lymphocytes as CD3(+)CD4(-)CD8(-) gamma delta T cell receptor-positive T cells. The results indicate that this novel and noninvasive assay is a direct and reliable method of characterizing EBV-infected lymphocytes that can be used not only to diagnose EBV infection but also to clarify the pathogenesis of EBV-associated diseases.