title: Application of flow cytometric in situ hybridization assay to Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases.
abstract: Epstein-Barr virus (EBV) infects various types of lymphocytes and is associated with not only B cell-origin lymphoma, but also T or natural killer cell lymphoproliferative diseases (T/NK LPD). Recently, we established a novel assay to identify EBV-infected cells using FISH. Using this assay, dual staining with antibodies to both surface antigens and an EBV-encoded small RNA (EBER) probe can be performed. In the present study, we applied this recently developed FISH assay to EBV-associated T/NK LPD to confirm its diagnostic utility. Using FISH, we prospectively analyzed peripheral blood from patients with suspected EBV-associated T/NK LPD. The results were compared with those obtained using immunobead sorting followed by quantitative PCR. In all, 26 patients were included study. Using FISH, 0.15-67.0% of peripheral blood lymphocytes were found to be positive for EBER. Dual staining was used to determine EBER-positive cell phenotypes in 23 of 26 subjects (88.5%). In five of seven patients with hydroa vacciniforme-like lymphoma (an EBV-positive cutaneous T cell lymphoma), EBER-positive cells were identified as CD3(+) CD4(-) CD8(-) TCRγδ(+) T cells. Furthermore, in a 25-year-old male patient with systemic EBV-positive T cell LPD, two lymphocyte lineages were positive for EBER: CD4(+) CD8(-) and CD4(-) CD8(+) T cells. Thus, we confirmed that our newly developed assay is useful for quantifying and characterizing EBV-infected lymphocytes in EBV-associated T/NK LPD and that it can be used not only to complement the pathological diagnosis, but also to clarify the pathogenesis and to expand the spectrum of EBV-associated diseases.
authors.affiliation: Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Kawabe
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ito
authors.forename: Kensei
authors.initials: K
authors.lastname: Gotoh
authors.forename: Seiji
authors.initials: S
authors.lastname: Kojima
authors.forename: Kimikazu
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kinoshita
authors.forename: Seiko
authors.initials: S
authors.lastname: Iwata
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Nishiyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D020031:Epstein-Barr Virus Infections / Q000150:complications / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D019672:Genes, T-Cell Receptor; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007223:Infant; D007694:Killer Cells, Natural / Q000276:immunology; D008232:Lymphoproliferative Disorders / Q000175:diagnosis* / Q000473:pathology / Q000821:virology; D008297:Male; D008875:Middle Aged; D011446:Prospective Studies; D055815:Young Adult