Application of flow cytometric in situ hybridization assay to E pstein– B arr virus‐associated T /natural killer cell lymphoproliferative diseases

E pstein– B arr 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 CD 3 + CD 4 − CD 8 −  TCR γδ + T cells. Furthermore, in a 25‐year‐old male patient with systemic EBV ‐positive T cell LPD , two lymphocyte lineages were positive for EBER : CD 4 + CD 8 − and CD 4 − CD 8 + 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. ( Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02305.x, 2012)