Immunologic and Virologic Analyses in Pediatric Liver Transplant Recipients with Chronic High Epstein‐Barr Virus Loads

Abstract

EBV-infected cells in the blood of chronic high EBV load carriers expressed a highly restricted set of latency genes, suggesting that the EBV-infected cells escaped from a T cell response.

Application of flow cytometric in situ hybridization assay to Epstein–Barr virus‐associated T/natural killer cell lymphoproliferative diseases

“…Purified cells were analyzed by real-time PCR and compared with PBMCs. (27,28) Southern blotting with a terminal repeat probe was used to assess EBV clonality, as described previously. (29) Determination of TCR gene rearrangement.…”

EBV-associated T/NK–cell lymphoproliferative diseases in nonimmunocompromised hosts: prospective analysis of 108 cases

“…This study was approved by the institutional review board of Nagoya University Graduate School of Medicine. From 1998 to 2010, patients whose samples were sent to Nagoya University Graduate School of Medicine for determination of the EBVinfected cell phenotype and who fulfilled the following criteria were prospectively enrolled in this study: (1) EBV-associated T/NK-LPD suspected or diagnosed based on clinical and/or histopathological findings; (2) high EBV load detected in PBMCs by quantitative PCR (Ն 10 2.5 copies/g of EBV-DNA) 12,32 ; and (3) EBV infection in T or NK cells in the peripheral blood confirmed by either immunobead sorting followed by quantitative PCR [34][35] or FISH. 36 Exclusion criteria were: (1) pathologically defined ENKL, 5 ANKL, 37 or peripheral T-cell lymphoma (PTCL) 38 ; (2) On entry into the study, peripheral blood was collected and sent to Nagoya University Graduate School of Medicine to examine EBV-DNA quantification and EBV-infected cell determination along with detailed clinical data.…”

“…For the former method, PBMCs were fractionated into CD3 ϩ , CD4 ϩ , CD8 ϩ , CD16 ϩ , CD19 ϩ , CD56 ϩ , TCR␣␤ ϩ , and TCR␥␦ ϩ cells using an immunobead method (IMag Cell Separation System; BD Biosciences) that resulted in 97%-99% purity. [34][35] Purified cells were analyzed by real-time quantitative PCR. The infected-cell phenotypes were determined in comparison with unfractionated (whole) PBMCs, as described previously.…”

“…The infected-cell phenotypes were determined in comparison with unfractionated (whole) PBMCs, as described previously. [34][35] For example, patients were defined as CD3 ϩ when CD3 ϩ cells contained higher amounts of EBV DNA than whole PBMCs. The FISH assay was performed as described previously.…”

“…In 1998, we established an EBV-DNA quantification system using real-time PCR, [32][33] which allowed for the determination of the phenotype of EBV-infected cells in the peripheral blood with the combination of fractionation to the lymphocyte subset. 12,[34][35] More recently, we developed the simultaneous staining method for surface antigens and nuclear EBV-encoded small RNA (EBER) to more precisely determine EBV-infected cell phenotypes. 36 Using these techniques, we enrolled and prospectively followed patients with definitive cases of EBV ϩ T/NK-LPDs in 1998.…”