Department of Pediatric Hematology and Oncology, University of Giessen, Germany.
J Clin Virol. 2012 Mar;53(3):186-94. doi: 10.1016/j.jcv.2011.11.010. Epub 2011 Dec 17.
Epstein-Barr Virus (EBV) a gamma-herpes virus is associated with a spectrum of lymphoid and epithelial malignancies including posttransplant lymphoproliferative disorders (PTLD). EBV-load measurement has been shown to be important for the monitoring of these patients. However, in contrast to the viral quantification of human immunodeficiency virus or human hepatitis C virus, the EBV-load measurement has not been completely standardized as yet.
In this study, we compared the EBV DNA levels in whole blood (WB), plasma, peripheral mononuclear cells (PBMC) and B-cells (BC) in children and adolescents after heart transplantations (HTx) and allogeneic hematopoietic stem cell transplantations (HSCT).
In a period of 2 years (from May 2007 to May 2009) we collected 547 samples of 96 cardiac transplant recipients and 248 samples of 37 patients who underwent HSCT. For EBV DNA quantification we used a duplex real-time PCR (ABI Prism 7500, Applied Biosystems). Additionally, EBV-load of PBMC and BC were normalized with respect to endogenous cell DNA.
In both patient populations we found no significant difference of test sensitivity for the EBV detection. In PBMC as well as BC, there was a high correlation between the analysis of cells with and without normalization in both populations. Spearman's correlation coefficient ρ between PBMC without and PBMC with normalization was ρ=0.98 (P<0.0001) in patients after HTx and ρ=0.99 (P<0.0001) in patients after HSCT. Correlation between BC with and without normalization was ρ=0.98 (P<0.0001) in patients after HTx and ρ=0.995 (P<0.0001) in patients after HSCT. When comparing the different blood compartments for EBV quantification in both populations, the strongest correlations were found between the EBV DNA levels in WB and PBMC (HTx: ρ=0.93, P<0.0001; HSCT: ρ=0.81, P<0.0001) followed by PBMC and BC (HTx: ρ=0.87, P<0.0001; HSCT: ρ=0.81, P<0.0001) as well as WB and BC (HTx: ρ=0.86, P<0.0001; HSCT: ρ=0.75, P<0.0001). In contrast, the correlation coefficients between plasma and the other blood compartments (WB as well as PBMC or BC) were lower. Six patients developed seven episodes of PTLD (five patients after HTx and one after renal transplantation). Analyzing the different blood compartments, we found that a threshold of WB ≥20,000EBV-copies/ml and plasma ≥1000EBV-copies/ml had the highest sensitivities and specificities (WB: sensitivity 100%, specificity 87% and plasma: sensitivity 88%, specificity 98%).
Normalization towards an endogenous control does not seem to be necessary for EBV quantification in peripheral blood. The analysis of whole blood correlates well with B-cells and PBMC. Routine screening of EBV DNA in whole blood appeared to be a useful tool supplemented by EBV-load measurement in plasma to discriminate chronic high EBV-load carrier without risk for PTLD from those who are at risk for PTLD. Values in whole blood higher than 20,000EBV-copies/ml WB and plasma values higher than 1000EBV-copies/ml plasma indicated PTLD in our series.
爱泼斯坦-巴尔病毒（EBV）是一种γ疱疹病毒，与包括移植后淋巴组织增生性疾病（PTLD）在内的一系列淋巴和上皮恶性肿瘤有关。已经证明 EBV 负荷测量对于这些患者的监测很重要。然而，与人类免疫缺陷病毒或丙型肝炎病毒的病毒定量相比，EBV 负荷测量尚未完全标准化。
在这项研究中，我们比较了心脏移植（HTx）和异基因造血干细胞移植（HSCT）后儿童和青少年全血（WB）、血浆、外周单核细胞（PBMC）和 B 细胞（BC）中的 EBV DNA 水平。
在 2 年的时间内（2007 年 5 月至 2009 年 5 月），我们收集了 96 例心脏移植受者的 547 份样本和 37 例接受 HSCT 的患者的 248 份样本。我们使用双工实时 PCR（ABI Prism 7500，Applied Biosystems）对 EBV DNA 进行定量。此外，还将 PBMC 和 BC 的 EBV 负荷与内源性细胞 DNA 进行了归一化。
在这两个患者群体中，我们发现 EBV 检测的测试灵敏度没有显著差异。在 PBMC 和 BC 中，在两个群体中，分析未经归一化和经归一化的细胞之间存在高度相关性。在 HTx 后患者中，未经归一化的 PBMC 和经归一化的 PBMC 之间的 Spearman 相关系数 ρ 为 ρ=0.98（P<0.0001），在 HSCT 后患者中 ρ=0.99（P<0.0001）。BC 未经归一化和经归一化之间的相关性 ρ=0.98（P<0.0001），在 HTx 后患者中 ρ=0.995（P<0.0001），在 HSCT 后患者中 ρ=0.995（P<0.0001）。在比较两个群体中 EBV 定量的不同血液隔室时，发现 WB 和 PBMC 之间的 EBV DNA 水平（HTx：ρ=0.93，P<0.0001；HSCT：ρ=0.81，P<0.0001）之间存在最强的相关性，其次是 PBMC 和 BC（HTx：ρ=0.87，P<0.0001；HSCT：ρ=0.81，P<0.0001）以及 WB 和 BC（HTx：ρ=0.86，P<0.0001；HSCT：ρ=0.75，P<0.0001）。相比之下，血浆与其他血液隔室（WB 以及 PBMC 或 BC）之间的相关系数较低。六名患者发生了七次 PTLD 发作（五名 HTx 后患者和一名肾移植后患者）。分析不同的血液隔室，我们发现 WB≥20,000 EBV 拷贝/ml 和血浆≥1000 EBV 拷贝/ml 的阈值具有最高的灵敏度和特异性（WB：灵敏度 100%，特异性 87%和血浆：灵敏度 88%，特异性 98%）。
对于外周血中的 EBV 定量，归一化似乎不是必需的。全血分析与 B 细胞和 PBMC 相关性良好。全血 EBV DNA 的常规筛查似乎是一种有用的工具，辅以血浆中的 EBV 负荷测量，可区分无 PTLD 风险的慢性高 EBV 负荷携带者与有 PTLD 风险的携带者。在我们的系列中，WB 中 EBV 拷贝数高于 20,000 拷贝/ml 和血浆中 EBV 拷贝数高于 1,000 拷贝/ml 提示发生了 PTLD。
