Comparison of six different specimen types for Epstein-Barr viral load quantification in peripheral blood of pediatric patients after heart transplantation or after allogeneic hematopoietic stem cell transplantation

BackgroundEpstein-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.ObjectivesIn 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).Study designIn 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.ResultsIn 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 ≥ 20,000 EBV-copies/ml and plasma ≥ 1000 EBV-copies/ml had the highest sensitivities and specificities (WB: sensitivity 100%, specificity 87% and plasma: sensitivity 88%, specificity 98%).ConclusionNormalization 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 20,000 EBV-copies/ml WB and plasma values higher than 1000 EBV-copies/ml plasma indicated PTLD in our series.