Patients at risk for development of posttransplant lymphoproliferative disorder: Plasma versus peripheral blood mononuclear cells as material for quantification of Epstein-Barr viral load by using real-time quantitative polymerase chain reaction

Background. Early diagnosis of Epstein-Barr virus (EBV)-associated posttran splant lymphoproliferative disorder (PTLD) is required to detect a stage of disease that is more likely to respond to treatment. Elevated levels of EB V DNA were found in peripheral blood of patients at the onset of PTLD. Methods. To compare plasma and peripheral blood mononuclear cells (PBMCs) a s material for real-time quantitative polymerase chain reaction (RQ-PCR) me asurement of Epstein-Barr viral load, we used two sets of primers and probe s specific for the BAM HI-K or RAM HI-W region of the EBV genome. Results. Patients with PTLD had a median viral load of 19,200 EBV genomes/m ug DNA (n=9) or 3,225 EBV genomes/100 mul plasma (n=5), being significantly higher compared with immunosuppressed patients with primary (n=9) or react ivated (n=20) EBV infection or immunosuppressed patients without serologica l signs of active EBV infection (n=67) (P <0.001). Hence, a value of greate r than 5,000 EBV genomes/mug PBMC DNA was considered as a diagnostic parame ter for PTLD with a sensitivity and specificity of 1.00 or 0.89, respective ly. When plasma was analyzed, however, a value of greater than 1,000 EBV ge nomes/100 ttl plasma had both a sensitivity and specificity of 1.00 for the diagnosis of PTLD. During remission of PTLD, viral load was more effective ly cleared in plasma compared with PBMCs. In plasma of nonimmunosuppressed individuals, even a qualitative detection of EBV-related sequences was sens itive and specific for the diagnosis of primary EBV infection, whereas for analysis of PBMC DNA a quantitative parameter had to be considered to diffe rentiate healthy individuals (< 100 EBV genomes/mug PBMC DNA) from patients with primary EBV infection (> 100 EBV genomes/mug PBMC DNA). Conclusion. Although both PBMCs and plasma were useful as material for EBV- specific RQ-PCR in immunosuppressed patients and nonimmunosuppressed indivi duals, the specificity of analysis seemed to be higher if plasma was taken for analysis.