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 posttransplant lymphoproliferative disorder (PTLD) is required to detect a stage of disease that is more likely to respond to treatment. Elevated levels of EBV DNA were found in peripheral blood of patients at the onset of PTLD.


METHODS
To compare plasma and peripheral blood mononuclear cells (PBMCs) as material for real-time quantitative polymerase chain reaction (RQ-PCR) measurement of Epstein-Barr viral load, we used two sets of primers and probes specific for the BAM HI-K or BAM HI-W region of the EBV genome.


RESULTS
Patients with PTLD had a median viral load of 19,200 EBV genomes/microg DNA (n=9) or 3,225 EBV genomes/100 microl plasma (n=5), being significantly higher compared with immunosuppressed patients with primary (n=9) or reactivated (n=20) EBV infection or immunosuppressed patients without serological signs of active EBV infection (n=67) (P<0.001). Hence, a value of greater than 5,000 EBV genomes/microg PBMC DNA was considered as a diagnostic parameter for PTLD with a sensitivity and specificity of 1.00 or 0.89, respectively. When plasma was analyzed, however, a value of greater than 1,000 EBV genomes/100 microl plasma had both a sensitivity and specificity of 1.00 for the diagnosis of PTLD. During remission of PTLD, viral load was more effectively cleared in plasma compared with PBMCs. In plasma of nonimmunosuppressed individuals, even a qualitative detection of EBV-related sequences was sensitive and specific for the diagnosis of primary EBV infection, whereas for analysis of PBMC DNA a quantitative parameter had to be considered to differentiate healthy individuals (< 100 EBV genomes/microg PBMC DNA) from patients with primary EBV infection (>100 EBV genomes/microg PBMC DNA).


CONCLUSIONS
Although both PBMCs and plasma were useful as material for EBV-specific RQ-PCR in immunosuppressed patients and nonimmunosuppressed individuals, the specificity of analysis seemed to be higher if plasma was taken for analysis.
Wagner
H J
HJ
Department of Pediatrics, Medical University of Lübeck, Germany. hjwagner@usa.net.
Wessel
M
M
Jabs
W
W
Smets
F
F
Fischer
L
L
Offner
G
G
Bucsky
P
P
D000293:Adolescent; D000328:Adult; D001769:Blood / Q000821:virology; D002648:Child; D002675:Child, Preschool; D003199:Computer Systems; D003430:Cross-Sectional Studies; D004279:DNA, Viral / Q000097:blood; D020031:Epstein-Barr Virus Infections / Q000097:blood / Q000150:complications* / Q000821:virology; D005260:Female; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D016030:Kidney Transplantation / Q000009:adverse effects*; D008232:Lymphoproliferative Disorders / Q000209:etiology* / Q000821:virology*; D008297:Male; D009000:Monocytes / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012307:Risk Factors; D012680:Sensitivity and Specificity; D019562:Viral Load
D004279:DNA, Viral