A Multicenter Comparison of Different Real-Time PCR Assays for the Quantitative Detection of Epstein Barr Virus (EBV)

Departments of Pathology and Biostatistics, St. Jude Children's Research Hospital, Memphis, TN, University of Minnesota Medical Center, Minneapolis, MN, Department of Microbiology, Diagnostic Laboratory Services, Inc. and the Queens and Kuakini Health Systems, Honolulu, HI, Viromed (LabCorp) Laboratories Minnetonka, MN, Department of Pathology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, Department of Pathology, Johns Hopkins Medicine, Baltimore, MD, Departments of Pediatrics and Pathology, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, PA., University of Utah School of Medicine, Salt Lake City, UT, University of Minnesota Medical Center, Fairview, Minneapolis, MN, ARUP Institute for Clinical & Experimental Pathology, Salt Lake City, UT

^* To whom correspondence should be addressed. Email: balfo001{at}umn.edu.

	Abstract

Quantification of Epstein-Barr virus (EBV) in peripheral blood is important for the diagnosis and management of serious EBV diseases including post-transplant lymphoproliferative disorder. A variety of PCR-based methods are currently in use; however, there is little information on their comparability. This study assessed the relative performance of different quantitative assays. A multicenter comparative study was performed at eight sites using three panels consisting of serial dilutions of quantified EBV DNA and extracts from a total of 19 whole blood specimens. Samples were distributed and tested blindly. Instrumentation, probe chemistries, amplification targets, and other test-related aspects varied considerably between laboratories. Each laboratory's calibration curve indicated strong evidence of a consistent log-linear relationship between viral load and cycle threshold (Ct), suggesting that intra-laboratory tracking of a given patient would yield similar relative quantitative trends among the participating test sites. There was strong concordance among laboratories with respect to qualitative test results; however, marked quantitative discordance was seen. For most samples, the across-laboratory interquartile range of the reported viral load (in copies/µl) was roughly 0.6 log-units and for one sample the overall range was approximately 4.2 log-units. While intra-laboratory tracking of patients may yield similar results, these data indicate a need for caution when attempting to compare clinical results obtained at different institutions and suggest the potential value to be gained by more standardized testing methodology.