MULTICENTER QUALITY CONTROL STUDY OF QUANTIFICATION OF EPSTEIN-BARR VIRUS DNA LOAD IN WHOLE BLOOD SPECIMENS BY THE FRENCH ANRS WORKING GROUP AC11

Aim: The French ANRS (Agence Nationale de Recherche sur le Sida) Epstein-Barr virus (EBV) Working Group organized a multicenter quality control program for EBV DNA quantification in whole blood by real-time PCR, before initiating new large-scale EBV studies in HIV-infected patients.

Methods: The study included two testing rounds in March 2006 and May 2007. Twelve testing centers evaluated a total of 12 and 14 whole blood specimens from patients with low, medium and high EBV DNA loads. Seven laboratories used manual silica based extraction and five automated extraction with the MagNAPure LC instrument™. The quantification step was performed on the Roche LightCycler instrument (N= 9), the ABIPrism 7700 instrument (N= 2), or the Rotorgene instrument (N= 1), according to methods using in-house quantification PCRs or the Roche LightCycler EBV Quantification kit. The range of acceptable values for each sample was defined as the geometric mean +/- 1 standard deviation, and delta log was calculated between high and low range value. In addition to the quantification standards usually used in each laboratory, a commercial quantification standard was simultaneously distributed with the second panel.

Results: The median delta-log was 0.73 log units for the first panel. Indeed, It was less than 0.73 (0.67) in 7/12 laboratories (in-house methods) and more than 0.73 (0.82) with commercially available kits (N=5). Lowest values (2.72 log copies/mL) were only detected with in-house PCR (N=4). Analyses of the second panel showed approximately the same results (median 0.76 log units). We found no improvement when using the commercial quantification standard.

Conclusions: Our data, established from various techniques of extraction, probes chemistry and PCR platforms, showed good correlation between the twelve participant centers. In our hands, quantification with a common standard did not improve the standardisation between the twelve laboratories. Moreover, in-house PCRs seemed to be more sensitive and efficient than the commercial kit used in this study.