Multi-site PCR-based CMV viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: A report of the association for molecular pathology

Viral load (VL) assessment of cytomegalovirus (CMV) by real-time PCR is an important tool for diagnosing and monitoring CMV viremia in patients with compromised immune systems. We report results from a sample exchange organized by members of the Association for Molecular Pathology that compared PCR results from 23 laboratories; 22 such laboratories used a laboratory-developed real-time PCR assay and one laboratory used a competitive PCR assay. The samples sent to each laboratory were comprised of a dilution panel of CMV virion-derived reference materials that ranged from 0 to 500,000 copies/ml. Accuracy, linearity, and intralaboratory precision were established for the different laboratory-developed assays. Overall, PCR results were linear for each laboratory (R² > 0.97 in all but two). While 13 laboratories showed no significant quantitative assay bias, 10 laboratories reported VLs that were significantly different compared with expected values (bias range, -0.82 to 1.4 logs). The intralaboratory precision [mean coefficient of variance of 2% to 5% (log-scale)] suggested that changes in VLs of less than 3- to five-fold may not be significantly different. There was no significant association between laboratory-specific technical variables (PCR platform, calibrator, extraction method) and assay linearity or accuracy. These data suggested that, within each laboratory, relative VL values were linear, but additional method standardization and a CMV DNA reference standard are needed to allow laboratories to achieve comparable numeric results.