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Journal of Molecular Diagnostics 2009, Vol. 11, No. 2
Copyright © 2009 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2009.080097

Special Article

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

Daynna J. Wolff^*, Denise LaMarche Heaney^*, Paul D. Neuwald^*, Kathleen A. Stellrecht^* and Richard D. Press^*¶

From the CMV Working Group * of the Association for Molecular Pathology Clinical Practice Committee; the Medical University of South Carolina, Charleston, South Carolina; AcroMetrix Corp., Benicia, California; the Albany Medical Center, Albany, New York; and the Oregon Health & Science University, ^¶ Portland, Oregon

Viralload (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 fivefold 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.