LETTER
Quantitation of Hepatitis C Virus RNA
Judith C. Wilber, PhD, and
Mickey S. Urdea, PhD
15 July 1996 | Volume 125 Issue 2 | Page 155
TO THE EDITOR:
Gretch and colleagues [1] compared two quantitative hepatitis C virus (HCV) RNA assays: the branched DNA (bDNA) assay, commonly available in research laboratories, and an in-house quantitative competitive polymerase chain reaction (PCR) test.
The authors reported a possible "prozone" effect and variable results in a small subset of samples obtained from patients with end-stage liver disease and from liver transplant recipients. Our internal technical services group was interested in studying the problem, but the samples were unavailable for further study. The authors' laboratory has not encountered the problem again. Samples with particulate matter, for example, may yield variable results because of problems in dilution, independent of the assay used. The authors reported excellent reproducibility in all other samples in the study (the coefficients of variation were usually less than 10% within run and 23% between kits).
The quantitative PCR-measured "molecules" are referred to as the quantitative standard, even though the value was determined by end-point dilutional analysis, an imprecise method. The term "equivalent," as used in bDNA analyses, compares light counts derived from a sample with rigorously derived molecular standards [2].
The bDNA assay uses a four-point standard curve. The protocol and good laboratory practice do not allow quantification outside the standard curve regardless of the linearity above that value. If a result is higher than the highest calibrator, the sample should be diluted and repeated or reported as 
5.7 x 107 Eq/mL. The quantitative PCR used in Gretch and colleagues' study was reported to be linear to 1010 molecules per milliliter, but the authors use an upper cutoff of 7.5 log or greater (3.2 x 107 molecules per milliliter or 2.7 x 106 Eq/mL using the 0.5-log reported difference between bDNA and quantitative PCR). The lowest calibrator in the first-generation bDNA assay was 3.5 x 105 Eq/mL. It is not appropriate to assess sensitivity below the quantitation limit. Recent modifications to the assay, however, have increased the overall sensitivity to over 95% (Detmer J. Unpublished data).
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Author and Article Information
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Chiron Corp., Emeryville, CA 94608
1. Gretch D, dela Rosa C, Carithers RL Jr, Willson RA, Williams B, Corey L. Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications. Ann Intern Med. 1995; 123:321-9.
2. Collins ML, Zayati C, Detmer JJ, Daly B, Kolberg JA, Cha TA, et al. Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays. Anal Biochem. 1995; 226:120-9.
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