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This communication takes note of unexpected factors that can influence the results of RT-PCR in quantitation of copy numbers such as in determination of viral loads and in viral identification. We show that the presence of serum separator gel in authorized collection tubes for hepatitis C (HCV) viral load determinations causes underestimation of viral loads by blocking viral diffusion into plasma and that the presence of more than one targeted virus in a multiplex RT-PCR viral assay, while not affecting analytical specificity, results in raising of the minimal detectable viral titer and therefore a decreased analytical sensitivity. Our results suggest the possibility that HCV samples should be placed in cell lysis buffer for full viral load determination and that multiplex assays should be carefully validated and modified if necessary to minimize loss of sensitivity.