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Hepatitis C virus (HCV) quantification is used as a prognostic marker for treatment success. In a routine clinical laboratory some infinitesimal sample handling factors can contribute to variability and loss of precision in HCV quantification. This may include blood collection tubes, blood drawing procedure, sample processing and storage temperatures. In current study blood was collected in tubes with different anticoagulant type (spray vs. liquid), group 1, blood was drawn with possible suck of methylated spirit through needle (experimental group) while avoiding the methylated spirit suck (control group) group 2, plasma separation was delayed from 0 to 60 min for group 3, plasma storage at different temperatures group 4. All samples were analyzed using Corbett research real time PCR system using AJ Roboscreen Kit. Mean viral load difference between spray vs. liquid was found 3.6 × 10(5) IU/ml (p < 0.001). Methylated spirit inhibited the viral load quantification with a value of 4.8 × 10(5) IU/ml (p < 0.001). Mean viral load difference was found 1.2 × 10(5) IU/ml (p < 0.05). Delay in centrifugation from 0 to 60 min and plasma placement at 25 °C for 15 min before freezing had no effect (p = 0.5996). Plasma storage temperature at -80 and -20 °C did not affect significantly on RNA levels (p > 0.05). In conclusion blood collection tubes and procedures can be a key factor in variability of results, that might affect the treatment response decision.