Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA

Abstract

Background/Aims: Both HCV RNA viral load and HCV genotype have been described as important predicting factors determining the response to interferon in chronic hepatitis C. To investigate whether processing and storage conditions might influence the stability and could alter the concentration of the HCV RNA in serum, quantification of HCV RNA was performed by branched DNA assay. Methods: We studied serum samples obtained from seven patients with histologically proven chronic hepatitis C. These were subjected to the following physical conditions: (1) immediate quantification, (2) storage at room temperature for 5 days, (3) storage at 4°C for 5 days, (4) storage at −20°C for 5 days, (5) storage at −80°C for 5 days, (6) five freeze-thaw cycles, (7) blood unspun for 4 h at room temperature then centrifuged and stored at −80°C for 5 days, (8) storage at 4°C for 6 months, (9) storage at −20°C for 6 months, (10) storage at −80°C for 6 months. Results: A loss of 100% HCV RNA titers was observed after storage at RT for 5 days and then storage at 4°C for 6 months. A surprising decrease of HCV RNA titer (15.6%) was observed in sera stored for 5 days at −20°C. Five freeze-thaw cycles resulted in a 16% decrease of the HCV RNA level. When centrifugation was performed after a 4 h delay at room temperature, a significant loss of HCV RNA titers of 29.5% was observed. Long-term stability (6 months) was observed at −80°C with a slight loss of about of 10% HCV RNA titers, but a significant decrease in HCV RNA of 23% was observed at −20°C. The reproducibility of the bDNA assay on five patient samples was performed eight times in duplicate and showed an average coefficient of variation of 9.1%. Conclusions: These data confirm the importance of storage and handling in measuring the amount of HCV RNA in clinical samples.