Quantification of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA often is performed in specimens that have been frozen and thawed more than once. To ensure optimal therapeutic and prognostic value, it is important to establish whether viral load measurements are affected by repeated freeze-thaw (FT) cycles. We therefore evaluated the effect of multiple FT cycles on HBV DNA and HCV RNA quantification by testing serum specimens subjected to one (baseline), two, four, and eight FT cycles with the appropriate Chiron Quantiplex assay. Linear regression analysis showed minor increases of 1.7% per FT cycle for both HBV DNA and HCV RNA. The rise in HCV RNA levels was more pronounced among low-concentration samples, since further analysis revealed an increase of 3.2% per FT cycle among samples with 0.2 to 3.86 Meq of HCV RNA per ml. Given that the coefficient of variation for the Quantiplex assays is generally 10 to 15%, the minor increases in HBV DNA and HCV RNA levels with progressive FT cycles for the specimens tested were recognized only because analysis of variance revealed a statistically significant trend (P < 0.05). Due to the minor statistical trend, the clinical impact for individual patient specimens is likely to be limited, but it may deserve further study. In conclusion, the concentration of HBV DNA and HCV RNA in serum specimens subjected to up to eight short-term FT cycles was stable.
Quantification of hepatitis B virus (HBV)DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log 10 HBV DNA dynamic range, were thawed and stored at ؊70, 4, 23, 37, and 45°C (؎1.5°C) for 0, 24, 72, and 120 h (؎2 h) and were refrozen at ؊70°C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4°C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at <4°C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45°C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at <4°C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of >20% at 23 or 37°C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at ؊70 or 4°C.
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