Stability of hepatitis B virus pregenomic RNA in plasma specimens under various temperatures and storage conditions

BACKGROUND: Hepatitis B virus (HBV) pregenomic RNA (pgRNA) has gained increasing attention owing to its role in replication of covalently closed circular DNA (cccDNA) in HBV. This marker has the potential to be used in clinical programs aimed to manage HBV infections. However, several reports on HBV pgRNA levels in clinical cases have conflicting results. RNA is easily degraded when exposed to heat and other environmental stressors. However, the stability of HBV pgRNA, during blood sample collection before the standard automated quantification, has never been estimated. This study aimed to demonstrate the effect of two different temperature conditions and storage durations on the stability of HBV pgRNA. METHOD: Blood from forty patients with chronic hepatitis B infection, who also showed evidence of active HBV DNA replication, was collected and processed within 2 h of collection. Plasma from each patient was divided and stored at 4 °C and 25 °C (room temperature) for six different storage durations (0, 2, 6, 12, 24, and 48 h) and subsequently transferred to −80 °C for storage. The effect of multiple cycles of freezing and thawing of plasma at −20 °C or −80 °C was evaluated using samples from ten patients. Quantification of pgRNA from the samples was performed simultaneously, using the digital polymerase chain reaction (dPCR) method. The differences in pgRNA levels at baseline and each time point were compared using generalized estimating equation (GEE). A change greater than 0.5 log(10) copies/mL of pgRNA is considered clinically significant. Statistical analyses were conducted using Stata 16.0. RESULTS: The mean HBV pgRNA level in the initially collected plasma samples was 5.58 log(10)copies/mL (ranging from 3.08 to 8.04 log(10) copies/mL). The mean pgRNA levels in samples stored for different time periods compared with the initial reference sample (time 0) significantly decreased. The levels of pgRNA for 6, 12, 24, and 48 h of storage reduced by −0.05 log(10) copies/mL (95% confidence interval (CI) −0.095 to −0.005, p = 0.03), −0.075 log(10) copies/mL (95% CI [−0.12 to −0.03], p = 0.001), −0.084 log(10) copies/mL (95% CI [−0.13 to −0.039], p = < 0.001), and −0.120 log(10) copies/mL (95% CI [−0.17 to −0.076], p = < 0.001), respectively. However, these changes were below 0.5 log(10) copies/mL and thus were not clinically significant. Compared with the samples stored at 4 °C, there were no significant differences in pgRNA levels in samples stored at 25 °C for any of the storage durations (−0.01 log(10) copies/mL; 95% CI [−0.708 to 0.689], p = 0.98). No significant difference in the levels of pgRNA was observed in the plasma samples, following four freeze-thaw cycles at −20 °C and −80 °C. CONCLUSION: The plasma HBV pgRNA level was stable at 4 °C and at room temperature for at least 48 h and under multiple freeze-thaw cycles. Our results suggest that pgRNA is stable during the process of blood collection, and therefore results of pgRNA quantification are reliable.