Dataset Information

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

ABSTRACT:

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 log₁₀ 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 log₁₀copies/mL (ranging from 3.08 to 8.04 log₁₀ 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 log₁₀ copies/mL (95% confidence interval (CI) -0.095 to -0.005, p = 0.03), -0.075 log₁₀ copies/mL (95% CI [-0.12 to -0.03], p = 0.001), -0.084 log₁₀ copies/mL (95% CI [-0.13 to -0.039], p = < 0.001), and -0.120 log₁₀ copies/mL (95% CI [-0.17 to -0.076], p = < 0.001), respectively. However, these changes were below 0.5 log₁₀ 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 log₁₀ 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.

SUBMITTER: Rattanachaisit P

PROVIDER: S-EPMC8052958 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Cold chain maintaining is least stable at its end, where domestic storage often represents one of the most critical links because of storage time and inappropriate temperatures, increasing the risk of food-borne outbreaks in domestic households. Considering the time-temperature profile of refrigerators as a food safety indicator, the purpose of this study is to gain insight into refrigeration temperatures in parallel with refrigerator and household characteristics that could potentially influence the refrigeration temperatures. During a 24 h period in 15-min intervals, internal temperature of the test product, refrigerator air and ambient air temperatures were measured with one penetration and two air probes coupled with a data logger. The internal temperature of the test product was measured with pre-prepared "Karlsruhe Test Material", which had thermal properties similar to those of lean beef. Refrigerator and household characteristics were collected with a predefined observational sheet and short, structured questionnaire. In total, 50 households and their refrigerators were included. Gaps related to the cold storage and cross-contamination were observed. Temperature displays were present in 16% while control thermometers were not observed at all; 20% of the refrigerators enabled 24 h average internal temperature lower than 4 °C, 30% between 4 and 6 °C and 50% over 6 °C. Refrigerator age, type and load were observed but had no significant impact, which suggests thermostat setting as a key factor influencing refrigerator temperatures. Food distribution inside refrigerators was related to the refrigerator load with significant risk for cross-contamination in overpacked refrigerators. High temperatures combined with a non-systematic distribution of food in the refrigerator, expired dates of durability, and non-systematic cleaning strategies allow favourable preconditions for food infections occurring at the end of the food supply chain. Tailored acceleration of existing food safety messages could and should help consumers to minimise food safety risks, improve food quality, and reduce food wastage.

Project description:S-cysteinylated albumin and methionine-oxidized apolipoprotein A-I (apoA-I) have been posed as candidate markers of diseases associated with oxidative stress. Here, a dilute-and-shoot form of LC-electrospray ionization-MS requiring half a microliter of blood plasma was employed to simultaneously quantify the relative abundance of these oxidized proteoforms in samples stored at -80 °C, -20 °C, and room temperature and exposed to multiple freeze-thaw cycles and other adverse conditions in order to assess the possibility that protein oxidation may occur as a result of poor sample storage or handling. Samples from a healthy donor and a participant with poorly controlled type 2 diabetes started at the same low level of protein oxidation and behaved similarly; significant increases in albumin oxidation via S-cysteinylation were found to occur within hours at room temperature and days at -20 °C. Methionine oxidation of apoA-I took place on a longer time scale, setting in after albumin oxidation reached a plateau. Freeze-thaw cycles had a minimal effect on protein oxidation. In matched collections, protein oxidation in serum was the same as that in plasma. Albumin and apoA-I oxidation were not affected by sample headspace or the degree to which vials were sealed. ApoA-I, however, was unexpectedly found to oxidize faster in samples with lower surface-area-to-volume ratios. An initial survey of samples from patients with inflammatory conditions normally associated with elevated oxidative stress-including acute myocardial infarction and prostate cancer-demonstrated a lack of detectable apoA-I oxidation. Albumin S-cysteinylation in these samples was consistent with known but relatively brief exposures to temperatures above -30 °C (the freezing point of blood plasma). Given their properties and ease of analysis, these oxidized proteoforms, once fully validated, may represent the first markers of blood plasma specimen integrity based on direct measurement of oxidative molecular damage that can occur under suboptimal storage conditions.

Project description:BackgroundHerbaria are becoming increasingly important as archives of biodiversity, and play a central role in taxonomic and biogeographic studies. There is also an ongoing interest in functional traits and the way they mediate interactions between a plant species and its environment. Herbarium specimens allow tracking trait values over time, and thus, capturing consequences of anthropogenic activities such as eutrophication. Here, we present an open, reproducible, non-destructive workflow to collect leaf trait data from herbarium specimens using near-infrared spectroscopy (NIRS), and a proof of concept for the reliability of this approach.ResultsWe carried out three experiments to test the suitability of non-destructive NIRS methods to predict leaf traits both for fresh and dried leaves: (1) With a fertilization experiment, we studied whether NIRS was able to capture changes in leaf N and leaf P during a fertilization experiment and we compared contents predicted by NIRS with results obtained from regular wet lab methods. Calibration models for leaf nitrogen and phosphorus contents had a quality of R2 = 0.7 and 0.5, respectively. We fitted calibration models for NIRS readings on fresh and dried leaf samples, both of which produced equally precise predictions compared to results from wet lab analyses. (2) We tested the effect of herbarium conservation on NIRS readings by simulating them through the application of six treatments combining freezing, drying and pesticide spraying in a factorial scheme and comparing these with untreated samples. No consistent changes were observed in the spectra quality before and after the simulated herbarium conditions. (3) Finally, we studied the effect of specimen storage duration using specimens from a 2018 study which were re-analyzed and compared with spectra obtained in 2021. No consistent changes in spectra were observed after the storage period.ConclusionsThe results demonstrate the reliability of NIRS to measure leaf N and P on herbarium samples. Together with the calibration method and dataset presented here, they provide a toolset allowing researchers to study the development of leaf traits and their response to environmental changes over decades and even centuries in a fast and non-destructive manner.