Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay

Abstract

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.

Effect of Multiple Freeze-Thaw Cycles on Hepatitis B Virus DNA and Hepatitis C Virus RNA Quantification as Measured with Branched-DNA Technology

“…Reliable assessment of the effects of multiple FT cycles on viral load measurements requires the following: (i) a quantitative assay capable of generating a reproducible relationship between viral load and the assay's output signal, (ii) testing of sufficient numbers of specimens (such that changes in viral load measurements due to FT cycling can be distinguished from inter-and intra-assay variability), (iii) appropriate statistical analysis of the data, and (iv) a descriptive end point that reflects a clinically relevant change. In this study, we have applied all of these criteria in evaluating the stability of HBV DNA and HCV RNA in serum specimens subjected to multiple FT cycles-the standardized and highly reproducible Quantiplex assays were used to measure viral load; sufficient numbers of specimens were assessed to achieve a relevant 95% confidence interval or its equivalent; both scattergram and linear regression analyses were used to evaluate the data; and, consistent with our earlier studies on HBV DNA and HCV RNA stability (10,11), we arbitrarily chose a Ն20% change in viral load as an indication of clinical relevance.…”

“…The raw data were evaluated by examining the mean levels of HBV DNA and HCV RNA after one (baseline), two, four, and eight FT cycles for each patient. All assay data were natural log transformed prior to analysis, and changes in viral nucleic acid concentration were evaluated by scattergram and linear regression analysis as described previously (10,16). Sufficient numbers of specimens were assessed to achieve a relevant 95% confidence interval or its equivalent.…”

“…On the other hand, if multiple FT cycles have no impact on HCV RNA and HBV DNA stability, there may be less need to aliquot serum when it is first collected, thereby saving valuable laboratory time and resources. Earlier studies have examined the effect of frozen storage on the integrity of HCV RNA (3,6,7,15,17) and HBV DNA (10) in serum. Some studies have included an analysis of the stability of HCV RNA in specimens subjected to multiple FT cycles (4,6,7,12,15,17); however, these studies were limited by the small number of samples tested, the use of nonstandardized assays, the lack of a clinically relevant end point, and/or the lack of rigorous statistical analysis.…”

Molecular methods used in clinical laboratory: prospects and pitfalls

“…Thus, analysis can have a significant impact on patient management and disease control through early detection of disease clusters or outbreaks. This molecular typing has added a new dimension to studying the epidemiology of communicable diseases, by the recognition of unsuspected transmission and defining likely locations or modes of transmission, as well as quantification of the extent of transmission (Oiu et al , 1992; Esteban et al , 1996; Harpaz et al , 1996; Krajden et al , 1998; Harrington & Bishai, 2004; Krajden, 2005).…”

Blood‐borne viruses and their survival in the environment: is public concern about community needlestick exposures justified?

“…22 HBV DNA in serum samples subjected to up to eight shor t-term freeze-thaw cycles is stable. 23 HBV DNA integrity in separated serum specimens is preser ved for at least f ive days when specimens are stored at -70°C or 4°C. Reduction in titer was least at 4°C and increased with storage at higher temperatures (23°C, 37°C and 45°C).…”