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-log10 HBV DNA dynamic range, were thawed and stored at -70, 4, 23, 37, and 45 degrees C (+/-1.5 degrees C) for 0, 24, 72, and 120 h (+/-2 h) and were refrozen at -70 degrees 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 degrees C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at < or =4 degrees C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45 degrees 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 < or =4 degrees C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of > or =20% at 23 or 37 degrees 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 degrees C.

FIG. 1

Scattergrams showing the range of quantitation of HBV DNA in PF specimens at each time point. The four incubation temperatures tested (±1.5°C) are shown in different plots: (A) 4°C; (B) 23°C; (C) 37°C; (D) 45°C. Each dot represents the observed quantity of HBV DNA (in megaequivalents per milliliter). Hollow dots indicate samples tested at the Toronto Hospital and solid dots indicate samples tested at Covance Laboratories. The horizontal line which bisects the diamonds represents the average of the data points at the respective time points, and the vertical apex of each diamond represents the 95% CI for the average value on each day (18). The horizontal line representing the average of the data points at time zero has been extended to day 5 in order to demonstrate the baseline average (panel B does not demonstrate the two high-value datum points present on panels A and C for days 1 and 3 because values for patient 23 were >3,000 Meq/ml at the 23°C incubation temperature).

FIG. 2

HBV DNA degradation trends by linear regression analysis. Each line represents the linear regression at the indicated incubation temperature (±1.5°C). Percentages to the right of each line represent the amount of HBV DNA after 5 days compared to the initial amount. At 45°C approximately 20% of the HBV DNA is lost within 24 h.