Serum hepatitis B DNA: stability in relation to multiple freeze–thaw procedures

Quantitation of hepatitis B virus (HBV) DNA is often performed in specimens that have been frozen and thawed more than once. It is important to establish whether viral load measurements are affected by repeated freeze–thaw cycles. The effect of multiple freeze–thaw cycles on HBV DNA quantitation was carried out by testing serum specimens subjected to 1 (baseline) to 10 cycles with the appropriate Digene Hybrid Capture System. Five HBV DNA-positive samples were selected at random from sera with concentrations ranging from 7 pg/ml to 3529 pg/ml and they were frozen and thawed up to 10 cycles and then tested for changes in HBV DNA levels. Negative control and positive standards were tested in triplicate; and all specimens were tested in duplicate. The stability of HBV DNA in serum was evaluated by scattergram analysis by determining the number of samples showing a ≥20% change in HBV DNA levels after freeze–thaw cycles. With the exception of one sample (7 pg/ml) 10 cycles of freezing and thawing did not change significantly the HBV DNA quantity in any of the samples tested. The results showed that the quantity of HBV DNA in four of five serum specimens subjected up to 10 freeze–thaw cycles was stable.