Stability of Hepatitis C Virus, HIV, and Hepatitis B Virus Nucleic Acids in Plasma Samples after Long-Term Storage at −20 ^o C and −70 ^o C

Abstract

The storage of biological samples may affect detection of viral nucleic acid, yet the stability of viral nucleic acid at standard laboratory storage temperatures (؊70°C and ؊20°C) has not been comprehensively assessed. Deterioration of viral RNA and DNA during storage may affect the detection of viruses, thus leading to an increased likelihood of false-negative results on diagnostic testing. The viral loads of 99 hepatitis C virus (HCV), 41 HIV, and 101 hepatitis B virus (HBV) patient samples were measured before and after storage at ؊20°C and ؊70°C for up to 9.1 years using Versant branched DNA assays, Cobas Monitor assays, and/or AmpliPrep/AmpliScreen assays. Clinical samples stored at ؊20°C for up to 1.2 years and at ؊70°C for up to 9 years showed a statistically significant difference from baseline with respect to HCV RNA titer, although this difference was not greater than 0.5 log 10 unit. The concentration of HIV RNA in clinical samples stored at ؊20°C for 2.3 years and at ؊70°C for up to 9.1 years did not differ significantly from the baseline viral load. HBV DNA-positive clinical samples stored at ؊20°C for up to 5 years and at ؊70°C for up to 4 years differed significantly in viral load. In all studies, however, the loss of viral load of HCV, HIV, or HBV in clinical samples tested after storage at ؊20°C and ؊70°C for up to 9 years ranged from 0.01 to 0.35 log 10 IU/ml and did not exceed 0.5 log 10 , which is the estimated intra-assay variation for molecular tests. Hence, the loss was considered of minimal clinical impact and adequate for the detection of HCV, HIV-1, and HBV nucleic acids using nucleic acid assays for the assessment of the infectious risk of cell, blood, and tissue donors.

The impact on blood donor screening for human immunodeficiency virus, hepatitis C virus, and hepatitis B virus using plasma from frozen‐thawed plasma preparation tubes

“…Most studies have demonstrated that when plasma is frozen in separate aliquots there is minimal degradation of viral DNA or RNA over time . This, however, was not the case for HCV RNA in this study, where we observed a 50% decrease in viral load, or for HBV DNA, where the viral load increased.…”

“…However, there are few published studies describing the effect of freezing and thawing samples in PPTs, especially in terms of viral stability or qualitative detection of viral nucleic acids and antibodies and/or antigens. Where studies are available, either they are limited to a short storage period, restricted to one or two viral analytes, or they examine only plasma or serum aliquots . We therefore conducted this study to demonstrate that frozen and thawed plasma stored for long periods in a PPT continue to provide reliable qualitative blood donor NAT and serology screening results for HIV, HCV, and HBV for up to 36 months after freezing.…”

“…Where studies are available, either they are limited to a short storage period, restricted to one or two viral analytes, or they examine only plasma or serum aliquots. 5,9,10 We therefore conducted this study to demonstrate that frozen and thawed plasma stored for long periods in a PPT continue to provide reliable qualitative blood donor NAT and serology screening results for HIV, HCV, and HBV for up to 36 months after freezing.…”

“…However, this study was performed over a short time period and only included HCV and HBV serum samples removed from the RBC clot within 4 hours of collection. The stability of HCV, HIV, and HBV nucleic acid after long‐term storage in plasma aliquots has also been investigated, and although a small decrease in viral load was observed, the reduction did not exceed the estimated intra‐assay variation for molecular testing …”

“…The stability of HCV, HIV, and HBV nucleic acid after long-term storage in plasma aliquots has also been investigated, and although a small decrease in viral load was observed, the reduction did not exceed the estimated intra-assay variation for molecular testing. 10 In Australia, all blood donations are screened for the presence of HIV and HCV antibodies, HBV surface antigen (HBsAg), HIV and HCV RNA, and HBV DNA. 11 In 2012 a national sample archive was established for the purpose of retrospective testing and confirmation of test results.…”