Biological qualification of blood units: considerations about the effects of sample's handling and storage on stability of nucleic acids.

BACKGROUND: In transfusional setting introduction of nucleic amplification technique (NAT) for HBV-DNA, HCV-RNA and HIV-RNA in biological qualification of blood units suggest some problems. At first the opportunity to operate on mini-pool, at second the need to store the samples at +4 degrees C. The authors therefore have tried to estimate the impact of these conditions on the operativity of NAT testing in the transfusional setting. METHODS: The following parameters has been estimated: distribution of viral-load in untreated subjects, stability of nucleic acids during storage at +4 degrees C, stability of nucleic acids after repeated cycles of freezing and defrosting, robustness of the test to the cross-contamination, definition of the detection-limit (95%). Quantitative tests has been performed by using the following kits: Cobas Amplicor HBV Monitor, Cobas Amplicor HCV Monitor, Cobas Amplicor HIV Monitor; the qualitative tests has been performed by using the following kits: Ampliscreen HBV, Ampliscreen HCV 2,0, Ampliscreen HIV 1,5 all supplied by Roche Molecular System (Brancburg, NJ). RESULTS: Viral load in untreated subjects showed wide variation for HBV, HCV and HIV. HBV has been demonstrated much stable to the conservation +4 degrees C also until 168 h while for HCV and HIV a greater decrease of the viral-load was observed. For all and three virus the conservation to +4 degrees C until 72 h does not seem to involve meaningful fall in the viral-load. A remarkable reduction of the viral-load has been observed after five cycles of freezing and defrosting. All the tests showed a good robustness to cross-contamination. The detection-limit (95%) was 8 U/ml for HBV, 21 U/ml for HCV and 27 copy/ml for HIV. CONCLUSIONS: Samples for NAT testing, can be stored until 72 h to +4 degrees C without appreciable lowering of the viral-load. Repeated cycles of changes of state should be avoided. The tests showed a good robustness to cross-contamination. NAT tests for biological qualification of blood units had a minimal sensibility around 50 (copy/unit/ml). In our experience the detection-limit (95%) was 21 U/ml for HCV, 27 copies/ml for HIV, 8 U/ml for HBV. The availability of NAT test for HBV-DNA, HCV-RNA e HIV-RNA, sensitive and reliable, together with epidemiological data, suggest the opportunity to place side by side, in the biological qualification of the blood units, to add the tests for HBV-DNA and HIV-RNA to the test for HCV-RNA mandatory by low, in Italy in the biological qualification of blood units.

The stability of hepatitis C virus RNA after storage at +4 degrees C.

One of the major issues in nucleic acid testing is how to store blood samples to obtain reliable results. We therefore studied hepatitis C virus (HCV) RNA concentration in samples after storage at +4 degrees C or freezing and thawing. Six HCV RNA-positive, untreated subjects were studied. Blood samples were collected from these subjects in plasma preparation tubes. The HCV RNA concentration was analysed after storage at +4 degrees C for 168 h or after five freeze-thaw cycles. For HCV RNA quantification we used a qualitative and a quantitative commercial test. After 168 h of storage at +4 degrees C, the HCV RNA concentration was similar to that observed at time-point 0 (5.025 log vs 5.492 log). In one sample we observed a significant fall in HCV RNA concentration. After five freeze-thaw cycles, the HCV RNA concentration was lower than that observed at time-point 0 (4.454 log vs 5.492 log) and in four samples we observed a significant fall in HCV RNA concentration. Our data suggest that HCV RNA is stable in whole blood samples stored at +4 degrees C for 168 h. Based on our results, we conclude that the standard procedures for transport of blood samples (at room temperature for a maximum of 5 h) and storage schedules (+4 degrees C for a maximum of 48 h) can be maintained without compromising the quality of results.