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.