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.
Gessoni
G
G
Regione Veneto, A-ULS 14 Chioggia Clinical Pathology Department, Ospedale Civile, Via Madonna Marina 500, 30015 Chioggia VE, Italy. ggessoni@asl14chioggia.veneto.it
Barin
P
P
Valverde
S
S
Giacomini
A
A
Di Natale
C
C
Orlandini
E
E
Arreghini
N
N
De Fusco
G
G
Frigato
A
A
Fezzi
M
M
Antico
F
F
Marchiori
G
G
D001793:Blood Preservation / Q000379:methods*; D004279:DNA, Viral / Q000302:isolation & purification*; D015658:HIV Infections / Q000097:blood / Q000517:prevention & control; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification; D006509:Hepatitis B / Q000097:blood / Q000517:prevention & control; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification; D006526:Hepatitis C / Q000097:blood / Q000517:prevention & control; D006801:Humans; D009696:Nucleic Acids / Q000097:blood*; D020871:RNA Stability; D012367:RNA, Viral / Q000302:isolation & purification*; D012034:Refrigeration / Q000592:standards / Q000706:statistics & numerical data*; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors; D019562:Viral Load / Q000706:statistics & numerical data
D004279:DNA, Viral; D009696:Nucleic Acids; D012367:RNA, Viral