Gessoni G, Barin P, Valverde S, Giacomini A, Di Natale C, Orlandini E, Arreghini N, De Fusco G, Frigato A, Fezzi M, Antico F, Marchiori G
Regione Veneto, A-ULS 14 Chioggia Clinical Pathology Department, Ospedale Civile, Via Madonna Marina 500, 30015 Chioggia VE, Italy.
Transfus Apher Sci. 2004 Jun;30(3):197-203. doi: 10.1016/j.transci.2003.11.010.
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.
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).
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.
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.
在输血领域，将核酸扩增技术（NAT）用于血液制品的生物学检测中检测乙肝病毒脱氧核糖核酸（HBV-DNA）、丙肝病毒核糖核酸（HCV-RNA）和人类免疫缺陷病毒核糖核酸（HIV-RNA）存在一些问题。首先是对混合样本进行检测的可行性，其次是样本需要在4℃下保存。因此，作者试图评估这些条件对输血领域NAT检测操作的影响。
评估了以下参数：未治疗患者的病毒载量分布、核酸在4℃保存期间的稳定性、反复冻融循环后核酸的稳定性、检测对交叉污染的耐受性、检测限（95%）的定义。定量检测使用了以下试剂盒： Cobas Amplicor HBV Monitor、Cobas Amplicor HCV Monitor、Cobas Amplicor HIV Monitor；定性检测使用了以下试剂盒：Ampliscreen HBV、Ampliscreen HCV 2.0、Ampliscreen HIV 1.5，均由罗氏分子系统公司（新泽西州布兰堡）提供。
未治疗患者的病毒载量在乙肝、丙肝和艾滋病毒方面显示出很大差异。已证明乙肝病毒在4℃保存至168小时也非常稳定，而丙肝和艾滋病毒的病毒载量则有较大下降。对于所有三种病毒，在4℃保存至72小时似乎不会导致病毒载量显著下降。经过五个冻融循环后，病毒载量显著降低。所有检测对交叉污染均显示出良好的耐受性。乙肝的检测限（95%）为8 U/ml，丙肝为21 U/ml，艾滋病毒为27拷贝/ml。
用于NAT检测的样本可以在4℃下保存72小时，而病毒载量不会明显降低。应避免反复的状态变化循环。检测对交叉污染显示出良好的耐受性。血液制品生物学检测的NAT检测最低灵敏度约为50（拷贝/单位/毫升）。根据我们的经验，丙肝的检测限（95%）为21 U/ml，艾滋病毒为27拷贝/ml，乙肝为8 U/ml。乙肝病毒脱氧核糖核酸、丙肝病毒核糖核酸和人类免疫缺陷病毒核糖核酸的NAT检测灵敏且可靠，结合流行病学数据，表明在意大利血液制品生物学检测中，在进行丙肝病毒核糖核酸强制检测的同时，有必要增加乙肝病毒脱氧核糖核酸和人类免疫缺陷病毒核糖核酸检测。
Zotero 插件