Forcić D, Zgorelec R, Branović K, Kosutić-Gulija T, Santak M, Mazuran R
Department of Molecular Biomedicine, Institute of Immunology Inc, Zagreb, Croatia.
Transfus Apher Sci. 2001 Jun;24(3):269-78. doi: 10.1016/s1473-0502(01)00069-6.
The risks of transmitting viral infection by blood and products derived from plasma have long been known and still remain an area of concern. Blood banks and transfusion centres are faced with the imminent introduction of nucleic acid amplification testing (NAT) of plasma pools as used by the plasma industry. In this paper, we show a part of our results of a validation study of an in-house method for routine polymerase chain reaction (PCR) screening for hepatitis C virus (HCV) RNA in plasma pools and the results of testing 2,718 anti-HCV negative plasma pools for the presence of HCV RNA. The European Committee for Proprietary Medical Products (CPMP) recommended that from 1 July 1999, only batches derived from plasma pools tested and found non-reactive for HCV RNA, using validated test methods of suitable sensitivity and specificity, should be batch released by authorities. The quality and efficiency of NAT detection of HCV RNA is among others influenced by the efficacy of RNA isolation, the primer selection and the use of control samples. Using modern molecular biology techniques (sensitive and specific in-house amplification methods for detection of HCV RNA and automated sequencing), we analysed samples of plasma pools from different Croatian transfusion centres. By detection of HCV RNA in an NIBSC working reagent (genotype 3) and a Pelispy HCV RNA run control (genotype 1) we determined a high reproducibility and sensitivity (below 100 International Units (IU)/ml) for our in-house method. By direct sequencing PCR cDNAs we proved the specificity of the test system and the possibility of determining the HCV genotype when the method was used for PCR screening of HCV RNA in single donations. Of 2,718 anti-HCV negative plasma pools we have found that 2.1$ were HCV RNA positive. Results of our investigation confirm the necessity of testing HCV RNA in plasma pools to further increase the safety of human plasma-derived drugs.
通过血液及血浆衍生产品传播病毒感染的风险早已为人所知，至今仍是一个令人担忧的领域。血库和输血中心面临着即将引入血浆行业所采用的血浆池核酸扩增检测（NAT）的情况。在本文中，我们展示了一项内部方法验证研究的部分结果，该方法用于常规聚合酶链反应（PCR）筛查血浆池中丙型肝炎病毒（HCV）RNA，以及对2718份抗-HCV阴性血浆池进行HCV RNA检测的结果。欧洲 proprietary 药品委员会（CPMP）建议，自1999年7月1日起，只有使用经验证的具有适当灵敏度和特异性的检测方法检测且HCV RNA呈非反应性的血浆池衍生批次，才可由当局进行批次放行。HCV RNA的NAT检测质量和效率尤其受到RNA分离效果、引物选择和对照样品使用的影响。我们使用现代分子生物学技术（用于检测HCV RNA的灵敏且特异的内部扩增方法和自动测序），分析了来自克罗地亚不同输血中心的血浆池样本。通过检测NIBSC工作试剂（基因型3）和Pelispy HCV RNA运行对照（基因型1）中的HCV RNA，我们确定了我们的内部方法具有高重现性和灵敏度（低于100国际单位（IU）/毫升）。通过对PCR cDNA进行直接测序，我们证明了检测系统的特异性以及在该方法用于单份献血中HCV RNA的PCR筛查时确定HCV基因型的可能性。在2718份抗-HCV阴性血浆池中，我们发现2.1%为HCV RNA阳性。我们的调查结果证实了检测血浆池中HCV RNA对于进一步提高人血浆衍生药物安全性的必要性。
