Schottstedt V, Tuma W, Bünger G, Lakenberg P
DRK-Blutspendedienst Nordrhein-Westfalen, Institut Hagen, Deutschland.
Beitr Infusionsther Transfusionsmed. 1997;34:21-5.
We adapted the PCR method to screen up to 3,000 blood donations per day for HBV, HCV, and HIV-1 contamination. Concerning logistics: The first step is the generation of 3 identical microtiter plates (PT) by using the self-validated automatic sample processor with disposable tips. Using the first PT, we pooled up to 600 aliquots taken from blood donations which are serological negative and free for clinical usage according to actual federal regulations. In the case of a positive PCR pool result the viremic donation is identified by 2 additional PCR pool testing steps with smaller pool sizes using the second and third PT. All described steps are supported by electronic data processing. The PCR-method: After virus concentration by ultracentrifugation and--in case of HCV and HIV-1--additional reverse transcription PCR-amplifications were performed. PCR in two genomic regions are done for each virus. Laser-induced fluorescence detection after polyacrylamide gel electrophoresis and computer analysis were used to check the amplification products. Using this approach, a virus-containing donation can be detected in up to 599 negative samples with following sensitivity: HBV and HIV-1, 1,000-1,500 genome equivalents/ml; HCV, 2,000-2,500 genome equivalents/ml, thus sensitivity being in the range of commercial available PCR kits when testing nonpooled samples. The sensitivity was validated by using national and international available standards with known virus genome concentrations. All processing steps are checked using different controls such as, e.g., negative, positive, premix controls, reporter virus, inhibition controls. Routinely employing this validated methodology, we investigated 327,013 donations until the end of June 1996. During this survey, we found at least 16 virus-containing donations which are negative in corresponding serological tests and would have been transfused (4 HBV-, 13 HCV-, 0 HIV-1-containing donations), including one later seroconversion for HBV and one for HCV. Using our adapted PCR-methodology, it seems possible to shorten the diagnostic window periods with acceptable costs for large transfusion centers (15 DM per donation for all 3 viruses including all steps and investments). Therefore, PCR seems to become a new method of choice to prevent transfusion transmitted infections.
我们采用了聚合酶链反应（PCR）方法，每天对多达3000份献血进行筛查，以检测是否受到乙肝病毒（HBV）、丙肝病毒（HCV）和人类免疫缺陷病毒1型（HIV-1）的污染。关于后勤保障：第一步是使用配备一次性吸头的自动样本处理器，制备3个相同的微量滴定板（PT）。使用第一个PT，我们将根据实际联邦法规血清学检测呈阴性且可供临床使用的献血样本中的多达600份等分试样混合在一起。如果PCR混合检测结果呈阳性，则使用第二个和第三个PT，通过另外两个池大小更小的PCR混合检测步骤来确定病毒血症献血样本。所有上述步骤均由电子数据处理提供支持。PCR方法：通过超速离心进行病毒浓缩后，对于丙肝病毒和人类免疫缺陷病毒1型，还要进行额外的逆转录PCR扩增。针对每种病毒，在两个基因组区域进行PCR。使用聚丙烯酰胺凝胶电泳后的激光诱导荧光检测和计算机分析来检查扩增产物。采用这种方法，在多达599份阴性样本中可以检测到一份含有病毒的献血样本，其灵敏度如下：乙肝病毒和人类免疫缺陷病毒1型为1000 - 1500个基因组当量/毫升；丙肝病毒为2000 - 2500个基因组当量/毫升，因此在检测非混合样本时，其灵敏度与市售PCR试剂盒相当。通过使用具有已知病毒基因组浓度的国内和国际标准对灵敏度进行了验证。所有处理步骤均使用不同的对照进行检查，例如阴性对照、阳性对照、预混对照、报告病毒对照、抑制对照。截至1996年6月底，我们采用这种经过验证的方法对327013份献血进行了调查。在此次调查中，我们发现至少16份含有病毒的献血样本在相应的血清学检测中呈阴性，这些样本本应被输血（4份含乙肝病毒、13份含丙肝病毒、0份含人类免疫缺陷病毒1型的献血样本），其中包括1份后来出现乙肝病毒血清转化和1份丙肝病毒血清转化的样本。使用我们改进的PCR方法，对于大型输血中心而言，似乎有可能以可接受的成本（包括所有步骤和投资，每份献血检测三种病毒的成本为15德国马克）缩短诊断窗口期。因此，PCR似乎将成为预防输血传播感染的一种新的首选方法。
