Zucker K, Cirocco R, Roth D, Olson L, Burke G W, Nery J, Esquenazi V, Miller J
Department of Surgery, University of Miami School of Medicine, Florida.
Transplantation. 1994 Mar 27;57(6):832-40. doi: 10.1097/00007890-199403270-00011.
The safety of the use of kidneys procured from a hepatitis C virus (HCV)-positive cadaver donors in transplantation has recently been the subject of controversy. One factor that is important in determining the transmission of the virus and/or viral liver disease is the total viral inoculum to which the renal allograft recipient is exposed as a result of the transplant. We have studied the effect of a standard pulsatile renal preservation procedure and variations of it on the number of viral copies in organs from HCV-positive donors. An HCV-RNA quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was utilized with a recombinant competitive inhibitor substrate added after cDNA synthesis with the PCR primers within the relatively non-polymorphic 5' untranslated region of the HCV genome. Additionally, strain specificity was found to be detectable using a modification of the technique of restriction fragment-length polymorphism (RFLP-PCR), so that virus from the organ donor could be specifically identified and quantified in the recipient. It was observed that standard preservation procedures using pulsatile perfusion were able to eliminate 75% of the virus from the organ in 20 hr. By modifying this procedure to include additional wash steps and a second pulsatile perfusion, greater than 99% of the virus could be eliminated from the kidney. In a related study, we used quantitative PCR to study requirements for filtration of the virus using HCV-positive serum. It was found that a high-flow-rate ultrafilter with a molecular weight cut-off (MWCO) of 300,000 daltons placed in series to the preservation apparatus was very efficient in eliminating the virus from perfusion solution in less than 2 hr. It can therefore be proposed that with the use of these molecular techniques, pulsatile perfusion coupled with additional viral depletion steps (dilution, and/or filtration) may allow the practical reduction of HCV transmission risk in recipient follow-up studies. The means are thereby presented for similar manipulation of other known or, as yet, unknown transmissible agents.
使用丙型肝炎病毒（HCV）阳性尸体供体的肾脏进行移植的安全性近来一直是争议的焦点。在确定病毒传播和/或病毒性肝病方面，一个重要因素是肾移植受者因移植而接触到的病毒接种总量。我们研究了标准的脉动式肾脏保存程序及其变体对HCV阳性供体器官中病毒拷贝数的影响。采用HCV-RNA定量逆转录-聚合酶链反应（RT-PCR）方法，在HCV基因组相对非多态性的5'非翻译区内用PCR引物合成cDNA后添加重组竞争性抑制剂底物。此外，发现使用限制性片段长度多态性技术（RFLP-PCR）的改良方法可检测菌株特异性，从而能够在受者中特异性鉴定和定量来自器官供体的病毒。据观察，使用脉动灌注的标准保存程序能够在20小时内从器官中清除75%的病毒。通过修改该程序，增加额外的冲洗步骤和第二次脉动灌注，可以从肾脏中清除超过99%的病毒。在一项相关研究中，我们使用定量PCR研究使用HCV阳性血清过滤病毒的要求。结果发现，一个分子量截留值（MWCO）为300,000道尔顿的高流量超滤器串联在保存装置上，在不到2小时的时间内就能非常有效地从灌注液中清除病毒。因此可以提出，通过使用这些分子技术，脉动灌注结合额外的病毒清除步骤（稀释和/或过滤）可能会在受者后续研究中切实降低HCV传播风险。由此提供了对其他已知或未知可传播病原体进行类似处理的方法。
