Beld M, Habibuw M R, Rebers S P, Boom R, Reesink H W
Department of Medical Microbiology, University of Amsterdam, the Netherlands.
Transfusion. 2000 May;40(5):575-9. doi: 10.1046/j.1537-2995.2000.40050575.x.
The objective of this study was the evaluation of NAT technology for the detection of HCV RNA in plasma pools according to the recommendations of the Paul Ehrlich Institute (5000 IU/mL/donation) and the Committee for Proprietary Medical Products (100 IU/mL/manufacturing pool).
Serial dilutions of both the EUROHEP standard (3,800 genome equivalents [geq]/mL; HCV genotype 1) and the World Health Organization (WHO) international standard (100,000 IU/mL; HCV genotype 1) were made in S/D plasma (ESPEP plasma, OctaPharma), which was nonreactive in serologic tests. Serial dilutions of plasma (2 mL) were used for extraction of HCV RNA with an automated version of a nucleic acid isolation method (NucliSens Extractor, Organon Teknika). HCV RNA was co-extracted from 2 mL of plasma, together with 84 copies of an in vitro-synthesized single-strand RNA serving as internal extraction control (IC) to monitor the efficiency of extraction and PCR. Amplification and detection of both HCV RNA and IC RNA were performed with an automated PCR system and a qualitative HCV assay (COBAS Amplicor 2.0 HCV, Roche Diagnostics).
A cutoff value of 16 geq per mL (10/10 runs [100% hit rate]) was found by using the EUROHEP standard, whereas the WHO international standard had a cutoff value of approximately 12 IU per mL (10/10 runs [100% hit rate]). The IC had a cutoff value of approximately 17.5 copies per mL (6/6 runs [100% hit rate]). Forty-two copies per mL of IC RNA were found in 282 of 284 runs (99% hit rate). The negative controls (ESDEP plasma) were negative in all experiments. Experiments with pool sizes of 12, 24, 48, and 96 using serial dilutions of the WHO international standard revealed a cutoff value of 8 IU per mL (100% hit rate). The EUROHEP standard and the WHO international standard were detected with a 50 percent detection endpoint of 5.2 geq per mL and 1.5 IU per mL, respectively.
This test system (NucliSens Extractor, and the COBAS Amplicor 2.0 HCV assay) revealed a high sensitivity for HCV RNA; considering the proposed requirements for sensitivity of NAT assays for the detection of HCV RNA in donor plasma, pool sizes of about 400 donors are possible. These endpoint results indicated that 1 IU is equal to about 3.4 geq.
本研究的目的是根据保罗·埃利希研究所（每单位献血5000国际单位/毫升）和专利医疗产品委员会（每生产池100国际单位/毫升）的建议，评估核酸扩增技术（NAT）检测血浆混合样本中丙型肝炎病毒（HCV）RNA的效果。
在血清学检测呈阴性的S/D血浆（ESPEP血浆，奥克塔法玛公司）中，对欧洲肝脏研究学会（EUROHEP）标准品（3800基因组当量/毫升；HCV 1型）和世界卫生组织（WHO）国际标准品（100000国际单位/毫升；HCV 1型）进行系列稀释。取2毫升血浆系列稀释样本，采用核酸分离方法的自动化版本（NucliSens核酸提取仪，奥加农公司）提取HCV RNA。从2毫升血浆中同时提取HCV RNA和84个拷贝的体外合成单链RNA作为内源性提取对照（IC），以监测提取和PCR的效率。使用自动化PCR系统和定性HCV检测方法（COBAS Amplicor 2.0 HCV，罗氏诊断公司）对HCV RNA和IC RNA进行扩增和检测。
使用EUROHEP标准品时，每毫升的临界值为16基因组当量（10次运行中有10次[100%命中率]），而WHO国际标准品的临界值约为每毫升12国际单位（10次运行中有10次[100%命中率]）。IC的临界值约为每毫升17.5个拷贝（6次运行中有）。在284次运行中的282次（99%命中率）中，每毫升IC RNA检测到42个拷贝。所有实验中的阴性对照（ESDEP血浆）均为阴性。使用WHO国际标准品系列稀释样本进行的池容量为12、24、48和96的实验显示，临界值为每毫升8国际单位（100%命中率）。EUROHEP标准品和WHO国际标准品的50%检测终点分别为每毫升5.2基因组当量和1.5国际单位。
该检测系统（NucliSens核酸提取仪和COBAS Amplicor 2.0 HCV检测方法）对HCV RNA显示出高灵敏度；考虑到对检测献血者血浆中HCV RNA的NAT检测灵敏度的建议要求，约400名献血者的混合样本量是可行的。这些终点结果表明，1国际单位约等于3.4基因组当量。 6次[100%命中率]）
