Evaluation of automated RNA-extraction technology and a qualitative HCV assay for sensitivity and detection of HCV RNA in pool-screening systems

BACKGROUND: The objective of this study was the evaluation of NAT technolog y for the detection of HCV RNA in plasma pools according to the recommendat ions of the Paul Ehrlich Institute (5000 IU/mL/donation) and the Committee for Proprietary Medical Products (100 IU/mL/manufacturing pool). STUDY DESIGN AND METHODS: Serial dilutions of both the EUROHEP standard (3, 800 genome equivalents [geq]/mL; HCV genotype 1) and the World Health Organ ization (WHO) international standard (100,000 IU/mL; HCV genotype 1)were ma de in S/D plasma (ESPEP plasma, OctaPharma), which was nonreactive in serol ogic tests. Serial dilutions of plasma (2 mt) were used for extraction of H CV RNA with an automated version of a nucleic acid isolation method (NucliS ens Extractor, Organon Teknika). HCV RNA was cc-extracted from 2 mt of plas ma, together with 84 copies of an in vitro-synthesized single-strand RNA se rving as internal extraction control (IC) to monitor the efficiency of extr action 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). RESULTS: A cutoff value of 16 geq per mt (10/10 runs [100% hit rate]) was f ound by using the EUROHEP standard, whereas the WHO international standard had a cutoff value of approximately 12 IU per mt (10/10 runs [100% hit rate ]). The IC had a cutoff value of approximately 17.5 copies per mt (6/6 runs [100% hit rate]). Forty-two copies per mt of IC RNA were found in 282 of 2 84 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 mt (100% hit rate). The EUROHEP standard and the WHO internati onal standard were detected with a 50 percent detection endpoint of 5.2 geq per mt and 1.5 IU per mt, respectively. CONCLUSION: This test system (NucliSens Extractor, and the COBAS Amplicor 2 .0 HCV assay) revealed a high sensitivity for HCV RNA; considering the prop osed requirements for sensitivity of NAT assays for the detection of HCV RN A in donor plasma, pool sizes of about 400 donors are possible. These endpo int results indicated that 1 IU is equal to about 3.4 geq.