European multi-centre validation study of NucliSens Extractor in combination with HCV Amplicor 2.0 assay for HCV-NAT screening of plasma pools

• Published in: Biologicals Vol. 27; no. 4; pp. 303 - 314
• Main Authors: Cuypers, H T, van Dijk, R, Viret, J F, Schottstedt, V, Lankinen, M, Da Silva Cardoso, M, Lelie, P N
• Format: Journal Article
• Language: English
• Published: England 01-12-1999
• Subjects: Europe; Evaluation Studies as Topic; Genotype; Guidelines as Topic; Hepacivirus - genetics; Hepacivirus - isolation & purification; Hepatitis C virus; Humans; Nucleic Acid Amplification Techniques; nucleic acid amplification technology; Plasma - virology; Reference Standards; Reproducibility of Results; RNA, Viral - blood; RNA, Viral - genetics; World Health Organization; Europe
• ISSN: 1045-1056
• DOI: 10.1006/biol.1999.0225

The NucliSens Extractor in combination with the 2.0 version of the Roche Cobas HCV Amplicor test has been validated by five European blood screening laboratories in a multi-centre study. For testing the performance characteristics of this HCV-NAT method, the European Pharmacopoeia validation guidelines were followed. The CLB VQC reference reagents were used for testing robustness and sensitivity. After a technical improvement in the extraction stations, the NucliSens Extractor appeared to be contamination-free as was proved by testing negative controls alternating with samples containing a high HCV-RNA concentration. The Pelicheck HCV-RNA genotype 1 dilution panel was tested 74 times in the five laboratories and an overall 95% detection limit of 80 genome equivalents (geq)/ml was found. In one laboratory the Pelicheck panel was tested in 25 runs and here a 95% detection limit of 32 geq/ml was achieved. In this laboratory the Pelispy HCV-RNA run control samples of 140 geq/ml were consistently picked up in all extractor stations. In addition the laboratories have tested a WHO HCV-RNA genotype 1 standard dilution series 39 times and a Pelicheck HCV-RNA genotype 3 reference panel in 32 test runs. The limiting dilution analysis enabled us to compare the detection efficiency of the NucliSens-Amplicor method for the genoype 1 and genotype 3 isolates and to calibrate the reference reagents against each other. The combined Nuclisens-Amplicor method was found to detect the genotype 3 isolate in the Pelicheck HCV-RNA panels with 2-3 fold lower efficiency than the genotype 1 standard (assuming that the historical calibration of the genotype 3 against the genotype 1 standard is correct). In this study of a single method 1 IU of the WHO HCV-RNA standard was found to be equivalent to 5.1 geq of the VQC HCV-RNA standard (95% confidence intervals 3.1-9.1 geq). To avoid confusion with the use of the CLB VQC reagents we accept the NIBSC collaborative study in which calibration by a variety of methods showed that the Pelispy 380 geq/ml run control is equivalent to 100 IU/ml of the WHO standard. This multi-centre validation study demonstrates that the 95% detection limit of the NucliSens HCV Amplicor method lies far below the detection limits required by the international regulatory bodies.