Validation of the NucliSens Extractor in combination with the hepatitis C virus Cobas Amplicor 2.0 assay in four laboratories in the Netherlands utilizing nucleic acid amplification technology for blood screening

Background and Objectives Since July 1 1999, four laboratories in the Nethe rlands have been routinely screening plasma minipools for the release of la bile blood components utilizing hepatitis C virus nucleic acid amplificatio n technology (HCV NAT). This report describes the performance evaluation of the HCV NAT method and the quality control results obtained during 6 month s of routine screening. Materials and Methods Plasma minipools of 48 donations were prepared on a T ecan Genesis robot. HCV RNA was isolated from 2 ml of plasma by using the N ucliSens Extractor and amplified and detected with the Cobas HCV Amplicor 2 .0 test system. For validation of the test system the laboratories used vir al quality control (VQC) reagents of CLB. Results Initial robustness experiments demonstrated consistent detection of PeliSpy HCV RNA samples of 140 genome equivalents/ml (geq/ml) in each stat ion of the installed Nuclisens Extractors. Further 'stress' tests with a hi ghly viraemic sample of approximate to 5.10(6) geq/ml did not contaminate n egative samples processed on all Extractor stations in subsequent runs. In the validation period prior to July 1999, 1021 pools were tested with the f ollowing performance characteristics: 0.1%, initially false reactive; 0.89% , failure of internal control detection; 0.97%, no eluate generated by the Extractor; and 100% reactivity of the PeliSpy 140 geq/ml control in 176 Ext ractor runs and a 98% reactivity rate of the PeliSpy 38 geq/ml control in 1 02 test runs. By testing the PeliCheck HCV RNA genotype I dilution panels 4 9 times, an overall 95% detection limit of 30 geq/ml (approximate to8 IU/ml ) and a 50% detection limit of 5 geq/ml was found by the four laboratories. In the first 6 months of routine screening, the minimum requirement for in valid results (2%) was exceeded with some batches of silica and NucliSens E xtractor cartridges. From November 1999 to February 2000, the manufacturer (Organon Teknika) improved the protocol for silica absorption of the Nuclis ens Extractor - the cartridge design as well as the software of the Extract or. During the next 6 months of observation in 2000, the percentages of fal se initial reactives and invalids were 0.05% and 1.4%, respectively, in 896 2 pools tested. Of these invalid results, 0.74% and 0.66% were caused by Ex tractor failure and negative internal control signals, respectively. The Pe liSpy HCV RNA 'stop or go' run control of 140 geq/ml was 100% reactive, but invalid in 16/1375 (1.2%) of cases. The PeliSpy run control of 38 geq/ml f or monitoring sensitivity of reagent batches was reactive in 95% of 123 sam ples tested. Conclusions Each of the four HCV NAT laboratories in the Netherlands have a chieved similar detection limits that are well below the sensitivity requir ements of the regulatory bodies. After improvement of the NucliSens Extract or procedure, the robustness of the test system has proved to be acceptable for routine screening and timely release of all labile blood components.