Laboratory Issues

The use of internal controls for a test increases the confidence in negative HCV RNA test results and eliminates concerns that the test is falsely negative because of the presence of amplification inhibitors in the sample or poor recovery of the viral RNA. In a large clinical trial of the Amplicor HCV test v2.0, the failure rate for the internal control was only 1.1%.66 False-negative results may occur with PCR-based tests if blood samples are collected through central lines due to contamination with heparin. False-negative HCV RNA results also may occur if the specimen was not processed or stored appropriately due to the lability of the viral RNA. Serum or plasma should be separated from the cellular components of blood within 6 hours of collection to avoid significant loss of HCV RNA. Once separated, the sample is stable for 3 days at 4°C. Storage for longer periods should be at -70°C.

False-positive HCV RNA tests can result from contamination of the sample with HCV target RNA or amplicon. The widely used Amplicor HCV test employs the uracil-N-glycolase/dUTP protocol and an equivocal zone for low-level results to limit the number of false-positive test results. Real-time RT-PCR tests for HCV RNA eliminate the risk of amplicon carryover contamination because both amplification and detection are accomplished in a sealed reaction tube.

Much has been written about the lack of agreement and the genotype bias of early commercially available versions of the HCV quantitative assays. The development the WHO First International HCV RNA standard and its acceptance by the manufacturers of these assays as a calibrator was a significant advance in HCV RNA quantitation.94 As a result, viral load values obtained with current versions of the Amplicor Monitor and Versant quantitative tests are in much better agreement and are largely interchangeable. The remaining disagreement probably results from differences in the dynamic ranges of the assays. The agreement between results for high viral load samples can be improved if the samples are diluted 1: 100 prior to testing with the Amplicor Monitor assay.

Both the current versions of the Amplicor Monitor and Versant quantitative tests are free of significant genotype bias due to improvements in assay design. The Versant test is more amenable to high-volume testing and is less labor-intensive than the Amplicor Monitor test, while the Amplicor Monitor test may have advantages for laboratories with a lower test volume. Recently a sample preparation instrument, the COBAS Ampliprep, was developed to automate the cumbersome manual sample preparation method used for the Amplicor Monitor test. The automated sample preparation protocol compared favorably to the manual protocol for use with the COBAS Amplicor HCV Monitor test v2.0 in a recent clinical evaluation.95

Unfortunately, there is a trade-off between sensitivity and dynamic range for most of the HCV RNA tests currently used in clinical laboratories. As a result, most laboratories have separate qualitative and quantitative tests. The analytical sensitivities of the Amplicor Monitor and Versant quantitative tests are inadequate for assessing ETR or for reliable diagnostic use for some untreated chronically infected patients with low viral loads. Maintaining multiple assay formats for the same virus is costly for the laboratory and often confusing to the ordering physician. The best hope for unifying the HCV RNA assay platforms lies in the real-time PCR methods, which combine the sensitivity required for diagnosis and assessment of treatment response with the broad dynamic range required for viral load determinations.

Genotyping schemes based on variable subgenomic regions such as E1, core, and NS5B provide enough resolution to reliably determine HCV types and subtypes; however, the 5' UTR is too conserved for accurate discrimination of all subtypes.59 Nevertheless, the conserved nature of this region makes it the preferred target for pangenomic HCV RNA detection tests, and sequence analysis of amplicons from these tests is an efficient way to genotype HCV in a clinical laboratory since both tests can be completed with the product from a single amplification reaction. The genotyping results obtained using 5' UTR amplicons are highly accurate at the genotype level but are not accurate for identifying different subtypes.48,82,96

The widespread use of tests not cleared by the FDA for HCV RNA quantitation and genotyping has placed an increased burden on clinical laboratories to verify the performance characteristics of these tests prior to clinical use. When validating HCV tests, laboratories should take advantage of the published evaluations and commercially available panels for HCV RNA quantitation and genotyp-ing to streamline the verification process.

The College of American Pathologists has a well-established proficiency-testing program for laboratories performing tests for detection, quantitation, and characterization of HCV RNA. These surveys have shown a steady improvement in the performance of laboratories over time that probably reflects progress in both the available technologies and laboratory practices.

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