Clinical Performance Evaluation of Four Automated Chemiluminescence Immunoassays for Hepatitis C Virus Antibody Detection

Assay systems.Four automated CLIAs were compared, the Elecsys Anti-HCV assay on the Cobas e 411 analyzer (Roche Diagnostics, Mannheim, Germany), the Architect Anti-HCV assay on the Architect i2000 system (Abbott Laboratories, Abbott Park, IL), the Vitros Anti-HCV assay on the Vitros ECiQ Immunodiagnostic System (Ortho-Clinical Diagnostics, Raritan, NJ), and the Access HCV Ab PLUS assay (Bio-Rad Laboratories, Redmond, WA) on the UniCel DxI 800 analyzer (Beckman-Coulter, Fullerton, CA). The characteristics of the four reagents and the technical specifications of each instrument are summarized in Table 1.

Precision tests of four CLIAs.The reproducibility of each CLIA was determined by using a modified form of the EP5-A2 protocol of the Clinical and Laboratory Standards Institute (7). Experiments were performed twice a day for 10 days in duplicate with negative and positive quality control materials recommended by each manufacturer.

CLIA screening.A total of 400 consecutive unselected fresh serum samples sent daily to our laboratory underwent anti-HCV testing with four CLIAs. We prospectively collected HCV-positive samples screened by Genedia HCV ELISA 3.0 (Greencross Life Science, Seoul, Korea) from December 2007 to April 2008. These sera, which had been stored frozen at −20°C, were then tested with the four CLIAs and for HCV RNA. However, there were limitations in sample volume; the Vitros and Access assays were performed with only 127 and 140 samples, respectively. The four anti-HCV CLIAs were carried out according to the manufacturers' instructions.

Confirmation of results.Samples showing positive results from any CLIA were investigated by medical record review and confirmatory testing. The medical record review involved the examination of any clinical or laboratory evidence of chronic HCV infection. These samples were investigated for the presence of clinical conditions that could interfere with the accuracy of CLIA results such as autoimmune disease, dialysis, pregnancy, bacterial infection, and rheumatoid factor.

Measurements of HCV RNA were performed with samples with undetermined HCV status by medical record review and any discrepancy in the CLIA results. HCV RNA was detected by the COBAS Amplicor HCV 2.0 qualitative assay and the COBAS TaqMan HCV assay (both from Roche Diagnostics). In the COBAS Amplicor HCV 2.0 assay, the viral genome was extracted with the HCV Specimen Preparation Kit; details of the assay have been described elsewhere (25). The lower detection limit of the COBAS Amplicor HCV 2.0 assay is 50 IU/ml. In the COBAS TaqMan HCV assay, the viral genome was extracted by automated COBAS AmpliPrep instruments and HCV RNA was amplified and detected with the COBAS TaqMan Analyzer (24). The lower detection limit of the COBAS TaqMan HCV assay is 15 IU/ml.

Recombinant immunoblot assays (RIBA) were additionally performed with samples with HCV RNA-negative results by using LG HCD Confirm (LG Life Sciences, Seoul, Korea) according to the manufacturer's instruction.

Precision.The precision of each CLIA was evaluated by using the commercial negative and positive control materials recommended by each manufacturer. Each control was run twice a day for 10 days in duplicate (40 runs in 10 days). Precision results for each CLIA are shown in Table 2. Intra-assay and total percent coefficient of variation values for positive controls ranged from 2.8 to 4.7% and 3.5 to 5.7%, respectively. Due to low mean values for the negative control, intra-assay and total percent coefficient of variation values for negative controls ranged from 5.5 to 9.1% and 7.2% to 10.2%, respectively.

Comparison of CLIA results.The overall correlation of the results of the four CLIAs is shown in Table 3. The agreement between the results of the Elecsys, Architect, and Vitros CLIAs ranged from 97.1 to 98.1%. The agreement between the results of the Access CLIA and the other CLIAs ranged from 94.5 to 97.0%.

There was a limitation on sample volume; the Access CLIA could not be performed with every sample, and the percent agreement ranged from 94.5 to 97.0%, slightly lower than that of the others. Among 267 samples assayed by all of the CLIAs (Table 4), the overall concordance rate was 94.0% (251/267). Sixteen samples showing discrepant results were confirmed negative by either the COBAS Amplicor HCV 2.0 qualitative assay or the COBAS TaqMan HCV assay for detection of HCV RNA. Furthermore, an additional confirmatory RIBA was performed with 14 of 16 HCV RNA-negative samples; the remaining 2 samples could not be confirmed by RIBA due to lack of sample volume. Ten samples were RIBA negative, and four were RIBA indeterminate.

Clinical specificity.To assess the specificity of each CLIA, we confirmed HCV infectious status with the following algorithm. Among 267 samples assayed by all CLIAs, 160 showing negative results in all CLIAs were categorized as “screening test negative” and did not undergo any supplemental testing as recommended by the CDC (4). Among 107 samples showing positive results in any of the CLIAs, 54 were further investigated based on clinical data and medical record review as described above due to insufficient sample volume as a result of the consumption of large volumes during comparison experiments and repeat tests. Therefore, 30 samples were further categorized as “clinically confirmed positive” and 24 in which HCV status could not be confirmed were excluded from the specificity analysis.

The other 53 samples were tested for HCV RNA. If HCV RNA test were negative, a further confirmatory RIBA was performed. Any samples showing indeterminate HCV RNA or RIBA results were excluded from the specificity analysis. The specificity of each CLIA ranged from 96.5 to 98.8% (Table 5). There were no false-negative results from any CLIA, making the sensitivity of each assay 100% in our experiment.

Detection of HCV RNA in relation to CLIA s/co ratios.We analyzed the s/co ratio result of samples confirmed by HCV RNA and RIBA or confirmed by retrospective medical record review (clinically confirmed positive). The number of cases with HCV infection increased in relation to the s/co ratio (Table 6). In the Vitros assay, only 1 (5.9%) of 17 cases with an s/co ratio of <8.0 had HCV infection. In the Architect and Access assays, 1 (7.1%) of 14 and 1 (5.9%) of 17 cases with an s/co ratio of <3.0 had HCV infection, respectively. However, in the Elecsys, we could not precisely identify the s/co ratio predictive of HCV infection negativity for more than 95% of the samples due to the paucity of samples with a low s/co ratio.

The guidelines for laboratory testing and result reporting of antibodies to HCV from the CDC (4) include an option for reflex supplemental testing based on screening test-positive s/co ratios. Use of s/co ratios could minimize the amount of supplemental testing that needs to be performed while improving the reliability of reported test results. In short, screening test-positive results are classified as having high s/co ratios if their ratios are at or above a predetermined value that predicts a supplemental test (HCV RNA or RIBA) positive result for ≥95% of the samples tested. However, only the Vitros Anti-HCV assay has been approved by the FDA and an s/co ratio of 8.0 was set as the screening test positive value to determine the need for reflex supplemental tests. With our data, we set the cutoff s/co ratios as follows: Elecsys assay, ≥200 (89 [95.7%] of 93); Architect assay, ≥3 (93 [94.9%] of 98); Vitros assay, ≥7.0 (67 [95.7%] of 70); Access assay, ≥3 (90 [94.7%] of 95).