The clinical significance of specimens with low sample-to-cutoff (S/Co) ratios in the Ortho VITROS chemiluminescence assay (CIA) for detection of antibodies to hepatitis C virus (HCV) was evaluated. In one study of 482 CIA-reactive samples, none of the 83 samples with S/Co ratios of <5 was HCV RNA positive. In a subsequent study, 332 samples with S/Co ratios of between 1 and 20 were tested with the recombinant immunoblot assay (RIBA). None of the 163 samples with S/Co ratios of <5 was RIBA positive, 83% were RIBA negative, and 28 samples (18%) were RIBA indeterminate. HCV RNA and/or clinical evidence of hepatitis was not found in the 27 indeterminate cases examined. These results show that over 99% of samples with very low S/Co ratios (<5) have no evidence of HCV infection. Therefore, we suggest that the HCV antibody testing algorithm for the VITROS assay might be modified to eliminate supplemental testing of samples with very low S/Co ratios.Assays for the detection of hepatitis C virus (HCV) antibodies have high false-positive rates, particularly in low-prevalence populations. Therefore, other tests such as recombinant immunoblot assay (RIBA) or HCV RNA PCR are used to confirm positive HCV antibody screening tests. To facilitate usage of the supplemental testing, the Centers for Disease Control and Prevention (CDC) published guidelines that incorporate anti-HCV assay signal-to-cutoff (S/Co) ratios into reflex testing algorithms for HCV antibody testing, in order to provide a more systematic approach for the laboratory diagnosis of HCV and minimize the number of specimens that require supplemental testing (2). Data obtained with three anti-HCV screening assays were used for these recommendations: two enzyme immunoassays (EIAs) (Abbott HCV EIA 2.0 and ORTHO HCV version 3.0 enzyme-linked immunosorbent assay) and one enhanced chemiluminescence immunoassay (CIA) (VITROS anti-HCV assay; Ortho-Clinical Diagnostics).The VITROS anti-HCV CIA has recently been shown to be at least as specific and sensitive as conventional EIAs (7, 9), and use of this CIA has been increasing. However, detailed studies of the CIA S/Co ratio value predictive of a positive supplemental HCV test have been limited (7, 9). The CDC guidelines have suggested reflex supplemental testing for samples with S/C ratios of Ͻ8.0 in the VITROS anti-HCV assay based on the evaluation of a total of 1,326 reactive samples, with only 142 of these specimens having S/Co ratios of Յ7.9 (2).We started using the VITROS anti-HCV assay shortly after its Food and Drug Administration approval in August 2001 and have prospectively applied reflex supplemental testing to all samples with S/Co ratios of between 1.00 and 20.0 in the setting of a population of U.S. veterans (8). The objectives of the present study were (i) to determine the S/Co ratio predictive of detectable HCV RNA; (ii) to evaluate, in samples with low S/Co ratios (between 1 and 20), the relationship between S/Co ratios and the number and types of bands detected by RIBA; (iii) to assess, for samples with i...