False positive reactions in anti-HCV screening assays is a well-known problem and an obstacle to improve assays’ performance all along. In the present study, we found that the average false positive rate of anti-HCV screening assays was as high as 52.54%, which meant large amount of donations were discarded and qualified donors were deferred unfortunately. It is reported in Uganda, 7.6% (76/1000) were serologically reactive by Ortho, but none were confirmed [8]. Similarly, Schroter M, et al [9]also found false positive results by widely used anti-HCV EIAs were at least 10%, which, compared to other virus screening assays, was unacceptable.
Whereas the false positive rate in this study was much higher than the two reports above, which might partly result from the serological screening strategy in China. Use of two EIAs no doubt would relatively increase the sensitivity but sacrifice the specificity to a certain extent. In the next place, samples used in screening tests were directly from blood collection tubes, while samples in the plasma panel were from plasma bag, which contained about 20% blood preserving fluid, so that diluting the samples and resulting in missing detection of some weak positives. Besides, researchers also found in low risk populations, 40–50% of screening reactives were negative in supplemental immunoblot tests, which was similar to our results [10].
Secondly, performance analysis of 10 different assays indicated that generally all assays’ performance was worse than reported [11–19] or claimed by their instructions, which may be mainly due to sample source. Large amount of false positive samples increased the difficulty of testing a lot and false positive results of an assay during the first screening are likely to be false positives again. Specifically, Roche got the highest sensitivity (98.47%) and Youden’s index (0.961) but specificity (97.61%) was lower than four EIAs (KHB 98.23%, Wantai (indirect) 98.23%, Wantai (sandwich) 98.03% and Livzon (sandwich) 98.13%). The other CLIA, Abbott didn’t perform well: it had the lowest sensitivity, but its results were highly comparable to those of Ortho, which also has been shown in other studies [17–20]. Therefore, Two CLIAs especially in the respect of specificity didn’t show much better performance than EIAs, no matter imported assays or domestic ones.
Since the strategy of two EIAs together using in China, we then analyzed the combined performance of any two assays. Interestingly, not two assays both with good sensitivity had the best combined sensitivity, but Ortho/Abbott together with InTec had a 100% sensitivity indicating these two assays just made up for each other's missing detection. In the terms of specificity, though use of two assays would definitely decrease detection specificity, we found that Wantai (indirect) together with Livzon (sandwich) got a 97.80% specificity, even better than only using one assay (InTec 92.94%, Ortho 90.13%, Livzon(indirect) 93.15%, Murex 97.20%, Roche 97.61%, Abbott 93.25%).
Analysis of assays’ performance also suggested that confirmation of positive HCV screening results by supplemental tests is necessary. Usually, we use RIBA and/or NAT as a way to confirm screening reactive samples. However, RIBA as a confirmatory method for serological screening test of HCV, is too expensive and laborious to be used in every laboratory in China and these confirmatory tests often gives many RIBA indeterminate and/or HCV RNA negative results, indicating the donor may be a resolved HCV infection, infection with a different genotype, early seroconversion, occult HCV infection or just non-specific reactivity [21], which makes us much difficult to estimate the donations’ true status. Like our results, we got 85 (6.49%, 85/1,309) RIBA indeterminate/HCV RNA negative samples, and most (53/85, 62.35%) were due to isolated reactivity to antigens from the core1 region of the HCV genome. We also found these indeterminate samples showed quite different average S/CO ratio detected by Roche compared with RIBA positives (4.84 vs 19.36, P < 0.0001), higher S/CO ratios than RIBA negatives (4.84 vs 2.94, P = 0.020). Similarly to Kiely P’s results [21], the antigen distribution and band intensity were different between indeterminate samples and RIBA positives. All these results indicated RIBA indeterminate samples were a special group different from negative and positives.
Finally, we talked about the relationship between PPV and S/CO ratio on two CLIAs. When S/CO went to 8.2 on Roche and 4.2 on Abbott, the PPV could achieve more than 95%. The S/CO ratio of ARCHITECT anti-HCV established for FDA–approved is more than 5.0 [5], similar to our results. But there is no such approved ratio on Elecsys Anti-HCV II. Some studies suggested that the value predictive of a true positive ≥ 95% of the time with the assay could be set at an S/CO ratio of 20.0, much higher than our study [13, 15], which meant it was required to verify the value in further study.