Serologic Tests

Enzyme Immunoassay

Persons suspected to have hepatitis C should be tested for anti-HCV as an initial screening test. Anti-HCV is detected by enzyme immunoassay (EIA). The third-generation test (EIA-3) used today is more sensitive and specific than previous ones. As with all enzyme immunoassays, however, false-positive results are occasionally a problem with the EIA-3. Additional or confirmatory testing is often helpful.
The best approach to confirm the diagnosis of hepatitis C is to test for HCV RNA using a sensitive assay such as polymerase chain reaction (PCR) or transcription-mediated amplification (TMA). The presence of HCV RNA in serum indicates an active infection.
Testing for HCV RNA is also helpful in patients in whom EIA tests for anti-HCV are unreliable. For instance, immunocompromised patients may test negative for anti-HCV despite having HCV infection because they may not produce enough antibodies for detection with EIA. Likewise, patients with acute hepatitis may test negative for anti-HCV when first tested. Antibody is present in almost all patients by 1 month after onset of acute illness; thus, patients with acute hepatitis who initially test negative may need follow-up testing. In these situations, HCV RNA is usually present and confirms the diagnosis.

Recombinant Immunoblot Assay

Immunoblot assays can be used to confirm anti-HCV reactivity. These tests are also called “Western blots”; serum is incubated on nitrocellulose strips on which four recombinant viral proteins are blotted. Color changes indicate that antibodies are adhering to the proteins. An immunoblot is considered positive if two or more proteins react and is considered indeterminate if only one positive band is detected. In some clinical situations, confirmatory testing by immunoblotting is helpful, such as for the person with anti-HCV detected by EIA who tests negative for HCV RNA. The EIA anti-HCV reactivity could represent a false-positive reaction, recovery from hepatitis C, or continued virus infection with levels of virus too low to be detected (the last occurs only rarely when sensitive PCR or TMA assays are used). If the immunoblot test for anti-HCV is positive, the patient has most likely recovered from hepatitis C and has persistent antibody. If the immunoblot test is negative, the EIA result was probably a false positive.
Immunoblot tests are routine in blood banks when an anti-HCV-positive sample is found by EIA. Immunoblot assays are highly specific and valuable in verifying anti-HCV reactivity. Indeterminate tests require further follow-up testing, including attempts to confirm the specificity by repeat testing for HCV RNA.