Hepatitis viruses constitute a major public health problem because of the morbidity and mortality associated with the acute and chronic consequences of these infections. New immunization strategies have been developed to eliminate the spread of hepatitis B virus (HBV) and hepatitis A virus (HAV) in the United States. Recommendations have also been developed for the elimination of hepatitis C virus (HCV) infection. Because of the high rate of asymptomatic infection with these viruses, information about the prevalence of these diseases is needed to monitor prevention efforts. By testing a nationally representative sample of the U.S. population, NHANES will provide the most reliable estimates of age-specific prevalence needed to evaluate the effectiveness of the strategies to prevent these infections. In addition, NHANES provides the means to better define the epidemiology of other hepatitis viruses. NHANES testing for markers of infection with hepatitis viruses has been used to determine secular trends in infection rates across most age and racial/ethnic groups, and has provided a national picture of the epidemiologic determinants of these infections.
In August 2025, a new variable (SSLBDHD) was added based on retesting of the original 2011-2012 samples using a different assay after concerns were raised about potential false positive results during original testing (see Description of Laboratory Methodology section for additional information).
All participants aged 6 years or older are eligible to be tested.
Serum specimens are processed, stored, and shipped to the Division of Viral Hepatitis, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention. Detailed specimen collection and processing instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM).
Hepatitis B core antibody (anti-HBc)
The VITROS Anti-HBc assay is performed using the VITROS Anti-HBc Reagent Pack and VITROS Immunodiagnostic Products Anti-HBc Calibrator on the VITROS ECi/ECiQ or VITROS 3600 Immunodiagnostic System.
A competitive immunoassay technique is used. This involves the reaction of anti-HBc in the sample with hepatitis B core antigen (HBcAg) coated wells. Unbound sample is removed by washing. Horseradish peroxidase (HRP)-labeled antibody conjugate (mouse monoclonal anti-HBc) is then allowed to react with the remaining exposed HBcAg on the well surface. Unbound conjugate is removed by washing.
The bound HRP conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent increases the level and duration of the light produced. The light signals are read by the VITROS ECi/ECiQ or VITROS 3600 Immunodiagnostic System. The amount of HRP conjugate bound is indicative of the concentration of anti-HBc present in the sample.
Hepatitis B surface antigen (HBsAg)
The VITROS HBsAg assay is performed using the VITROS HBsAg Reagent Pack and VITROS Immunodiagnostic Products HBsAg Calibrator on the VITROS ECi/ECiQ or VITROS 3600 Immunodiagnostic System.
An immunometric technique is used. This involves the simultaneous reaction of HBsAg in the sample with mouse monoclonal anti-HBs antibody coated onto the wells and a horseradish peroxidase (HRP)-labeled mouse monoclonal anti-HBs antibody in the conjugate. Unbound conjugate is removed by washing.
A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent increases the level and duration of the light produced. The light signals are read by the VITROS ECi/ECiQ or VITROS 3600 Immunodiagnostic System. The amount of HRP conjugate bound is indicative of the level of HBsAg present in the sample.
Hepatitis D antibody
(anti-HDV) - original testing
The DiaSorin
ETI-AB-DELTAK-2 enzyme immunoassay used for qualitative anti-HDV determination
is a simultaneous competitive assay. Anti-HDV present in the sample and labeled
anti-HDV antibodies compete for a fixed quantity of HDAg bound to the solid
phase. The quantity of enzyme tracer bound to the solid phase and consequently
the enzyme activity is inversely proportional to the anti-HDV concentration
present in samples or controls.
Enzyme activity is measured by adding a colorless chromogen/substrate solution.
The enzyme action on the chromogen/substrate produces a color which is measured
with a photometer.
There were no changes (from the previous 2 years of NHANES) to equipment, lab
methods or lab site.
April 2025 anti-HDV retesting for 2007-2008, 2009-2010, 2011-2012, 2013-2014, 2015-2016, and 2017-2018 cycles
From 2007-2008 through 2017-2018, NHANES anti-HDV testing was performed with a DiaSorin enzyme immunoassay designated as Research Use Only in the United States because it was not approved for clinical use by the US Food and Drug Administration. Concerns about high rates of false positivity from this assay were raised after NHANES results showed a higher-than-expected number of positive results for hepatitis D antibody for certain cycles of data collection (Soriano, et al. 2019). The DiaSorin assay was withdrawn from the market in 2019.
Unpublished replication of hepatitis D antibody testing results by CDC’s Division of Viral Hepatitis (DVH) staff confirmed that the reported high prevalence of HDV antibody was not due to incorrect analysis of data or to outlying or influential survey weights. Therefore, in April 2025, samples from 2007-2008 through 2017-2018 were retested. Retesting eligibility included participants with reactive/positive test results for hepatitis B surface antigen during 2007-2008 through 2017-2018 (because Hepatitis D viral infection and replication is only possible with co-infection with hepatitis B - see below “Analytic Notes” session) and with available surplus serum samples.
Retesting was performed with a different CLIA-validated hepatitis D virus antibody assay, International Immunodiagnostics HDV Ab (IID HDV Ab) ELISA, that was approved for use at CDC in 2023. This assay is a competitive enzyme immunoassay (EIA) for the determination of antibodies to HDV in human serum with a “one-step” methodology. Antibodies to HDV, if present in the sample, compete with a virus-specific polyclonal IgG/IgM (Total antibodies), labeled with Horseradish peroxidase (HRP), for a fixed amount of recombinant HDV protein coated on the microplate. The concentration of the bound enzyme on the solid phase is inversely proportional to the amount of HDV antibodies in the sample and its activity is detected by adding the chromogen/substrate in the second incubation.
Hepatitis D Antibody (April 2025) - Retesting Method (January 2026)
Hepatitis B Core Antibody (September 2013)
Hepatitis B surface antigen (September 2013)
Hepatitis D antibody (September 2013)
The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.
The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Amendments mandates. Detailed quality control and quality assurance instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM).
A detailed description of the quality assurance and quality control procedures can be found on the NHANES website.
Refer to the 2011-2012 Laboratory Data Overview for general information on NHANES laboratory data.
There are over 800 laboratory tests performed on NHANES participants. However, not all participants provided biospecimens or enough volume for all the tests to be performed. The specimen availability can also vary by age or other population characteristics. Analysts should evaluate the extent of missing data in the dataset related to the outcome of interest as well as any predictor variables used in the analyses to determine whether additional re-weighting for item non-response is necessary. Please refer to the Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues. The Analytic Guidelines are available on the NHANES website.
Demographic and Other Related Variables
The analysis of NHANES 2011-2012 laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2011-2012 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample weight variables. The Fasting Questionnaire File includes auxiliary information such as fasting status, the time of venipuncture, and the conditions precluding venipuncture. The demographics and fasting questionnaire files may be linked to the laboratory data file using the unique survey participant identifier (i.e., SEQN).
The age range and constraints for hepatitis B and D testing are as follows:
Hep B
The hepatitis B core antibody test is performed on all examinees 6 years old or older while the hepatitis B surface antibody test is performed on all examinees 2 years old or older. The Hepatitis B surface antigen is tested only when the Hepatitis B core antibody test is positive. Participant results are coded positive for surface antigen if the surface antigen test is positive; they are coded negative for surface antigen if the test for surface antigen is negative or if the test for hepatitis B core antibody is negative.
Hep D
Hepatitis D virus (HDV), or delta infection, occurs only in the presence of acute or chronic HBV infection because delta requires HBV components to enter liver cells, assemble, and make new virus. In NHANES, the test for antibody to HDV is performed on participants 6 years of age or older who test positive for anti-HBc and HBsAg. Participant results were coded positive for hepatitis D antibody if the HDV antibody test was positive; they were coded negative for hepatitis D antibody if the HDV antibody test was negative, or if no test for HDV antibody was performed because the anti-HBc or HBsAg tests were negative.
For the results from 2025 retesting, the following codes were used:
0 = HBsAg+ participants with no stored serum available, thus no re-testing could be performed.
1 = HBsAg+ participants with stored serum available and re-tested as positive (reactive) to the hepatitis D antibody.
2 = HBsAg+ participants with stored serum available and re-tested as negative (non-reactive) to the hepatitis D antibody.
6 = HBsAg- participants who were originally not tested and presumed negative to the hepatitis D antibody; no re-testing was performed because presumed negative.
. = Participants with a missing blood sample for the original testing were coded as missing for the re-testing as well.
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 1 | Positive | 468 | 468 | |
| 2 | Negative | 6598 | 7066 | |
| 3 | Indeterminate | 0 | 7066 | |
| . | Missing | 755 | 7821 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 1 | Positive | 43 | 43 | |
| 2 | Negative | 7022 | 7065 | |
| 3 | Indeterminate | 0 | 7065 | |
| . | Missing | 756 | 7821 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 1 | Positive | 5 | 5 | |
| 2 | Negative | 7060 | 7065 | |
| 3 | Indeterminate | 0 | 7065 | |
| . | Missing | 756 | 7821 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 0 | Hep B sAg positive, no surplus sample | 8 | 8 | |
| 1 | Hep B sAg positive, reactive | 1 | 9 | |
| 2 | Hep B sAg positive, non-reactive | 34 | 43 | |
| 6 | Hep B sAg negative, not retested | 7022 | 7065 | |
| . | Missing | 756 | 7821 |