National Health and Nutrition Examination Survey

Tissue transglutaminase assay (IgA-TTG) and the IgA endomysial antibody assay (IgA EMA) estimates the prevalence of celiac disease in the United States population. Celiac disease is an intolerance to dietary glutens that has protean manifestations. In population surveys in other countries, it is found in about 0.5 to 1 percent of the population. Celiac disease may be as common in the United States, but it has not been adequately examined. Advances in diagnostic testing now allow accurate disease prevalence estimates using two step serological testing. To provide reliable population estimates, four to six years of data collection are required.

Examined participants aged 6 years and older were eligible.

Tissue transglutaminase assay (IgA-TTG)

The laboratory method is an Enzyme-Linked Immunosorbant Assay (ELISA). This ELISA method is for the semi-quantitative detection of IgA antibodies to tissue transglutaminase (endomysium) in human serum. Microwells are pre-coated with recombinant human tTG antigen. The calibrators, controls and diluted patient samples are added to the wells and autoantibodies recognizing the tTG antigen bind during first incubation. Unbound proteins are removed during washing and purified peroxidase conjugate is added which binds to the captured human autoantibody; excess is washed. TMB substrate is added which gives a blue reaction product, intensity is proportional to the concentration of autoantibody in the sample. Phosphoric acid is added to stop the reaction which gives the yellow color. Plate is read at 450 nm.

Endomysial antibody assay (IgA EMA)

Endomysial antibodies of the IgA subclass present in the serum bind to the reticulin component of the endomysium of the smooth muscle in monkey esophagus tissue and can be detected by indirect immunofluorescence. Substrate-bound IgA antibody is identified with a fluorescein-conjugated anti-human IgA (Fab2-specific) conjugate. Positive staining is identified as a reticulated lace-like pattern on perimucosal smooth muscle bands. Sera positive for endomysial antibody are tittered to end-point dilutions.

Refer to the Laboratory Method Files section for detailed laboratory procedure manual(s) of the methods used.

There were no changes to the lab method, lab equipment, or lab site for this component in the NHANES 2013-2014 cycle.

Endomysial Antibody Assay (EMA) (October 2015)

Tissue Transglutaminase Assay (IgA) (October 2015)

Serum specimens were processed, stored and shipped to the Mayo Clinic, Rochester, Minnesota for analysis.

Detailed instructions on specimen collection and processing are discussed in the NHANES Laboratory Procedures Manual (LPM). Vials are stored under appropriate frozen (–30°C) conditions until they are shipped to Mayo Clinic for testing.

The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed QA/QC instructions are discussed in the NHANES LPM.

Mobile Examination Centers (MECs)

Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured quality assurance evaluation during unscheduled visits to evaluate both the quality of the laboratory work and the quality-control procedures. Each laboratory staff member is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff member. Formal retraining sessions are conducted annually to ensure that required skill levels were maintained.

Analytical Laboratories

NHANES uses several methods to monitor the quality of the analyses performed by the contract laboratories. In the MEC, these methods include performing blind split samples collected on “dry run” sessions. In addition, contract laboratories randomly perform repeat testing on 2% of all specimens.

NCHS developed and distributed a quality control protocol for all the contract laboratories which outlined the use of Westgard rules (Westgard et al, 1981) when running NHANES specimens. Progress reports containing any problems encountered during shipping or receipt of specimens, summary statistics for each control pool, QC graphs, instrument calibration, reagents, and any special considerations are submitted to NCHS quarterly. The reports are reviewed for trends or shifts in the data. The laboratories are required to explain any identified areas of concern.

All QC procedures recommended by the manufacturers were followed. Reported results for all assays meet the Mayo Clinic’s quality control and quality assurance performance criteria for accuracy and precision.

The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.

Refer to the 2013-2014 Laboratory Data Overview for general information on NHANES laboratory data.

Demographic and Other Related Variables

The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The NHANES 2013-2014 Demographics File contains demographic data, health indicators, and other related information collected during household interviews as well as the sample design variables. The recommended procedure for variance estimation requires use of stratum and PSU variables (SDMVSTRA and SDMVPSU, respectively) in the demographic data file.

The Fasting Questionnaire File includes auxiliary information such as fasting status, the time of venipuncture, and the conditions precluding venipuncture.

This laboratory data file can be linked to the other NHANES data files using the unique survey participant identifier (i.e., SEQN).

Detection Limits

Since this data is reported as qualitative data the use of lower LLODs isn’t applicable.

Exam sample weights should be used for analyses. Please refer to the NHANES Analytic Guidelines and the on-line NHANES Tutorial for further details on the use of sample weights and other analytic issues.