National Health and Nutrition Examination Survey

The objectives of this component are:

1. To provide data for monitoring secular trends in measures of nutritional status in the U.S. population;
2. To evaluate the effect of people's habits and behaviors such as physical activity and the use of alcohol, tobacco, and dietary supplements on people's nutritional status; and
3. To evaluate the effect of changes in nutrition and public health policies including welfare reform legislation, food fortification policy, and child nutrition programs on the nutritional status of the U.S. population.

These data will be used to estimate deficiencies and toxicities of specific nutrients in the population and subgroups, to provide population reference data, and to estimate the contribution of diet, supplements, and other factors to serum levels of nutrients. Data will be used for research to further define nutrient requirements as well as optimal levels for disease prevention and health promotion.

Participants aged 20 years and older, who do not meet any of the exclusion criteria are eligible.

The Elecsys Vitamin B12 assay employs a competitive test principle using intrinsic factor specific for vitamin B12. The fully automated electrochemiluminescence immunoassay “ECLIA”) is intended for use on Elecsys and cobas e immunoassay analyzers. The total duration of the assay is 27 minutes. The 1st step is to incubate 15 uL of sample with the vitamin B12 pretreatment 1 and pretreatment 2 to release bound vitamin B12. The 2nd incubation adds the ruthenium labeled intrinsic factor to the pretreated sample causing a vitamin B12-binding protein complex to form; the amount of which is dependent upon the analyte concentration in the sample. During the 3rd incubation, streptavidin-coated microparticles and vitamin B12 labeled with biotin are added and the still-vacant sites of the ruthenium labeled intrinsic factor become occupied. This forms a ruthenium labeled intrinsic factor-vitamin B12 biotin complex. The entire complex becomes bound to the solid phase via interaction of biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. Results are determined via a calibration curve which is instrument specifically generated by a 2 point calibration and a master curve provided via the reagent barcode.

Vitamin B12 was last tested in 2005-2006 by the Bio-Rad Quantiphase II Folate/Vitamin B12 radio assay. The test was added back into the 2011-2012 cycle using the Elecsys Vitamin B12 assay.

A detailed description of the laboratory method used can be found on the NHANES website.

Serum specimens are processed, stored, and shipped to the Division of Environmental Health Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention for analysis.

Detailed specimen collection and processing instructions are discussed in the NHANES LPM. Vials are stored under appropriate frozen (–20°C) conditions until they are shipped to National Center for Environmental Health for testing.

This file contains no top coding.

One derived variable was created in this data file. The formula for the derivation is as follows:

• The vitamin B12 in pg/mL was converted to pmol/L by multiplying by 0.738.

Detailed instructions on specimen collection and processing can be found on the NHANES website.

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 Laboratory/Medical Technologists Procedures Manual (LPM).

A detailed description of the quality assurance and quality control procedures can be found on the NHANES website.

Vitamin B12 for 2011–2012:

Serum vitamin B12 was not measured in 2007–2010 and there was a change in vitamin B12 methods from 2005–2006 to 2011–2012. From 1999–2006, the Bio-Rad Quantaphase II radioassay was used, but the manufacturer discontinued this assay in 2006. In 2011–2012, serum vitamin B12 was measured in adults 20 years and older using the fully automated Roche electrochemiluminescence immunoassay on the Elecsys 170. 

The two methods have similar imprecision (<5% CV) and similar limits of detection (LOD): 20 pg/mL for the Bio-Rad assay and 30 pg/mL for the Roche assay. The weighted distributions of the 2005–2006 and 2011–2012 datasets were slightly different (Table 1). Using weighted analysis, 3.06% of the vitamin B12 results by the Bio-Rad assay compared to 1.78% of the vitamin B12 results by the Roche assay were ≤200 pg/mL, a value commonly used as a cutoff for inadequate vitamin B12 status. These small differences suggest that the datasets from these two survey periods are not directly comparable and an adjustment is required to account for method differences.

Table 1.  Weighted geometric mean, standard error of the geometric mean and selected percentiles for vitamin B12 (pg/mL) in 20 years and older NHANES participants

* Method-adjusted to the Roche E-170 assay

To assess method differences, a crossover study was performed between these two assays on 284 specimens. Four samples with results exceeding the upper calibration range (2000 pg/mL) were excluded from the data analysis. The distributions of vitamin B12 were positively skewed. The Roche values were higher (mean: 629, median: 593 pg/mL) compared to the Bio-Rad values (mean: 569, median: 529 pg/mL), but the range of values was similar (Roche 94-1736 pg/mL, Bio-Rad 81-1688 pg/mL) and the Pearson correlation coefficient between the two methods was high (r = 0.98). Because Deming regression considers errors in both variables, we recommend that data users employ the Deming regression equations to assess time trends.

Roche vitamin B12 = 10**(0.97*log₁₀(Bio-Rad vitamin B12) + 0.14 pg/mL)

Bio-Rad vitamin B12 = 10**(1.03*log₁₀(Roche vitamin B12) – 0.14 pg/mL)

Applying this equation to the 2005–2006 vitamin B12 data to forward regress the Bio-Rad assay to make it comparable to the 2011-2012 Roche assay, resulted in a similar weighted distribution (Table 1). Furthermore, 1.54% of the vitamin B12 results by the adjusted Bio-Rad assay were ≤200 pg/mL, which is similar to the prevalence determined with the Roche assay (1.78%). Regression analysis based on the crossover study was therefore able to correct for method differences.

Refer to the 2011-2012 Laboratory Data Overview for general information on NHANES laboratory data.

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).