Table of Contents

Component Description

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.

Eligible Sample

Participants aged 6 years and older who do not meet any of the exclusion criteria are eligible.

Description of Laboratory Methodology

The Diasorin (formerly Incstar) 25-OH- Vitamin D assay consists of a two-step procedure. The first procedure involves an extraction of 25-OH-D and other hydroxylated metabolites from serum with acetonitrile. Following extraction, the treated sample is assayed by using an equilibrium RIA procedure. The RIA method is based on an antibody with specificity to 25-OH-D. The sample, antibody, and tracer are incubated for 90 min at 20-25 °C. Phase separation is accomplished after a 20-minute incubation at 20-25 °C with a second antibody-precipitating complex. A NSB buffer is added after this incubation and prior to centrifugation to aid in reducing non-specific binding. More detailed information about the Diasorin RIA method can be found on the NHANES Web site in the Laboratory Procedures Manuals.

Data Processing and Editing

Serum specimens are processed, stored, and shipped to the Division of 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 Laboratory/Medical Technologists Procedures Manual (LPM). Vials are stored under appropriate frozen (–20°C) conditions until they are shipped to National Center for Environmental Health for testing.

Detailed instructions on specimen collection and processing can be found at the NHANES web page.

Laboratory Quality Assurance and Monitoring

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). Read the LABDOC file for detailed QA/QC protocols.

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

Analytic Notes

Revised Analytical Note for NHANES 2000-2006 and NHANES III (1988-1994) 25-Hydroxyvitamin D Analysis (Revised November 2010)

Data Advisory

The purpose of this revised note is to inform users that serum 25-hydroxyvitamin D (25(OH)D) data from NHANES 2003-2004 and 2005-2006 have been adjusted for assay drifts, as described below. In addition, this note modifies the previous analytical note about two issues that should be addressed when analyzing 25(OH)D data from NHANES 2000-2006 and NHANES III (1988-1994). First, data users should be aware that the 25(OH)D data from the NHANES 2003-2006 were most likely affected by drifts in the assay performance (method bias and imprecision) over time. These assay drifts are likely due to reagent and calibration lot changes in the reformulated DiaSorin assay and may affect comparability, and therefore the interpretability of NHANES data from 2000-2006. The 2003-2004 and 2005-2006 25(OH)D data are being adjusted to account for these assay drifts. The new 2003-2004 and 2005-2006 adjusted 25(OH)D data files replaced the unadjusted 25(OH)D files in November 2010. NCHS recommends that the adjusted data rather than the previously available unadjusted data files be used for all analyses of 25(OH)D.

Second, users are cautioned about making direct comparisons between serum 25(OH)D measurements from NHANES 2000-2006 and measurements obtained in NHANES III (1988-1994). NHANES III 25(OH)D data must be adjusted in order to make a valid comparison to the NHANES 2000-2006 survey years due to a reformulation of the DiaSorin radioimmunoassay (RIA) kit that resulted in a shift in assay results between the two time periods. Therefore, users of these various NHANES data sets are cautioned that changes in 25(OH)D results over the time period 1988-2006 are affected by the two methodological issues described above and both should be considered when evaluating whether, and how much, differences over time are due to true changes in the vitamin D status of the US population.


Measurements of serum 25(OH)D were performed as part of the nutrition biomarker component of NHANES III (1988-1994) and in the years 2000-2006 of NHANES. These 25(OH)D data are available on public use data files on the NCHS/NHANES website for NHANES III and NHANES 2001-2002, 2003-2004 and 2005-2006. The 25(OH)D data collected in 2000 are available through the NCHS Research Data Center (not available in public data sets) because of a disclosure risk of confidential information for a single-year data release. Additional variables of interest relative to 25(OH)D, such as geography, are only accessible through the NCHS Research Data Center (due to increased disclosure risk) and are subject to the analytic limitations of data used in that setting. Readers should be aware that all issues discussed below in regard to the publicly available data for 2001-2006 also apply to the controlled-access data from 2000. 

Measurements of serum 25(OH)D were performed in NHANES III (1988-1994) and NHANES 2000-2006, at the National Center for Environmental Health, CDC, Atlanta, GA using the DiaSorin RIA kit (Stillwater MN). The DiaSorin assay kit had been reformulated by the manufacturer in 1998 by introducing an antibody that provided improved binding and altering the washing solution to reduce non-specific binding.


Impact of assay reformulation on serum 25(OH)D measurements from NHANES III

Steps were taken to address the changes in assay between NHANES III and NHANES 2000-2006. To assess the magnitude of assay changes that might have an impact on any observed trends in serum 25(OH)D in the population, in 2004 the CDC laboratory reanalyzed a subset of 150 banked serum samples from NHANES III using the reformulated version of the RIA assay (Yetley EA, et al., 2010). The serum samples were selected to represent the entire distribution of serum 25(OH)D values in NHANES III. The NHANES III results as measured with the reformulated assay were regressed on the NHANES III values obtained with the original assay for these 150 specimens. The average difference between the reformulated and original RIA was -12% and is described by the following equation:

NHANES III 25(OH)Dreformulated RIA assay = (0.8429* NHANES III 25(OH)Doriginal RIA assay) + 2.5762 nmol/L (r = 0.8966).

This adjustment equation was generated after first accounting for the assay drift occurring during 2003-2004 with the reformulated DiaSorin assay (Yetley EA, et al., 2010). The above equation allows an approximation of NHANES III results to the level of the reformulated assay used in NHANES 2000-2006.

Impact of assay drift on serum 25(OH)D measurements from the NHANES 2000-2006

In addition to the assay reformulation, the CDC laboratory observed drifts in the serum 25(OH)D assay performance (as reflected in QC pool shifts in the mean, up or down, by up to 10%) over the period of 2003-2006. The variation in 25(OH)D values appeared to be due to method variation that probably resulted from reagent and calibration lot-to-lot variation. Two approaches for adjusting the 2003-2006 25(OH)D were tried (Yetley EA, et al., 2010): 1) a statistical adjustment model based on quality control pool data that encompassed the period of method drift, and 2) a statistical model based on participant 25(OH)D values, transformed by the natural logarithm, and an analysis of covariance model to control for time effects of assay fluctuations. Both methods gave approximately the same statistical adjustment of the 25(OH)D data during 2003-2006. It was decided to use the model based on quality control pool data because the results should be independent of any empirical trend in the sample participant data.

 The following weighted descriptive data and selected percentiles (calculated using SAS version 9 proc surveymeans) show the effect of the adjustment for 2003-2004 and 2005-2006:

Years Adjusted N Mean 5th 25th 50th 75th 95th
2003-2004 No 8294 25.3 9.9 18.3 24.2 30.6 41.4
2003-2004 Yes 8298* 24.3 9.9 17.9 23.3 29 38.9
2005-2006 No 8306 22.2 8.3 15.9 21.3 27 36
2005-2006 Yes 8306 23.9 8.8 17.1 23.1 28.8 38.5

*4 more participants were added for 2003-2004 that were inadvertently missed in the unadjusted data.

As seen above, the 2003-2004 25(OH)D data were adjusted to lower values and would result in higher prevalence estimates of 25(OH)D deficiency. In contrast, the 2005-2006 data were adjusted to higher values and would result in lower prevalence estimates of 25(OH)D deficiency.

Future Plans

The National Institute of Standards and Technology (NIST) has released standard reference materials for 25(OH)D assays with certified values assigned by use of isotope dilution tandem mass spectrometry (LC-MS/MS) candidate reference measurement procedures in July 2009 (Phinney KW, 2008).  CDC intends to generate regression equations that will permit the adjustment of the 25(OH)D data from various NHANES survey years to the NIST accuracy-based standard by reanalyzing subsets of specimens from NHANES 1988-1994 and 2001-2006 using LC-MS/MS. This will improve the ability to interpret the 25(OH)D data for all types of analyses, including comparisons between NHANES 2001-2006 and NHANES III. When these equations become available, this analytical note will be updated with a revised analytical note.


Codebook and Frequencies

SEQN - Respondent sequence number

Variable Name:
SAS Label:
Respondent sequence number
English Text:
Respondent sequence number.
Both males and females 6 YEARS - 150 YEARS

LBXVID - Vitamin D (ng/mL)

Variable Name:
SAS Label:
Vitamin D (ng/mL)
English Text:
Vitamin D (ng/mL)
Both males and females 6 YEARS - 150 YEARS
Code or Value Value Description Count Cumulative Skip to Item
3 to 80 Range of Values 7807 7807
. Missing 840 8647