The objectives of the folate component are to: 1) provide data for monitoring secular trends in measures of nutritional status in the U.S. population; 2) evaluate the effect of people's habits and behaviors, such as physical activity and the use of alcohol, tobacco, and dietary supplements on nutritional status; and 3) 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. Total folate measurement provides information on the folate status of the individual, where serum folate is an indicator of short-term status and red blood cell (RBC) folate is an indicator of long-term status.
These data will be used to estimate deficiencies and toxicities of specific nutrients in the population and subgroup, 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 in research to further define nutrient requirements as well as optimal levels for disease prevention and health promotion.
All examined female participants aged 12-49 years, and other examined participants (all males as well as females 1-11 years and 50+ years) one year and older from a one-half subsample were eligible.
Population folate status in the NHANES 2017-2018 survey cycle was assessed by a combination of two analytical methods: whole-blood folate was measured by microbiologic assay, while serum folate forms were measured by isotope-dilution high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), this is the same method used in NHANES 2011-2016. RBC folate was then calculated using the data from both assays.
Whole Blood Folate
Microbiological assays have been used for many years to estimate the concentration of folate in blood and other tissues. In the 1990s, more robust and reliable procedures were developed that use microtiter plates for higher throughput and a cryopreserved antibiotic resistant microorganism to avoid having to work under aseptic conditions. The described procedure is an adaptation of this method, and was introduced by the National Center for Environmental Health (NCEH) to NHANES in 2007. Diluted whole blood is added to an assay medium containing Lactobacillus rhamnosus (formerly known as Lactobacillus casei) (NCIB 10463) and all of the nutrients necessary for the growth of L. rhamnosus except folate. The inoculated medium is incubated for 45 hours at 37oC. Since the growth of L. rhamnosus is proportional to the amount of total folate present in whole blood sample; the folate level can be assessed by measuring the turbidity of the inoculated medium at 590 nm in a PowerWave X340 Microplate reader (Bio-Tek Instrument). The assay was calibrated with 5-methyl-tetrahydrofolate from Merck Cie (Eprova).
Serum Total Folate
Serum total folate was calculated as the sum of individual folate forms. Five folate forms, 5-methyl-tetrahydrofolate, folic acid, 5-formyl-tetrahydrofolate, tetrahydrofolate, and 5,10-methenyl-tetrahydrofolate are measured by isotope-dilution high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (Fazili, et al. 2013). The assay is performed by combining specimen (150 µL serum) with an ammonium formate buffer and an internal standard mixture. Sample extraction and clean-up is performed by automated 96-probe solid phase extraction (SPE) using 96-well phenyl SPE plates and takes ~1 h for a 96-well plate. Folate forms are separated within 6 min using isocratic mobile phase conditions and measured by LC-MS/MS. Quantitation is based on peak area ratios interpolated against a five-point aqueous linear calibration curve using 1/x2 weighting.
Refer to the Laboratory Method Files section for a detailed description on the laboratory methods used.
There were no changes to the lab method, lab equipment, or lab for this component during the NHANES 2017-2018 cycle.
Folate - RBC (July 2020)
Whole blood and blood serum are processed, stored, and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 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 National Center for Environmental Health 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 QA evaluation during unscheduled visits to evaluate both the quality of the laboratory work and the QC 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 during “dry run” sessions. In addition, contract laboratories randomly perform repeat testing on 2% of all specimens.
NCHS developed and distributed a QC 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 Division of Laboratory Sciences’ QA/QC performance criteria for accuracy and precision, similar to the Westgard rules (Caudill, et al. 2008).
The data were reviewed. Incomplete data or improbable values were sent to the performing laboratory for confirmation.
One variable was created in this data file. The variable (LBDRFO) was created using the following formula:
LBDRFO: The RBC folate value in nmol/L RBC (LBDRFOSI) was converted to ng/mL RBC (LBDRFO) by dividing LBDRFOSI by 2.265 (rounded to 3 significant figures).
Red blood cell (RBC) folate in NHANES 2017-2018 was calculated from the whole blood folate concentration as measured by microbiologic assay by adjusting for RBC volume and correcting for serum total folate concentration, which was calculated as the sum of individual folate forms. The amounts of individual serum folate forms were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For folate forms with results lower than limit of detection (LOD), an imputed value of LOD divided by the square root of 2 was used. Serum folate forms used to calculate serum total folate concentration were: 5-methyl-tetrahydrofolate, folic acid, 5-formyl-tetrahydrofolate, tetrahydrofolate, and 5,10-methenyl-tetrahydrofolate. For more detailed information regarding folate forms data in NHANES 2017-2018, please refer to the documentation accompanying the Folate Forms – Serum (FOLFMS_J) file.
Refer to the 2017-2018 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. For example, in 2017-2018, approximately 80% of children aged 1-17 years who were examined in the MEC provided a blood specimen through phlebotomy, while 95% of examined adults age 18 and older provided a blood specimen. 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 NHANES Analytic Guidelines and the on-line NHANES Tutorial for details on the use of sample weights and other analytic issues.
Subsample Weights
The analytes included in this dataset were measured in all examined female participants aged 12-49 years, and in a one-half subsample of other examined participants one year and older. Special sample weights are required to analyze these data properly. Variable (WTFOL2YR) encoding of the specific sample weights for this subsample is included in this data file and should be used when analyzing these data. These special sample weights were created to account for the subsample selection probability, as well as the additional nonresponse to these lab tests. Therefore, if participants were eligible for the subsample, but did not provide a blood specimen, they would have the sample weight value assigned as “0” in their records.
Demographic and Other Related Variables
The analysis of NHANES laboratory data must be conducted using the appropriate survey design and demographic variables. The 2017-2018 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
An exact lower limit of detection (LLOD) for RBC folate cannot be calculated because the value is a composite of whole blood folate, serum folate, and hematocrit. Therefore, there is no LLOD for the calculated value of RBC folate. Furthermore, the LOD of this method for whole blood folate depends on the dilution factor (i.e., LOD = 44 nmol/L whole blood if whole blood hemolysate is only diluted 1/40; assuming a hematocrit of 40%, this would correspond to a RBC folate concentration of 110 nmol/L RBC.
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 3748.376146 to 903457.15327 | Range of Values | 4620 | 4620 | |
| 0 | No Lab Result | 472 | 5092 | |
| . | Missing | 0 | 5092 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 50.3 to 2590 | Range of Values | 4582 | 4582 | |
| . | Missing | 510 | 5092 |
| Code or Value | Value Description | Count | Cumulative | Skip to Item |
|---|---|---|---|---|
| 114 to 5870 | Range of Values | 4582 | 4582 | |
| . | Missing | 510 | 5092 |