• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Method Files
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • LBDRF7SI - Total Folate, RBC (nmol/L)
  • LBXRF1SI - 5-Methyl-tetrahydrofolate, RBC (nmol/L)
  • LBDRF1LC - 5-Methyl-tetrahydrofolate, RBC cmt
  • LBXRF2SI - Folic acid, RBC (nmol/L)
  • LBDRF2LC - Folic acid, RBC cmt
  • LBXRF3SI - 5-Formyl-tetrahydrofolate, RBC (nmol/L)
  • LBDRF3LC - 5-Formyl-tetrahydrofolate, RBC cmt
  • LBXRF4SI - Tetrahydrofolate, RBC (nmol/L)
  • LBDRF4LC - Tetrahydrofolate, RBC cmt
  • LBXRF5SI - 5,10-Methenyl-tetrahydrofolate (nmol/L)
  • LBDRF5LC - 5,10-Methenyl-tetrahydrofolate, RBC cmt
  • LBXRF6SI - Mefox oxidation product, RBC (nmol/L)
  • LBDRF6LC - Mefox oxidation product, RBC cmt

National Health and Nutrition Examination Survey

2019-March 2020 Data Documentation, Codebook, and Frequencies

Folate Forms - Total & Individual – Washed RBCs (FFMR_K_R)

RDC Only Data File: FFMR_K_R.xpt

First Published: May 2022

Last Revised: NA

Due to disclosure issues, this dataset is not available publicly. Access may be obtained through the NCHS Research Data Center (https://www.cdc.gov/rdc).

Component Description

The NHANES program suspended field operations in March 2020 due to the coronavirus disease 2019 (COVID-19) pandemic. As a result, data collection for the NHANES 2019-2020 cycle was not completed. Data collected in 2019-March 2020 can be accessed as convenience samples through the NCHS Research Data Center (RDC). Any analyses based solely on the 2019-March 2020 data would not be generalizable to the U.S. civilian non-institutionalized population. Please refer to the Analytic Notes section for more details on the use of the data.

Folate belongs to the group of water-soluble B vitamins that occur naturally in food. Prolonged folate deficiency leads to megaloblastic anemia. Low folate status has been causally linked to an increased risk in women of reproductive age to have an offspring with neural tube defects.  Low folate status also increases plasma homocysteine levels, a potential risk factor for chronic diseases, such as cardiovascular disease or cognitive function. Potential roles of folate and other B vitamins in modulating the risk for diseases (e.g., heart disease, cancer, and cognitive impairment) are under investigation. While serum folate is an indicator of recent intake, red blood cell (RBC) folate is an indicator of long-term status.

These data will be used to estimate deficiencies and other health effects 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 the 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. Data will also be used 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.

Eligible Sample

Examined participants aged 6 years and older in the NHANES 2019-March 2020 convenience sample were eligible.

Description of Laboratory Methodology

RBC folate status can be determined directly by measuring folate forms in washed RBCs (Stamm et al. 2018; Fazili et al. 2021). The direct measurement requires the addition of exogenous γ-glutamyl hydrolase (exo-GGH) to deconjugate folate polyglutamates to monoglutamates. It also requires the measurement of hemoglobin (Hb) in the RBC lysate to correct for residual moisture in the packed RBCs. By using the mean corpuscular hemoglobin content (MCHC), RBC folate can be calculated.

Five folate forms, 5-methyltetrahydrofolate, folic acid, tetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methenyltetrahydrofolate, and one oxidation product of 5-methyltetrahydrofolate called MeFox (pyrazino-s-triazine derivative of 4-α-hydroxy-5-methyltetrahydrofolate) 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 of RBC lysate) with an internal standard mixture containing exo-GGH enzyme (5 µg per mL of WB lysate or RBC lysate) and incubation at room temperature for 30 min to deconjugate folate polyglutamates to monoglutamates prior to folate extraction. Ammonium formate buffer (1%) is added to the samples and extraction and clean-up is performed by automated 96-probe solid phase extraction (SPE) using 96-well phenyl SPE plates which takes ~1 h for a 96-well plate. Folate forms are separated within 4 min using isocratic mobile phase conditions and measured by LC-MS/MS (7 min to next injection). Quantitation is based on peak area ratios interpolated against a five-point aqueous linear calibration curve using 1/x² weighting.

Hb is measured by a cyanide-free variation of the cyanmethemoglobin method, using the low toxicity compound sodium lauryl sulfate (SLS) to generate a stable Hb-SLS complex (Oshiro et al. 1982; Hamaguchi et al. 1992). Hb-SLS is measured spectrophotometrically at 535 nm in a microplate reader after addition of SLS solution to RBC lysate which forms a color reaction; the Hb-SLS concentration is calculated using a 7-point calibration curve.

RBC folate form concentrations (nmol/L) measured in a RBC lysate are calculated as RBC folate form = (RBC lysate folate form / Hb-SLS) * MCHC.

Refer to the Laboratory Method Files section for a detailed description on the laboratory methods used.

This is a new component in the 2019-2020 survey cycle.

Laboratory Method Files

Folate Forms – RBCs (May 2022)

Laboratory Quality Assurance and Monitoring

Washed red blood cell specimens 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 the National Center for Environmental Health for testing.

The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Amendments. 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 testing 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).

Data Processing and Editing

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

Analytic Notes

The COVID-19 pandemic required suspension of NHANES 2019-2020 field operations in March 2020 after data were collected in 18 of the 30 survey locations in the 2019-2020 sample. Data collection was cancelled for the remaining 12 locations. Calculation of survey weights for this partial cycle is not possible due to incomplete data collection. Therefore, data from survey components that were only collected in 2019-March 2020 are made available as convenience samples through NCHS's Research Data Center (RDC) because unbiased estimates for the NHANES target population cannot be produced with these samples.

For survey components conducted in both 2017-2018 and 2019-2020 cycles, data collected from 2019 to March 2020 were combined with data from 2017 to 2018 to form a nationally representative sample of NHANES 2017-March 2020 pre-pandemic data. Please see the NHANES 2017-March 2020 pre-pandemic data page for detailed information on this combined sample.

Refer to the 2019-2020 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 2019-2020, approximately 71% of children aged 1-17 years who were examined in the MEC provided a blood specimen through phlebotomy, while 94% of examined adults aged 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 further details on the use of sample weights and other analytic issues.

RBC Folate Forms for NHANES 2019–2020

In NHANES 2019–2020, a comprehensive list of RBC folate forms were measured by isotope-dilution high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (Table 1). Total RBC folate (LBDRF7SI) was calculated by adding LBXRF1SI to LBXRF5SI. LBXRF6SI was not included in the total folate calculation, because this folate form is not biologically active and due to evidence that it may already be present in vivo (Pfeiffer, et al. 2015). An imputed value of LOD divided by the square root of 2 was used for individual folate forms with results that were < LOD. No RBC folate form concentration was calculated when the Hb or MCHC value was missing. No RBC total folate concentration was calculated when one or multiple RBC folate form results were missing.

Table 1.  Folate forms measured by LC-MS/MS

Demographic and Other Related Variables

The analysis of NHANES laboratory data may require additional demographic variables. The NHANES 2019-March 2020 Demographics File contains demographic data, health indicators, and other related information collected during household interviews. 

This laboratory data file can be linked to the Demographics file and other NHANES data files in the 2019-March 2020 convenience sample using the unique survey participant identifier (i.e., SEQN).

Detection Limits

The detection limits were constant for all of the analytes in the data set. Two variables are provided for each of these analytes. The variable name ending in “LC” (ex., LBDRF1LC) indicates whether the result was below the limit of detection: the value “0” means that the result was at or above the limit of detection, “1” indicates that the result was below the limit of detection. The other variable prefixed LBX (ex., LBXRF1SI) provides the analytic result for that analyte. For analytes with analytic results below the lower limit of detection (ex., LBDRF1LC=1), an imputed fill value was placed in the analyte results field. This value is the lower limit of detection divided by the square root of 2 (LLOD/sqrt[2]).

The lower limit of detection (LLOD, in nmol/L) for the 6 folate forms are shown below. Because total folate is calculated from the sum of folate forms RF1 to RF5, a lower limit of detection does not apply.

Variable Name	Analyte Description	LLOD
LBXRF1SI	5-Methyltetrahydrofolate, RBC	0.09
LBXRF2SI	Folic acid, RBC	0.05
LBXRF3SI	5-Formyltetrahydrofolate, RBC	0.07
LBXRF4SI	Tetrahydrofolate, RBC	0.13
LBXRF5SI	5,10-Methenyltetrahydrofolate, RBC	0.11
LBXRF6SI	MeFox, RBC	0.09
LBDRF7SI	Total folate, RBC	n/a

References

• Caudill, S.P., Schleicher, R.L., Pirkle, J.L. Multi-rule quality control for the age-related eye disease study. Statist. Med. (2008) 27(20):4094-40106.
• Fazili Z, Paladugula N, Zhang M, and Pfeiffer CM. The stability of folate forms in RBC lysates compared to whole blood lysates during frozen storage over 24 months and comparison of total folate between LC-MS/MS and microbiologic assay. J Nutr. 2021;151:2852-60.  
  
• Fazili Z, Whitehead RD Jr, Paladugula N, Pfeiffer CM. A high-throughput LC-MS/MS method suitable for population biomonitoring measures five serum folate vitamers and one oxidation product. Anal Bioanal Chem. 2013;405:4549–60.
  
• Hamaguchi Y, Oshiro I, Maeda J. A study on reaction mechanism of sodium lauryl sulfate-hemoglobin (Hb-SLS), Part 1. Rinsho Byori 1992;40(6):649-54 (In Japanese).
• Oshiro I, Takenaka T and Maeda J. New method for hemoglobin determination by using sodium lauryl sulfate (SLS). Clin. Biochem 1982;15(1):83-8.
• Pfeiffer C, Sternberg M, Fazili M, Lacher D, Zhang M, Johnson C, Hammer H, Baily R, Rader J, Yamini S, Berry RJ, Yetley E. British Journal of Nutrition (2015) 113:1965:1977.
• Stamm R, Fazili Z, Pfeiffer CM. Addition of exogenous γ-glutamyl hydrolase eliminates the need for lengthy incubation of whole blood lysate for quantitation of folate vitamers by high-performance liquid chromatography-tandem mass spectrometry. Curr Dev Nutr. 2018;2:1–9.
• Westgard J.O., Barry P.L., Hunt M.R., Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem (1981) 27:493-501.
  

Codebook and Frequencies