National Health and Nutrition Examination Survey

Vitamin B12 (B12) is an essential cofactor for two enzymes involved in one-carbon metabolism: methylmalonyl CoA mutase (reduced function of this enzyme results in increased serum methylmalonic acid (MMA) levels) and methionine synthetase (this enzyme catalyzes the remethylmation of homocysteine to methionine) (IOM 1998). A serum B12 level below the normal expected range may indicate B12 deficiency. However, a B12 level within the low normal range does not exclude B12 deficiency; symptomatic patients need to be further evaluated with MMA, folic acid, and homocysteine (Allen 2008; Cordero, et al. 2008)

A chronic dietary deficiency of either folate or vitamin B12 causes macrocytic anemia, although strict dietary deficiencies are rare. Most people who develop a vitamin B12 deficiency have an underlying stomach or intestinal disorder that limits the absorption of vitamin B12. Subtly reduced cognitive function resulting from early vitamin B12 deficiency is sometimes the only symptom of these intestinal disorders. Untreated deficiencies will lead to megaloblastic anemia and vitamin B12 deficiency results in irreversible central nervous system degeneration. Hematologic signs, however, are not always present in vitamin B12 deficiency and hematologic signs and neurologic abnormalities can be inversely correlated (Baik, et al. 1999).

Examined participants aged 19 years and older were 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 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.

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

There were no changes to lab method, equipment or lab site.

Vitamin B12 (May 2016)

Serum samples 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 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 arediscussed in the NHANES LPM.

Mobile Examination Centers (MECs)

Laboratory team performance is monitored using several techniques. NCHS and contract consultants use a structured competency assessment evaluation during  visits to evaluate both the quality of the laboratory work and the quality-control procedures. Each laboratory staff person is observed for equipment operation, specimen collection and preparation; testing procedures and constructive feedback are given to each staff. Formal retraining sessions areconducted 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 2.0% of all specimens.

NCHS developed and distributed a quality control protocol for all CDC and contract laboratories, which outlined the Westgard rules (Westgard et al, 1981) used 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 Environmental Health Sciences’ quality control and quality assurance 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.

Two  derived variables were created in this data file. The formula for the derivation is as follows:

The vitamin B12 data (LBXB12) was adjusted using a Deming regression = LBDB12.
The vitamin B12 in pg/mL (LBDB12) was converted to pmol/L (LBDB12SI) by multiplying by 0.738.

Vitamin B12 for 2013-2014

Laboratory bench and “blind” quality control pools showed significant shifts during NHANES 2013-2014. These quality control shifts were associated with reagent lot-to-lot variations. It was necessary to adjust the vitamin B₁₂ data using mean quality control pool data using a Deming regression.

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

The analysis of NHANES 2013-2014 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 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).

Detection Limits

The detection limits were constant for this analyte in the data set.

The lower limit of detection (LLOD, in pg/mL) for vitamin B₁₂:

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.