Laboratory Procedure Manual - Centers for Disease Control and Prevention

Laboratory Procedure Manual

Analyte: Vitamin B12 (B12)

Matrix:

Serum

Method: Roche E-170 Vitamin B12 "ECLIA"

Method No: 4009.03

Revised:

As performed by:

Nutritional Biomarkers Branch Division of Laboratory Sciences National Center for Environmental Health

Contact: James L. Pirkle, M.D., Ph.D.

Director, Division of Laboratory Sciences

Important Information for Users CDC periodically refines these laboratory methods. It is the responsibility of the user to contact the person listed on the title page of each write-up before using the analytical method to find out whether any changes have been made and what revisions, if any, have been incorporated.

Public Release Data Set Information

This document details the Lab Protocol for testing items listed.

Data File Name Variable Name

SAS Label

VITB12_H

LBDB12

Vitamin B12 (pg/mL)

Total Vitamin B12 in Serum NHANES 2013-2014

Page 1 of 13

1. Summary of Clinical Relevance and Test Principle

A. Clinical relevance

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) [1]. 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 [2, 3].

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 [4].

B.Test principle

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 [5].

2. Safety Precautions

Consider all specimens potentially positive for infectious agents including HIV, hepatitis B and hepatitis C. We recommend that the hepatitis B vaccination series for all the analysts working with whole blood and/or serum. Observe universal precautions; wear protective gloves, laboratory coats, and safety glasses during all steps of this method. Discard any residual sample material by autoclaving after analysis is completed. Place all disposable plastic, glassware, and paper (pipette tips, vials, gloves, etc.) in a biohazard autoclave bag and keep these bags in appropriate containers until sealed and autoclaved. Wipe down all work surfaces with 10% bleach solution when work is finished.

Total Vitamin B12 in Serum

NHANES 2013-2014

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Handle acids and bases with extreme care; they are caustic and toxic. Handle organic solvents only in a well-ventilated area or, as required, under a chemical fume hood.

Reagents and solvents used in this study are listed in Section 6. Material safety data sheets (MSDSs) for all chemicals are readily accessible as hard copies in the lab.

3. Computerization and Data System Management

A. During sample preparation and analysis, samples are identified by their sample ID. The sample ID is a number that is unique to each sample that links the laboratory information to demographic data recorded by those who collected the sample.

B. Calculation of B12 concentration is accomplished with the software on the Roche E-170 and generated data is transferred to the DLS network where it is saved. The result file is imported into a database for review of the patient data, statistical evaluation of the QC data, and approval of the results. See "SOP Computerization and Data System Management" for a step-by-step description of data transfer, review and approval.

C. NHANES data is transmitted electronically on a regular basis (approximately weekly for 3-week turnaround analytes). Abnormal values are confirmed by the analyst, and codes for missing data are entered by the analyst and are transmitted as part of the data file. NCHS makes arrangements for the abnormal report notifications to the NCHS Survey Physician.

D. The data file and results file from the instrument workstation are typically backed up daily to the Roche/Hitachi USB Memory Stick for long-term storage. This is the responsibility of the analyst under the guidance of the project lead person. Files stored on the DLS network are automatically backed up nightly by ITSO support staff.

4. Specimen Collection, Storage, and Handling Procedures: Criteria for Specimen Rejection

A. For best results, a fasting sample should be obtained, but fasting is not required.

B. Specimens for B12 analysis may be fresh or frozen serum or plasma. Serum specimens may be collected with regular red-top Vacutainers or tubes containing separating gel and plasma specimens may be collected with Na-heparin or K3-EDTA as an anticoagulant.

C. A routine test requires 150 ?L for the sample cup. For every additional B12 test performed from the same container, an additional 15 ?L of samples is required. Thus, a 500 ?L serum sample is preferable to allow for repeat analyses. The appropriate amount of serum is dispensed into a Nalge cryovial or other plastic screw-capped vials labeled with the participant's ID.

D. Specimens collected in the field should be kept cold and protected from light. After processing, specimens should be frozen and shipped on dry ice by overnight mail. Once received, they should be stored at ................
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