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Test ID: B12 Vitamin B12 Assay, Serum

Reporting Name

Vitamin B12 Assay, S

Useful For

Investigation of macrocytic anemia


Workup of deficiencies seen in megaloblastic anemias

Clinical Information

Vitamin B12 (cobalamin) is necessary for hematopoiesis and normal neuronal function. In humans, it is obtained only from animal proteins and requires intrinsic factor (IF) for absorption. The body uses its vitamin B12 stores very economically, reabsorbing vitamin B12 from the ileum and returning it to the liver; very little is excreted.


Vitamin B12 deficiency may be due to lack of IF secretion by gastric mucosa (eg, gastrectomy, gastric atrophy) or intestinal malabsorption (eg, ileal resection, small intestinal diseases).


Vitamin B12 deficiency frequently causes macrocytic anemia, glossitis, peripheral neuropathy, weakness, hyperreflexia, ataxia, loss of proprioception, poor coordination, and affective behavioral changes. These manifestations may occur in any combination; many patients have the neurologic defects without macrocytic anemia.


Pernicious anemia is a macrocytic anemia caused by vitamin B12 deficiency that is due to a lack of IF secretion by gastric mucosa.


Serum methylmalonic acid and homocysteine levels are also elevated in vitamin B12 deficiency states.


A serum vitamin B12 level less than 180 ng/L may cause megaloblastic anemia and peripheral neuropathies.


Vitamin B12 levels less than 150 ng/L are considered evidence of vitamin B12 deficiency. Follow-up with a test for antibodies to intrinsic factor (IFBA / Intrinsic Factor Blocking Antibody, Serum) is recommended to identify this potential cause of vitamin B12 malabsorption. For specimens without antibodies and the patient is symptomatic, follow-up testing for vitamin B12 tissue deficiency may be indicated. Consider analysis of methylmalonic acid (MMAS / Methylmalonic Acid, Quantitative, Serum) and/or homocysteine (HCYSP / Homocysteine, Total, Plasma).


Patients with serum vitamin B12 levels between 150 and 400 ng/L are considered borderline deficient and should be evaluated further by functional tests for vitamin B12 deficiency. Plasma homocysteine measurement (HCYSP / Homocysteine, Total, Plasma) is a good screening test where a normal level effectively excludes vitamin B12 and folate deficiency in an asymptomatic patient. However, the test is not specific, and many situations can cause an increased level. In contrast, an increased serum methylmalonic acid (MMAS / Methylmalonic Acid, Quantitative, Serum) level is more specific for cellular-level B12 deficiency and is not increased by folate deficiency.


In patients being evaluated for vitamin B12 deficiency who have intrinsic factor blocking antibodies (IFBA), false elevations of vitamin B12 may occur due to IFBA interference, potentially obscuring a physiological deficiency of vitamin B12. If observed vitamin B12 concentrations are discordant with clinical presentation, measurement of methylmalonic acid (MMAS / Methylmalonic Acid, Quantitative, Serum) should be considered.


For more information, see Vitamin B12 Deficiency Evaluation.

Report Available

1 to 3 days

Day(s) Performed

Monday through Friday

Clinical Reference

1. Babior BM: The megaloblastic anemias. In: Williams WJ, Beutler E, Lichtman MA et al, eds. Hematology. 5th ed. McGraw-Hill; 1995:471-490

2. Roberts NB, Taylor A, Sodi R: Vitamins and trace elements. In: Rifai N, Horvath AR, Wittwer CT, eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 37

3. Klee GG: Cobalamin and folate evaluation: measurement of methylmalonic acid and homocysteine vs vitamin B12 and folate. Clin Chem. 2000 August;46(8 Pt 2):1277-1283

4. Allen LH, Miller JW, de Groot L, et al. Biomarkers of nutrition for development (BOND): Vitamin B-12 review. J Nutr. 2018;148(suppl_4):1995S–2027S. doi: 10.1093/jn/nxy201

5. Wolffenbuttel BHR, Wouters HJCM, Heiner-Fokkema MR, van der Klauw MM: The many faces of cobalamin (vitamin B12) deficiency. Mayo Clin Proc Innov Qual Outcomes. 2019;3(2):200-214 Published 2019 May 27. doi: 10.1016/j.mayocpiqo.2019.03.002

6. Hannibal L, Lysne V, Bjorke-Monsen AL, et al: Biomarkers and algorithms for the diagnosis of vitamin B12 deficiency [published correction appears in Front Mol Biosci. 2017 Aug 08;4:53]. Front Mol Biosci. 2016;3:27. doi: 10.3389/fmolb.2016.00027

7. Green R, Kinsella LJ: Current concepts in the diagnosis of cobalamin deficiency. Neurology. 1995;45:1435-1440

8. Lahner E, Annibale B: Pernicious anemia: new insights from a gastroenterological point of view. World J Gastroenterol. 2009 Nov 7;15(41):5121-5128

9. Bizzaro N, Antico A: Diagnosis and classification of pernicious anemia. Autoimmun Rev. 2014;13(4-5):565-568

10. Toh BH: Pathophysiology and laboratory diagnosis of pernicious anemia. Immunol Res. 2017;65(1):326-330

Method Name

Immunoenzymatic Assay

Specimen Type


Ordering Guidance

Ask patients if they have received a vitamin B12 injection or radiolabeled vitamin B12 injection within the last 2 weeks. Patient results will not reflect deficiency or malabsorption after recent B12 injection. If patient has received such an injection within the past 2 weeks, this test should not be ordered.


This test provides a measurement of serum vitamin B12 level only. For a more comprehensive workup, order ACASM / Pernicious Anemia Cascade, Serum, which initiates testing with measurement of vitamin B12. Depending of the vitamin B12 concentration, testing for intrinsic factor blocking antibody, gastrin, and methylmalonic acid may be added.

Necessary Information

Ask patients if they have received a vitamin B12 injection within the last 2 weeks. Patient results will not reflect deficiency or malabsorption after recent B12 injection. If patient has received an injection within the past 2 weeks, this test should not be ordered.

Specimen Required

Patient Preparation: This test should not be performed on patients who have received a vitamin B12 injection or radiolabeled vitamin B12 injection within the previous 2 weeks.


Preferred: Serum gel

Acceptable: Red top

Specimen Volume: 0.6 mL

Collection Instructions:

1. Serum gel tubes should be centrifuged within 2 hours of collection.

2. Red-top tubes should be centrifuged and the serum aliquoted into a plastic vial within 2 hours of collection.

Specimen Minimum Volume

0.5 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Serum Refrigerated (preferred) 7 days
  Frozen  90 days

Special Instructions

Reference Values

180-914 ng/L

Test Classification

This test has been cleared, approved, or is exempt by the US Food and Drug Administration and is used per manufacturer's instructions. Performance characteristics were verified by Mayo Clinic in a manner consistent with CLIA requirements.

CPT Code Information


LOINC Code Information

Test ID Test Order Name Order LOINC Value
B12 Vitamin B12 Assay, S 2132-9


Result ID Test Result Name Result LOINC Value
B12 Vitamin B12 Assay, S 2132-9
Mayo Clinic Laboratories | Neurology Catalog Additional Information:

mml-Behavioral, mml-Movement-Disorders, mml-Demyelinating-Diseases, mml-Neuromuscular, mml-Pediatric, mml-Spinal-Cord, mml-Neurometabolic, mml-Neuro-ophthalmology