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Test Code Vit B12 Vitamin B12 Assay, Serum

Reporting Name

Vitamin B12 Assay, S

Useful For

Investigation of macrocytic anemia


Workup of deficiencies seen in megaloblastic anemias

Advisory Information

This test provides a measurement of serum B12 level only. If a more comprehensive workup is preferred, consider ACASM / Pernicious Anemia Cascade.


The ACASM cascade simplifies the evaluation of B12 deficiency and ensures that additional testing is performed in patients with a decreased vitamin B12 level. The cascade begins with serum B12 measurement. If the vitamin B12 level is less than 150 ng/L, intrinsic factor-blocking antibody (IFBA) testing is automatically performed. If the IFBA test is negative or indeterminate, gastrin level is evaluated. If the serum vitamin B12 level is 150 to 400 ng/L, methylmalonic acid is measured. If the methylmalonic acid is greater than 0.40 units/L, IFBA testing is performed. If the serum vitamin B12 level is above 400 ng/L, no further testing is performed. See Vitamin B12 Deficiency Evaluation algorithm in Special Instructions.

Necessary Information

Ask patients if they have received a vitamin B12 injection within the last 2 weeks. If patient has received an injection within the past 2 weeks, this test cannot be ordered.

Specimen Required


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 aliquoted within 2 hours of collection.

Specimen Type


Specimen Minimum Volume

0.5 mL

Specimen Stability Information

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

Day(s) and Time(s) Performed

Monday through Friday; 5 a.m.-12 a.m.

Saturday; 6 a.m.-6 p.m.

Performing Laboratory

Mayo Medical Laboratories in Rochester

Method Name

Immunoenzymatic Assay

Method Description

The instrument used is a Beckman Coulter DXI 800. The Access Vitamin B12 assay is a competitive-binding immunoenzymatic assay. A sample is added to a reaction vessel along with alkaline potassium cyanide and dithiothreitol. This treatment denatures B12 binding proteins and converts all forms of vitamin B12 to the cyanocobalamin form. After neutralization, intrinsic factor-alkaline phosphatase conjugate and paramagnetic particles coated with goat-antimouse IgG:mouse monoclonal anti-intrinsic factor are added to the sample. Vitamin B12 in the sample binds to the intrinsic factor conjugate, preventing the conjugate from binding to the solid phase anti-intrinsic factor. After incubation in a reaction vessel, materials bound to the solid phase are held in a magnetic field, while unbound materials are washed away. The chemiluminescent substrate Lumi-Phos 530 is added to the vessel and light generated by the reaction is measured with a luminometer. The light production is inversely proportional to the concentration of vitamin B12 in the sample. The amount of analyte in the sample is determined by means of a stored, multipoint calibration curve.(Instruction manual: Beckman Coulter Assay Manual 2015, Beckman Coulter Inc, Brea, CA)

Reference Values

180-914 ng/L

Test Classification

This test has been cleared or approved by the U.S. 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

Special Instructions

Reject Due To


Mild OK; Gross reject


Mild OK; Gross OK





Testing Algorithm

See Vitamin B12 Deficiency Evaluation in Special Instructions.


If not ordering electronically, complete, print, and send a Benign Hematology Test Request Form (T755) with the specimen (