Clinical Lab Handbook

Container/Tube: SST

Alternate Specimen: Lithium Heparin, Gel

Specimen Volume: 5 mL of serum

Collection Instructions: Allow specimen to clot for 30 minutes. Spin specimen in centrifuge.

Do not use turbid specimens.

Chemiluminescence Immunoassay

A competitive binding immunoassay technique is used, which depends on a competition between vitamin B12 present in the sample with a horseradish peroxidase (HRP)-labeled vitamin B12 conjugate for a limited number of binding sites on a biotinylated Intrinsic Factor present in the liquid phase. Vitamin B12 present in patient samples is released from its endogenous binding proteins by alkaline denaturation. Biotinylated Intrinsic Factor conjugate is added and incubated with the treated neutralized sample. An aliquot of the treated sample is transferred into a streptavidin coated well and B12‑HRP conjugate added. Following a competitive binding reaction, the vitamin B12 Intrinsic Factor complexes are captured by streptavidin on the wells. Unbound materials are removed by washing.

The bound HRP conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent, is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent (a substituted acetanilide) increases the level of light produced and prolongs its emission. The light signals are read by the system. The amount of HRP conjugate bound is indirectly proportional to the concentration of vitamin B12 present.

Reduced concentrations of vitamin B12 may indicate the presence of vitamin dependent anemia. Elevated concentrations of vitamin B12 have been associated with pregnancy, the use of oral contraceptives and multivitamins and in myeloproliferative diseases, such as chronic granulocytic leukemia and myelomonocytic leukemia. An elevated concentration of vitamin B12 is not known to cause clinical problems. Measurement of vitamin B12 is intended to identify and monitor vitamin B12 deficiency. This can arise from the following; (1) defect in the secretion of Intrinsic Factor, resulting in inadequate absorption from food (pernicious anemia); (2) gastrectomy and malabsorption due to surgical resection; and (3) a variety of bacterial or inflammatory diseases affecting the small intestine.

239-931 pg/mL

Monday through Sunday

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