Vitamin B12

Vitamin B12 is found in animal source foods. A chronic dietary deficiency of vitamin B12 contributes to failure to thrive in infants and to neurologic disorders among all age groups. It is one nutrient deficiency that causes macrocytic anaemia. Although strict dietary deficiencies are rare among populations consuming a Western diet, some population groups consume minimal or no animal source foods due to abject poverty, to religion or to other customs 1, 2. More common amongst the elderly, vitamin B12 deficiency may also result from inability to absorb vitamin B12 based on an underlying disorder of the stomach or intestine, such as hypertrophy of the intestines, reduced gastric acidity, lack of intrinsic factor, or an interference with medications. The autoimmune condition known as pernicious anaemia, most commonly experienced by elderly populations, is a rare but important disease that inhibits absorption of vitamin B12 and will result in deficiency if untreated 3.

The most commonly used biomarker to assess vitamin B12 status in population-based surveys is serum vitamin B12 (total cobalamin) 3.

Specimen collection and management: Serum samples are obtained by centrifugation from whole blood collected by venipuncture. The whole blood needs to be protected from light and processed to serum within a few days of blood collection. Serum vitamin B12 is stable for at least one week at 4˚C, and for at least one year at -20˚C 4.

Biomarker analysis: Serum vitamin B12 is commonly measured via a competitive protein-binding assay on a fully-automated clinical analyser using commercial assay kits. The required analysis volume is typically around 25 µL, however a minimum specimen volume of 150 µL may be needed to fill the sample cup for the clinical analyser. The product sheet for the intended assay will specify the specimen matrix requirements and should be consulted before deciding on the method and ordering survey supplies. Serum is the preferred matrix, since not all assays can utilize EDTA or heparin plasma. Appropriate quality control measures must be followed to ensure high quality results. The assay kits include calibration materials, and in many cases they also include quality control materials. It is nonetheless recommended to establish “in-house” quality control materials that can be tracked over a longer period to verify that the method did not shift over time. The method imprecision is typically 5–10%. A WHO developed serum-based international standard (IS 03/178) is available through the NIBSC to verify method accuracy. However, not every assay may be able to use this material because the assay performance may differ between native patient samples and reference materials that have undergone some processing. Moderate assay differences can be observed in proficiency testing programmes, such as the US College of American Pathologists (CAP) Ligand Survey 5. CDC’s Performance Verification Program for Serum Micronutrients 6 covers serum vitamin B12 and CDC also offers quality control materials for this analyte to support in-house quality assurance programmes for laboratories engaged in public health work 7.

Approximate budget requirements for analysis: The cost for a clinical analyser can vary widely but is usually around US$ 100 000. The cost for materials and supplies is approximately US$ 3–5 per sample.

Interpretation of results: To estimate vitamin B12 deficiency at the individual level, the WHO recommended cutoff value is 203 pg/mL (150 pmol/L) 8. This is the point where the slope of the relationship between serum vitamin B12 and methylmalonic acid changes and where serum methylmalonic acid concentrations rise steeply in response to decreasing serum vitamin B12 concentrations. This is nearly identical to the clinically derived cutoff value of 200 pg/mL (148 pmol/L), below which there are often metabolic abnormalities present. Serum vitamin B12 concentrations between 200 and 300 pg/mL are frequently characterized as “subclinical” deficiency, or at risk of deficiency (depletion), but it is less clear whether these concentrations have a negative health impact 1, 8.

  1. Allen LH. How common is vitamin B-12 deficiency. Am J Clin Nutr. 2009;89:693S–6S. doi:10.3945/ajcn.2008.26947A.  2

  2. Stabler SP, Allen RH. Vitamin B12 deficiency as a worldwide problem. Annu Rev Nutr. 2004;24:299–326. 

  3. Allen LH, Miller JW, de Groot L, Rosenberg IH, Smith AD, Refsum H 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.  2

  4. Drammeh BS, Schleicher RL, Pfeiffer CM, Jain RB, Zhang M, Nguyen PH. Effects of delayed specimen processing and freezing on serum concentrations of selected nutritional indicators. Clin Chem. 2008;54:1883–91. doi: 10.1373/clinchem.2008.108761. 

  5. Proficiency testing [website]. Northfield (IL): US College of American Pathologists (CAP); 2020 (, accessed 14 June 2020). 

  6. Performance Verification Program for Serum Micronutrients [website]. Atlanta: US Centers for Disease Control and Prevention (CDC); 2019 (, accessed 14 June 2020). 

  7. Quality control materials for serum micronutrients [website]. Atlanta: US Centers for Disease Control and Prevention (CDC); 2019 (, accessed 11 May 2020). 

  8. de Benoist B. Conclusions of a WHO Technical Consultation on folate and vitamin B12 deficiencies. Food Nutr Bull. 2008;29(2 Suppl):S238-44.  2