assessment, investigation and diagnosis of B12 deficiency

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  • clinical

  • diagnosis of megaloblastic anaemia:
    • FBC - macrocytic anaemia; a thrombocytopaenia may occur secondary to ineffective megakaryopoiesis
    • blood film - macrocytosis, hypersegmented neutrophils
    • bone marrow - megaloblastic, erythropoiesis, giant metamyelocytes

  • measurement of B12 / folate levels - in B12 deficiency red cell folate levels are low (B12 required in synthesis) but serum folate is generally normal or high (1)

  • measurement of B12 absorption - Schilling test - very rarely undertaken now (3)

  • other investigations to help define the cause of the vitamin B12 deficiency
    • thyroid function tests and anti-thyroid antibodies
    • test for coeliac disease
      • tissue transglutaminase (tTG)
    • tests for generalised malabsorption (if symptoms are suggestive) - faecal tests are generally only requested by a gastroenterologist/after gastroenterological advice
      • serum
        • calcium and vitamin D
        • folate
        • ferritin
      • faecal
        • fats
        • elastase

  • presence of intrinsic factor antibodies in serum
    • positive in 50-60% of patients with pernicious anaemia (1)
    • the presence of intrinsic factor (IF) is diagnostic of pernicious anaemia but negative intrinsic factor antibodies does not exclude pernicious anaemia (due to the test’s low sensitivity (50-60%)) - gastric parietal cell (GPC) antibodies are seen in 95% of cases of pernicious anaemia and, although there is an overlap with other autoimmune diseases and with normal individuals, a negative result makes pernicious anaemia unlikely (1)
      • note a review stated testing for anti-gastric parietal cell antibodies is not recommended because of the variable specificity of 50-100% (4)
    • a positive anti-GPC and/or anti-IF antibody test does need repeating (3)

  • urinary methylmalonyl CoA urinary excretion is increased in B12 deficiency - B12 is the co-enzyme in the conversion reaction of methylmalonyl CoA to succinyl CoA


  • the clinical picture is the most important factor in assessing the results of the serum vitamin B12. Definitive cut off points for clinical and subclinical deficiency are not possible. Bear in mind:
    • the test measures total, not metabolically active vitamin B12
    • levels are not easily correlated with clinical symptoms, although patients with vitamin B12 levels <100ng/L almost always have clinical or metabolic evidence of vitamin B12 deficiency, and <150ng/l usually do
    • in most patients with clinically significant vitamin B12 deficiency, the serum level is below 200ng/L but clinically significant vitamin B12 deficiency may be present even when levels are in the normal range, especially in elderly patients (2)
    • about a third of patients with B12 deficiency may not have macrocytosis (4)


  1. Pulse 2004; 64(35):88.
  2. NHS Wiltshire CCG. Investigation and treatment of Vitamin B12 (cobalamin) deficiency in primary care
  3. (accessed 25/4/2020)
  4. Royal United Hospital Bath NHS Trust. Guidelines for the Investigation & Management of vitamin B12 deficiency (accessed 25/4/2020).
  5. Mohamed M et al. Pernicious anaemia. BMJ 2020;369:m1319.

Last edited 04/2020 and last reviewed 04/2020