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Neurological disease associated with vitamin B12 deficiency may be irreversible; therefore, early detection is critical in preventing permanent neurological damage.

Serum vitamin B12 is a standard initial diagnostic test.[1]​ Low serum vitamin B12 (generally <148 picomols/L [<200 picograms/mL]) in the presence of specific clinical symptoms provides evidence of deficiency. Supplementary tests, including markers of tissue deficiency (homocysteine, methylmalonic acid [MMA], and holotranscobalamin), improve earlier diagnosis, and may be considered.

Optimal use of serum vitamin B12 and tissue markers is undefined, but general guidance will help the clinician to determine whether a patient has true vitamin B12 deficiency.

At-risk groups

The clinician should take certain risk factors into account when considering who should be tested for vitamin B12 deficiency.

Advancing age increases risk.[11][12][13][14][15][16]​​ Older patients who present with clinical features of deficiency may have vitamin B12 levels within the reference range; further testing may be warranted.[1][64]​​​​

Chronic use of certain medicines (specifically, metformin, H2 receptor antagonists, proton-pump inhibitors, and anticonvulsants) can also place a patient at risk of deficiency.[31][32][37]​​​[38][50][51][53]​​

Recreational nitrous oxide (N₂O) misuse may increase the risk of vitamin B12 deficiency.[39][40]​​

Patients with chronic gastrointestinal (GI) illnesses that can cause malabsorption or inadequate absorption, including Crohn's disease and coeliac disease, and those with a history of upper GI surgery, including gastrectomy, gastric bypass, or ileectomy, should be tested for vitamin B12 deficiency.[27][45][46][47]

Vegans or strict vegetarians who do not take additional dietary supplementation are at risk of developing vitamin B12 deficiency.[26][48]

Vitamin B12 deficiency may be seen in 20% to 30% of women during pregnancy, and is particularly common if the woman is vegetarian or vegan.[11][29]​ 

Symptoms and signs

Patients with unexplained neurological disease (specifically, decreased vibration sense, gait abnormalities, and peripheral neuropathies) should be tested for vitamin B12 deficiency. Neuropsychiatric complaints such as depression and dementia may alert the clinician in to an occult vitamin B12 deficiency.[11]​​

Late signs of vitamin B12 deficiency include angular cheilitis, glossitis of the tongue, and signs of frank anaemia and thrombocytopenia.

Initial diagnostic testing

Serum vitamin B12 remains a useful initial diagnostic test due to its widespread availability and familiarity. However, caution must be used when interpreting the values, as there are no well-defined cut-offs for deficiency.[65]

The likelihood of vitamin B12 deficiency can be defined according to the serum vitamin B12 level as follows:[1][2]

  • Probable vitamin B12 deficiency: <148 picomols/L (<200 picograms/mL)

  • Possible vitamin B12 deficiency: 148 to 258 picomols/L (201 to 350 picograms/mL)

  • Unlikely vitamin B12 deficiency: >258 picomols/L (>350 picograms/mL).

Vitamin B12 level >258 picomols/L (>350 picograms/mL) does not exclude vitamin B12 deficiency, particularly if pernicious anaemia is suspected. Spuriously normal or high serum vitamin B12 levels have been reported in patients with pernicious anaemia due to anti-intrinsic factor antibody (anti-IFAB) interference in laboratory assays.[3][4][5][6]

A full blood count with peripheral smear is useful to determine whether there is evidence of macrocytosis and frank anaemia, leukopenia, or thrombocytopenia. This suggests a more severe and prolonged vitamin B12 deficiency. However, a normal mean corpuscular volume (MCV), haemoglobin, and haematocrit are not useful to rule out vitamin B12 deficiency, as many patients with vitamin B12 deficiency may have normal haematological parameters.

A peripheral smear may show the classic hypersegmented polymorphonucleated cells and megalocytes found in severe vitamin B12 deficiency with associated macrocytic anaemia, but is not sensitive to early vitamin B12 deficiency.

In the era of folic acid fortification, concomitant folate deficiency is rare. In parts of the world where nutritional deficiencies are common, testing for concomitant folic acid deficiency and treatment can help clarify whether true vitamin B12 deficiency co-exists.

A reticulocyte count may also be considered to differentiate B12 deficiency from haemolytic anaemia. Vitamin B12 deficiency is associated with a low reticulocyte index, whereas haemolytic anaemia is associated with a high reticulocyte index.

Clinical assessment of deficiency severity

The severity of the deficiency can be graded clinically as follows:

  • Mild to moderate neurological manifestations: peripheral neuropathy (e.g., dysaesthesia/paraesthesia), polyneuropathy, and depression.

  • Mild to moderate haematological manifestations: usually asymptomatic with normal haematocrit and an MCV that is at the upper limit of the normal range or mildly elevated.

  • Severe neurological manifestations: sub-acute combined spinal degeneration, dementia, or cognitive impairment. Sub-acute combined spinal degeneration is progressive neurological degeneration of the posterior and lateral columns of the spinal cord; patients present with ataxia, decreased vibration sense, muscle weakness, and hyper-reflexia.

  • Severe haematological manifestations: pancytopenia and marked symptomatic anaemia.

Confirmatory diagnostic testing: serum vitamin B12 <200 picograms/mL

Confirmatory diagnosis is generally unnecessary and empiric treatment should begin. Clinical and serologic response in follow-up confirms vitamin B12 deficiency.[1][2]

Confirmatory diagnostic testing: serum vitamin B12 201 to 350 picograms/mL

Methylmalonic acid (MMA) can be very sensitive for vitamin B12 deficiency but falsely high levels can occur in renal disease.[1] Additionally, an abnormal MMA level is undefined. Patients with serum vitamin B12 201 to 350 picograms/mL together with an elevated MMA can be considered to have probable vitamin B12 deficiency. Diagnosis is confirmed if vitamin levels normalise and serum vitamin B12 rises with adequate vitamin B12 treatment.

MMA

Can be elevated (i.e., >0.4 micromol/L) with folate deficiency. Elevated MMA may be spurious and requires subsequent follow-up to determine whether MMA normalises with adequate treatment. Results should be interpreted with caution in those with renal disease as this can elevate MMA levels.[1]

Homocysteine

Can be elevated (i.e., >15 micromol/L [>2.03 mg/dL]) with folate deficiency, hypothyroidism, and vitamin B12 deficiency.[1] Patients with elevated homocysteine related to vitamin B12 deficiency (when folate deficiency and hypothyroidism are ruled out) should have normalisation with empirical vitamin B12 treatment.

Holotranscobalamin (hTC)

This is transcobalamin bound to vitamin B12, and can be a measure of the true functional serum vitamin B12 levels. Several studies have reported greater diagnostic accuracy with the hTC assay than with other assays measuring markers of vitamin B12 deficiency.[66] hTC may be the first marker to be detected with vitamin B12 deficiency. Levels of hTC <35 picograms/L can be consistent with vitamin B12 deficiency.[1][67][68][69]

Confirmatory diagnostic testing: serum vitamin B12 >350 picograms/mL

Patients with pernicious anaemia may have spuriously normal or high serum vitamin B12 levels.[3][4][5][6]

If pernicious anaemia is suspected in patients with normal or high serum vitamin B12 levels (>350 picograms/mL), further testing for MMA, homocysteine, and hTC should be carried out to determine if vitamin B12 deficiency is present.[1]

Determining the underlying cause of vitamin B12 deficiency

Once the diagnosis of vitamin B12 deficiency is established, an aetiology should be sought. While treatment remains the same, vitamin B12 deficiency can lead the astute clinician to discover an underlying malabsorption process such as coeliac disease or Crohn's disease.

Pernicious anaemia can be determined by testing for the following:

  • Anti-IFAB: only 50% sensitive, but highly specific for pernicious anaemia.[2] Testing for anti-IFAB should be done before initiating vitamin B12 replacement therapy because high vitamin B12 levels may lead to false positive results.[70][71]

  • Antiparietal cell (APC) antibody: highly sensitive (85%), but has low specificity for pernicious anaemia because APC antibodies may be elevated in atrophic gastritis.[2]

  • Fasting serum gastrin levels rise in gastric achlorhydria and can signify pernicious anaemia.[72]

Venepuncture and phlebotomy animated demonstration
Venepuncture and phlebotomy animated demonstration

How to take a venous blood sample from the antecubital fossa using a vacuum needle.

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