What initial investigations should I perform for a patient presenting with macrocytic anaemia to differentiate between B12 and folate deficiency?

Guideline-aligned answer with reasoning, red flags and references. Clinically reviewed by Dr Kola Tytler MBBS CertHE MBA MRCGP.

Posted: 16 August 2025Updated: 16 August 2025 Guideline-Aligned (High Confidence) Clinically Reviewed
Dr Kola Tytler MBBS CertHE MBA MRCGPClinical Lead • iatroX

For a patient presenting with macrocytic anaemia, the initial investigations to differentiate between vitamin B12 deficiency and folate deficiency involve specific blood tests.

  • Initial Blood Tests:
    • For suspected vitamin B12 deficiency, the initial test should be either total B12 (serum cobalamin) or active B12 (serum holotranscobalamin) .
    • It is also important to check serum folate levels ,,.
    • Blood samples for diagnostic tests should be taken before starting any vitamin B12 replacement .
    • It is crucial to check vitamin B12 levels in all people before starting folic acid treatment, as folic acid can improve wellbeing, mask an underlying B12 deficiency, and allow neurological disease to develop .
  • Further Differentiating Tests (if initial results are indeterminate or for specific scenarios):
    • If initial total or active B12 test results are indeterminate, or if symptoms of vitamin B12 deficiency persist, a further test to measure serum methylmalonic acid (MMA) concentrations can be considered . Elevated MMA is indicative of vitamin B12 deficiency .
    • Plasma homocysteine can also be used as an initial test in specific cases, such as suspected vitamin B12 deficiency caused by recreational nitrous oxide use . However, when interpreting plasma homocysteine results, it is important to consider that folate deficiency can also increase these levels .

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