What blood tests are used to diagnose a suspected vitamin B12 (cobalamin) deficiency in a patient, particularly in high-risk groups such as the elderly, vegetarians, and individuals with gastrointestinal disorders like celiac disease or Crohn's disease?

Blood Tests for Vitamin B12 Deficiency

Serum vitamin B12 is the primary initial test for diagnosing B12 deficiency, with levels <180 pg/mL (133 pmol/L) confirming deficiency, while borderline results (180-350 pg/mL) require methylmalonic acid (MMA) measurement to identify functional deficiency. 1, 2

Initial Diagnostic Approach

Start with serum total vitamin B12 as the first-line test, which costs approximately £2 with rapid turnaround in local laboratories. 1 This test should be ordered in patients presenting with:

• Hematologic findings: Anemia, macrocytosis (elevated MCV), or megaloblastic changes on blood smear 1, 3
• Neurological symptoms: Peripheral neuropathy, paresthesias, ataxia, cognitive difficulties, memory problems, or "brain fog" 1, 2
• Other manifestations: Glossitis, unexplained fatigue, visual problems, or depression 1, 2

Interpretation Algorithm

Step 1: Serum B12 Level Interpretation

• <180 pg/mL (<133 pmol/L): Confirms B12 deficiency—proceed directly to treatment 1, 2
• 180-350 pg/mL (133-258 pmol/L): Indeterminate range—measure MMA to confirm functional deficiency 1, 2
• >350 pg/mL (>258 pmol/L): Makes deficiency unlikely, but consider functional markers if high clinical suspicion persists 1

Critical limitation: Standard serum B12 testing misses functional deficiency in up to 50% of cases. The Framingham Study demonstrated that while 12% had low serum B12, an additional 50% had elevated MMA indicating metabolic deficiency despite "normal" serum levels. 1

Step 2: Confirmatory Testing with Methylmalonic Acid

When B12 results fall in the indeterminate range (180-350 pg/mL), measure serum MMA to identify functional B12 deficiency that reflects actual cellular B12 status. 1, 2

• MMA >271 nmol/L: Confirms functional B12 deficiency with 98.4% sensitivity 1
• MMA ≤271 nmol/L: Makes B12 deficiency unlikely 1

MMA is superior to homocysteine for B12 deficiency diagnosis because it is more specific—MMA is elevated in only 12.2% of folate-deficient patients versus 91% for homocysteine. 1 However, both MMA and homocysteine can be falsely elevated in hypothyroidism, renal insufficiency, and hypovolemia, requiring cautious interpretation in these conditions. 1

Alternative and Supplementary Tests

Active B12 (Holotranscobalamin)

Active B12 measures the biologically active form available for cellular use and is more accurate than total B12, but costs £18 per test with longer turnaround times. 1, 4 Interpretation:

• <25 pmol/L: Confirms deficiency 1
• 25-70 pmol/L: Indeterminate—requires MMA testing 1
• >70 pmol/L: Makes deficiency unlikely 1

Homocysteine

Homocysteine >15 μmol/L supports B12 deficiency diagnosis, though it is less specific than MMA and can be affected by renal impairment, folate deficiency, and other B vitamins. 1 The target for optimal cardiovascular outcomes is homocysteine <10 μmol/L. 1

Interpretation pattern for combined testing:

• Elevated homocysteine + elevated MMA = B12 deficiency 1
• Elevated homocysteine + normal MMA = folate deficiency (or MTHFR dysfunction) 1

Complete Initial Laboratory Panel

In addition to B12 testing, obtain the following baseline tests to establish the full clinical picture and identify complications: 5

• Complete blood count (CBC): Assess for megaloblastic anemia, macrocytosis, pancytopenia 1, 3
• Peripheral blood smear: Evaluate for hypersegmented neutrophils, megaloblastic changes 1
• Liver profile and albumin: Assess nutritional status and rule out liver disease 5
• Iron studies: Identify concurrent iron deficiency (ferritin up to 100 μg/L may still indicate iron deficiency in inflammatory conditions) 1
• Folate level: Check concurrently, as folate deficiency can coexist and must never be treated before B12 deficiency 1, 6
• Renal function: Interpret MMA and homocysteine results appropriately 1

High-Risk Populations Requiring Screening

Testing should be considered in patients with at least one risk factor, even without overt symptoms: 3, 2

• Age >75 years: 18.1% have metabolic B12 deficiency; 25% of those ≥85 years have B12 <170 pmol/L 1
• Gastrointestinal conditions: Ileal resection >20 cm, Crohn's disease with ileal involvement >30-60 cm, celiac disease, atrophic gastritis, pernicious anemia 5, 1, 6
• Post-bariatric surgery: All patients require screening due to reduced gastric acid and intrinsic factor 1
• Medication use: Metformin >4 months, proton pump inhibitors or H2 blockers >12 months 1, 3, 2
• Dietary factors: Vegans, strict vegetarians 3, 2
• Autoimmune conditions: Autoimmune hypothyroidism (28-68% prevalence of B12 deficiency), type 1 diabetes 1, 4

For Crohn's disease patients specifically, routine blood tests including vitamin B12 should be conducted at diagnosis to establish baseline values, with annual screening for those with ileal involvement. 5

Etiologic Testing After Confirming Deficiency

Once B12 deficiency is confirmed, identify the underlying cause to determine appropriate long-term management:

• Intrinsic factor antibodies: Test for pernicious anemia (positive in 50-70% of cases) 1
• Gastric parietal cell antibodies: Less specific but may support diagnosis of autoimmune gastritis 1
• Gastrin levels: Markedly elevated levels (>1000 pg/mL) indicate pernicious anemia 1
• Helicobacter pylori testing: Evaluate for atrophic gastritis 2
• Tissue transglutaminase (tTG) antibodies and total IgA: Screen for celiac disease, particularly in patients with autoimmune thyroid disease (found in 13.3% of B12-deficient hypothyroid patients) 1

Critical Pitfalls to Avoid

Never rely solely on serum B12 to rule out deficiency, especially in patients >60 years where metabolic deficiency is common despite normal serum levels. 1 Elderly populations have significantly higher rates of metabolic deficiency—18.1% of patients >80 years have metabolic B12 deficiency even with "normal" serum B12. 1

Never administer folic acid before treating B12 deficiency, as folic acid may mask the anemia while allowing irreversible neurological damage to progress (subacute combined degeneration of the spinal cord). 1, 6

Do not stop B12 supplementation before testing if the patient is already taking it. Instead, measure MMA as the primary test, which reflects actual cellular B12 status regardless of supplementation. 1 If the patient is not currently taking supplements, start with total serum B12 or active B12 as the initial test. 1

Recognize that macrocytosis precedes anemia in B12 deficiency, with elevated MCV often being the earliest laboratory sign appearing before anemia develops. 1 However, one-third of B12-deficient patients may not have megaloblastic anemia. 1

Cost-Effectiveness Considerations

MMA testing is cost-effective at £3,946 per quality-adjusted life year when B12 results are indeterminate, but using MMA as first-line screening is not cost-effective due to expense (£11-80 per test) and processing delays. 1 The recommended approach is to start with serum B12 (£2), then add MMA for indeterminate results. 1