What is the recommended workup to assess vitamin B12 deficiency?

Assessment of Vitamin B12 Deficiency

Start with total serum vitamin B12 as the first-line test, then add methylmalonic acid (MMA) testing only when B12 results fall in the indeterminate range (180–350 pg/mL) to confirm functional deficiency. 1

Initial Testing Strategy

First-Line Test Selection

• Measure total serum vitamin B12 first because it costs approximately £2 per test with rapid turnaround in local laboratories, making it the most cost-effective initial screening tool. 1
• Active B12 (holotranscobalamin) is an alternative first-line test that measures the biologically active form available for cellular use, but costs £18 per test with longer processing times due to external laboratory requirements. 1, 2
• Do not use MMA as a first-line screening test because universal MMA screening is not cost-effective due to expense (£11–£80 per test) and processing delays. 1

Interpretation of Total Serum B12 Results

• < 180 pg/mL (< 133 pmol/L): Definite deficiency—initiate treatment immediately without additional testing. 1, 3
• 180–350 pg/mL (133–258 pmol/L): Indeterminate range—proceed to MMA measurement to confirm functional deficiency. 1, 3
• > 350 pg/mL (> 258 pmol/L): Deficiency unlikely; consider MMA only if clinical suspicion remains high despite normal levels. 1

Interpretation of Active B12 (Holotranscobalamin)

• < 25 pmol/L: Confirmed deficiency—start treatment immediately. 1
• 25–70 pmol/L: Indeterminate—order MMA to clarify functional status. 1
• > 70 pmol/L: Deficiency unlikely; no further B12-specific testing required. 1

Confirmatory Testing with Methylmalonic Acid

When to Order MMA

• Measure MMA only when initial B12 results are indeterminate (180–350 pg/mL for total B12 or 25–70 pmol/L for active B12). 1
• MMA testing in this targeted manner yields a cost-effectiveness of £3,946 per quality-adjusted life year. 1
• MMA detects an additional 5–10% of patients with functional B12 deficiency who have low-normal serum B12 levels. 1

MMA Interpretation

• MMA > 271 nmol/L confirms functional B12 deficiency with 98.4% sensitivity, making it highly reliable for confirming cellular B12 deficiency. 1
• MMA is more specific than homocysteine for B12 deficiency, with MMA elevated in only 12.2% of folate-deficient patients versus 91% for homocysteine. 1

Important Caveats for MMA Testing

• Both MMA and homocysteine can be falsely elevated in hypothyroidism, renal insufficiency, and hypovolemia—interpret cautiously in these conditions. 1
• Standard serum B12 testing misses functional deficiency in up to 50% of cases; the Framingham Study found that 12% had low serum B12, but an additional 50% had elevated MMA indicating metabolic deficiency despite "normal" serum levels. 1

Complete Blood Count Findings

• Check a complete blood count (CBC) with differential to assess for megaloblastic anemia, although anemia may be absent in one-third of B12 deficiency cases. 1
• Macrocytosis (MCV > 98 fL) is often the earliest laboratory sign of B12 deficiency, appearing before anemia develops. 1
• Hypersegmented neutrophils on peripheral smear indicate a megaloblastic pattern from impaired DNA synthesis. 1

Additional Diagnostic Tests for Underlying Cause

When B12 Deficiency is Confirmed

• Test for intrinsic factor antibodies if pernicious anemia is suspected; positive antibodies indicate lifelong treatment with intramuscular B12 is necessary. 1
• Measure gastrin levels if pernicious anemia is suspected, with markedly elevated levels (> 1,000 pg/mL) indicating the condition. 1
• Screen for Helicobacter pylori and autoantibodies associated with autoimmune gastritis in patients without a clear cause of deficiency. 3

High-Risk Populations Requiring Screening

• Age ≥ 75 years: 18.1% of patients > 80 years have metabolic B12 deficiency. 1, 2
• Metformin use > 4 months: Associated with approximately three-fold increased risk of B12 deficiency. 1, 2
• PPI or H2 blocker use > 12 months: Impairs B12 absorption. 1, 2
• Gastric or small intestine resections: Ileal resection > 20 cm requires lifelong supplementation. 1
• Inflammatory bowel disease: Crohn's disease affecting > 30–60 cm of terminal ileum warrants annual screening. 1
• Autoimmune conditions: Thyroid disease, type 1 diabetes, and Sjögren syndrome have 28–68% prevalence of B12 deficiency. 1
• Vegans or strict vegetarians: No dietary source of B12. 1, 4

Clinical Symptoms Warranting Testing

• Neurological manifestations: Symmetric paresthesia, balance issues, impaired gait, proprioception impairment, peripheral neuropathy. 1, 2
• Cognitive symptoms: Difficulty concentrating, short-term memory loss, "brain fog," personality changes. 1, 2
• Visual problems: Blurred vision, optic atrophy, visual field loss related to optic nerve dysfunction. 1, 2
• Hematologic findings: Anemia, macrocytosis on CBC. 1, 2
• Oral symptoms: Glossitis, glossodynia, fissured tongue. 1
• Mood disturbances: Depression is a common clinical association with cobalamin deficiency. 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
• Do not postpone treatment in patients with B12 < 180 pg/mL and macrocytic anemia—immediate treatment is mandated regardless of pending ancillary test results. 1
• Never administer folic acid before treating B12 deficiency, as it may mask anemia while allowing irreversible neurological damage to progress. 1, 5
• Do not stop B12 supplements before testing—instead, measure MMA as the primary test when patients are already taking supplements, as this functional marker reflects actual cellular B12 status regardless of supplementation. 1
• Megaloblastic anemia can be masked by concurrent folic acid supplementation. 2

Practical Diagnostic Algorithm

1. Identify patients at risk based on age, medications, gastrointestinal conditions, or clinical symptoms. 1, 2
2. Order total serum B12 as the initial test (or active B12 if available and cost is not prohibitive). 1
3. If B12 < 180 pg/mL: Diagnose deficiency and initiate treatment immediately without additional testing. 1
4. If B12 180–350 pg/mL: Order MMA; if MMA > 271 nmol/L, confirm functional B12 deficiency and treat. 1
5. If B12 > 350 pg/mL but high clinical suspicion persists: Consider MMA testing on a case-by-case basis, particularly in elderly patients or those with neurological symptoms. 1
6. Check CBC with differential to assess for macrocytosis and hypersegmented neutrophils. 1
7. Evaluate underlying cause with intrinsic factor antibodies, gastrin levels, H. pylori testing, and autoimmune gastritis antibodies. 1, 3

Special Considerations for Specific Populations

• Post-bariatric surgery patients: Require 1,000 mcg/day oral or 1,000 mcg/month IM indefinitely; deficiencies can occur when serum concentrations are 300 pmol/L (approximately 406 pg/mL). 1
• Autoimmune hypothyroidism: Screen all patients at diagnosis and annually thereafter due to 28–68% prevalence of B12 deficiency. 1
• Post-stroke patients: 17.3% have biochemical or metabolic deficiency; serum B12 < 258 pmol/L with elevated homocysteine or MMA defines metabolic deficiency. 1
• HIV disease: Associated with B12 deficiency and warrants testing. 2

Monitoring After Diagnosis

• Recheck B12 levels after 3–6 months of treatment to confirm normalization. 1
• Monitor hematocrit and reticulocyte counts daily from the fifth to seventh days of therapy, then frequently until hematocrit is normal. 5
• Target homocysteine < 10 μmol/L for optimal cardiovascular outcomes. 1
• Continue annual B12 screening in high-risk populations even after initial treatment. 1