How do I assess folate status?

Assessing Folate Status

Measure both serum folate and red blood cell (RBC) folate simultaneously, along with vitamin B12 and homocysteine levels, to comprehensively assess folate status. 1, 2

Primary Laboratory Tests

Serum Folate

• Serum folate reflects recent dietary intake (past few days to weeks) and serves as the earliest indicator of altered folate exposure 1, 3
• Normal threshold: ≥10 nmol/L (approximately 4.4 ng/mL), with optimal levels ≥13.0 ng/mL 3
• Serum folate below 10 nmol/L confirms deficiency 1
• Should be measured in a fasting state for most accurate assessment 4

Red Blood Cell (RBC) Folate

• RBC folate is the preferred marker for long-term folate status as it reflects folate accumulation during erythropoiesis over the preceding 3 months and tissue stores 1, 2
• Normal threshold: ≥340 nmol/L 1, 2
• RBC folate below 340 nmol/L confirms deficiency 1, 2
• Less influenced by recent dietary intake compared to serum folate 5, 6

Essential Concurrent Testing

Vitamin B12 (Cobalamin)

• Always measure vitamin B12 simultaneously with folate to avoid missing combined deficiencies 2, 3
• Both deficiencies cause megaloblastic anemia and elevated homocysteine 1, 2
• High folate can mask B12 deficiency by correcting anemia while allowing irreversible neurological damage to progress 1, 2, 3

Methylmalonic Acid (MMA)

• MMA differentiates isolated folate deficiency from B12 deficiency 1, 2
• MMA remains normal in isolated folate deficiency but is elevated in B12 deficiency 1, 2
• Measure when B12 is borderline (<148 pmol/L) or low 7

Homocysteine

• Plasma homocysteine serves as a functional marker of folate status since remethylation of homocysteine to methionine is folate-mediated 1, 2
• Elevated in both folate and B12 deficiency 1, 2
• Levels >15 µmol/L indicate functional deficiency 7, 3
• Also affected by vitamins B2, B6, and renal impairment 1

Laboratory Methods

Gold Standard: Microbiological Assay

• Lactobacillus rhamnosus microbiological assay (MBA) is the gold standard for folate measurement 1, 2
• Responds to all active monoglutamate forms of folate 1, 2
• Measures total folate present in the sample 1, 2
• Requires careful pre-analytical handling: protect samples from light, temperature, pH changes, and oxygen 1
• Use ascorbic acid to protect folate from oxidation 1
• May be affected by antibiotics that inhibit L. rhamnosus 1

Alternative Methods

• Protein binding assays are widely available but have different affinities for different folate forms and greater analytical variability 1, 8
• Liquid chromatography-mass spectrometry (LC-MS) enables quantification of individual folate forms with good sensitivity but requires expensive equipment and experienced staff 1

Clinical Testing Algorithm

Initial Assessment

1. Order simultaneously: serum folate, RBC folate, vitamin B12, and homocysteine 2, 3
2. If macrocytic anemia is present (high MCV), add complete blood count with reticulocyte count 2
3. If B12 is borderline or low, add MMA to confirm functional B12 deficiency 2, 7

Follow-Up Monitoring

• Recheck folate levels within 3 months after initiating supplementation to verify normalization 3
• In conditions with increased folate requirements (chronic infection, alcoholism, anticonvulsant therapy), monitor every 3 months until stable, then annually 3
• Verify normalization of blood picture and resolution of clinical symptoms 3

Critical Pitfalls to Avoid

Missing B12 Deficiency

• Never supplement folate without checking B12 first 2, 7, 3
• If elevated folate is found with low or borderline B12, stop folic acid immediately and initiate urgent B12 replacement (1000 µg IM every other day for one week, then monthly for life) 7

Timing and Sample Handling

• Folate forms are susceptible to degradation—ensure proper sample collection and processing 1
• Fasting samples preferred for serum folate 4
• RBC folate requires hemolysis of whole blood with ascorbic acid solution after collection 1

Interpretation Errors

• Serum folate <8 µg/L (18 nmol/L) shows significant effect on homocysteine and should be considered the decision limit for folate depletion 4
• Haematological abnormalities require profound folate deficiency (serum folate <6 µg/L or 14 nmol/L) 4