Megaloblastic Anemia


Indications for Testing

Laboratory Testing

  • CBC
    • Blood smear – may view macro ovalocytes, anisocytosis, and hypersegmented nuclei in white blood cells 
    • Hemoglobin and hematocrit – if anemia not present, evaluate for nonmegaloblastic causes of macrocytosis
  • Reticulocyte count
    • Usually low; if elevated, proceed with hemolytic evaluation
  • B12 and folate levels – use to differentiate single and combined defect
    •  Folate levels
      • Low folate suggests folate deficiency
        • Folate deficiencies are unusual – folate testing may not be necessary
      • Serum RBC folate – acceptable alternative test to serum folate testing
      • Suggest concurrent testing with B12
    • B12 levels
      • B12 <100 pg/mL – B12 deficiency confirmed; consider evaluation for pernicious anemia
      • B12 100-400 pg/mL – order methylmalonic acid (MMA) 
        • MMA elevated – B12 deficiency likely; consider pernicious anemia
        • MMA normal – not pernicious anemia
      • B12 >400 pg/mL – B12 deficiency unlikely; folate deficiency likely
        • If clinical suspicion of B12 deficiency remains – order MMA and check homocysteine levels
          • MMA and homocysteine elevated – B12 deficiency confirmed
    • Normal B12 and folate levels – consider bone marrow biopsy
  • Parietal cell antibody (PCA) and intrinsic factor (IF)-blocking antibody testing (see Megaloblastic Anemia Testing algorithm)
    • PCA – not as helpful as IF-blocking test; lacks specificity
    • Intrinsic factor positive – pernicious anemia confirmed
    • Intrinsic factor negative – order PCA
      • PCA positive – pernicious anemia confirmed
      • PCA negative – order gastrin (serum)
        • Gastrin <100 pg/mL – not pernicious anemia
        • Gastrin >100 pg/mL – pernicious anemia (indirect confirmation)

Differential Diagnosis

  • Nonmegaloblastic etiologies
    • Alcoholism
    • Medication-induced macrocytosis
    • Liver disease-associated macrocytosis
    • Hypothyroidism
    • Infiltrative disorders of the bone marrow
    • Nitrous oxide abuse
    • Splenectomy-induced macrocytosis
  • Megaloblastic etiologies
    • See information in Clinical Background section

Clinical Background

Megaloblastic anemias are a group of macrocytic anemias in which the bone marrow shows megaloblastic erythropoieses.


  • Prevalence – macrocytosis occurs in 2-4% of the population
  • Age – usually occurs in older adults


  • Anemia
    • Male – HgB <13 g/dL 
    • Female – HgB <12 g/dL 
  • Macrocytosis – mean corpuscular volume >100 fL


  • Vitamin B12 (cobalamin) deficiency
    • Malabsorption
      • Absence of intrinsic factor (IF)
    • Achlorhydria 
      • Most common in the elderly and patients on acid suppression
    • Postgastrectomy syndrome
    • Intestinal stasis due to anatomic lesions
    • Ileal abnormalities  
      • Tropical sprue
      • Inherited B12 disorders
  • Folic acid deficiency
  • Combined deficiencies of folic acid and cobalamin are not uncommon


  • Pernicious anemia – most common cause of B12 deficiency
    • Absence of IF
      • Autoimmune destruction of parietal cell antibodies (PCA) is most common etiology
      • 80% have PCA; ≥50% have IF-blocking antibodies

Clinical Presentation

  • Symptoms
    • Often based on the presence of anemia
    • Pale skin, anorexia, sore tongue, numbness, paresthesias
    • >50% present without anemia and have few symptoms
    • Pernicious anemia – increased incidence in patients with other autoimmune diseases such as Graves disease, vitiligo, hypoparathyroidism, Addison disease, Hashimoto disease


  • Oral – folate replacement
  • Parenteral or intranasal – B12 replacement (B12 cannot be replaced orally)
    • In pernicious anemia, lifelong replacement is necessary

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
CBC with Platelet Count 0040002
Method: Automated Cell Count

Screen for anemia and assess for macrocytosis

Vitamin B12 and Folate 0070160
Method: Quantitative Chemiluminescent Immunoassay

First-line test to differentiate between single and combined defect in macrocytic anemia

Vitamin B12 with Reflex to Methylmalonic Acid, Serum (Vitamin B12 Status) 0055662
Method: Quantitative Chemiluminescent Immunoassay/Quantitative High Performance Liquid Chromatography-Tandem Mass Spectrometry

Use in workup of megaloblastic anemia when B12 is 100-400 pg/mL

Renal failure may increase serum methylmalonic acid (MMA) concentrations in patients who do not have vitamin B12 deficiency

Folate, RBC 0070385
Method: Quantitative Chemiluminescent Immunoassay
Acceptable alternative to serum folate    
Intrinsic Factor Blocking Antibody 0070210
Method: Qualitative Enzyme-Linked Immunosorbent Assay

Use to confirm pernicious anemia as etiology of megaloblastic anemia

Gastric Parietal Cell Antibody, IgG 0050596
Method: Semi-Quantitative Enzyme-Linked Immunosorbent Assay

Use to confirm pernicious anemia as etiology of megaloblastic anemia

Gastrin 0070075
Method: Quantitative Chemiluminescent Immunoassay

Use to confirm pernicious anemia as etiology of megaloblastic anemia

Methylmalonic Acid, Serum or Plasma (Vitamin B12 Status) 0099431
Method: Quantitative Liquid Chromatography-Tandem Mass Spectrometry

Evaluate patients with megaloblastic anemia and normal B12 levels with neurological symptoms present and suspicion for B12 deficiency

Homocysteine, Total 0099869
Method: Quantitative Enzymatic

Evaluate patients with megaloblastic anemia and normal B12 levels with neurological symptoms present and suspicion for B12 deficiency

False elevations of plasma or serum homocysteine may occur if the plasma or serum is not promptly separated from the cells at the time of collection

Additional Tests Available
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
Vitamin B12  0070150
Method: Quantitative Chemiluminescent Immunoassay

Preferred test is B12 reflex to MMA

Folate, Serum 0070070
Method: Quantitative Chemiluminescent Immunoassay

Preferred test is B12 & folate panel

Vitamin B12 Binding Capacity 0070260
Method: Quantitative Radioimmunoassay