Megaloblastic Anemia

  • Diagnosis
  • Algorithms
  • Background
  • Lab Tests
  • References
  • Related Content

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
      • Not necessary testing for most patients due to folate supplementation (Gudgeon, 2015; Gilfix, 2014)
      • 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

Megaloblastic Anemia Testing Algorithm

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) (pernicious anemia)
    • 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 of other autoimmune diseases such as Graves disease, vitiligo, hypoparathyroidism, Addison disease, Hashimoto disease

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

CBC with Platelet Count 0040002
Method: Automated Cell Count

Vitamin B12 and Folate 0070160
Method: Quantitative Chemiluminescent Immunoassay

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


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

Intrinsic Factor Blocking Antibody 0070210
Method: Qualitative Enzyme-Linked Immunosorbent Assay

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

Gastrin 0070075
Method: Quantitative Chemiluminescent Immunoassay

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

Homocysteine, Total 0099869
Method: Quantitative Enzymatic

Additional Tests Available

Vitamin B12  0070150
Method: Quantitative Chemiluminescent Immunoassay


Aids in detection of vitamin B12 deficiency in individuals with macrocytic or unexplained anemia, or unexplained neurologic disease

Folate, Serum 0070070
Method: Quantitative Chemiluminescent Immunoassay


Aids in detection of folate deficiency

Preferred test is B12 & folate panel

Vitamin B12 Binding Capacity 0070260
Method: Quantitative Radioimmunoassay

Vitamin B12 Deficiency Panel 2012276
Method: Quantitative Gas Chromatography/Mass Spectrometry 


Not recommended for initial testing in suspected B12 deficiency; may be useful when B12 and MMA results alone are equivocal

Panel includes methylmalonic acid, 2-methylcitric acid, homocysteine, and cystathionine

General References

Bizzaro N, Antico A. Diagnosis and classification of pernicious anemia. Autoimmun Rev. 2014; 13(4-5): 565-8. PubMed

Ford J. Red blood cell morphology. Int J Lab Hematol. 2013; 35(3): 351-7. PubMed

Galloway M, Hamilton M. Macrocytosis: pitfalls in testing and summary of guidance. BMJ. 2007; 335(7625): 884-6. PubMed

Gilfix B. Utility of measuring serum or red blood cell folate in the era of folate fortification of flour. Clin Biochem. 2014; 47(7-8): 533-8. PubMed

Gudgeon P, Cavalcanti R. Folate testing in hospital inpatients. Am J Med. 2015; 128(1): 56-9. PubMed

Kaferle J, Strzoda C. Evaluation of macrocytosis. Am Fam Physician. 2009; 79(3): 203-8. PubMed

Oberley M, Yang D. Laboratory testing for cobalamin deficiency in megaloblastic anemia. Am J Hematol. 2013; 88(6): 522-6. PubMed

Stabler S. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013; 368(2): 149-60. PubMed

References from the ARUP Institute for Clinical and Experimental Pathology®

Miller J, Garrod M, Rockwood A, Kushnir M, Allen L, Haan M, Green R. Measurement of total vitamin B12 and holotranscobalamin, singly and in combination, in screening for metabolic vitamin B12 deficiency. Clin Chem. 2006; 52(2): 278-85. PubMed

Owen W, Roberts W. Comparison of five automated serum and whole blood folate assays. Am J Clin Pathol. 2003; 120(1): 121-6. PubMed

Medical Reviewers

Last Update: December 2015