Megaloblastic anemia, a group of diseases characterized by large red blood cells (RBCs), is a form of macrocytic anemia. The condition results from impaired DNA synthesis and consequent defective erythrocyte maturation. Megaloblastic anemia is common in adults older than 60 years (a demographic group that continues to grow in the United States) and is associated with significant morbidity. Causes of megaloblastic anemia include vitamin B12 (cobalamin) or folate (vitamin B9) deficiency and specific drugs. Vitamin B12 deficiency and associated megaloblastic anemia may be caused by pernicious anemia, in which a lack of intrinsic factor (IF) prevents sufficient absorption of vitamin B12. The initial evaluation for megaloblastic anemia includes a CBC and review of a peripheral blood smear. Additional testing for vitamin B12 and/or folate deficiency and antibody testing for pernicious anemia may be performed based on the results of these initial tests.
Quick Answers for Clinicians
Patients with megaloblastic anemia may present with symptoms of anemia such as pale skin and fatigue. In severe cases, atrophy of mucous membranes may occur and result in pain in the mouth; infants may exhibit failure to thrive. There may be jaundice as a result of hemolysis. Patients with megaloblastic anemia due to vitamin B12 deficiency may also present with symptoms of peripheral neuropathy, such as dysesthesia and hypoesthesia. In severe cases, ataxia and other severe peripheral and central nervous system manifestations (eg, changes in mental status) of prolonged vitamin B12 deficiency may be present. However, some patients may be asymptomatic, and certain laboratory features of megaloblastic anemia may be masked by concurrent iron deficiency or microcytic anemia.
An evaluation for megaloblastic anemia is often prompted by CBC results. CBC findings may include anemia (hemoglobin <13 g/dL in men and <12 g/dL in women) with macrocytosis (mean corpuscular volume >100 fL). A peripheral blood smear may reveal macro-ovalocytes, anisocytosis, poikilocytosis, and hypersegmented neutrophils, a specific indicator of megaloblastic anemia.
Causes of megaloblastic anemia include vitamin B12 or folate deficiency (as a result of decreased intake, decreased absorption, or increased demand), specific drugs, inborn errors of metabolism, myelodysplastic syndromes, and problems with DNA synthesis (eg, from chemotherapy). However, folate fortification programs are increasingly common and have greatly reduced the prevalence of folate deficiency. There are numerous less common causes, including nitrous oxide exposure and Diphyllobothrium latum infection. Because vitamin deficiency is a major cause of megaloblastic anemia, older individuals, persons who are malnourished, those with alcohol use disorder, and individuals who consume a vegetarian or vegan diet are at increased risk for megaloblastic anemia.
Drugs that induce megaloblastic anemia typically interfere with vitamin B12 or folate absorption or metabolism. These drugs include immunomodulators and antineoplastic agents that act on purine metabolism or pyrimidine synthesis (eg, methotrexate, fluorouracil); antibiotics, hormones, or tuberculosis treatments that interfere with absorption (eg, estrogens, tetracyclines, isoniazid); antimalarial agents; and many others. A detailed medical history is therefore useful in ruling out medication-related megaloblastic anemia.
Indications for Testing
Testing for megaloblastic anemia is used to identify the etiology of macrocytosis and/or clinical features of megaloblastic anemia (eg, neurologic symptoms).
For information on the initial evaluation of anemia, see the ARUP Consult Anemia topic.
Testing for megaloblastic anemia is often prompted by the identification of macrocytosis from a CBC; if neurologic symptoms or other clinical features prompt testing, a CBC should be performed. Hemoglobin and hematocrit can be measured to confirm anemia. Once macrocytic anemia has been confirmed, a peripheral smear is recommended. Anisocytosis, poikilocytosis, macro-ovalocytes, and hypersegmented neutrophils may be present. However, these peripheral smear findings are not diagnostic. A reticulocyte count is also recommended. The reticulocyte count is generally low in megaloblastic anemia; therefore, if reticulocytosis is present, consider a workup for hemolysis (including lactate dehydrogenase and bilirubin tests).
If megaloblastic anemia is suspected based on clinical features and the results of the CBC and peripheral smear, evaluation for possible drug-induced megaloblastic anemia through a detailed medical history is recommended. If the megaloblastic anemia is not drug induced, testing for vitamin deficiencies should be considered.
Evaluation for Vitamin Deficiency
Vitamin B12 and/or folate deficiency may result in megaloblastic anemia. Because vitamin B12 deficiency is much more common than folate deficiency, the evaluation should commence with tests for vitamin B12, or a combined vitamin B12/folate test can be used.
Vitamin B12 Deficiency Testing
Testing for vitamin B12 deficiency begins with a serum concentration test. Follow-up testing depends on the result of this test. The patient’s use of vitamin B12 injections should be considered when interpreting test results, given that serum vitamin B12 concentration may be affected by injections administered within approximately 2 weeks of specimen collection.
|Serum Vitamin B12 Concentration||Interpretation||Next Steps|
|<200 pg/mL||Vitamin B12 deficiency is probable||Evaluation for pernicious anemia is recommended|
|200-400 pg/mL||Results are borderline||Perform MMA and homocysteine tests; consider evaluation for pernicious anemia|
|>400 pg/mL||Vitamin B12 deficiency is unlikelya||Consider testing for folate deficiency|
aIf suspicion for vitamin B12 deficiency persists in a patient with a vitamin B12 concentration >400 pg/mL, consider MMA and homocysteine tests.
MMA, methylmalonic acid
The MMA test is a sensitive and specific indicator of vitamin B12 deficiency that can be used if the serum vitamin B12 concentration is borderline. An elevated MMA concentration (>0.4 µmol/L) confirms vitamin B12 deficiency. Plasma homocysteine levels may also be increased in vitamin B12 deficiency, although this test is not specific. If vitamin B12 deficiency is confirmed, evaluation for pernicious anemia is recommended. For additional information on vitamin B12 and MMA tests, see the ARUP Consult Vitamins topic.
Folate Deficiency Testing
Testing for folate deficiency should begin with a serum or plasma folate test. A serum or plasma folate concentration <4 µg/L indicates folate deficiency. If results are not conclusive, an RBC folate test can be performed, followed by a homocysteine test if the concentration of folate is borderline. Although the homocysteine test is not specific, an increased homocysteine level is consistent with folate deficiency. For additional information on these tests, see the ARUP Consult Vitamins topic.
Evaluation for Pernicious Anemia
The majority of patients with pernicious anemia have antibodies for parietal cells and IF. In patients with confirmed vitamin B12 deficiency, testing for pernicious anemia is recommended. In patients with borderline vitamin B12 and MMA test results, this testing is considered optional.
Intrinsic Factor Blocking Antibody Testing
Antibodies to IF are specific to pernicious anemia and are present in the majority of patients. A positive IF antibody test confirms pernicious anemia.
Parietal Cell Antibody Testing
Parietal cell antibody tests are more sensitive than IF antibody tests, but parietal cell antibodies are less specific to pernicious anemia and may be seen in chronic gastritis. A positive parietal cell antibody test following a negative IF test in the appropriate clinical context confirms the diagnosis of pernicious anemia.
Serum Gastrin Testing
Gastrin concentration is generally elevated in pernicious anemia but may be increased in other conditions as well. Gastrin testing can be considered to indirectly confirm pernicious anemia if a parietal cell antibody test is negative but suspicion for pernicious anemia persists.
The Schilling test is an obsolete test that measures the enteral absorption of vitamin B12; it is not generally available. The deoxyuridine suppression test uses radioactive deoxyuridine to assess vitamin B12 and folate status. This test may be useful in some patients if other tests fail to diagnose vitamin B12 or folate deficiency. Bone marrow biopsy may also be considered if other tests fail to yield a diagnosis.
ARUP Laboratory Tests
Use to evaluate cellular morphology
Aids in the detection of vitamin B12 deficiency
For patients with known risk factors for folate deficiency, consider Vitamin B12 and Folate test
Preferred reflex test for detection of vitamin B12 deficiency in individuals with macrocytic or unexplained anemia
Quantitative Chemiluminescent Immunoassay/Quantitative High Performance Liquid Chromatography-Tandem Mass Spectrometry
Use to evaluate vitamin B12 deficiency
Quantitative Liquid Chromatography-Tandem Mass Spectrometry
Use to confirm vitamin B12 or folate deficiency
Use to detect vitamin B12 or folate deficiency
Aids in the detection of vitamin B9 (folate) deficiency
Use to confirm pernicious anemia as etiology of megaloblastic anemia
Qualitative Enzyme-Linked Immunosorbent Assay
Use to evaluate pernicious anemia or immune-mediated deficiency of vitamin B12
Semi-Quantitative Enzyme-Linked Immunosorbent Assay
Indirectly confirms pernicious anemia
Lanier JB, Park JJ, Callahan RC. Anemia in older adults. Am Fam Physician. 2018;98(7):437-442.
Green R, Datta Mitra A. Megaloblastic anemias: nutritional and other causes. Med Clin North Am. 2017;101(2):297-317.
Practical Diagnosis of Hematologic Disorders - 5th edition
Kjeldsberg, CR, ed. Practical Diagnosis of Hematologic Disorders. 5th ed. American Society for Clinical Pathology; 2010.
Hesdorffer CS, Longo DL. Drug-induced megaloblastic anemia. N Engl J Med. 2016;374(7):696-697.
Bizzaro N, Antico A. Diagnosis and classification of pernicious anemia. Autoimmun Rev. 2014;13(4-5):565-568.
Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood. 2017;129(19):2603-2611.
Oberley MJ, Yang DT. Laboratory testing for cobalamin deficiency in megaloblastic anemia. Am J Hematol. 2013;88(6):522-526.
Socha DS, DeSouza SI, Flagg A, et al. Severe megaloblastic anemia: vitamin deficiency and other causes. Cleve Clin J Med. 2020;87(3):153-164.
Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013;368(2):149-160.
Reflex pattern – if vitamin B12 is <300 pg/mL, methylmalonic acid, serum will be added