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
and/or 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 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.
Testing Strategy Based on Serum Vitamin B12 ConcentrationSerum Vitamin B12 ConcentrationInterpretationNext Steps<200 pg/mLVitamin B12 deficiency is probableEvaluation for pernicious anemia is recommended200-400 pg/mLResults are borderlinePerform MMA and homocysteine tests; consider evaluation for pernicious anemia>400 pg/mLVitamin B12 deficiency is unlikelyaConsider testing for folate deficiencyaIf 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
Source: Green, 2017
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 is recommended. For additional information on vitamin B12 and MMA tests, see the ARUP Consult 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 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.
Other Testing
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
Initial Evaluation
Use to evaluate cellular morphology
3001947 Blood Smear with Interpretation 3001947 Method Cytochemical Stain
Evaluation for Vitamin Deficiency
Aids in the detection of vitamin B12 deficiency
For patients with known risk factors for folate deficiency, consider Vitamin B12 and Folate test
0070150 Vitamin B12 0070150 Method Quantitative Chemiluminescent Immunoassay
Preferred reflex test for detection of vitamin B12 deficiency in individuals with macrocytic or unexplained anemia
0055662 Vitamin B12 with Reflex to Methylmalonic Acid, Serum (Vitamin B12 Status) 0055662 Method Quantitative Chemiluminescent Immunoassay/Quantitative High Performance Liquid Chromatography-Tandem Mass Spectrometry
Reflex pattern – if vitamin B12 is <300 pg/mL, methylmalonic acid, serum will be added
Use to evaluate vitamin B12 deficiency
0099431 Methylmalonic Acid, Serum or Plasma (Vitamin B12 Status) 0099431
Method
Quantitative Liquid Chromatography-Tandem Mass Spectrometry
Use to confirm vitamin B12 or folate deficiency
0099869 Homocysteine, Total 0099869 Method Quantitative Enzymatic Assay
Use to detect vitamin B12 or folate deficiency
0070160 Vitamin B12 and Folate 0070160 Method Quantitative Chemiluminescent Immunoassay
Aids in the detection of vitamin B9 (folate) deficiency
0070070 Folate, Serum 0070070 Method Quantitative Chemiluminescent Immunoassay
0070385 Folate, RBC 0070385 Method Quantitative Chemiluminescent Immunoassay
Evaluation for Pernicious Anemia
Use to confirm pernicious anemia as etiology of megaloblastic anemia
0070210 Intrinsic Factor Blocking Antibody 0070210 Method Qualitative Enzyme-Linked Immunosorbent Assay
Use to evaluate pernicious anemia or immune-mediated deficiency of vitamin B12
0050596 Gastric Parietal Cell Antibody, IgG 0050596 Method Semi-Quantitative Enzyme-Linked Immunosorbent Assay
Indirectly confirms pernicious anemia
0070075 Gastrin 0070075 Method Quantitative Chemiluminescent Immunoassay
References
30252420
Lanier JB, Park JJ, Callahan RC. Anemia in older adults. Am Fam Physician. 2018;98(7):437-442.
28189172
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.
26886541
Hesdorffer CS, Longo DL. Drug-induced megaloblastic anemia. N Engl J Med. 2016;374(7):696-697.
24424200
Bizzaro N, Antico A. Diagnosis and classification of pernicious anemia. Autoimmun Rev. 2014;13(4-5):565-568.
Additional Resources
Green R. Vitamin B12 deficiency from the perspective of a practicing hematologist. Blood. 2017;129(19):2603-2611.28360040
Oberley MJ, Yang DT. Laboratory testing for cobalamin deficiency in megaloblastic anemia. Am J Hematol. 2013;88(6):522-526.23423840
Socha DS, DeSouza SI, Flagg A, et al. Severe megaloblastic anemia: vitamin deficiency and other causes. Cleve Clin J Med. 2020;87(3):153-164.32127439
Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013;368(2):149-160.23301732
Related Information From ARUP Laboratories
Topics From ARUP Consult
Anemia
Hemolytic Anemias
Iron Deficiency Anemia
Vitamins - Deficiency and Toxicity
Selected Scholarly Publications From ARUP Laboratories
Kushnir MM, Nelson GJ, Frank EL, et al. High-throughput analysis of methylmalonic acid in serum, plasma, and urine by LC-MS/MS. Method for analyzing isomers without chromatographic separation. Methods Mol Biol. 2016;1378:159-173.26602128
Merrigan SD, Owen WE, Straseski JA. Performance characteristics of the ARCHITECT Active-B12 (Holotranscobalamin) assay. Clin Lab. 2015;61(3-4):283-288.25974994
Merzianu M, Groman A, Hutson A, et al. Trends in bone marrow sampling and core biopsy specimen adequacy in the United States and Canada: multicenter study. Am J Clin Pathol. 2018;150(5):393-405.30052721
Miller JW, Garrod MG, Rockwood AL, et al. Measurement of total vitamin B12 and holotranscobalamin, singly and in combination, in screening for metabolic vitamin B12 deficiency. Clin Chem. 2006;52(2):278-285.16384886
Owen WE, Roberts WL. Comparison of five automated serum and whole blood folate assays. Am J Clin Pathol. 2003;120(1):121-126.12866382
Related Algorithms
Megaloblastic Anemia Testing Algorithm
Educational Videos From ARUP Laboratories
Diagnostic Approach to Anemia
75 min
Medical Experts
Contributor
Doyle
Kelly Doyle, PhD, DABCC, FAACC
Associate Professor of Pathology (Clinical), University of Utah
Medical Director, Special Chemistry and Endocrinology, ARUP Laboratories
Contributor
Frank
Elizabeth L. Frank, PhD, DABCC
Professor of Pathology (Clinical), University of Utah
Medical Director, Analytic Biochemistry, Calculi and Manual Chemistry; Co-Medical Director, Mass Spectrometry, ARUP Laboratories