Folate Deficiency Workup

Updated: Jul 20, 2022
  • Author: Katherine Coffey-Vega, MD; Chief Editor: Emmanuel C Besa, MD  more...
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Laboratory Studies

Serum folate and serum cobalamin  testing

As the initial test, ruling out cobalamin deficiency is very important because deficiency of folic acid and vitamin B12 produce overlapping neurologic manifestations, and both cause megaloblastic anemia, but folate treatment will not improve neurologic abnormalities due to cobalamin deficiency. [48] The reference range for serum cobalamin is 200-900 pg/mL. 

The usual reference range of serum folate is 2.5-20 ng/mL. By statistical definition, 2-5% of healthy individuals will have a serum folate level of less than 2.5 ng/mL; hence, the serum folate level cannot be used alone to establish the diagnosis of folate deficiency. Therefore, the serum folate test is definitive only when the level is greater than 5.0 ng/mL, which rules out folate deficiency. Otherwise, additional follow-up tests include serum homocysteine (reference range 5-16 mmol/L), which is elevated in vitamin B12 and folate deficiency, and serum methylmalonic acid (reference range 70-270 mmol/L), which is elevated in vitamin B12 deficiency only.

In most cases, serum folate testing alone is sufficient for assessment of folate status, but if there is strong clinical suspicion of folate deficiency and the serum folate level is normal and cobalamin deficiency has been ruled out, the red blood cell (RBC) folate level may be measured. [49]  RBC folate levels tend to reflect long-term folate status rather than acute changes in folate that are reflected in serum folate levels. However, many confounding factors, such as transfused red cells, can make this unreliable as a test for folate deficiency states. 

Other than folate or cobalamin deficiency, the only other confounding causes for elevation of these compounds include renal failure, intravascular volume depletion, and some rare inborn errors of metabolism involving folate or cobalamin-dependent enzymes.



Bone marrow biopsy and aspirate may show a hypercellular bone marrow with a megaloblastic maturation of cells (see the slides below). This cannot be differentiated from changes observed with vitamin B12 deficiency.

Histologically, the megaloblastosis caused by foli Histologically, the megaloblastosis caused by folic acid deficiency cannot be differentiated from that observed with vitamin B-12 deficiency.
Peripheral smear of blood in a patient with pernic Peripheral smear of blood in a patient with pernicious anemia. Macrocytes are observed and some of the red blood cells show ovalocytosis. A 6-lobed polymorphonuclear leucocyte is present.
Bone marrow aspirate from a patient with untreated Bone marrow aspirate from a patient with untreated pernicious anemia. Megaloblastic maturation of erythroid precursors is shown. Two megaloblasts occupy the center of the slide with a megaloblastic normoblast above.