At a Glance

With the FDA-mandated folic acid supplementation of many grain products, true folate deficiency is now much less common in the United States. Nutritional folate deficiency can develop quite rapidly and produce subnormal folate levels in a matter of weeks. In some instances, this negative folate balance is transient, such as following acute alcohol intoxications and in hospitalized patients. If the negative folate balance persists for a 3-4 months, tissue folate stores are completely depleted.

Reduced dietary intake and alcoholism should always be considered as conditions leading to folate deficiency. Chronic diseases, institutionalization in nursing homes or psychiatric facilities, and advanced old age are also associated with reduced dietary intake and folate deficiency.

Folate deficiency may also be a consequence of intestinal malabsorption, such as in celiac disease and inflammatory bowel disease.

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Folate deficiency can be drug-induced and should be considered in patients treated with methotrexate, phenytoin, trimethoprimand pyrimethamine.

Increased folate requirements are associated with pregnancy and lactation (from 200-400 microg/day to 500-800 microg/day), chronic hemolysis and skin diseases characterized by massive exfoliation.

In developing countries, the most common cause of megaloblastic anemia is insufficient folate intake in pregnant women. In addition, this is associated with increased birth defects, which can be prevented if the daily folate intake is 400 µg or greater.

Folate and B12 deficiency may both be present in the same patient, so in the work-up of a possible folate deficiency, B12 metabolism should always be assessed.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

The following tests should be ordered:

Complete blood count (CBC): Diagnosis of folate deficiency should be considered in the presence of anemia, macrocytosis (MCV > 100 fL with ovalocytosis, possibly in conjunction with thrombocytopenia and neutropenia progressing to pancytopenia), and neutrophil hypersegmentation (at least 5% of neutrophils with five lobes or at least 1% with six lobes). Reticulocytosis is absent. Direct measurement of folate levels in serum/plasma and of relevant metabolites is required to confirm a diagnostic suspicion of folate deficiency. In many cases, final confirmation of the diagnosis requires demonstrating normalization of biochemical markers of folate deficiency following folate therapy.

Serum folate: National Health and Nutrition Examination Surveys (NHANES)/Center for Disease Control (CDC) criteria for folate deficiency use a cut-off value for serum folate of 3.0 ng/ml. Values above 4 ng/ml (9.1 nmol/L) effectively rule out folate deficiency.

Red cell folate assay: This assay has traditionally been recommended based on the fact that it is not affected by recent intake of folate-containing food and thus is more representative of the true tissue folate content. Values below 140 ng/ml have been interpreted as diagnostic of folate deficiency. However, more recent studies show no additional value for ordering a red blood cell (RBC) folate assay, with the serum folate being diagnostic in most conditions. RBC folate can also decrease in the presence of B12 deficiency.

Serum/plasma B12 levels: B12 deficiency is highly unlikely when B12 values in plasma are greater than 300 pg/ml (221 pmol/L).

Metabolic intermediates assays: These assays are useful in cases with equivocal folate levels. Serum homocysteine is elevated in folate deficiency, whereas serum/urine metylmalonic acid is in the normal range. These assays are helpful in the differential diagnosis with B12 deficiency, which is associated with increases in both serum homocysteine and methylmalonic acid.

Increased LDH and bilirubin: These (mostly direct) confirm the presence of ineffective erythropoiesis due to folate deficiency, which results in altered maturation of erythroid precursors.

Bone marrow aspirate and biopsy: Marrow is hypercellular with characteristic features of megaloblastic erythropoiesis and presence of large metamyelocytes. The bone marrow does not allow one to distinguish folate from B12 deficiency and should be reserved for cases in which there is a suspicion of an underlying hematological malignancy.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Macrocytosis is a common finding in adult patients, with an estimated incidence around 2-3%. The majority of these cases do not have anemia. Causes other than folate deficiency include B12 deficiency, azathioprine therapy, chemotherapy, elevated alcohol intake and/or liver dysfunction, hypothyroidism, myelodysplastic syndrome and multiple myeloma. Macrocytosis (MCV > 100 fL) may be masked or absent in the presence of concomitant iron deficiency or alpha/beta thal traits, although these cases tend to have an increased red cell distribution width (RDW).

A serum folate sample should be taken before any meal, since serum folate may transiently normalize following a folate-containing meal. Similar effects have also been described following blood/plasma transfusions. Folate levels can also be below the normal range due to low intake in the preceding few days, during pregnancy, as a consequence of increased alcohol intake or of therapy with anticonvulsant drugs such as carbamazepine, phenobarbital and phenytoin.

Serum/plasma homocysteine values are elevated in the presence of hereditary homocysteinemia.

Serum/plasma/urinary methylmalonic acid (MMA) levels remain elevated during the first year of life. Cutoff values have varied from 210 to 480 nmol/L, with the most commonly used being around 270 nmol/L. Renal failure is associated with increased values of plasma MMA, whereas methylmalonic aciduria increased both serum and urine MMA.

What Lab Results Are Absolutely Confirmatory?

Normalization of homocysteine following folate supplementation is a strong additional confirmation of the diagnosis of folate deficiency. Incomplete therapeutic response is associated with only transient normalization of these parameters. In many instances, normalization of HC provides the most convincing proof of the presence of folate deficiency.

The reticulocyte count promptly increases after treatment, peaking at 1 week following folate administration. A blunted response may be indicative of the presence of iron deficiency.

Additional Issues of Clinical Importance

Hereditary folate malabsorption (HFM) is a rare disease characterized by impaired folate absorption and central nervous system (CNS) transport into the cerebrospinal fluid (CSF), which becomes apparent a few months after birth. In these patients, a cerebral folate deficiency appears with normal blood folate levels.

Severe megaloblastic anemia can be misdiagnosed as erythroleukemia. In rare cases, acute myelogenous leukemia can be misdiagnoses as megaloblastic anemia.

Iron deficiency may be masked in the presence of severe folate deficiency. A blunted reticulocyte response following folate administration is an important sign of this possibility. Further indication may be provided by an incomplete normalization of cellular indices 8 weeks after treatment. Iron studies should be considered and iron supplements can be given prophylactically in conjunction with folate administration.

Infections with Helicobacter Pylori have been associated with B12 and folate deficiencies and should be considered in the presence of upper GI symptoms.