Megaloblastic Anemia Workup

Workup for megaloblastic anemia may include a complete blood count (CBC) and peripheral smear; lactate dehydrogenase (LDH), indirect bilirubin, iron, and ferritin assays; tests for cobalamin deficiency (eg, serum cobalamin, Schilling test, protein-bound absorption test); tests for folate deficiency (eg, serum folate and red blood cell [RBC] folate); diagnostic imaging (for possible blind loop syndrome); and bone marrow aspiration and biopsy.

Initial studies include a complete blood cell (CBC) count, RBC indices, platelet count, a peripheral smear, differential WBC count, and a reticulocyte count.

A CBC and platelet count would confirm an anemia and pancytopenia that are characteristic of severe megaloblastosis. RBC indices (an elevated mean corpuscular volume) would indicate macrocytosis.

A peripheral smear would demonstrate macroovalocytes that are characteristic of megaloblastosis. They should be distinguished from macrocytes that are not oval and occur in liver disease, hemolytic anemia, and in patients with increased RBC production.

The smear might also reveal megaloblasts and hypersegmented neutrophils that contain 5 or more lobes. Normal neutrophils contain 3-4 lobes. The smear might demonstrate RBCs with multiple Howell-Jolly bodies that represent nuclear fragments.

Macrocytosis due to cobalamin or folate deficiencies may be masked in patients with iron deficiency. However, hypersegmentation of neutrophils can persist in iron deficiency.

The reticulocyte count can be low despite hemolysis. This dissociation is characteristic of intramedullary hemolysis.

Note the image below.

Lactate hydrogenase (LDH) and indirect bilirubin levels are increased due to intramedullary hemolysis. The LDH is usually markedly increased in severe megaloblastic anemia.

Serum vitamin B-12 and RBC folate levels

Blood should be drawn prior to transfusions, meals, and therapy to achieve accurate results. Serum folate values are affected by meals and can be become normal after a single hospital meal. In contrast, RBC folate levels are not affected by diet since folate content is established early in RBC development. However, RBC folate is less sensitive for detecting a deficiency than serum folate. Serum for folate and cobalamin should be frozen and stored prior to meals or therapy if the tests cannot be performed within a reasonable timeframe. Serum vitamin B-12 and folate levels are usually low in cobalamin and folate deficiency but false positive and negatives can occur.

Serum homocysteine and methylmalonic acid levels

The former is elevated in both cobalamin and folate deficiencies, while the later is elevated only in cobalamin deficiency. These tests can be used if the clinical presentation and serum vitamin B-12 and folate levels are ambiguous.

Parietal cell and intrinsic factor antibodies

The former is present in 90% of patients with pernicious anemia, but it can also is found in thyroid disease and other autoimmune disorders. Anti-intrinsic factor antibodies (type I and II) are highly specific for pernicious anemia. However, these tests are not commonly ordered.

Schilling test

A Schilling test (a radiometric test) is given in 3 parts and can distinguish between pernicious anemia, a failure of cobalamin uptake in the terminal ileum, and a blind loop syndrome. The test may not be available at many hospitals. The 3 parts to the Schilling test are as follows:

1. First, radioactive cyanocobalamin is given orally and its urinary secretion is measured to estimate the ability to take up cobalamin. Low secretion suggests either pernicious anemia, an abnormality in the terminal ileum, or a blind loop syndrome.
2. The second part is performed in the same manner, except that intrinsic factor is given orally along with radioactive cyanocobalamin. If intrinsic factor restores cobalamin uptake, the patient most likely has pernicious anemia. If not, an abnormality in the terminal ileum or a blind loop syndrome might be present.
3. In the third phase, the patient is treated with antibiotics before the administration of radioactive cyanocobalamin. If antibiotics restore cobalamin uptake, the patient most likely has a blind loop syndrome.

Problems

The main difficulty with the Schilling test is inadequate collection of urine samples in patients who are either noncompliant or have renal failure. Another issue is that severe folate deficiency affects ileal mucosa and therefore can block cobalamin uptake. Therefore, a Schilling test would be more reliable after patients have been treated with folate and cobalamin so that the ileal mucosa’s ability to take up cobalamin has been restored. A Schilling test can be performed in patients treated with cobalamin.

This is useful if the clinical presentation and results from routine diagnostic tests are ambiguous.

Serum ferritin and iron studies are performed. It is important to establish baseline iron levels prior to treatment since iron is consumed when patients are treated with cobalamin and folate. Therefore, the baseline value can be helpful in predicting the need for iron therapy in patients who are being treated with cobalamin and folate.

A bone marrow aspiration is useful to confirm the diagnosis, to rule out myelodysplasia, and to assess the iron stores. The bone marrow is hypercellular with erythroid hyperplasia. Erythroid precursors have megaloblastic features in that they are larger than normoblastic cells and they have immature nuclear development. Megaloblastic changes are most prominent in more mature RBC precursors. Cytoplasmic maturation is normal, but nuclear remnants, Howell-Jolly bodies, may be present in the cytoplasm. Giant bands (neutrophils) can be present. Megakaryocytes may be large and hyperlobulated.

Bone marrow megaloblastic changes are reversed within 12 hours after treatment with cobalamin or folate, and bone marrow morphology appears to be normal within 2-3 days. Therefore, a bone marrow aspiration should be performed as soon as possible and preferably before therapy.

With peripheral smears, macroovalocytes and hypersegmented neutrophils are present. Megaloblasts may be present. RBCs may have multiple Howell-Jolly bodies in their cytoplasm. Pancytopenia is a common finding.

Abdominal radiographs, upper and lower gastrointestinal series, and computed tomography (CT) scans may be useful for detecting and evaluating strictures and other gastrointestinal tract abnormalities that could cause a blind loop syndrome.

Other tests that may be considered include the following:

• With cobalamin deficiency: Tests and diagnostic studies can be performed for the detection and evaluation of other autoimmune disorders. Regional ileitis, fish tapeworm infection, Zollinger-Ellison syndrome, and pancreatitis should be ruled out.
• With folate deficiency: Diagnostic studies can be performed to detect malnutrition, sprue, chronic hemolysis, and exfoliative dermatitis.
• Tests relevant for the diagnosis and evaluation of inborn errors that cause or are associated with cobalamin or folate deficiency may be warranted.