Megaloblastic Anemia Workup

The anemia should be characterized. Pancytopenia and systemic impairment should be evaluated. The etiology of megaloblastosis should be identified.

Initial workup for megaloblastic anemia should include the following assays:

• Complete blood count (CBC)
• Red blood cell (RBC) indices
• Peripheral blood smear
• Reticulocyte count
• Lactate dehydrogenase (LDH) 
• Indirect bilirubin
• Iron and ferritin
• Serum cobalamin
• Serum folate, and possibly RBC folate 

The LDH level is usually markedly increased in severe megaloblastic anemia. Reticulocyte counts are inappropriately low, representing lack of production of RBCs due to massive intramedullary hemolysis. These findings are characteristics of ineffective hematopoiesis that occurs in megaloblastic anemia as well as in other disorders such as thalassemia major.

Peripheral smear morphology

A peripheral smear may reveal macroovalocytes, a characteristic of megaloblastosis (see the image below). They should be distinguished from macrocytes that are not oval, which can occur in liver disease and hypothyroidism. Polychromatophilic macrocytes, reticulocytes, and immature RBCs can be seen in hemolytic anemia and disorders associated with increased RBC production.

Peripheral smear of blood from a patient with pernicious anemia. Macrocytes are observed, and some of the red blood cells show ovalocytosis. A 6-lobed polymorphonuclear leukocyte is present.

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Single and multiple Howell-Jolly bodies, nuclear fragment, may be seen in RBCs. Cabot rings, remnants of mitotic spindles, may also be present in RBCs.

Nucleated RBCs and megaloblasts can be seen.

Smears may reveal hypersegmented neutrophils if at least 5% neutrophils have 5 or more lobes. Normal neutrophils contain 3-4 lobes.

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

Bone marrow aspiration

Bone marrow aspiration is usually not needed to make the diagnosis of vitamin B-12 deficiency. However, it can help rule out myelodysplasia and assess iron stores. The bone marrow is hypercellular with erythroid hyperplasia. Erythroid precursors have megaloblastic features being larger than normoblastic cells. In addition, nuclear maturation is immature relative to cytoplasmic maturation. Megaloblastic changes are most prominent in more mature RBC precursors. Giant bands, neutrophil precursers, 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, bone marrow aspiration, if necessary, should be performed as soon as possible and preferably before therapy.

Serum B-12 (cobalamin)

Reference range: 200-900 pg/mL

• Borderline: 180-250 pg/mL
• Associated with anemia and neuropathy: < 180 mg/L
• Diagnostic of B-12 deficiency: < 150 mg/L

The serum cobalamin level can be normal in the following circumstances:

• In some inborn areas of cobalamin deficiency
• Transcobalamin II (TC II) deficiency
• Cobalamin deficiency due to nitrous oxide

Serum cobalamin levels may be low in the following circumstances:

• Pregnancy
• Oral contraceptives
• Transcobalamin I (TC I) deficiency
• Severe folic acid deficiency
• Patients taking large doses of ascorbic acid

Serum folate

Folate reference range in adults: 2-20 ng/mL

• Folate deficiency likely (overlap with normal): < 2.5 ng/mL 

The following should be considered:

• Effect of diet: A single meal may falsely elevate serum folate levels to normal. Hence, blood should be drawn prior to transfusions, meals, and therapy to achieve accurate results.
• Hemolysis: Might cause false positive results

RBC folate

Reference range for adults: >140 ng/mL

• Not affected by diet and reflects tissue stores (folate content is established early in RBC development)
• Affected by hemolysis
• Low in severe B-12 deficiency
• Test is intricate and expensive

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.

Lab tests to confirm and distinguish B-12 and folate deficiencies

Serum homocysteine and methylmalonic acid (MMA) levels are helpful confirmatory tests for cobalamin and folate deficiencies. Both are increased in cobalamine deficiency. Homocysteine but not MMA is increased in folate deficiency. Homocysteine and MMA levels should be used if the clinical presentation and serum vitamin B-12 and folate levels are ambiguous.

The MMA level can be increased in the following circumstances:

• End-stage renal disease

• Inborn error of methylmalonic acid metabolism

Serum homocysteine can be increased in the following circumstances:

• Homocystinuria

• Hyperhomocysteinemia

• MTHFR C677T

Serum homocysteine can be decreased in the following circumstances:

• A high rate of conversion back to methionine

• Low production

• A high rate of conversion of into sulfite/sulfate etc

Intrinsic factor (IF) blocking and parietal cell and antibodies

IF antibodies, type 1 and type 2, occur in 50% of patients with pernicious anemia and are specific for this disorder. Therefore, they can be used to confirm the diagnosis of pernicious anemia. Parietal cell antibody occurs in 90% of patients with pernicious anemia but can also occur in thyroid disease and other autoimmune disorders. Therefore, parietal cell antibodies are not specific for pernicious anemia.

A valuable test that is no longer available (Schilling test)

The value of a Schilling test (a radiometric test) is that it can confirm B-12 deficiency, can be done after patient has been given B-12 therapy, and can distinguish between pernicious anemia and failure in transport or ileal uptake. Unfortunately, the test is no longer available at most 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 cobalamin uptake. Low urinary secretion suggests pernicious anemia, a failure in intestinal transport, defective uptake of cobalamin in the terminal ileum, or a blind loop syndrome.

2. The second part is performed in the same manner, except that IF is given orally along with radioactive cyanocobalamin. If IF restores cobalamin uptake, the patient most likely has pernicious anemia. If not, an abnormality in cobalamin intestinal transport, defective absorption in the terminal ileum, or a blind loop syndrome might be responsible for the deficiency.

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.

If the results of the evaluation are ambiguous, a clinical trial of the effects of cobalamin therapy may be indicated. However, a clinical trial of folate is contraindicated if cobalamin deficiency has not been ruled out. The administration of folate to patients with cobalamin deficiencies may precipitate or worsen neurologic impairment.

Baseline iron studies and serum ferritin should be obtained since they may predict the need for iron therapy since iron stores can be consumed during cobalamin or folate therapy.

Radiographic imaging of the upper and lower gastrointestinal tract may be useful for detecting abnormalities that could cause a blind loop syndrome. These procedures also may detect defects in the terminal ileum that might interfere with cobalamin absorption.

Other tests that may be considered include the following:

• With cobalamin deficiency, evaluate and rule out autoimmune disorders, Zollinger-Ellison syndrome, pancreatic insufficiency, fish tapeworm infestation, Imerslund-Grasbeck syndrome, Crohn disease, or ileal scarring.

• With folate deficiency, evaluate evidence for malnutrition and alcoholism, sprue, chronic hemolysis, and exfoliative dermatitis.

• Megaloblastic anemia