Pernicious Anemia Workup

Workup for pernicious anemia may include a peripheral blood smear; indirect bilirubin and lactate dehydrogenase assays; evaluation of gastric secretions; cobalamin, methylmalonic acid, and homocysteine assays; the Schilling test; a clinical trial of vitamin B-12; and bone marrow aspiration and biopsy.

The peripheral blood usually shows a macrocytic anemia with a mild leukopenia and thrombocytopenia. The mean cell volume (MCV) and mean cell hemoglobin (MCH) are increased, with a mean corpuscular hemoglobin concentration (MCHC) within the reference range (see the image below). The leukopenia and thrombocytopenia usually parallel the severity of the anemia.

The peripheral smear shows oval macrocytes, hypersegmented granulocytes, and anisopoikilocytosis. In severe anemia, red blood cell inclusions may include Howell-Jolly bodies, Cabot rings, and punctate basophilia. The macrocytosis can be obscured by the coexistence of iron deficiency, thalassemia minor, or inflammatory disease.

The indirect bilirubin level may be elevated because pernicious anemia is a hemolytic disorder associated with increased turnover of bilirubin. The serum lactic dehydrogenase (LDH) concentration usually is markedly increased.

Increased values for other red blood cells, enzymes, and serum iron saturation also are observed. The serum potassium, cholesterol, and skeletal alkaline phosphatase often are decreased.

Total gastric secretions are decreased to about 10% of the reference range. Most patients with pernicious anemia are achlorhydric, even with histamine stimulation. Intrinsic factor (IF) is either absent or markedly decreased.

The serum cobalamin level is low in patients with pernicious anemia. However, it may be within the reference range in certain patients with other forms of cobalamin deficiency, such as some inborn areas of cobalamin deficiency, transcobalamin II (TCII) deficiency, and cobalamin deficiency due to nitrous oxide.

Conversely, serum cobalamin levels may be low in patients who are pregnant, have transcobalamin I (TCI) deficiency, have severe folic acid deficiency, or have taken large doses of ascorbic acid.

Screening of individuals who are older has shown that 10-20% have low serum cobalamin levels, and half of these patients have increased levels of homocysteine and methylmalonic acid, indicating a tissue cobalamin deficiency.

Elevated serum methylmalonic acid and homocysteine levels are found in patients with pernicious anemia. They probably are the most reliable test for cobalamin deficiency in patients who do not have a congenital metabolism disorder (see the table below). In the absence of an inborn error of methylmalonic acid metabolism, methylmalonic aciduria is a sign of cobalamin deficiency.

Table 1. Serum Methylmalonic Acid and Homocysteine Values Used in Differentiating Between Cobalamin and Folic Acid Deficiency (Open Table in a new window)

The Schilling test measures cobalamin absorption by assessing increased urine radioactivity after an oral dose of radioactive cobalamin. The test is useful in demonstrating that the anemia is caused by an absence of IF and is not secondary to other causes of cobalamin deficiency (see the table below). It is also useful for identifying patients with classic pernicious anemia, even after they have been treated with vitamin B-12. Not all medical centers have the facilities to perform a Schilling test.

Table 2. Schilling Test Results (Open Table in a new window)

The test is performed by administering 0.5-2.0 mCi of radioactive cyanocobalamin in a glass of water to patients who have fasted. Two hours later, the patient is injected with 1 mg of unlabeled vitamin B-12 to saturate circulating transcobalamins. A 24-hour urine sample is collected, and the radioactivity in the specimen is measured and compared to a standard.

Specimens with less than 7% excretion represent abnormal findings and indicate that poor absorption of the oral test dose occurred. If abnormal low values are obtained, a stage II Schilling test is performed. In this test, 60 mg of active hog IF is administered with the oral test dose to determine if this enhances the absorption of vitamin B-12. If poor absorption of vitamin B-12 is normalized, the patient presumably has classic pernicious anemia.

If poor absorption is observed in a stage II test, other causes of vitamin B-12 malabsorption must be sought. Performance of a stage I Schilling test after 5 days of tetracycline therapy is used to exclude a blind loop as the etiology for cobalamin deficiency (stage III). Similarly, if administration of trypsin or pancreatic enzyme with the radiolabeled test dose corrects the absorption of vitamin B-12, pancreatic disease (stage IV) should be suspected.

False-positive Schilling test results are observed in patients with incomplete 24-hour urine collections or renal insufficiency. False-positive results are also observed when inactive IF is used. Finally, false-positive results may occur because of neutralization of the IF in the stage II test by any IF antibodies in the stomach and severe ileal megaloblastosis.

Occasionally, cobalamin deficiency and a normal stage I Schilling test result are observed. Patients with these findings can absorb vitamin B-12 in the fasting state, but not when it is presented with food. Adding the radiolabeled vitamin B-12 to egg white and testing the absorption usually reveals this cause of cobalamin deficiency.

Intramuscular (IM) administration of 1000 µg of vitamin B-12 can be used as a clinical trial for suspected cobalamin deficiency. Subjectively, patients who are cobalamin deficient usually begin to experience a marked sense of well-being within 24 hours after administration. Objectively, administration of cobalamin produces a marked reticulocytosis, which reaches its maximal level 5-7 days after the injection; correction of the anemia occurs in about 3 weeks (see the image below).

Bone marrow aspiration and biopsy can be performed for histologic examination. The bone marrow biopsy and aspirate usually are hypercellular and show trilineage differentiation. Erythroid precursors are large and often oval (see the image below).

The nucleus is large and contains coarse motley chromatin clumps, providing a checkerboard appearance. Nucleoli are visible in the more immature erythroid precursors. An imbalance in the rate of maturation of the nucleus relative to the cytoplasm exists, leading to disassociation between the maturity of the nucleus and the hemoglobinization of the orthochromic megaloblastic normoblasts.

Giant metamyelocytes and bands are present, and the mature neutrophils and eosinophils are hypersegmented. Imbalanced growth of megakaryocytes is evidenced by hyperdiploidy of the nucleus and the presence of giant platelets in the smear. Lymphocytes and plasma cells are spared from the cellular gigantism and cytoplasmic asynchrony observed in other cell lineages.

The bone marrow histology in cobalamin deficiency is similar to that in folic acid deficiency. Significant changes in the histology have been observed within 12 hours after appropriate treatment is initiated. The megaloblastic changes due to cobalamin deficiency can be reversed by pharmacologic doses of folic acid. However, folic acid therapy may worsen the neurologic consequences of cobalamin deficiency, despite the hematologic improvement.