Pernicious Anemia Workup

Updated: Dec 28, 2017
  • Author: Paul Schick, MD; Chief Editor: Emmanuel C Besa, MD  more...
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Approach Considerations

The workup for pernicious anemia may include the following:

  • Peripheral blood smear
  • Indirect bilirubin and lactate dehydrogenase assays
  • Evaluation of gastric secretions
  • Serum cobalamin, folic acid, methylmalonic acid, and homocysteine assays
  • Schilling test (no longer available in most medical centers)
  • A clinical trial of vitamin B12
  • Bone marrow aspiration and biopsy

Peripheral Blood Smear

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.

Peripheral smear of blood from a patient with pern 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 leucocyte is present.

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.


Indirect Bilirubin and Serum Lactate Dehydrogenase

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

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.


Evaluation of Gastric Secretions

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.


Serum Cobalamin

Serum cobalamin reference ranges may vary slightly among different laboratories, but are generally from 200–900 pg/mL. Values of 180-250 pg/mL are considered bordeline, while less than 150 pg/mL is considered diagnostic of vitamin B12 deficiency.

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 falsely low in patients who are pregnant or taking oral contraceptives, have multiple myeloma, have transcobalamin I (TCI) deficiency, have severe folic acid deficiency, or have taken large doses of ascorbic acid. [15]

Serum cobalamin levels can be in the low reference range in patients with clinical vitamin B12 deficiency. In these cases, elevated levels of methylmalonic acid and total homocysteine can confirm the diagnosis. [16] Screening of older individuals 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.



Serum Folic Acid, Methylmalonic Acid, and Homocysteine

A serum folic acid assay is useful for ruling out folic acid deficiency. The reference range is 2.5-20 ng/mL. Blood should be drawn before patients have a single hospital meal since food can restore serum folic acid levels to normal. Red blood cell folic acid level is not influenced by food. (For more information, see Megaloblastic Anemia and Folic Acid Deficiency).  

A significantly decreased serum cobalamin level along with a typical clinical presentation, a characteristic peripheral smear, and an increased indirect bilirubin and LDH level is sufficient evidence for the diagnosis of a megaloblastic anemia.

Serum methylmalonic acid and homocysteine tests are important confirmatory tests but are not first-line tests. 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)

Patient Condition Methylmalonic Acid Homocysteine
Healthy Normal Normal
Vitamin B12 deficiency Elevated Elevated
Folate deficiency Normal Elevated

Intrinsic Factor Antibodies

Intrinsic factor (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.

In one case report, the presence of IF antibodies was used to diagnose cobalamin deficiency in a patient with severe leukoencephalopathy. Interestingly, serum vitamin B12, homocysteine, and methylmalonic acid levels were normal. The patient responded to intense cobalamin therapy. [17]

Parietal cell antibodies occurs in 90% of patients with pernicious anemia. However, these antibodies are not specific for pernicious anemia.


Schilling Test

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 B12. The Schlling test is no longer available in most medical centers.

Table 2. Schilling test results (Open Table in a new window)

Patient Condition Stage I

No Intrinsic Factor

Stage II

Intrinsic Factor

Stage III


Stage IV

Pancreatic Extract

Healthy Normal
Pernicious anemia Low Normal
Bacterial overgrowth Low Low Normal
Pancreatic insufficiency Low Low Low Normal
Defect in ileum Low Low Low Low

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 B12 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 B12. If poor absorption of vitamin B12 is normalized, the patient presumably has classic pernicious anemia.

If poor absorption is observed in a stage II test, other causes of vitamin B12 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 B12, 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 B12 in the fasting state, but not when it is presented with food. Adding the radiolabeled vitamin B12 to egg white and testing the absorption usually reveals this cause of cobalamin deficiency.


Clinical Trial of Vitamin B12

Intramuscular (IM) administration of 1000 µg of vitamin B12 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).

Response to therapy with cobalamin (Cbl) in a prev Response to therapy with cobalamin (Cbl) in a previously untreated patient with pernicious anemia. A reticulocytosis occurs within 5 days after an injection of 1000 mcg of Cbl and lasts for about 2 weeks. The hemoglobin (Hgb) concentration increases at a slower rate because many of the reticulocytes are abnormal and do not survive as mature erythrocytes. After 1 or 2 weeks, the Hgb concentration increases about 1 g/dL per week.

Bone Marrow Aspiration and Biopsy

Bone marrow aspiration and biopsy can provide complementary information, with the aspirate revealing the numerical and cytological features of marrow cells, while the biopsy shows the spatial relationships between cells and the overall marrow structure.  [18] The bone marrow biopsy and aspirate specimens usually are hypercellular and show trilineage differentiation. Erythroid precursors are large and often oval (see the image below).

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.

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.