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Pernicious Anemia Workup

  • Author: Paul Schick, MD; Chief Editor: Emmanuel C Besa, MD  more...
 
Updated: Aug 05, 2015
 

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
  • Intrinsic factor antibody assay
  • Schilling test (no longer available in most medical centers)
  • A clinical trial of vitamin B12
  • Bone marrow aspiration and biopsy
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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.

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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.

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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.

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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.[9]

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.[10] 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.

 

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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
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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.[11]

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

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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



Antibiotic



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.

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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.
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Bone Marrow Aspiration and Biopsy

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).

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.

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Contributor Information and Disclosures
Author

Paul Schick, MD Emeritus Professor, Department of Internal Medicine, Jefferson Medical College of Thomas Jefferson University; Research Professor, Department of Internal Medicine, Drexel University College of Medicine; Adjunct Professor of Medicine, Lankenau Hospital

Paul Schick, MD is a member of the following medical societies: American College of Physicians, American Society of Hematology

Disclosure: Nothing to disclose.

Coauthor(s)

Marcel E Conrad, MD Distinguished Professor of Medicine (Retired), University of South Alabama College of Medicine

Marcel E Conrad, MD is a member of the following medical societies: Alpha Omega Alpha, American Association for the Advancement of Science, American Association of Blood Banks, American Chemical Society, American College of Physicians, American Physiological Society, American Society for Clinical Investigation, American Society of Hematology, Association of American Physicians, Association of Military Surgeons of the US, International Society of Hematology, Society for Experimental Biology and Medicine, SWOG

Disclosure: Partner received none from No financial interests for none.

Chief Editor

Emmanuel C Besa, MD Professor Emeritus, Department of Medicine, Division of Hematologic Malignancies and Hematopoietic Stem Cell Transplantation, Kimmel Cancer Center, Jefferson Medical College of Thomas Jefferson University

Emmanuel C Besa, MD is a member of the following medical societies: American Association for Cancer Education, American Society of Clinical Oncology, American College of Clinical Pharmacology, American Federation for Medical Research, American Society of Hematology, New York Academy of Sciences

Disclosure: Nothing to disclose.

Acknowledgements

David Aboulafia, MD Medical Director, Bailey-Boushay House, Clinical Professor, Department of Medicine, Division of Hematology, Attending Physician, Section of Hematology/Oncology, Virginia Mason Clinic; Investigator, Virginia Mason Community Clinic Oncology Program/SWOG

David Aboulafia, MD is a member of the following medical societies: American College of Physicians, American Medical Association, American Medical Directors Association, American Society of Hematology, Infectious Diseases Society of America, and Phi Beta Kappa

Disclosure: Nothing to disclose.

Troy H Guthrie, Jr, MD Director of Cancer Institute, Baptist Medical Center

Troy H Guthrie, Jr, MD is a member of the following medical societies: American Federation for Medical Research, American Medical Association, American Society of Hematology, Florida Medical Association, Medical Association of Georgia, and Southern Medical Association

Disclosure: Nothing to disclose.

Francisco Talavera, PharmD, PhD Adjunct Assistant Professor, University of Nebraska Medical Center College of Pharmacy; Editor-in-Chief, Medscape Drug Reference

Disclosure: Medscape Salary Employment

References
  1. Hoffman R, Benz EJ, Furie B, Shattil SJ. Hematology: Basic Principles and Practice. Philadelphia, Pa: Churchill Livingstone; 2009.

  2. Elmadfa I, Singer I. Vitamin B-12 and homocysteine status among vegetarians: a global perspective. Am J Clin Nutr. 2009 May. 89(5):1693S-1698S. [Medline].

  3. Andrès E, Vogel T, Federici L, Zimmer J, Ciobanu E, Kaltenbach G. Cobalamin deficiency in elderly patients: a personal view. Curr Gerontol Geriatr Res. 2008. 848267. [Medline]. [Full Text].

  4. Bizzaro N, Antico A. Diagnosis and classification of pernicious anemia. Autoimmun Rev. 2014 Apr-May. 13(4-5):565-8. [Medline].

  5. Murphy G, Dawsey SM, Engels EA, Ricker W, Parsons R, Etemadi A, et al. Cancer Risk After Pernicious Anemia in the US Elderly Population. Clin Gastroenterol Hepatol. 2015 Jun 14. [Medline].

  6. Chan JC, Liu HS, Kho BC, Lau TK, Li VL, Chan FH, et al. Longitudinal study of Chinese patients with pernicious anaemia. Postgrad Med J. 2008 Dec. 84(998):644-50. [Medline].

  7. Venkatesh P, Shaikh N, Malmstrom MF, Kumar VR, Nour B. Portal, superior mesenteric and splenic vein thrombosis secondary to hyperhomocysteinemia with pernicious anemia: a case report. J Med Case Rep. 2014 Aug 25. 8:286. [Medline]. [Full Text].

  8. Kocaoglu C, Akin F, Caksen H, Böke SB, Arslan S, Aygün S. Cerebral atrophy in a vitamin B12-deficient infant of a vegetarian mother. J Health Popul Nutr. 2014 Jun. 32(2):367-71. [Medline]. [Full Text].

  9. Centers for Disease Control and Prevention. Vitamin B12 Deficiency: Detection and Diagnosis. CDC. Available at http://www.cdc.gov/ncbddd/b12/detection.html. June 29, 2009; Accessed: August 5, 2015.

  10. Stabler SP. Clinical practice. Vitamin B12 deficiency. N Engl J Med. 2013 Jan 10. 368(2):149-60. [Medline].

  11. Graber JJ, Sherman FT, Kaufmann H, Kolodny EH, Sathe S. Vitamin B12-responsive severe leukoencephalopathy and autonomic dysfunction in a patient with "normal" serum B12 levels. J Neurol Neurosurg Psychiatry. 2010 Dec. 81(12):1369-71. [Medline].

  12. Erkurt MA, Aydogdu I, Dikilitas M, Kuku I, Kaya E, Bayraktar N, et al. Effects of cyanocobalamin on immunity in patients with pernicious anemia. Med Princ Pract. 2008. 17(2):131-5. [Medline].

  13. Zhang J, Field CJ, Vine D, Chen L. Intestinal Uptake and Transport of Vitamin B12-loaded Soy Protein Nanoparticles. Pharm Res. 2014 Oct 16. [Medline].

  14. Andres E, Serraj K. Optimal management of pernicious anemia. J Blood Med. 2012. 3:97-103. [Medline]. [Full Text].

  15. Favrat B, Vaucher P, Herzig L, et al. Oral vitamin B12 for patients suspected of subtle cobalamin deficiency: a multicentre pragmatic randomised controlled trial. BMC Fam Pract. 2011 Jan 13. 12:2. [Medline]. [Full Text].

 
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Pernicious anemia. The structure of cyanocobalamin is depicted. The cyanide (Cn) is in green. Other forms of cobalamin (Cbl) include hydroxocobalamin (OHCbl), methylcobalamin (MeCbl), and deoxyadenosylcobalamin (AdoCbl). In these forms, the beta-group is substituted for Cn. The corrin ring with a central cobalt atom is shown in red and the benzimidazole unit in blue. The corrin ring has 4 pyrroles, which bind to the cobalt atom. The fifth substituent is a derivative of dimethylbenzimidazole. The sixth substituent can be Cn, CC3, hydroxycorticosteroid (OH), or deoxyadenosyl. The cobalt atom can be in a +1, +2, or +3 oxidation state. In hydroxocobalamin, it is in the +3 state. The cobalt atom is reduced in a nicotinamide adenine dinucleotide (NADH)–dependent reaction to yield the active coenzyme. It catalyzes 2 types of reactions, which involve either rearrangements (conversion of l methylmalonyl coenzyme A [CoA] to succinyl CoA) or methylation (synthesis of methionine).
Pernicious anemia. Inherited disorders of cobalamin (Cbl) metabolism are depicted. The numbers and letters correspond to the sites at which abnormalities have been identified, as follows: (1) absence of intrinsic factor (IF); (2) abnormal Cbl intestinal adsorption; and (3) abnormal transcobalamin II (TC II), (a) mitochondrial Cbl reduction (Cbl A), (b) cobalamin adenosyl transferase (Cbl B), (c and d) cytosolic Cbl metabolism (Cbl C and D), (e and g) methyl transferase Cbl utilization (Cbl E and G), and (f) lysosomal Cbl efflux (Cbl F).
Pernicious anemia. Cobalamin (Cbl) is freed from meat in the acidic milieu of the stomach where it binds R factors in competition with intrinsic factor (IF). Cbl is freed from R factors in the duodenum by proteolytic digestion of the R factors by pancreatic enzymes. The IF-Cbl complex transits to the ileum where it is bound to ileal receptors. The IF-Cbl enters the ileal absorptive cell, and the Cbl is released and enters the plasma. In the plasma, the Cbl is bound to transcobalamin II (TC II), which delivers the complex to nonintestinal cells. In these cells, Cbl is freed from the transport protein.
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.
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.
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.
Table 1. Serum Methylmalonic Acid and Homocysteine Values Used in Differentiating Between Cobalamin and Folic Acid Deficiency
Patient Condition Methylmalonic Acid Homocysteine
Healthy Normal Normal
Vitamin B12 deficiency Elevated Elevated
Folate deficiency Normal Elevated
Table 2. Schilling test results
Patient Condition Stage I



No Intrinsic Factor



Stage II



Intrinsic Factor



Stage III



Antibiotic



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
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