Macrocytic Anemia

Diagnostic algorithm for macrocytic anemia

MCV = mean corpuscular volume; MDS = myelodysplastic syndrome; MMA = methylmalonic acid.

Evaluation of Macrocytic Anemia

• A side effect of certain medications, such as those used to treat cancer, seizures and autoimmune disorders
Use of certain drugs, eg, hydroxyurea, zidovudine (AZT), and alcohol consumption are notoriously associated with macrocytosis and should be first considerations during evaluation of macrocytic anemia.

• The next step is to rule out nutritional causes (B12 or folate deficiency);
Check serum homocysteine, serum B12 and serum folate levels. we prefer to use serum homocysteine and serum B12 level for initial screening of vitamin B12 deficiency.
If 1 of the 2 tests has abnormal results, the serum methylmalonic acid level should be checked; an increased level strongly suggests B12 deficiency.
In patients with vitamin B12 deficiency, the next step is to screen for the presence of intrinsic factor antibodies.; if present, a working diagnosis of pernicious anemia (PA) is made. Otherwise, the Schilling test is performed to differentiate PA from primary intestinal malabsorptive disorders.

• Further investigation of macrocytic anemia that is neither drug-induced nor nutritional is simplified by subcategorizing the process into either a marked (MCV, >110 fL) or mild (MCV, 100-110 fL) subtype. In this instance, markedly macrocytic anemia is almost always associated with primary bone marrow disease, whereas mildly macrocytic anemia also can be associated with more benign conditions.

Drugs that may induce macrocytosis.

■ Chemotherapeutic agents
• Cyclophosphamide • Hydroxyurea • Methotrexate • Azathioprine • Mercaptopurine • Cladribine • Cytosine arabinoside • 5-Fluouracil
■ Antimicrobials
• Pyrimethamine • Sulfamethoxazole • Trimethoprim • Valacyclovir
■ Diuretics
• Triamterene
■ Antiretroviral
• Zidovudine • Stavudine
■ Anticonvulsant agents
• Phenytoin • Primidone • Valproic acid
■ Hypoglycemic
• Metformin
■ Anti-inflammatory
• Sulfasalazine
■ Other
• Nitrous oxide

Etiology

B12 deficiency: DNA synthesis requires cyanocobalamin (vitamin B-12) as a cofactor.
• Lack of intrinsic factor in patients who have who have pernicious anemia or undergone gastrectomy.
• Malabsorption of vitamin B-12 secondary to ileal bypass, ileal enteritis, sprue, small bowel bacterial overgrowth, tapeworm, familial factors, or drugs.
■ Folate deficiency: Folate also is needed as a cofactor in the synthesis of DNA
• Dietary deficiency
• Increased requirements of pregnancy
• Congenital deficiency
• Sprue
• Alcoholism
• Increased turnover due to conditions such as hemolysis or sickle cell disease, among others
■ Inherited disorders of DNA synthesis include the following:
• Lesch-Nyhan syndrome
• Deficient enzymes for folate metabolism
• Homocystinuria
■ Drug-induced macrocytosis is the most common cause in nonalcoholic patients. Usually, no associated anemia is present. The following categories of drugs are known to cause macrocytosis:
• Folate antagonists (eg, methotrexate)
• Purine antagonists (eg, 6-mercaptopurine [6-MP])
• Pyrimidine antagonists (eg, cytosine arabinoside [ara-C])
• Alkylating agents (eg, cyclophosphamide)
• Tyrosine kinase inhibitors (eg, sunitinib, and imatinib)
• Zidovudine (AZT)
• Trimethoprim
• Oral contraceptive pills
• Phenytoin
• Arsenic
■ Reticulocytosis may be due to posthemorrhagic blood loss or hemolysis.
■ Long-term alcohol intake directly affects bone marrow. This effect is not related to the presence of liver disease or vitamin deficiency and resolves only after months of abstinence from alcohol.
■ Refractory anemias of the following types may cause macrocytosis:
• Myelodysplastic anemias
• Myelophthisic anemias (marrow replacement by neoplasm, granuloma, or fibrosis)
• Aplastic anemia
• Acquired sideroblastic anemia
■ Macrocytosis in patients with COPD is attributed to excess cell water secondary to carbon dioxide retention.
■ Benign familial macrocytosis is an inherited syndrome in which patients have mild asymptomatic macrocytosis.
■ Macrocytosis of liver disease is secondary to increased cholesterol and phospholipids deposited on membranes of circulating erythrocytes. This deposition effectively increases the surface area of the erythrocyte.
■ Hypothyroidism is a manifestation of hormone deficiency. More commonly, hypothyroid patients exhibit a normocytic anemia.
■ Artifactual elevation of the MCV must be considered in certain patients, although this occurs less frequently with newer cell-counting machines. Hyperglycemia and cold agglutinins may cause artificially elevated MCVs.

Pathophysiology

Megaloblastic anemiais the result of impaired DNA synthesis. Although DNA synthesis is impaired, RNA synthesis is unaffected, leading to a buildup of cytoplasmic components in a slowly dividing cell. This results in a larger-than-normal cell. The nuclear chromatin of these cells also has an altered appearance.

DNA synthesis may also be delayed when certain chemotherapeutic agents are used, including folate antagonists, purine antagonists, pyrimidine antagonists, and even folate antagonist antimicrobials.
Hydroxyurea interferes with DNA synthesis, causing macrocytosis by which compliance with therapy may be monitored. Patient compliance with zidovudine, an agent used in the treatment of patients with HIV infection, may be monitored in the same way.

Nonmegaloblastic macrocytic anemias are those in which no impairment of DNA synthesis occurs. Included in this category are disorders associated with increased membrane surface area, accelerated erythropoiesis, alcoholism, and chronic obstructive pulmonary disease (COPD).
• Increased membrane surface area
Patients with hepatic disease and obstructive jaundice have macrocytosis that is secondary to increased deposition of cholesterol or phospholipids on the membranes of circulating red blood cells (RBCs). Similarly, in splenectomized patients, RBC membrane lipids that usually are removed during maturation in the spleen are not effectively removed, and the result is a larger-than-normal cell.
• Accelerated erythropoiesis
In patients with hemolytic anemia or posthemorrhagic anemia, the reticulocyte count increases. The reticulocyte, an immature RBC, is approximately 20% larger than the more mature RBC. When the reticulocyte is released prematurely from the marrow, its volume is averaged with the volume of the more mature RBC, and the resultant MCV is increased.
• Chronic alcoholism
Although the macrocytosis of alcoholism may be secondary to poor nutrition with a resulting folate or vitamin B-12 deficiency, it is more often due to direct toxicity of the alcohol on the marrow. The macrocytosis of alcoholism usually reverses only after months of abstinence from alcohol.
• COPD
The macrocytosis associated with COPD is attributed to excess cell water that is secondary to carbon dioxide retention.
• A murine study found that disruption of the Gardos channel (the erythrocyte Ca2+ -activated K+ channel [KCa3.1]) caused subtle erythrocyte macrocytosis and led to mild but progressive splenomegaly.

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Methylmalonic Acid (MMA) and Homocysteine Serum Concentrations
Cobalamin and folate are cofactors in several important metabolic pathways in the cell. The hydroxylated form of cobalamin plays an important role in the metabolism of homocysteine and MMA. The conversion of homocysteine to methionine requires both vitamin B12 and folate as cofactors. However, the metabolism of L-methylmalonyl CoA to succinyl CoA, an enzymatic pathway involved in oxidative phosphorylation reactions within the cell, only requires vitamin B12. These metabolites provide early information regarding the cellular state of vitamin B12 and folate and can be used to distinguish folate from vitamin B12 deficiency, since most patients with folate deficiency have normal MMA or mildly elevated levels. It should be kept in mind that nearly 50% of those with elevation of these metabolites will have normal serum vitamin B12 levels. This emphasizes the low sensitivity of using vitamin B12 levels, particularly in the presence of other signs or symptoms.

Previous studies on large groups of cobalamine and/or folate deficient patients have shown the ability of differentiating cobalamin deficiency from folate deficiency by measuring serum MMA and homocysteine levels. Both of these metabolites are elevated in cobalamin deficiency, with anemic cobalamin deficient patients showing marked elevations. However, MMA is more sensitive for identifying non-anemic cobalamin deficiency patients than homocysteine. In folate deficiency patients, serum homocysteine levels are markedly increased, while serum MMA levels are not elevated. Therefore, measuring the serum levels of these two metabolites not only helps in differentiating cobalamine deficiency from folate deficiency, but also provides a reliable degree of both sensitivity and specificity in diagnosing these important deficiency states.40 An important limitation that must be considered when measuring MMA is the presence of renal insufficiency, i.e., an elevated serum creatinine, and hypovolemia. Serum MMA will be elevated in patients with underlying renal dysfunction, decreasing its specificity and sensitivity in identifying patients with cobalamin deficiency. Similarly, hereditary homocysteinemia is a condition in which the serum homocysteine levels are elevated. Measurements of MMA levels are recommended when initial vitamin B12 and/or homocysteine levels are abnormal.

Holotranscobalamin II (holoTC II)
HoloTC II is an emerging marker that may be useful in establishing a diagnosis of early vitamin B12 deficiency in cases where there is a discordance between vitamin B12 levels and its metabolites, or in lieu of measuring vitamin B12, MMA and/or homocysteine concentrations.41 HoloTC II can also be used in cases of renal failure or myeloproliferative diseases in which vitamin B12 concentrations may be falsely elevated. HoloTC II is a metabolically active protein that transports cobalamin to cell membrane receptors. Its serum concentration can be used to measure the amount of vitamin B12 attached to the binding protein transcobalamin II. Compared to measurements of serum vitamin B12, holoTC II seems to have greater sensitivity and specificity. However, routine ordering of this test as part of the work-up to establish the diagnosis of vitamin B12 deficiency is not currently recommended.

There is currently no gold standard for diagnosing vitamin B12 deficiency. Until one is identified, it is recommended that when initial vitamin B12 values are low (i.e., <150 ng/L) and a vitamin B12 deficiency is suspected, the vitamin B12 level should be repeated on a separate occasion or tests should be used in combination (e.g., serum vitamin B12 level, MMA and homocysteine). Complete diagnostic testing should only be performed in cases of unexplained macrocytic anemia.