What is macrocytic anemia

The term macrocytosis refers to a blood condition in which red blood cells (RBCs) are larger than normal. Macrocytosis is reported in terms of mean corpuscular volume (MCV). Normal MCV values range from 80 to 100 femtoliters (fl) and vary by age and reference laboratory ¹⁾.

Macrocytosis can be identified by reviewing peripheral blood smears and/or by automated red blood cell indices. The peripheral blood smear is more sensitive than red blood cell indices for identifying early macrocytic changes because the mean corpuscular volume (MCV) represents the mean of the distribution curve and is insensitive to the presence of small numbers of macrocytes ²⁾. However, compared to the peripheral blood smear, mean corpuscular volume (MCV) may underestimate macrocytosis in over 30% of cases ³⁾.

Macrocytosis is a relatively common finding in the era of automated blood cell counters, with prevalence estimates ranging from 1.7% to 3.6% ⁴⁾. Its significance tends to be underestimated by physicians, since about 60% of patients present without associated anemia ⁵⁾, unless there are other accompanying abnormalities noted.

Macrocytic anemia describes an anemic state characterized by the presence of abnormally large red blood cells in the peripheral blood. This abnormality is usually recognized by the automated blood cell counter and confirmed on review of the peripheral blood smear. The cause of macrocytic anemia may be due to a variety of illnesses and demands further clinical and laboratory assessment.

Macrocytosis Without Anemia

Large circulating erythrocytes (red blood cells) are not always associated with a pathologic process or condition. In fact, red blood cells of newborns and infants tend to be larger (mean MCV = 108 fl) than normal adult red blood cells ⁶⁾ and large red blood cells can be seen during pregnancy in the absence of an obvious etiology. Macrocytosis without anemia may be a normal variant and is only noted as a result of repeated peripheral red blood cell indices in the absence of any known or existing clinical problems. In some instances this variation from normal can be found in other family members, which suggests a genetic predisposition, and requires no therapeutic intervention or further investigation ⁷⁾, ⁸⁾.

Macrocytic anemia causes

Macrocytic anemia can usually be divided into two categories:

1. Megaloblastic: Megaloblastic anemia is caused by deficiency or impairment of utilization of vitamin B12 or folate.
2. Nonmegaloblastic: Nonmegaloblastic anemia may be the result of liver dysfunction, alcoholism, myelodysplastic syndrome (MDS), or hypothyroidism.

This categorization is important and frequently aids in determining the etiology of the anemia. Additionally, a careful review of the peripheral blood smear noting the morphology of the red blood cells, as well as the other cellular elements and features on the smear, can provide important clues as to the etiology of the anemia.

Common causes of macrocytosis are different by region and setting. For example, in New York, 37% of cases diagnosed in hospitalized patients were medication related ⁹⁾. Antiretroviral therapy (ART) for human immunodeficiency virus (HIV) infections accounted for 13% ¹⁰⁾. In Finland, the common causes of macrocytic anemias were alcoholism (65%) ¹¹⁾ and vitamin B12 or folate deficiency (28%) ¹²⁾ in outpatients over 75 years of age.

Vitamin B12 deficiency is the most common cause of megaloblastic anemia. Vitamin B12 deficiency is caused by insufficient dietary intake, as in the cases of vegetarians or malnutrition, malabsorption due to the absence of intrinsic factor caused by pernicious anemia or following gastric surgery, congenital disorders, such as transcobalamin II deficiency, or exposure to nitrous oxide.

The result of one study, conducted in Japan, indicated that the most common cause of megaloblastic anemia is pernicious anemia (61%), followed by vitamin B12 deficiency due to gastrectomy (34%), vitamin B12 deficiency due to other causes (2%), and folate deficiency (2%) ¹³⁾. Vitamin B12 is contained in animal foods, and the daily intake is approximately 3‐30 μg. The daily required amount is approximately 1‐3 μg, and except for stomach or intestinal obstruction, or being a strict vegetarian, vitamin B12 deficiency is rare.

Vitamin B12 binds to intrinsic factor secreted by the gastric parietal cells, and it is absorbed in the terminal ileum. Once absorbed, vitamin B12 acts as a coenzyme in the enzymatic reaction that produces methionine from homocysteine. As a result, folic acid is converted into its active form. When vitamin B12 is deficient, active folic acid is also deficient. As a result, the intracellular reaction involving the coenzyme form of folic acid is affected. Thus, not only vitamin B12 but also folate deficiencies impair DNA synthesis. Because a large amount of vitamin B12 is stored in the liver, it takes 5‐10 years for clinical problems to manifest following decreased intake or absorption of vitamin B12 ¹⁴⁾.

Folic acid is contained in green vegetables and animal products, such as liver. The recommended dietary allowance of folic acid for adults is 240 μg a day, and an intake of around 400 μg each day is necessary for pregnant or lactating women. Folate deficiency may increase the risk of a congenital neural tube stenosis during pregnancy. Folic acid is absorbed in the upper jejunum by both passive diffusion and active uptake. Folate deficiency is caused by nutritional deficiency (eg, poor diet, alcoholism), malabsorption (eg, celiac disease, inflammatory bowel disease), increased requirements (eg, pregnancy, lactation, chronic hemolysis), or medication (eg, methotrexate, trimethoprim, phenytoin). Because serum folate levels fluctuate with dietary intake, measurement of red blood cell folate levels, which reflect folate stores in tissue, has been considered more reliable ¹⁵⁾. Patients are usually treated with oral folic acid if the cause of folate deficiency is nutritional deficiency or increased nutritional requirements.

The spectrum of causes associated with macrocytic anemia includes:

• Nutritional deficiencies (e.g., vitamin B12 and folate),
• Primary bone marrow disorders (e.g., myelodysplasia and leukemia) and other chronic illnesses (Table 1) and
• Drugs (Table 2).

Macrocytosis due to vitamin B12 or folate deficiency is a direct result of ineffective or dysplastic erythropoiesis. These important vitamins and cofactors are required for normal maturation of all cells. Marrow erythroblasts are no exception. When either of these two factors is deficient, red blood cell proliferation and maturation result in large erythroblasts with nuclear/cytoplasmic asynchrony. These abnormalities are caused by a defect in DNA synthesis that interferes with cellular proliferation and maturation. RNA synthesis and cytoplasmic components remain relatively unaffected. The marrow is hypercellular with all forms of the myeloid cell line being increased and erythroid elements being dominant on the marrow aspirate smear preparations. The erythroblasts become large, oval shaped and contain a characteristic immature, lacy nucleus. These bone marrow features are called “megaloblastic” and are highly suspicious of a vitamin B12 or folate deficiency. Megaloblastoid (megaloblastic-like) abnormalities of the marrow are frequently seen in other hematologic disorders not associated with vitamin B12 or folate deficiency, (e.g., myelodysplasia and leukemia) and a careful examination of the bone marrow is necessary to make this distinction.

Macrocytosis is frequently linked to alcoholism, with or without liver disease. In fact, it is purported to be one of the most common causes of nonmegaloblastic macrocytosis ¹⁶⁾.

Table 1. Common causes of macrocytosis

Drugs

Alcoholism

Reticulocytosis

Nonalcoholic and alcoholic liver disease

Hypothyroidism

Vitamin B12 deficiency

Folate deficiency

Multiple myeloma

Myelodysplastic syndromes

Aplastic anemia

Acute leukemia

[Source ¹⁷⁾]

Table 2. Drugs that may induce macrocytosis

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

[Source ¹⁸⁾]

Figure 1. Flowchart for the differential diagnosis of macrocytic anemias (Note: MDS = myelodysplastic syndrome)

[Source ²¹⁾]

Figure 3. Macrocytic anemia peripheral blood smear of a patient with liver disease showing predominantly round macrocytes (MCV = 114 fl), some targeted

[Source ²²⁾]

Reticulocyte Count

A reticulocyte count should be obtained if there is evidence of hemolysis on the peripheral smear, i.e., increased polychromasia, nucleated red blood cells, spherocytes or schistocytes ²³⁾. The presence of increased polychromasia of the macrocytes on the peripheral smear and a reticulocyte count of >10% should raise suspicion of hemolysis or an acute bleed. These large polychromatophilic erythrocytes noted on the peripheral smear represent reticulocytes, immature red blood cells that are larger than mature red blood cells, and are indicative of increased erythropoiesis or red blood cell production and, if present in increased number, can raise the MCV. Additionally, the reticulocyte maturation parameters performed on the peripheral blood may also be helpful to differentiate megaloblastic from nonmegaloblastic causes of the macrocytosis ²⁴⁾. An elevated reticulocyte maturation value is more suggestive of a megaloblastic rather than a non-megaloblastic anemia.

Bone Marrow Examination

Macrocytosis associated with a megaloblastic marrow is usually accompanied by anemia due to ineffective erythropoiesis. The bone marrow is hypercellular, showing evidence of abnormal proliferation and maturation of multiple myeloid cell lines. These abnormalities are most evident in the erythroid precursors with large megaloblastic erythroblasts present in increased numbers throughout the marrow. Similar morphologic abnormalities can be seen in the other myeloid elements, e.g., large or giant metamyelocytes and other granulocytic precursors. This ineffective erythropoiesis is accompanied by intramedullary hemolysis causing an elevated lactate dehydrogenase and indirect bilirubin in the serum ²⁵⁾. However, the reticulocyte count is low due to the abnormal maturation process. More severe degrees of abnormal proliferation and maturation are seen with myelodysplasia and myeloid leukemias. It is imperative that a hematologist or hematopathologist examine the marrow in order to appreciate these important, subtle, hematopoietic abnormalities. Patients with macrocytosis who are not anemic and have no other abnormalities noted on the peripheral blood smear do not usually need a bone marrow examination.

Investigation of Vitamin B12 and Folate Deficiencies

Macrocytosis is the earliest abnormality seen in complete blood counts of patients with folate or vitamin B12 deficiency. In patients with elevated MCV values, laboratory tests for vitamin B12 and folate deficiencies are routinely ordered by physicians, although these tests are limited by their low sensitivity and specificity. Like the red blood cell MCV, the lower limits of normal for vitamin B12 levels are not well defined ²⁶⁾.

Serum B12 Levels

Vitamin B12 levels may be reported as normal or elevated in myeloproliferative disorders, liver disease, congenital transcobalamin II deficiency, intestinal bacterial overgrowth and antecedent administration of vitamin B12 ²⁷⁾. Moreover, there are reports of falsely low vitamin B12 levels with folate deficiency, pregnancy, use of oral contraceptives, congenital deficiency of serum haptocorrins and multiple myeloma ²⁸⁾. The prevalence of vitamin B12 deficiency among the elderly ranges from 1.5% to 4.6% ²⁹⁾ and was reported to be as high as 15% in the population over the age of 60 years ³⁰⁾. The deficiency in many cases is associated with gastric achlorhydria, resulting in decreased synthesis and availability of intrinsic factor, a necessary binding protein that facilitates vitamin B12 absorption in the ileum. This constellation of events eventually leads to pernicious anemia and requires prompt intervention with exogenous vitamin B12 preparations. The diagnosis of pernicious anemia can be confirmed by identifying and measuring intrinsic antibody levels in the serum. Parietal cell antibodies, although not specific, are also commonly present. However, these tests are expensive and not always available to the practicing clinician.

Serum Folate Levels

Folic acid deficiency in the United States is extremely rare because of the fortification of foods ³¹⁾. Although tissue stores may be normal, serum folate levels can decrease within a few days of dietary folate restriction ³²⁾. Thus, patients should fast prior to testing for serum folate levels, as serum folate levels increase with feeding. Because of the high concentration of folate within the red blood cell, mild degrees of hemolysis can falsely elevate serum folate levels ³³⁾. Pregnancy, certain anticonvulsant drugs, and alcohol intake may also cause a decrease in folate serum levels despite adequate tissue stores. Serum folate levels tend to be increased in patients with vitamin B12 deficiency, presumably because of impairment of the methionine synthase pathway and accumulation of methyltetrahydrofolate, the principal form of folate in the serum ³⁴⁾.

Red blood cell Folate

Because of the limitations of measuring serum folate, RBC folate levels have been advocated as a more reliable source of measuring tissue stores of folate. RBC folate levels remain constant throughout the lifespan of the cell and are not affected by short-term dietary changes that can alter serum levels. However, assays for measuring RBC folate levels have also been fraught with unreliability.3,31–33 It should be noted that low RBC folate levels have been reported with alcohol use, pregnancy and anticonvulsant medications.34 Another important cause of low RBC folate levels is vitamin B12 deficiency.29,30,35 It is estimated that approximately 60% of patients with pernicious anemia have low RBC folate levels, presumably because vitamin B12 is necessary for normal transfer of methyltetrahydrofolate from plasma to RBCs.28,36

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 methylmalonic acid. 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 methylmalonic acid (MMA) or mildly elevated levels ³⁵⁾. It should be kept in mind that nearly 50% of those with elevation of these metabolites will have normal serum vitamin B12 levels ³⁶⁾. 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 methylmalonic acid (MMA) and homocysteine levels. Both of these metabolites are elevated in cobalamin deficiency, with anemic cobalamin deficient patients showing marked elevations. However, methylmalonic acid (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 methylmalonic acid (MMA) levels are not elevated ³⁷⁾. Therefore, measuring the serum levels of these two metabolites not only helps in differentiating cobalamine (vitamin B12) deficiency from folate deficiency, but also provides a reliable degree of both sensitivity and specificity in diagnosing these important deficiency states ³⁸⁾. An important limitation that must be considered when measuring methylmalonic acid (MMA) is the presence of renal insufficiency, i.e., an elevated serum creatinine, and hypovolemia. Serum methylmalonic acid (MMA) will be elevated in patients with underlying renal dysfunction, decreasing its specificity and sensitivity in identifying patients with cobalamin deficiency ³⁹⁾. Similarly, hereditary homocysteinemia is a condition in which the serum homocysteine levels are elevated. Measurements of methylmalonic acid (MMA) levels are recommended when initial vitamin B12 and/or homocysteine levels are abnormal.

Macrocytic anemia treatment

Traditionally, patients with vitamin B12 deficiency from any cause have received cyanocobalamin intramuscularly or subcutaneously 1000 μg/week for 1 month and monthly, thereafter. This time-honored method remains an acceptable form of treatment for all causes of vitamin B12 deficiency, particularly when cognitive impairment or neurologic disease is present. Alternatively, hydroxocobalamin given in the same dose every 1–3 months intramuscularly is also effective therapy. This form of cobalamin remains in the tissues longer than the cyanocobalamin forms and can, therefore, be given less frequently ⁴⁰⁾. In cases of deficiency due to inadequate intake, food-cobalamin malabsorption and pernicious anemia, oral cyanocobalamin administered at 1000–2000 μg/day for 1 month, followed by 125–500 μg/day is recommended and considered a safe and effective method of treatment ⁴¹⁾. Other oral administration regimens have demonstrated efficacy and have proven to be equally as effective as intramuscular administration ⁴²⁾. In some cases, nasal or sublingual cyanocobalamin may also be useful in replenishing vitamin B12 stores ⁴³⁾. Because of the inherent unreliability of measuring either serum or red blood cell folate, it is recommended that patients receiving treatment for vitamin B12 deficiency receive empiric folate supplementation of 400 μg/day to 1 mg/day ⁴⁴⁾. Maintenance therapy should continue until the underlying cause of the deficiency is corrected.

In folate deficiency, the serum folate level is very sensitive to dietary folate intake and responds well to short-term treatment. Long-term treatment is not warranted except with chronic conditions such as malnutrition, exfoliative dermatitis or hemolysis ⁴⁵⁾. A complete blood cell count 10–14 days after starting the treatment for vitamin B12 or folate deficiency should reveal a rise in hemoglobin and a decrease in MCV. A full hematologic response should occur within 8 weeks. During treatment, further monitoring of the complete blood cell count or measuring vitamin B12 and folate levels or their metabolites is not necessary ⁴⁶⁾. In patients taking long-term treatment for vitamin B12 deficiency, an annual complete blood cell count may be a reasonable consideration to monitor therapy.