Continuing Education Activity

Megaloblastic anemia (MA) encompasses a heterogeneous group of anemias characterized by the presence of large red blood cell precursors called megaloblasts in the bone marrow. This condition is due to impaired DNA synthesis, which inhibits nuclear division. Cytoplasmic maturation, mainly dependent on RNA and protein synthesis, is less impaired. This leads to an asynchronous maturation between the nucleus and cytoplasm of erythroblasts, explaining the large size of the megaloblasts. This activity reviews the cause and presentation of megaloblastic anemia and highlights the role of the interprofessional team in its management.

Objectives:

• Identify the etiology of megaloblastic anemia.
• Describe the diagnostic approach towards the evaluation of patients with megaloblastic anemia.
• Summarize the complications of megaloblastic anemia.
• Review the importance of improving care coordination among interprofessional team members to improve outcomes for patients affected by megaloblastic anemia.

Introduction

Megaloblastic anemia (MA) encompasses a heterogeneous group of macrocytic anemias characterized by the presence of large red blood cell precursors called megaloblasts in the bone marrow.[1] This condition is due to impaired DNA synthesis, which inhibits nuclear division. Cytoplasmic maturation, mainly dependent on RNA and protein synthesis, is less impaired. This leads to an asynchronous maturation between the nucleus and cytoplasm of erythroblasts, explaining the large size of the megaloblasts.[2] The process affects hematopoiesis as well as rapidly renewing tissues such as gastrointestinal cells. Megaloblastic anemia is most often due to hypovitaminosis, specifically vitamin B12 (cobalamin) and folate deficiencies, which are necessary for the synthesis of DNA.[3] Copper deficiency and adverse drug reactions (due to drug interference with DNA synthesis) are other well-known causes of megaloblastic anemia. A rare hereditary disorder known as thiamine-responsive megaloblastic anemia syndrome (TRMA) is also identified as a cause of megaloblastic anemia.[4] The list of drugs associated with the disease is long however, frequently implicated agents include hydroxyurea, chemotherapeutic agents, anticonvulsants, and antiretroviral therapy (ART) drugs.

Etiology

Deficiencies of vitamin B12 and folic acid are the leading causes of megaloblastic anemia. 

Folic acid is present in food such as green vegetables, fruits, meat, and liver. Daily adult needs range from 50 to 100 mcg. The recommended dietary allowance is 400 mcg in adults and 600 mcg in pregnant women.[5] Folic acid is mainly absorbed in the jejunum and the body stores around 5 mg of folate in the liver, which is enough for 3 to 4 months. Folic acid deficiency may be related to decreased intake in the case of alcohol use disorder or malnutrition (elderly patients, institutionalized patients, poverty, special diets, etc.), increased demand particularly in case of pregnancy, hemolysis, hemodialysis, and malabsorption (tropical sprue, celiac disease, jejunal resection, Crohn disease, etc.). In some cases, medications like anticonvulsants and anticancer agents cause megaloblastic anemia related to folate deficiency by affecting folate metabolism.

The primary dietary sources of cobalamin/vitamin B12 are meats, fish, eggs, and dairy products. Vegan diets are low in vitamin B12. However, not all patients following a vegan diet develop clinical evidence of deficiency. Vitamin B12 is first bound within the duodenum and jejunum to the intrinsic factor (IF) produced by gastric parietal cells and is then absorbed in the terminal ileum. The body stores 2 to 3 mg of vitamin B12 in the liver (sufficient for 2 to 4 years). The most frequent cause of vitamin B12 deficiency is pernicious anemia caused by autoimmune gastric atrophy, leading to decreased intrinsic factor production.[6] Vitamin B12 deficiency may also develop following gastrectomy, ileal resection, or ileitis of any cause. Other causes of impaired vitamin B12 absorption include Zollinger-Ellison syndrome, blind loop syndrome, fish tapeworm infestation, and pancreatic insufficiency.

Clinical copper deficiency can cause microcytic, normocytic, or macrocytic anemia and neutropenia. Copper deficiency also causes myelopathy and peripheral neuropathy. Bone marrow evaluation can reveal myelodysplasia and megaloblastic anemia. Treatment with copper replacement promptly reverses hematologic manifestations of the disease, although neurologic manifestation may take longer.[7]

In rare cases, MA is due to inherited problems:

• Thiamine-responsive megaloblastic anemia syndrome: An autosomal recessive disease characterized by megaloblastic anemia associated with diabetes mellitus and early-onset sensorineural hearing loss.[8] Mutations in the gene encoding a thiamine transporter (SLC19A2) are thought to be the cause of this disorder.[9] The disease manifests in early infancy and is treated with high-dose thiamine. 
• Inherited deficiency of intrinsic factor or the receptor in the intestines: Imerslund-Grasbeck syndrome or juvenile megaloblastic anemia is caused by biallelic mutations affecting the ileal receptor for the vitamin B12-IF complex. These patients also have proteinuria and abnormal vitamin D metabolism.[10][11]
• Some infants have congenital folate malabsorption syndrome.

Drug-induced megaloblastic anemia can occur from a variety of different medications via various different mechanisms. Drugs known to cause megaloblastic changes in the bone marrow include:[12]

• Allopurinol
• Azathioprine
• Capecitabine
• Cladribine
• Fludarabine
• Fluorouracil
• Gadolinium
• Gemcitabine
• Hydroxyurea
• Lamivudine
• Leflunomide
• Mercaptopurine
• Methotrexate
• Mycophenolate mofetil
• Trimethoprim
• Zidovudine

Drugs that reduce the intestinal absorption of folic acid or vitamin B12 and/or metabolism of these vitamins include:[12]

• Aminosalicylic acid
• Antacids and proton pump inhibitors
• Penicillin antibiotics
• Chloramphenicol
• Erythromycin
• Oral contraceptives
• Metformin
• Phenytoin
• Tetracyclines
• Valproic acid

Epidemiology

Megaloblastic anemia is not rare, but data are insufficient regarding its prevalence. Moreover, there is a variation amongst different populations, which may be dependent on universal supplementation of folate in dietary products, frequency of chronic illnesses, such as pernicious anemia and Helicobacter pylori (H. pylori) infection, and cultural/personal dietary practices. In a 2016 study from the Netherlands, evaluating more than 3000 patients with anemia, only 7.5% of the cases displayed macrocytic anemia. Approximately 1.4% of all the cases were due to vitamin B12 deficiency and 0.5% were due to folate deficiency. When compared to the macrocytic anemia subgroup, this translated to 18% and 6% prevalence for B12 deficiency and folate deficiency, respectively.[13] The incidence of megaloblastic anemia increases with age,[14] and elderly patients (older than 60 years of age) living in retirement facilities or eldercare centers seem to have a higher prevalence of megaloblastic anemia than their age-controlled peer group living independently.[15] Pernicious anemia is the most frequent cause of anemia related to cobalamin deficiency worldwide and usually occurs in individuals older than 40 years. The incidence of pernicious anemia is higher in the United Kingdom and Nordic countries than in other developed countries.[16]

The incidence of folate deficiency is low, especially in countries with universal supplementation of folate in dietary products. A 2015 report from Canada, reported a prevalence of 0.16% for folate deficiency in hospitalized patients, with alcohol use disorder, malabsorption syndromes, and decreased oral intake due to mental health diagnoses as the identified causes.[17]

Pathophysiology

The pathophysiology of this group of anemia is ineffective erythropoiesis secondary to intramedullary apoptosis of hematopoietic cell precursors, which results from DNA synthesis abnormalities. Both vitamin B12 and folate deficiencies may cause defective DNA synthesis. Subsequently, the nucleus and cytoplasm do not mature simultaneously. The cytoplasm (in which hemoglobin synthesis is unaltered) mature at the normal rate, and the nucleus (with impaired DNA synthesis) is not fully mature. The cells arrest in the DNA synthesis (S) phase and make DNA replication errors, which eventually leads to apoptotic cell death.[18]

The primary role of folate is to donate methyl groups in DNA synthesis. Vitamin B12 is a cofactor in the reaction that recycles 5-methyl-tetrahydrofolate back to tetrahydrofolate (THF). The generation of THF is coupled to the conversion of homocysteine to methionine. Lack of vitamin B12 causes folate to become trapped in the 5-methyl-THF form, and it also leads to a deficiency of methionine.[19] The donation of a methyl group or the "one-carbon metabolism" pathway is crucial for DNA synthesis.[18]

With altered DNA synthesis hematopoiesis is disrupted as hematopoietic precursor cells are rapidly dividing cells. As stated above, these deficiencies lead to arrested nuclear division without significant alteration in the cytoplasmic maturation cycle. Nucleated precursor cells in the bone marrow develop immature or morphologically abnormal nuclei and giant metamyelocytes, with macrocytic red blood cells and hypersegmented neutrophils on the peripheral blood smear.[18][20] Prolonged deficiency leads to intramedullary hemolysis of the developing erythropoietic precursor cells in the bone marrow.[20] Laboratory evaluation during this phase of the disease will reveal bone marrow hypercellularity and peripheral evidence of hemolysis with a low reticulocyte count.[20] The pathophysiology behind neuronal dysfunction associated with megaloblastic anemia is unclear.

The pathophysiology behind pernicious anemia involves autoantibodies against the intrinsic factor or gastric parietal cell antigens.[21][16] These antibodies are not sensitive or specific for pernicious anemia and cannot be used in isolation to make the diagnosis.[21]

Histopathology

In a myelogram, megaloblastosis presents as large red blood cells (megaloblasts) and hypersegmented neutrophils, which are detectable in a peripheral blood smear.  Poikilocytosis and anisocytosis are common due to ineffective erythropoiesis. The bone marrow evaluation shows hypercellularity with abnormal maturation and proliferation of red cell precursors. Erythroblasts show a failure of nuclei maturation, maintaining open or lax chromatin and normal mature cytoplasm.[22]

History and Physical

The most common presentation of megaloblastic anemia is an asymptomatic incidental finding on routine laboratory testing. Usually, anemia develops gradually, and symptoms are present only in severely anemic patients. Common symptoms include weakness, shortness of breath (primarily with exertion), palpitation, and lightheadedness. Physical examination may reveal pallor, tachycardia, functional heart murmur, Hunter glossitis, and splenomegaly. Jaundice can occur from intramedullary hemolysis.[23]

There are some minor differences between the clinical manifestations caused by cobalamin deficiency and folic acid deficiency. In vitamin B12 deficiency, neurological manifestations are observable. The main symptoms are paresthesia and balance disorders. Patients with vitamin B12 deficiency may present lancinating pains caused by peripheral neuropathy, mainly affecting the lower extremities. Less frequently, there may be a development of visual disturbances caused by optic atrophy. The clinical exam usually shows a loss of vibratory sense and proprioception with a positive Romberg test. Babinski reflex, hyporeflexia, and clonus are less frequent. Moreover, there are psychological disturbances that include a form of dementia. These neurological disorders may not be completely reversible after replacement therapy.[24]

Pernicious anemia frequently has associations with other autoimmune conditions such as autoimmune thyroid disease, type 1 diabetes, and vitiligo.[25]

Evaluation

Clinical suspicion for megaloblastic anemia should be high in patients with unexplained macrocytic (mean corpuscular volume [MCV] greater than 100 fL) anemia or hypersegmented neutrophils on a peripheral smear. An MCV of greater than 115 fL is more specific for vitamin B12 deficiency or folate deficiency than other causes of macrocytosis, however, a normal MCV does not rule out megaloblastic anemia.[24] A reticulocyte count is also indicated in the workup of this disease. In a patient with typical peripheral blood smear findings and a low reticulocyte count, the only testing needed is a serum vitamin B12 and folate level.[24] In a patient consuming a normal diet, folate levels can be omitted. In patients with suspected disorders of absorption or malnutrition such as excess alcohol consumption, both levels should be obtained. A B12 level above 300 pg/mL (above 221 pmol/L) is considered normal. A level between 200 to 300 pg/mL (148 to 221 pmol/L) is considered borderline and additional testing should be obtained to verify the diagnosis and elucidate the cause. A level below 200 pg/mL (below 148 pmol/L) is consistent with deficiency and further testing is only indicated if the route of administration of B12 supplementation needs clarification.[24] It is important to note that this assay is not accurate in patients with IF autoantibodies and will give false-negative results. Spuriously low serum vitamin B12 levels can occur in patients with multiple myeloma, HIV infection, pregnancy, oral contraceptive use, and diphenylhydantoin administration.[26] Falsely elevated B12 levels may be seen in patients with myeloproliferative neoplasm, alcoholic liver disease, and renal disease.[18] A folate level of 2 to 4 ng/mL (from 4.5 to 9.1 nmol/L) is considered borderline. A level below 2 ng/mL (below 4.5 nmol/L) is consistent with folate deficiency. 

Methylmalonic acid and homocysteine levels can be obtained in patients with borderline results from the above testing. They are often also ordered to confirm the diagnosis of B12 deficiency.[27] Methylmalonic acid can also help differentiate between vitamin B12 and folate deficiency as it is elevated in vitamin B12 deficiency but not in folate deficiency.[28] Falsely elevated levels of methylmalonic acid are seen in patients with renal insufficiency making the assay unreliable in these patients. Homocysteine is elevated in both vitamin B12 and folate deficiencies.[29]

In patients with evidence of B12 deficiency from the above testing, autoantibodies assays to test for antibodies against intrinsic factors should be obtained. Although the sensitivity of this assay is low, the specificity is quite high and a positive test is diagnostic of pernicious anemia.[18] Negative IF antibody titer can be seen in patients with autoantibodies to parietal cells. 

It is imperative to remember that vitamin B12 and folate deficiency testing should be done simultaneously to ensure both deficiencies are diagnosed if present. In cases where folate is replaced without vitamin B12 supplementation and underlying B12 deficiency, the neurologic manifestations of vitamin B12 deficiency will not be treated and may potentially get worse.[30]

The 2014 Guidelines from the British Committee for Standards in Haematology[23]

• Serum cobalamin and folate levels should be obtained simultaneously due to the close relationship in metabolism. (Grade 1A)
• Neurological symptoms due to vitamin B12 deficiency can occur in the absence of macrocytosis. In unexplained neurological symptoms consistent with B12 deficiency, cobalamin assays should be obtained. (Grade 1B)
• Methylmalonic acid and homocysteine levels should be obtained in patients with clinical suspicion of B12 deficiency but borderline serum levels. (Grade 2B)
• Homocysteine level is more sensitive for B12 deficiency but methylmalonic acid is more specific. Both have to be interpreted in relation to the patient's renal function.
• Holotranscobalamin (HoloTC) is the ‘active’ fraction of serum cobalamin and is more specific than serum cobalamin levels.[31] It may be used as a routine diagnostic laboratory in the future. (Grade 1B)
• All patients with clinical features suspicious for pernicious anemia should be tested for anti-IF antibodies regardless of cobalamin levels. (Grade 1A)
• Patients with low serum cobalamin levels without anemia or malabsorption syndromes to explain the result should be tested for anti-IF antibodies as they may have an early/latent presentation of pernicious anemia. (Grade 2A)
• Anti-gastric parietal cell antibody testing is not recommended. (Grade 1A)
• Red cell folate testing is not recommended in most cases. (Grade 1A)
• In the presence of strong clinical suspicion of folate deficiency and a normal serum level, a red cell folate assay can be obtained if cobalamin deficiency has already been ruled out. (Grade 2B

Treatment / Management

Vitamin B12 and folic acid can be given orally or parenterally. If there is no evidence of malabsorption, the generally preferred route for supplementation is oral.[23] It is important to remember that oral supplementation takes time and should not be used in cases where urgent supplementation is required. In asymptomatic cases oral supplementation is sufficient. In patients with neurologic symptoms or those with increased demand such as pregnancy and in infancy, vitamin B12 and folic acid supplementation should be initiated parenterally. Patients with symptomatic anemia may require a blood transfusion to relieve symptoms, as vitamin B12 and folic acid supplementation do not correct anemia rapidly. Vitamin B12 is also available in a sublingual formulation, which may be appropriate for patients with intestinal malabsorption syndromes.[23]

The recommended dose for vitamin B12 supplementation in children is 50 to 100 mcg parenterally once a week until the deficiency is corrected. They may require supplemental doses every month or every other month thereafter, depending on the formulation used (cyanocobalamin versus hydroxocobalamin). In adults, the recommended dose is 1000 mcg parenterally once a week until the deficiency is corrected, followed by supplemental doses every month or every other month. An oral vitamin B12 dose of 1000 mcg daily is equally effective as the above parenteral regimen, provided that there is no intestinal malabsorption issue.[18] A 2018 Cochrane review reported oral supplementation was equally effective in raising serum B12 levels as compared to intramuscular formulations, with the added benefit of it being a low-cost treatment.[32] The duration of treatment is dependent on the cause of the deficiency. If the root cause is correctable, supplementation can be stopped after serum B12 levels normalize. However, in cases with expected life-long deficiency (gastric bypass surgery patients, pernicious anemia, etc.) indefinite supplementation is warranted. 

The recommended dose for folic acid supplementation is 1 mg orally once a day until the deficiency is corrected. If the cause of this deficiency is correctable, supplementation can be stopped after repletion. However, in cases with nonreversible causes, indefinite supplementation is recommended.[23]

With adequate supplementation and bone marrow response, hemolytic markers (if intramedullary hemolysis is present) will improve within 1 week and serum hemoglobin/hematocrit levels will completely normalize within 1 to 2 months.[24] However, the neuropsychiatric symptoms take a longer period of time to recover (from 3 to 12 months) and according to some reports, there is transient clinical worsening of the neurological symptoms.[24] In some cases, the neurological symptoms may be irreversible. A 2006 observational study evaluating 57 patients with subacute combined degeneration reported only 14% clinical resolution after B12 treatment.[33] They did note that 86% of the patients had some clinical improvement. Subgroup analysis in the study revealed that absence of sensory dermatomal deficit, Romberg, and Babinski signs, age less than 50 years, and less than or equal to 7 segment involvement on magnetic resonance imaging, correlated with resolution of neurologic symptoms.[33] This highlights the importance of early diagnosis and treatment as patients with severe/prolonged neurological symptoms tend to have persistence of symptoms despite treatment.

Differential Diagnosis

The complete blood count may show macrocytosis in non-megaloblastic macrocytic anemias. Reticulocyte count will help distinguish between two primary conditions. If reticulocytosis is present, hemolytic anemia and acute hemorrhage are the two main conditions for which the clinician must look. If a reticulocytopenia is present, the underlying conditions may be evident in some cases, such as hypothyroidism, alcoholism, liver dysfunction, and certain drugs. In other cases, one should perform bone marrow aspiration provided that the investigations to exclude vitamin B12 or folate deficiency are carried out. Indeed, myelodysplastic disorders and sideroblastic anemia can manifest as refractory megaloblastic anemia.[34] Common clinical conditions to consider in patients who present with megaloblastic anemia include conditions that present with macrocytosis such as:

• Alcoholic hepatitis
• Atrophic gastritis
• Gastric cancer
• Celiac sprue
• Tropical sprue
• Myelodysplastic syndrome
• Aplastic anemia
• Acquired sideroblastic anemia
• Homocystinuria

Prognosis

The prognosis for megaloblastic anemia is favorable with proper identification of the precise etiology and the institution of appropriate treatment.[35] Hematologic abnormalities recover with adequate supplementation although neurologic manifestations show some delay in improvement.[33] Timely recognition and supplementation improve the prognosis of this disease, which may have little to no morbidity or mortality associated with it. There are some complications of the disease that can lead to poor outcomes in patients, such as gastric malignancy in patients with pernicious anemia as the cause of megaloblastic anemia. These outcomes, however, are associated with pernicious anemia itself, rather than the megaloblastic disease. Gastric malignancy associated with pernicious anemia is discussed below. 

Complications

Complications of megaloblastic anemia can vary according to specific etiology.[36] The most concerning complication of patients with megaloblastic anemia secondary to pernicious anemia are gastric malignancy. The incidence of gastric malignancy in patients with pernicious anemia was reported as 0.27% per patient-year and a sevenfold relative risk of gastric cancer in patients with pernicious anemia.[37] A recent study reported an increased risk of gastric adenocarcinoma and gastric carcinoid in patients with pernicious anemia.[38] Folate deficiency is associated with neural tube defects in the fetus.[39] This is a highly preventable complication with potentially devastating consequences, that can be eliminated with adequate supplementation during pregnancy. According to a 2018 study, inadequate folic acid intake or folate deficiency increases the risk for cancers of the head and neck, oral cavity and pharynx, esophagus, pancreas, bladder, and cervix.[40]

Deterrence and Patient Education

Patient education centers on resolving potential dietary deficiencies, addressing malabsorption issues, and working on other modifiable risk factors such as alcohol intake and medication regimens. Patients following a strictly vegan or Mediterranean diet are at high risk for vitamin B12 deficiency and should take oral supplementation on a regular basis.[41] Patients should be advised on moderation of alcohol intake and following a well-balanced diet to prevent nutritional causes of megaloblastic anemia.

In patients with gastrointestinal alterations or diseases as the cause of megaloblastic anemia, patient education on the cause of megaloblastic anemia and the importance of medication compliance is of the utmost importance. Patients need to be educated on the potentially irreversible neurologic complications of B12 deficiency and counseled on treatment compliance to prevent this outcome. Patients diagnosed with pernicious anemia, need counseling on self-monitoring of gastrointestinal symptoms, as they may be an early sign of gastric malignancy that will need urgent evaluation.

Patients taking medications that can potentially cause megaloblastic anemia, need to be counseled regarding vitamin supplementation while taking these medications to prevent megaloblastic anemia.

Enhancing Healthcare Team Outcomes

Megaloblastic anemia has several different causes. It generally has an excellent prognosis if diagnosed early and treated adequately. However, given multiple possible causes, medication interactions, and the need for long-term therapy in some cases, patients with megaloblastic anemia are best managed with an interprofessional team approach. A multifaceted treatment strategy is needed to prevent complications and treat patients with this disease.

The clinical nurse is essential in educating the patient about dietary recommendations and medication compliance to ensure patients follow the recommended treatment. Clinical pharmacists are crucial in preventing this disease, by ensuring adequate supplementation and monitoring of B12 and folic acid levels in patients taking medications known to cause megaloblastic anemia. The clinician needs to follow recommended treatment regimens and monitor serum levels for improvement to deter complications of this disease. A collaborative interprofessional team of clinicians, pharmacists, and nurses can help improve clinical outcomes in patients diagnosed with this disease.[Level 5]