Megaloblastic Anemia

Megaloblastic anemia is a subset of macrocytic anemias that arises because of impaired nucleic acid synthesis in erythroid precursors. This impairment leads to ineffective RBC production and intramedullary hemolysis that is characterized by large cells with arrested nuclear maturation. The most common causes are vitamin B12 and folic acid deficiencies, which can be due to low dietary intake, underlying malabsorptive conditions, and medications. Clinical presentation includes anemia and GI symptoms, with neurologic manifestations more commonly seen in B12 deficiency. Laboratory tests show macrocytic anemia (elevated mean corpuscular volume) and low B12 and folate levels. Confirmatory tests can be performed if levels are borderline. Treatment focuses on identifying the cause of the deficiency and replacing the deficient vitamin either orally or parenterally.

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Overview

Definition

Megaloblastic anemia is a subset of macrocytic anemias characterized by increased RBC size and an arrest in nuclear maturation arising from abnormal cell division in erythroid precursors.

Epidemiology

  • Folate deficiency has been less common in the United States owing to folic acid fortification of grain products and the use of prenatal vitamins.
  • The most common causes of megaloblastic anemia in developed countries are pernicious anemia (for vitamin B12 deficiency) and medications.

Causes

  • Vitamin B12 (cobalamin) deficiency:
    • Intrinsic factor deficiency (pernicious anemia)
    • Malabsorption (Crohn disease, celiac disease, chronic pancreatitis, prior gastric or ileal surgery)
    • Vegan diet (vegan)
    • Nitrous oxide abuse/toxicity
    • Diphyllobothrium latum (fish tapeworm) infestation 
    • Drugs (immunosuppressants, isoniazid, metformin, colchicine, H2 blockers, proton pump inhibitors)
    • HIV
    • Genetic disorders 
  • Folic acid deficiency: 
    • Increased demand: pregnancy, hemolytic anemia, chronic dermatitis, hemodialysis
    • Alcoholism
    • Dietary deficiency (restricted diets, countries without folate fortification of foods)
    • Drugs (antimetabolites such as methotrexate, tetracyclines, penicillins, nitrofurantoin, phenobarbital, phenytoin, trimethoprim)
    • Intestinal dysfunction: Malabsorption occurs in surgery (gastric bypass).
  • Rare disorders:
    • Orotic aciduria: Deficiency of uridine monophosphate synthase leads to ↓ de novo pyrimidine synthesis that is unresponsive to B12 and folate replacement.
    • Methylmalonic acidemia: inborn error of amino acid metabolism

Pathophysiology

Vitamin B12 and folic acid

  • Vitamin B12 (cobalamin):
    • Sources:
      • Animal products (meats, especially liver, dairy, eggs)
      • Fortified food
      • Not found in plants
    • Absorption:
      • B12 in food is bound to protein and gets bound to salivary haptocorrins in the stomach.
      • Absorption requires an acid environment (stomach) to be dissociated from protein and pancreatic proteases to cleave off the haptocorrins.
      • Once unbound, B12 binds to intrinsic factor produced by the gastric parietal cells.
      • B12–intrinsic factor complex is taken up in the ileum.
      • In the bloodstream, B12 is then endocytosed by cells. 
  • Vitamin B9 (folic acid):
    • Sources:
      • Plant products (especially dark green, leafy vegetables)
      • Animal products
    • Absorption:
      • Dependent on carrier systems
      • Absorbed in the jejunum (conjugase converts folate polyglutamate to monoglutamate)
      • Subsequently reduced to dihydrofolate → tetrahydrofolate (THF) → 5,10-methylene THF → L-5-methyl-THF (predominant plasma form)
      •  L-5-methyl-THF is taken up by cells.
  • Both are water-soluble (↓ risk of overdose, as they are excreted in the urine).
  • Vitamin deficiencies arise from conditions that affect intake and absorption and that interfere with other essential factors (i.e., intrinsic factor, carrier system).
Role of vitamin B12 and folic acid in DNA synthesis

Role of vitamin B12 and folic acid in DNA synthesis:

From the bottom:
Dietary folate is absorbed in the intestine in the form of 5-methyl-tetrahydrofolate (THF). Vitamin B12–dependent methionine synthetase converts 5-methyl-THF to THF. The same process generates methionine from homocysteine and methionine converts to S-adenosylmethionine or SAM (also necessary for DNA methylation).
The THF produced is converted to 5, 10-methylene-THF.
A methyl group is donated from methylene-THF to the 5-carbon of uridylate to form thymidylate.
As a consequence of donating the methyl group, methylene-THF becomes dihydrofolate.
Dihydrofolate is reduced by reductase to re-generate tetrahydrofolate.

Image by Lecturio.

B12 and folic acid in DNA synthesis

  • Both B12 and folic acid: 
    • Critical in the synthesis of nucleic acids
    • Act as cofactors in the synthesis of thymidylate, the rate-limiting step in DNA synthesis:
      • Folate supplies the methyl groups (via methylene-THF).
      • B12 is a cofactor in the reaction converting 5-methyl-THF to THF.
  • Methionine and folic acid cycle with DNA synthesis:
    1. Dietary folate is absorbed in the intestine in the form of 5-methyl-THF.
    2. Vitamin B12–dependent methionine synthetase converts 5-methyl-THF to THF.  
      • The same process generates methionine from homocysteine.
      • Methionine converts to S-adenosylmethionine (SAM) (in the methionine cycle), which is also necessary for DNA methylation.
    3. The THF produced is converted to methylene-THF. 
    4. A methyl group is donated from methylene-THF to the 5-carbon of uridylate to form thymidylate (deoxythymidine monophosphate). 
    5. As a consequence of donating the methyl group, methylene-THF becomes dihydrofolate. 
    6. Dihydrofolate is reduced to regenerate tetrahydrofolate.

Consequences of defective DNA synthesis

Rapidly dividing cells in the body are most sensitive to impaired DNA synthesis due to B12 and folate deficiency.

  • Impaired erythropoiesis:
    • Megaloblastic changes:
      • Slow nuclear division → immature, abnormal nuclei + normal cytoplasm → dyssynchrony between nuclear and cytoplasmic maturation
      • Increased mitotic figures → giant metamyelocytes + finely stippled, lacy nuclear chromatin pattern
    • Intramedullary hemolysis: 
      • Caused by premature death of abnormally developing erythroid precursors
      • Bone marrow becomes hypercellular owing to ongoing apoptosis → ↑ hemolysis, ↓ reticulocyte count
  • Reduced DNA methylation:
    • Significant epigenetic modification → methyl groups added to DNA → results in modification of gene expression
    • Results in defects in DNA repair and fragmentation
    • Enhances chance of translational errors including the possibility of malignant transformation
    • Believed to contribute to neuronal dysfunction in B12 deficiency:
      • ↓ Methylation of lipids and proteins (i.e., myelin basic protein) in the neurons
      • ↓ Myelin basic protein contributes to demyelination.

Clinical Presentation

General manifestations

  • B12 deficiency develops over years, whereas folate deficiency develops in weeks to months.
  • Depending on the degree of deficiency and time of onset, patients can be asymptomatic.
  • Signs and symptoms of anemia:
    • Fatigue
    • Shortness of breath 
    • Tachycardia
    • Palpitations
    • Pallor
    • Jaundice
  • GI symptoms (related to underlying GI conditions such as inflammatory bowel disease):
    • Diarrhea
    • Bloating
    • Epigastric/abdominal pain

Specific manifestations

  • Neurologic symptoms most commonly due to B12 deficiency: 
    • Subacute combined degeneration (classic finding):
      • Dorsal column: vibration, proprioception (wide-based gait)
      • Lateral corticospinal tracts: spasticity
      • Dorsal spinocerebellar: ataxia
    • Neuropathy: tingling, numbness
    • Psychosis, depression, irritability
    • Cognitive impairment, forgetfulness
    • Dementia
  • Additional findings in B12 deficiency:
    • Oral mucosa pathology (present in 50%–60% of patients):
      • Glossitis
      • Angular cheilitis
      • Recurrent oral ulcers
      • Diffuse erythematous mucositis/mucosal atrophy
      • Mouth soreness/burning sensation
    • Cutaneous hyperpigmentation
    • ↑ Risk of gastric cancer in pernicious anemia
  • Folate deficiency: ↑ risk of neural tube defects (congenital anomaly)
Peculiar cutaneous hyperpigmentation from cases with megaloblastic anemia

Hyperpigmentation in a patient with vitamin B12 deficiency

Image: “Peculiar cutaneous hyperpigmentation from cases with megaloblastic anemia” by Department of Pathology, Pondicherry Institute of Medical Sciences, Puducherry, India. License: CC BY 2.0

Diagnosis

History

  • Diet: vegan or vegetarian
  • Medical and social history: look for autoimmune disorders, alcoholism
  • Surgical history: gastric or ileal resection
  • GI symptoms
  • Neurologic symptoms
  • Medications

Laboratory tests

  • Hematologic tests:
    • ↓ Hemoglobin/hematocrit 
    • MCV > 100
    • Likely decreased leukocyte and/or platelet count
    • Low reticulocyte count
  • Cobalamin/B12 and folate levels :
    • Deficiency:
      • Low B12/cobalamin (normal, > 300 pg/mL)
      • Low folate levels (normal, > 4 ng/mL)
      • RBC folate can be an alternative for folate level (though not frequently used).
    • For borderline levels of B12 (200–300 pg/mL) or folate (2–4 ng/mL), proceed with obtaining homocysteine and methylmalonic acid (MMA) level:
      • Serum homocysteine level: elevated in both vitamin B12 and folate deficiency
      • Serum MMA level: elevated in vitamin B12 deficiency
  • Additional workup:
    • Pernicious anemia:
      • Anti-intrinsic factor antibodies: high specificity for pernicious anemia
      • Antiparietal cell antibodies: can be present in gastritis
      • ↑ Serum gastrin levels: not specific to pernicious anemia
    • Orotic aciduria: normal urine ammonia with ↑ orotic acid levels
  • Peripheral-blood smear:
    • Macrocytosis
    • Marked RBC size variation: anisocytosis
    • Abnormal and variable RBC morphology: poikilocytosis
    • Hypersegmented neutrophils:
      • ≥ 1% of neutrophils with 6 or more nuclear lobes, OR
      • ≥ 5% of neutrophils with 5 or more nuclear lobes

Additional tests

  • Bone marrow aspirate:
    • Not routinely done 
    • If done, findings include:
      • Hypercellularity
      • Erythroid predominance with decreased myeloid to erythroid ratio
      • Nuclear cytoplasmic dyssynchrony with immature nuclei combined with normal-appearing cytoplasm
      • Abnormal granulocytes: giant metamyelocytes and bands
      • Abnormally large megakaryocytes
      • In severe cases, dysplastic changes can be seen.
  • Gastric biopsy:
    • Not necessary for the diagnosis of pernicious anemia
    • If done, will reveal chronic autoimmune atrophic gastritis
  • Schilling test for pernicious anemia: 
    • Infrequently used
    • Supplanted by serologic testing for parietal cell and intrinsic factor antibodies
Bone marrow aspirate in megaloblastic anemia with cutaneous hyperpigmentation

Bone marrow biopsy:
A: Bone marrow aspirate
B: Hypercellularity and megaloblasts (arrow)
C: Giant abnormally shaped stab forms (arrow)

Image: “Bone marrow aspirate in megaloblastic anemia with cutaneous hyperpigmentation” by Department of Pathology, Pondicherry Institute of Medical Sciences, Puducherry, India. License: CC BY 2.0

Management

Principles

  • Patients can be asymptomatic or deficiency can be an incidental finding.
  • In the majority of cases, there is a gradual development of symptoms, so treatment can be given over weeks.
  • Intervention is needed urgently in for the following:
    • Symptomatic anemia
    • Neurologic/neuropsychiatric manifestations (as effects can be irreversible)
    • Pregnancy (fetus is affected)
    • Neonates and infants (growth is affected)

Treatment

  • Vitamin B12:
    • Intramuscular/parenteral route for neurologic symptoms or in patients with malabsorption or extensive gastric/bowel resections
    • Oral route for deficiency owing to low dietary intake or to continue replacement from parenteral route (if appropriate)
    • Watch for thrombocytosis and hypokalemia in severe cases.
  • Folic acid: 
    • Always check for concomitant B12 deficiency.
    • 1–5 mg/day, with the higher dose for pregnant women, alcoholics, patients on antiepileptic medications
  • Replacement can be lifelong if underlying condition is permanent (i.e., gastric bypass surgery).
  • As anemia resolves, iron levels may become depleted; monitor and replace as necessary.
  • Treatment of other associated conditions:
    • Pernicious anemia: in addition to replacement, screen for gastric cancer
    • Uridine triacetate: for orotic aciduria

Differential Diagnosis

  • Alcoholism and alcoholic liver disease: Alcohol is one of the top causes of macrocytosis, usually due to a toxic effect directly on the RBCs (without damage to DNA replication). Chronic alcoholism also leads to liver disease, ranging from a fatty liver to cirrhosis. Management aims at alcohol abstinence for reversal at certain stages; addressing contributing factors, such as viral infections or drugs; and minimizing damage to the hepatocytes.
  • Chronic liver disease: Cirrhosis is the late stage of hepatic necrosis and scarring. Chronic cellular damage causes extensive distortion of the normal hepatic architecture, which can lead to impairment of normal blood flow through the liver. Chronic liver disease of diverse etiologies can cause macrocytic anemia owing to effects in lipid composition of RBCs.
  • Myelodysplastic anemia: a group of hematologic malignancies due to germline mutations associated with one or more cytopenias, including macrocytic anemia: Dysplastic changes in the bone marrow biopsy, such as hyposegmented granulocytes and blasts, are seen. Cytogenetic analysis identifying mutations linked to myelodysplastic disease helps establish the diagnosis.
  • Aplastic anemia: a disorder of bone marrow failure characterized by absence of erythroid precursors due to exposure to drugs, radiation, chemicals, viruses, autoimmune disease, or genetic factors (hereditary or acquired): The bone marrow is hypocellular, with < 30% cellularity. Some cytopenias involve ≥ 2 cell lines. 
  • Hypothyroidism: a condition characterized by a deficiency of thyroid hormones: Iodine deficiency is the most common cause worldwide, but Hashimoto disease (autoimmune thyroiditis) is the leading etiology in non–iodine-deficient regions. Features of acquired hypothyroidism include fatigue, bradycardia, cold intolerance, and exertional dyspnea. Diagnosis is by thyroid function tests. Elevated thyroid-stimulating hormone and a low free thyroxine (T4) are noted. Treatment is with synthetic T4. Macrocytic anemia is seen in 55% of patients with hypothyroidism.

References

  1. Hesdorffer CS, Longo DL. (2015). Drug-induced megaloblastic anemia. N Engl J Med 373:1649–1658 https://pubmed.ncbi.nlm.nih.gov/26488695/
  2. Kim J, Kim MJ, Kho HS. (2016). Oral manifestations in vitamin B12 deficiency patients with or without history of gastrectomy. https://pubmed.ncbi.nlm.nih.gov/27234214/
  3. Means R,  Fairfield K. (2021). Clinical manifestations and diagnosis of vitamin B12 and folate deficiency. UpToDate. Retrieved April 4, 2021, from https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-vitamin-b12-and-folate-deficiency
  4. Means R, Fairfield K. (2021). Causes and pathophysiology of vitamin B12 and folate deficiencies. UpToDate. Retrieved April 5, 2021, from https://www.uptodate.com/contents/causes-and-pathophysiology-of-vitamin-b12-and-folate-deficiencies
  5. Means R, Fairfield K. (2021). Treatment of vitamin B12 and folate deficiencies. UpToDate. Retrieved April 5, 2021, from https://www.uptodate.com/contents/treatment-of-vitamin-b12-and-folate-deficiencies
  6. Socha DS, DeSouza SI, Flagg A, Sekeres M, Rogers HJ. (2020). Severe megaloblastic anemia: vitamin deficiency and other causes. Cleve Clin J Med https://pubmed.ncbi.nlm.nih.gov/32127439/

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