Definition/General
Introduction:
Vitamin B12 deficiency causes megaloblastic anemia characterized by impaired DNA synthesis and nuclear-cytoplasmic asynchrony
Bone marrow shows megaloblastic changes in all cell lines
The condition results from defective cobalamin metabolism affecting thymidine synthesis
Hypersegmented neutrophils and megaloblasts are pathognomonic features.
Origin:
Results from inadequate B12 intake, malabsorption, or metabolic defects
Pernicious anemia: autoimmune destruction of parietal cells
Dietary deficiency: strict vegetarianism without supplementation
Gastrectomy removes intrinsic factor source
Terminal ileal disease impairs B12 absorption
Bacterial overgrowth competes for B12.
Classification:
Pernicious anemia: autoimmune, intrinsic factor deficiency
Dietary deficiency: inadequate intake (vegans)
Malabsorption: gastrectomy, ileal resection, Crohn disease
Congenital disorders: intrinsic factor deficiency, transcobalamin deficiency
Drug-induced: metformin, proton pump inhibitors
Parasitic infection: Diphyllobothrium latum.
Epidemiology:
India: high prevalence (47% population deficient)
Vegetarian population: 70-80% affected
Age-related increase: 10-15% elderly affected
Pernicious anemia: 1-2% prevalence in elderly
Northern Europe: higher prevalence of pernicious anemia
Autoimmune diseases: increased association.
Clinical Features
Presentation:
Megaloblastic anemia with high MCV (>100 fL)
Pancytopenia in severe cases
Neurological symptoms (subacute combined degeneration)
Glossitis and oral ulceration
Jaundice from ineffective erythropoiesis
Thrombocytopenia with bleeding tendency.
Symptoms:
Fatigue and weakness (severe anemia)
Shortness of breath on exertion
Paresthesias in hands and feet
Loss of position and vibration sense
Memory loss and cognitive impairment
Depression and mood changes
Premature graying of hair.
Risk Factors:
Strict vegetarian diet without B12 supplementation
Advanced age with gastric atrophy
Autoimmune diseases (thyroiditis, diabetes type 1)
Family history of pernicious anemia
Gastrointestinal surgery (gastrectomy, ileal resection)
Inflammatory bowel disease affecting terminal ileum
Chronic alcoholism.
Screening:
Serum B12 levels (<200 pg/mL suggests deficiency)
Methylmalonic acid (elevated in B12 deficiency)
Homocysteine levels (elevated)
Intrinsic factor antibodies (pernicious anemia)
Parietal cell antibodies
Complete blood count with peripheral smear
Schilling test (historical).
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Gross Description
Appearance:
Bone marrow shows hypercellularity with increased M:E ratio
Megaloblastic changes visible in erythroid precursors
Giant metamyelocytes and band forms
Large, abnormal megakaryocytes may be present.
Characteristics:
Erythroid hyperplasia with megaloblastic morphology
Nuclear-cytoplasmic asynchrony: immature nuclei with mature cytoplasm
Open, lacy chromatin pattern in erythroblasts
Multinucleated erythroblasts common.
Size Location:
Increased cellularity throughout bone marrow spaces
Erythroid islands show prominent megaloblastic changes
Diffuse distribution of abnormal cells
Normal fat-to-cell ratio altered due to hyperplasia.
Multifocality:
Systemic effects: neurological, gastrointestinal, hematological
Peripheral blood: oval macrocytes, hypersegmented neutrophils
Nervous system: demyelination of posterior and lateral columns
Gastrointestinal tract: atrophic gastritis, glossitis.
Microscopic Description
Histological Features:
Megaloblastic erythropoiesis: enlarged erythroblasts with immature nuclei
Nuclear-cytoplasmic asynchrony: mature cytoplasm with open chromatin nuclei
Giant metamyelocytes in granulocytic series
Hypersegmented neutrophils with >5 nuclear lobes
Megaloblastic megakaryocytes with separated nuclear lobes.
Cellular Characteristics:
Megaloblasts: large erythroblasts with fine, open chromatin
Cytoplasm maturation exceeds nuclear maturation
Howell-Jolly bodies in erythroblasts
Nuclear fragmentation and karyorrhexis
Abnormal mitotic figures common.
Architectural Patterns:
Hypercellular bone marrow with reversal of normal M:E ratio
Erythroid predominance in cellular composition
Normal bone marrow architecture preserved
Ineffective erythropoiesis: increased apoptosis of erythroid precursors.
Grading Criteria:
Severity assessment: based on degree of megaloblastic changes
Mild: subtle nuclear-cytoplasmic asynchrony
Moderate: obvious megaloblastic changes, giant metamyelocytes
Severe: marked dysplastic changes, pancytopenia
Laboratory correlation: B12 levels, MCV values.
Immunohistochemistry
Positive Markers:
Glycophorin A: positive in megaloblastic erythroblasts
CD71 (transferrin receptor): increased expression
Ki-67: high proliferation index with increased apoptosis
Terminal deoxynucleotidyl transferase: may be positive in severe cases.
Negative Markers:
CD34: negative in megaloblasts (helps exclude MDS)
Myeloperoxidase: negative in erythroid cells
Lymphoid markers: negative
CD117: negative in megaloblasts.
Diagnostic Utility:
Morphological diagnosis: primarily based on nuclear-cytoplasmic asynchrony
Flow cytometry: may show increased immature cells
Cytogenetics: normal karyotype (excludes MDS)
Bone marrow iron: usually normal to increased
Reticulin stain: normal fiber pattern.
Molecular Subtypes:
Cobalamin deficiency: methylmalonic acid elevation
Folate deficiency: normal methylmalonic acid
Combined deficiency: both B12 and folate low
Metabolic defects: inherited disorders of B12 metabolism
Drug-induced: reversible with drug cessation.
Molecular/Genetic
Genetic Mutations:
GIF gene mutations: congenital intrinsic factor deficiency
CUBN mutations: Imerslund-Gräsbeck syndrome
AMN mutations: hereditary megaloblastic anemia
TCN2 mutations: transcobalamin II deficiency
MTHFR mutations: affect folate metabolism
Pernicious anemia: HLA-DQ2, HLA-DQ8 associations.
Molecular Markers:
Methylmalonic acid: markedly elevated (>400 nmol/L)
Homocysteine: elevated in both B12 and folate deficiency
Holotranscobalamin: early marker of B12 deficiency
Intrinsic factor antibodies: type I (blocking), type II (binding)
Gastrin levels: elevated in pernicious anemia.
Prognostic Significance:
Excellent response to B12 replacement therapy
Neurological damage: may be irreversible if prolonged
Reticulocyte response: begins 3-5 days after treatment
Hemoglobin normalization: 6-8 weeks with treatment
Lifelong therapy required for pernicious anemia
Gastric carcinoma risk: increased in pernicious anemia.
Therapeutic Targets:
Intramuscular B12: cyanocobalamin, hydroxocobalamin
Oral B12 supplementation: high-dose for dietary deficiency
Sublingual B12: alternative route
Folate supplementation: if combined deficiency
Treatment of underlying cause: bacterial overgrowth, malabsorption
Monitoring: complete blood count, B12 levels.
Differential Diagnosis
Similar Entities:
Folate deficiency (normal methylmalonic acid)
Myelodysplastic syndrome (dysplastic changes, cytogenetic abnormalities)
Acute leukemia (blasts present, immunophenotyping)
Hypothyroidism (macrocytic anemia)
Alcoholism (macrocytosis without megaloblasts)
Liver disease (target cells, normal B12).
Distinguishing Features:
B12 deficiency: elevated methylmalonic acid, neurological symptoms
Folate deficiency: normal MMA, no neurological involvement
MDS: dysplastic changes, cytogenetic abnormalities, refractory to vitamins
Hypothyroidism: normal nuclear morphology, elevated TSH
Alcoholism: round macrocytes, normal nuclear morphology.
Diagnostic Challenges:
Combined B12 and folate deficiency: requires both vitamin measurements
Early megaloblastic changes: subtle nuclear-cytoplasmic asynchrony
MDS vs
megaloblastic anemia: cytogenetics and vitamin levels crucial
Partial treatment: may mask typical morphology
Normal B12 with functional deficiency: MMA and homocysteine elevated.
Rare Variants:
Juvenile pernicious anemia: childhood onset, genetic predisposition
Congenital intrinsic factor deficiency: early presentation
Imerslund-Gräsbeck syndrome: proteinuria with megaloblastic anemia
Transcobalamin deficiency: normal B12 levels with megaloblastic anemia
Nitrous oxide exposure: inactivates B12, reversible megaloblastosis.
Sample Pathology Report
Template Format
Sample Pathology Report
Complete Report: This is an example of how the final pathology report should be structured for this condition.
Specimen Information
Bone marrow aspirate and biopsy from [site], adequate for evaluation
Cellularity
Hypercellular bone marrow ([percentage]%) with M:E ratio of [ratio] (erythroid predominance)
Megaloblastic Changes
Megaloblastic erythropoiesis with nuclear-cytoplasmic asynchrony and [severity] changes
Giant Forms
Giant metamyelocytes and hypersegmented neutrophils present
Iron Stores
Iron stores: Grade [1-3+], adequate to increased
Other Findings
Megakaryocytes: [normal/increased] with [normal/megaloblastic] features
Final Diagnosis
Bone marrow with megaloblastic changes consistent with vitamin B12 deficiency
Recommendations
Correlate with serum B12, methylmalonic acid, and homocysteine levels. Rule out pernicious anemia