Definition/General
Introduction:
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder characterized by deficiency of GPI-anchored proteins
Results from somatic mutations in PIGA gene
Leads to complement-mediated intravascular hemolysis
Bone marrow typically shows hypocellularity with erythroid hyperplasia
Diagnosis confirmed by flow cytometry showing CD55/CD59 deficiency.
Origin:
Results from somatic mutation in PIGA gene in hematopoietic stem cells
X-linked gene on chromosome Xp22.2 encoding phosphatidylinositol glycan class A protein
Loss of GPI anchor biosynthesis leads to deficiency of complement regulatory proteins
CD55 (DAF) and CD59 (MIRL) deficiency causes complement sensitivity
Clonal expansion occurs in bone marrow microenvironment.
Classification:
Classic PNH: hemolysis with minimal bone marrow failure
PNH in context of bone marrow failure: associated with aplastic anemia
Subclinical PNH: small clone without clinical symptoms
Type I cells: normal GPI expression
Type II cells: partial GPI deficiency
Type III cells: complete GPI deficiency.
Epidemiology:
Rare disease: incidence 1-2 per million annually
Peak age: 20-40 years
Equal sex distribution
Association with aplastic anemia: 20-25% of AA patients develop PNH
Geographic variation: higher prevalence in Southeast Asia
Median survival: 10-20 years without treatment.
Clinical Features
Presentation:
Chronic intravascular hemolysis with hemoglobinuria
Thrombotic complications (major cause of mortality)
Bone marrow failure (cytopenias)
Smooth muscle dystonia (esophageal spasm, erectile dysfunction)
Chronic kidney disease from hemosiderin deposition
Pulmonary hypertension.
Symptoms:
Dark urine especially in morning (hemoglobinuria)
Fatigue and weakness (anemia)
Shortness of breath
Abdominal pain (smooth muscle spasm)
Dysphagia (esophageal spasm)
Headaches
Erectile dysfunction in men.
Risk Factors:
Previous aplastic anemia
Myelodysplastic syndrome
Age 20-40 years
Asian ethnicity (higher prevalence)
Autoimmune conditions
Viral infections (hepatitis, EBV)
Drug exposure (chloramphenicol).
Screening:
Flow cytometry: CD55/CD59 expression on RBCs and neutrophils
Complement lysis tests: Ham test (acidified serum), sucrose lysis test
Lactate dehydrogenase: markedly elevated
Serum haptoglobin: absent or low
Urinalysis: hemoglobinuria, hemosiderinuria
Direct antiglobulin test: negative.
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Gross Description
Appearance:
Bone marrow shows variable cellularity from hypocellular to normocellular
Erythroid hyperplasia compensating for hemolysis
Dyserythropoietic changes may be present
Decreased megakaryocytes in some cases.
Characteristics:
Compensatory erythroid hyperplasia with increased M:E ratio
Normoblastic maturation typically preserved
Iron stores may be decreased due to chronic hemolysis
Reticulin fibrosis minimal in most cases.
Size Location:
Patchy distribution of hematopoietic cells
Areas of hypocellularity alternating with normal cellularity
Erythroid islands may be prominent
Fat spaces increased in hypocellular areas.
Multifocality:
Systemic hemolysis: intravascular red cell destruction
Thrombotic complications: hepatic veins (Budd-Chiari), cerebral, pulmonary
Kidney involvement: chronic kidney disease, hemosiderinuria
Smooth muscle involvement: gastrointestinal, genitourinary spasm.
Microscopic Description
Histological Features:
Variable bone marrow cellularity (20-80%)
Erythroid hyperplasia with normoblastic maturation
Dyserythropoietic changes: nuclear irregularities, multinucleation
Decreased granulopoiesis and reduced megakaryocytes
Increased plasma cells and lymphocytes may be present.
Cellular Characteristics:
Erythroblasts show relatively normal morphology with some dysplastic features
Nuclear-cytoplasmic synchrony preserved (unlike megaloblastic anemia)
Occasional megaloblastic changes due to folate deficiency
Granulocytes show normal maturation when present
Megakaryocytes morphologically normal but reduced in number.
Architectural Patterns:
Patchy cellularity with areas of hypocellularity
Erythroid predominance in cellular areas
Normal bone marrow architecture maintained
Minimal reticulin fibrosis
Occasional lymphoid aggregates.
Grading Criteria:
Cellularity assessment: age-adjusted percentage
Erythroid-to-myeloid ratio: typically increased
Dysplastic changes: mild to moderate in erythroid lineage
Clone size assessment: requires flow cytometry correlation
Bone marrow failure severity: based on peripheral blood counts.
Immunohistochemistry
Positive Markers:
Glycophorin A: positive in erythroid precursors
Myeloperoxidase: positive in granulocytes
CD61: positive in megakaryocytes
CD34: may be increased in severe cases.
Negative Markers:
CD55 (DAF): absent/reduced on PNH clone cells by flow cytometry
CD59 (MIRL): absent/reduced on PNH clone cells
CD14: absent/reduced on PNH monocytes
CD16: absent/reduced on PNH neutrophils.
Diagnostic Utility:
Flow cytometry: gold standard for PNH diagnosis
CD55/CD59 expression on RBCs, neutrophils, monocytes
FLAER (fluorescent aerolysin): highly specific for GPI deficiency
Clone size quantification: percentage of deficient cells
Multi-parameter analysis: different cell lineages.
Molecular Subtypes:
Type I cells: normal GPI-anchored protein expression
Type II cells: partial deficiency (intermediate fluorescence)
Type III cells: complete deficiency (negative fluorescence)
Clone size: <1% (subclinical), 1-10% (small), >10% (clinically significant)
Evolution over time: clone size may fluctuate.
Molecular/Genetic
Genetic Mutations:
PIGA gene mutations: >150 different mutations described
Nonsense mutations: most common type
Frameshift mutations: due to insertions/deletions
Splice site mutations: affect mRNA processing
Large deletions: rare but reported
X-linked inheritance pattern: hemizygous state in males.
Molecular Markers:
GPI-anchored proteins: CD55, CD59, CD14, CD16, CD24
FLAER binding: specific for GPI anchor detection
RT-PCR: can detect PIGA mutations
Chromosome studies: usually normal karyotype
Telomere length: often shortened (associated with bone marrow failure).
Prognostic Significance:
Clone size: >50% associated with higher thrombotic risk
Neutrophil clone size: correlates with clinical severity
Evolution to aplastic anemia: possible in some cases
Evolution to AML: rare (<5% of cases)
Thrombotic events: major predictor of survival
Treatment response: complement inhibition highly effective.
Therapeutic Targets:
Complement inhibition: eculizumab (anti-C5 monoclonal antibody)
Newer complement inhibitors: ravulizumab (longer half-life)
Supportive care: iron supplementation, folic acid
Anticoagulation: for thrombotic complications
Bone marrow transplantation: for severe bone marrow failure
Immunosuppression: for associated aplastic anemia.
Differential Diagnosis
Similar Entities:
Aplastic anemia (hypocellular bone marrow, pancytopenia)
Myelodysplastic syndrome (dysplastic changes, cytopenias)
Autoimmune hemolytic anemia (positive DAT, spherocytes)
Hereditary spherocytosis (family history, spherocytes)
Cold agglutinin disease (cold antibodies)
Microangiopathic hemolytic anemia (schistocytes).
Distinguishing Features:
PNH: CD55/CD59 deficiency by flow cytometry, negative DAT, intravascular hemolysis
Aplastic anemia: hypocellular marrow, normal complement proteins
AIHA: positive DAT, spherocytes, extravascular hemolysis
Hereditary spherocytosis: family history, osmotic fragility test
TTP/HUS: schistocytes, thrombocytopenia, organ dysfunction.
Diagnostic Challenges:
Small PNH clones: may be missed without sensitive flow cytometry
PNH with aplastic anemia: overlapping features require both diagnoses
Subclinical PNH: clone present but no clinical symptoms
Evolution over time: clone size may change, requiring monitoring
False negatives: technical issues with flow cytometry.
Rare Variants:
PNH with myelofibrosis: rare evolution
PNH with acute leukemia: transformation in <5% cases
Subclinical PNH: small clone without hemolysis
PNH in children: often associated with severe aplastic anemia
Pregnancy-associated PNH: increased thrombotic risk.
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
Bone marrow cellularity: [percentage]% (age-adjusted normal: [range]%)
Lineage Analysis
Erythroid: [percentage]%, Myeloid: [percentage]%, Megakaryocytes: [normal/decreased]
Dysplastic Changes
Mild dyserythropoietic changes with [specific features]
Flow Cytometry Correlation
CD55/CD59 deficient clone: [percentage]% of [cell types]
Iron Stores
Iron stores: [decreased/normal] consistent with chronic hemolysis
Final Diagnosis
Bone marrow findings consistent with PNH ([clone size]% CD55/CD59 deficient clone)
Recommendations
Clinical correlation with hemolysis markers. Monitor for thrombotic complications. Consider complement inhibitor therapy