Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare but serious acquired hemolytic anemia with periodic exacerbations. This anemia is caused by nonmalignant clonal expansion of ≥ 1 hematopoietic stem cells that have acquired a somatic mutation of the phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIG-A) gene. Clonal expansion of affected stem cells are deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Deficiency of the GPI-APs CD55 and CD59, which regulate complement, results in intravascular hemolysis. The classic triad of PNH is hemolytic anemia, marrow failure, and thrombophilia. Patients may be treated with the monoclonal antibody eculizumab or with stem cell transplantation, in addition to being treated for associated complications.

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Overview

Epidemiology

  • Prevalence: approximately 5 cases per million patients
  • Sex ratio: men = women
  • Ages affected: mostly young adults; also children and those > 70 years

Etiology

  • X-linked somatic mutation of the phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PIG-A) gene in a multipotent hematopoietic stem cell 
  • PIG-A gene: responsible for the 1st step in synthesis of glycosylphosphatidylinositol (GPI) 
  • GPI anchors dozens of proteins to the cell surface, including complement inhibitors (regulators) CD59 and CD55.
  • CD59: protects RBCs against membrane attack complex (MAC)–mediated intravascular hemolysis
  • CD55: protects RBCs against opsonization by complement C3b and subsequent extravascular hemolysis
  • RBCs may show partial or complete absence of GPI-anchored proteins (APs).
  • Benign clonal expansion of the mutated stem cells gives rise to CD59/CD55-deficient RBCs. 

Classification

Classic paroxysmal nocturnal hemoglobinuria (PNH): 

  • Clinical evidence of intravascular hemolysis: 
    • Reticulocytosis
    • ↑ Serum LDH 
    • ↑ Indirect bilirubin
    • Low serum haptoglobin
  • No evidence of a preexisting defined bone marrow abnormality

PNH in the setting of a specified bone marrow disorder: 

  • Clinical and laboratory evidence of hemolysis
  • Comorbid preexisting defined marrow abnormality (or history of one): 
    • Aplastic anemia
    • Myelodysplastic syndrome
    • Myelofibrosis

Subclinical PNH (PNH-sc): 

  • Comorbid preexisting defined marrow abnormality (or a history of one): 
    • Aplastic anemia
    • Myelodysplastic syndrome
    • Myelofibrosis
  • Small populations of GPI-AP–deficient erythrocytes and/or granulocytes detected by flow cytometric analysis 
  • No clinical or laboratory evidence of hemolysis

Pathophysiology

Genetics

PIG-A gene (normal physiology):

  • Located on the X chromosome 
  • Codes for GPI:
    • Anchors certain proteins to cell membrane
    • Anchors CD55 and CD 59 in multiple blood cell lines: 
      • Erythrocytes
      • Neutrophils
      • Monocytes
      • Platelets
    • CD55 and CD59 are complement-regulating surface proteins:
      • Prevent binding of complement to circulating blood cells
      • CD59 (also called membrane inhibitor of reactive lysis (MIRL)) prevents formation of MAC (prevents intravascular hemolysis).
      • CD55 (also called decay accelerating factor) prevents opsonization/complement-mediated blood cell destruction in the spleen (prevents extravascular hemolysis).

PIG-A gene (mutation):

  • Mutation of hematopoietic progenitor cells 
  • Results in clonal expansion of multiple blood cell lines:
    • Erythrocytes
    • Neutrophils
    • Monocytes
    • Platelets
  • Blood cells lack surface proteins CD55 and CD59:
    • Uncontrolled amplification of complement system
    • Intravascular and extravascular hemolysis ensue.
    • Multiple downstream pathologic manifestations

Bone marrow failure

  • Defined as peripheral cytopenias associated with deficient hematopoiesis
  • Present, to some degree, in all patients with PNH
  • Unknown mechanism of association between PNH and aplastic anemia (AA):
    • Aplastic anemia develops in 10%–20% of patients with PNH.
    • Paroxysmal nocturnal hemoglobinuria develops in 5% of patients with AA.
  • Bone marrow failure in PNH is highly variable: 
    • Mild decrease in hematopoietic stem cells (mild end of spectrum) 
    • Severe AA (severe end of spectrum)
  • Mechanism of bone marrow failure in PNH:
    • Proposed mechanism is complement-mediated destruction of hematopoietic precursor cells.
    • Still poorly understood

Anemia

  • Primary mechanism is intravascular hemolysis:
    • Circulating RBCs lack the protective effect of CD59
    • Complement factors assemble/activate MAC on RBC membrane and lyse the cell:
      • Hemoglobin released into circulation
      • Multiple downstream pathologic manifestations
  • Secondary mechanism is extravascular hemolysis:
    • Circulating RBCs lack the protective effect of CD55.
    • Complement opsonizes or accumulates on red cell membrane.
    • Opsonization marks red cells for phagocytic clearance in the spleen.
  • Other factors contributing to anemia:
    • Iron loss from excessive and chronic red cell lysis:
      • Some iron lost to hemoglobinuria
      • Some iron lost to tissue deposition
    • Lack of erythrocyte production:
      • Underlying bone marrow failure
      • Iron deficiency
Urine sample intravascular hemolysis with hemoglobinuria

Urine sample from a patient with PNH (left) and from a healthy control (right).
The patient’s urine is red because of intravascular hemolysis with hemoglobinuria. Hemolysis occurs throughout the day, not just at night.

Image: “Paroxysmal nocturnal hemoglobinuria in systemic lupus erythematosus: a case report” by Nakamura, N., et al. License: CC BY 2.0

Neutrophil involvement

Neutrophils also exhibit deficiency of GPI-APs:

  • Increased sensitivity to complement-mediated lysis
  • Once lysed, may release cytokines into circulation:
    • Further accelerate complement activity
    • Increased thrombogenic potential
  • Excessive neutrophil lysis may lead to increased susceptibility to infection.

Platelet involvement

Platelet-mediated factors include:

  • Nitrous oxide (NO) deficiency leads to platelet activation and aggregation.
  • Platelets deficient in GPI-linked proteins become activated by complement.
  • Lack of CD59 on platelet membranes induces platelet aggregation in particular.

Thrombophilia and thrombosis

Thrombosis in unusual sites (hepatic, mesenteric, cerebral, dermal veins) is typical of PNH.

  • NO-mediated factors: 
    • On red cell lysis, hemoglobin released into the circulation
    • Hemoglobin is bound to haptoglobin until haptoglobin is saturated.
    • After saturating haptoglobin, free hemoglobin binds to circulating NO, which:
      • Normally decreases endothelial adhesion
      • Normally prevents platelet activation and aggregation
      • Normally causes vasodilation
    • But if NO is depleted in the peripheral circulation:
      • ↓ NO enhances endothelial adhesion.
      • ↓ NO enhances platelet activation and aggregation.
      • ↓ NO causes vasoconstriction → exposure of tissue factor → triggers the extrinsic coagulation cascade.
  • Platelet-mediated factors:
    • NO deficiency leads to platelet activation and aggregation.
    • Platelets deficient in GPI-linked proteins become activated by complement.
    • Lack of CD59 on platelet membranes induces platelet aggregation.
  • Vascular factors:
    • Activation of complement on endothelial cell surfaces
    • Intravascular hemolysis may provide friable membrane surfaces on which coagulation may be initiated.
    • Slow flow: PNH thrombosis is far more common in the venous than in the arterial system, notably in large veins or where flow is slowest.
      • Hepatic
      • Portal
      • Splenic
      • Mesenteric
      • Pulmonary
      • Cerebral
      • Dermal
  • Other factors:
    • Unknown mechanism of increased procoagulant and fibrinolytic activity
    • White cell lysis may release vasoactive and/or thrombogenic molecules.

Renal involvement

Renal failure is progressive and multifactorial.

  • Acute tubular necrosis effects of heme and iron (pigment nephropathy)
  • Tubular obstruction with pigment casts
  • Decreased renal perfusion from renal vein thrombosis
  • Decreased renal perfusion from arterial thrombosis
  • Decreased renal perfusion from ↓ NO-mediated vasoconstriction

Smooth muscle dystonia

  • Upon red cell lysis, hemoglobin released into the circulation
  • Hemoglobin is bound to haptoglobin until haptoglobin is saturated.
  • After saturating haptoglobin, free hemoglobin binds to circulating NO, which:
    • Normally relaxes smooth muscle tone
    • Is depleted in the peripheral circulation
  • NO depletion leads to smooth muscle contraction.
  • Clinical manifestations depend on the smooth muscle population affected.

Paroxysms of hemolysis

Exacerbations of hemolysis may be due to:

  • Administration of iron to an iron-deficient patient: 
    • Erythrocytes lacking CD59/CD55 released into circulation
    • Intravascular/extravascular hemolysis ensues
  • Exogenous activation of the complement system:
    • Infection/sepsis
    • Surgery/trauma
    • Drugs

Antibody involvement

  • Coombs testing detects the presence of antibodies in autoimmune hemolytic anemias.
  • Untreated PNH is a Coombs-negative hemolytic anemia.
  • Coombs-positivity often occurs in patients with PNH treated with eculizumab:
    • Eculizumab is a monoclonal antibody that blocks complement-mediated formation of MAC:
      • Intravascular hemolysis does not occur.
      • This antibody is detected by the Coombs test.
    • Complement factors not consumed by MAC formation are free in the circulation and coat red cell surfaces lacking protection from CD55:
      • RBC is now opsonized by complement.
      • Opsonized RBCs undergo extravascular hemolysis.
PNH eculizumab

Paroxysmal nocturnal hemoglobinuria after eculizumab:
When C5 is blocked (inhibited) by eculizumab, C5 convertase cannot form C5b from the cleavage of C5. Membrane attack complex (MAC) is not formed and no intravascular hemolysis occurs. However, the C3 fragments that were released earlier in the activation process will coat the unprotected RBCs and cause extravascular hemolysis by the reticuloendothelial system (RES) in the liver and spleen.

Image by Lecturio. License: CC BY-NC-SA 4.0

Clinical Presentation

Hemolysis-associated symptoms

  • Hemoglobinuria:
    • Classically noticed at 1st morning void
    • Can occur at any time of day or night
    • Intermittent (paroxysmal) in nature
  • Jaundice/icterus
  • Heme/iron-related nephrotoxicity:
    • Acute renal failure
    • Chronic renal failure

Anemia-associated symptoms

  • Fatigue
  • Weakness
  • Dyspnea
  • Palpitations
  • Pallor
  • Tachycardia

Smooth muscle contraction–associated symptoms

These symptoms are due to decreased circulating NO:

  • Dysphagia (esophageal)
  • Nausea (intestinal)
  • Abdominal pain (intestinal)
  • Erectile dysfunction (microvascular/corpus cavernosa)
  • Pulmonary hypertension (pulmonary arterial)

Thrombosis-associated symptoms

  • Limb edema/discoloration/pain from deep vein thrombosis (DVT)
  • Pulmonary embolism
  • Cerebral vein/dural vein thrombosis leads to:
    • Focal neurologic deficit
    • Intracranial hypertension
    • Encephalopathy
  • Dermal vein thrombosis leads to:
    • Bruising 
    • Purpura fulminans
  • Hepatic/portal/mesenteric vein thrombosis leads to:
    • Abdominal pain
    • Splenomegaly
    • Budd-Chiari syndrome

Neutropenia-associated symptoms

  • Susceptibility to infection
  • Increased circulating cytokines leads to: 
    • Malaise/fatigue
    • Inflammation
    • Sepsis-like syndrome

Diagnosis

CBC

  • Anemia (↓ RBC count, ↓ hematocrit):
    • Variable, but generally present to some degree
    • May be subtle or pronounced depending on degree of hemolysis
    • May or may not be normocytic, microcytic, or macrocytic depending on degree of vitamin B12, folate, or iron deficiency
  • ↓ or normal neutrophil count
  • ↓ or normal platelet count
  • ↑ Free hemoglobin

Chemistry

  • ↑ AST/ALT (if hepatic congestion)
  • ↑ Creatinine (if renal toxicity)
  • ↑ Bilirubin 
  • ↑ LDH
  • ↓ Leukocyte alkaline phosphatase (LAP)

Urinalysis

  • Gross red or pink discoloration
  • Positive for hemoglobin
  • No RBCs on microscopy
  • +/– protein depending on renal integrity

Bone marrow analysis

  • GPI-AP deficiency can be demonstrated on bone marrow cells using flow cytometry.
  • Unnecessary for standard diagnosis of PNH
  • Needed for proper classification:
    • Evaluate for other marrow failure syndromes.
    • Evaluate for other clonal myelopathies.

Flow cytometry (FCM) performed on peripheral blood

  • Indicated for screening for:
    • Hemoglobinuria
    • Coombs-negative intravascular hemolysis 
    • Abnormally high serum LDH, especially with concurrent iron deficiency
    • Venous thrombosis involving unusual sites
    • AA (at diagnosis and once yearly)
    • Refractory anemia
    • Myelodysplastic syndrome
    • Episodic dysphagia or abdominal pain with evidence of intravascular hemolysis
  • Indicated for definitive diagnosis and monitoring of PNH:
    • Must show ≥ 5% population of RBCs that are deficient in CD55 and CD59 or ≥ 20% of CD55/CD59-deficient granulocytes to be diagnostic for PNH.
    • Patients with proven GPI-AP deficiency should be monitored yearly to track size of clonal population.

Management

Medical management

  • Eculizumab or ravulizumab: humanized monoclonal antibodies that bind to the complement component C5
  • Prevents cleavage of C5 and intravascular hemolysis by MACs 
  • Effect:
    • Elimination or marked reduction in blood transfusions 
    • Marked decrease in thrombotic complications
  • Administration: 
    • IV every 2 weeks for eculizumab 
    • IV every 8 weeks for ravulizumab 
  • Disadvantages:
    • Increased risk of life-threatening Neisseria infections, requiring pretreatment vaccination and daily antibiotics for prophylaxis
    • High cost: approximately $400,000 (U.S.)/year
  • Extravascular hemolysis will still continue, owing to opsonization of RBCs by C3 fragments.

Bone marrow transplantation

  • The only definitive cure, but has substantial intrinsic risks
  • Indicated mostly for: 
    • Severe AA
    • Severe myelodysplastic syndrome
    • PNH complications unresponsive to complement inhibitors (or if they are not available)

Supportive care

  • Always provided regardless of other therapies provided
  • RBC transfusions of leukocyte-reduced/filtered blood (used now routinely for all transfused blood): 
    • Decreases risk of antibody reaction to leukocyte antigens
    • Decreases complement activation
  • Vitamin B12/folic acid supplementation if ongoing hemolysis
  • Iron supplementation if iron deficient owing to hemoglobinuria
  • Anticoagulants in patients with history of or genetic predisposition to thrombosis: lifelong anticoagulation if high risk or if atypical thrombosis has occurred
  • Regular monitoring (every 6–12 months): 
    • CBC/DIFF
    • Reticulocyte count
    • LDH
    • Chemistry profile (electrolytes, renal function, hepatic function)
    • Iron studies (serum iron, total iron binding capacity, and ferritin)
    • FCM to determine any change in PNH clone size
    • Bone marrow biopsy if pancytopenia or suspected AA or myelodysplastic syndrome

Prognosis

  • Natural history, with only supportive treatment given: 
    • Median survival after diagnosis: 10–20 years
    • Major complications: 
      • Pancytopenia 
      • Thrombosis
      • Myelodysplastic syndrome
    • Causes of death: 
      • Venous thrombosis: number-one cause
      • Infection if severe neutropenia
      • AML
  • With complement inhibitors (eculizumab and ravulizumab), mortality appears similar to that for age-matched controls, but limited time data so far
  • Bone marrow transplantation: curative but significant inherent risks involved
  • Full, spontaneous recovery rarely occurs.

Differential Diagnosis

  • Hemolytic anemia (HA): hereditary disorders (e.g., glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary spherocytosis, sickle cell anemia) or acquired disorders (e.g., immune disorders, toxic chemicals and drugs, antiviral agents, physical damage, infections) that also present with hemolysis leading to anemia (but not with thrombosis and bone marrow failure). This is a broad category, as the hemolytic anemias each have a unique mechanism, presentation, and management. 
  • Aplastic anemia (AA): occurs as a result of hematopoietic stem cell damage. More than 10% of AA cases will develop into PNH. In addition, some people with PNH will develop AA. Like PNH, AA presents with bone marrow failure and low blood cell counts (but not thrombosis). Treatment for AA might include medications, blood transfusions, or stem cell transplantation.
  • Acute mesenteric ischemia: presents with severe acute abdominal pain in a patient with an identifiable source of thrombus/emboli obstructing the mesenteric vessels (e.g., preexisting atheroma, atrial fibrillation). Diagnosed with angiography. Treated with embolectomy or surgery to remove necrotic bowel. 
  • Paroxysmal cold hemoglobinuria: rare autoimmune hemolytic anemia seen in young children, triggered by infectious disease, neoplasm, or immune dysfunction. IgM antibodies bind to the RBCs in the cold and fix complement. Upon warming, the antibodies dissociate and complement lyses the RBCs, leading to extravascular hemolysis. Diagnosis is via Coombs testing. Treatment is preventive.
  • Portal vein obstruction: usually caused by primary thrombosis of the portal vein due to an inherited or acquired coagulopathy or associated with cirrhosis. Extrinsic obstruction also occurs. Correlative clinical findings raise clinical suspicion, but imaging (ultrasound or CT) showing a mass or thrombus is diagnostic. Treatment includes thrombectomy, excision of the mass/tumor, and/or anticoagulation. 
  • Renal vein thrombosis: The most common cause of renal vein thrombosis is nephrotic syndrome. Other causes include primary hypercoagulable disorders (such as PNH), renal tumors, extrinsic compression, trauma, and, rarely, inflammatory bowel disease. This condition presents with renal failure (acute or chronic). Diagnosed with imaging (ultrasound or CT). Treatment includes thrombectomy, excision of the mass/tumor, and/or anticoagulation.

References

  1. Luzzatto, L. (2018). Hemolytic anemias. In Jameson, J.L., et al. (Ed.), Harrison’s Principles of Internal Medicine, 20th ed. Vol 1. McGraw Hill. pp. 708–723. 
  2. Brodsky, R.A. (2019). Clinical manifestations and diagnosis of paroxysmal nocturnal hemoglobinuria. UpToDate. Retrieved December 5, 2020, from https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-paroxysmal-nocturnal-hemoglobinuria
  3. Braunstein, E.M. (2020). Paroxysmal nocturnal hemoglobinuria (PNH)—hematology and oncology. MSD Manual Professional Edition. Retrieved December 5, 2020, from https://www.msdmanuals.com/professional/hematology-and-oncology/anemias-caused-by-hemolysis/paroxysmal-nocturnal-hemoglobinuria-pnh
  4. Parker, C., Omine, M., Richards, S., et al. (2005). Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 106:3699–3709. https://doi.org/10.1182/blood-2005-04-1717

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