Paroxysmal Nocturnal Hemoglobinuria - PNH

Diagnosis

Indications for Testing

  • Unexplained hemoglobinuria
  • Coombs-negative hemolytic anemia
  • Unusual thrombotic sites (eg, Budd-Chiari, cerebral)
  • Thrombosis combined with intravascular hemolysis or cytopenias
  • Aplastic or hypoplastic anemia
  • Monitoring of individuals with confirmed paroxysmal nocturnal hemoglobinuria (PNH)

Laboratory Testing

  • Initial screen for hemolysis
    • CBC – overt hemolysis on smear (polychromasia); thrombocytopenia and leukopenia may also occur
    • Reticulocyte count – elevated
    • Lactate dehydrogenase (LD) – always elevated
    • Bilirubin – may be elevated
    • Haptoglobin – low
  • Ham and sugar tests are obsolete for the diagnosis of PNH due to their nonspecificity and low sensitivity
    • In the rare instance when congenital dyserythropoietic anemia type II is suspected, the Ham test is the preferred initial test
  • Flow cytometry analysis of granulocytes and erythrocytes – evaluate for presence of GPI-linked antigens
    • Gold standard for diagnosis of PNH
    • Combined WBC and RBC testing recommended for initial diagnosis
      • WBC testing – best to monitor the PNH clone size
        • Fluorescently labeled inactive toxin aerolysin (FLAER) included in WBC testing
      • RBC testing – more sensitive for detecting minor PNH clones
        • Best for PNH type II and type III quantification
  • PIGA gene mutation – confirms flow cytometry results but seldom necessary

Histology

  • Bone marrow biopsy
    • Indicated when pancytopenia is present to rule out other disorders or when marrow transplantation considered
    • Reveals normal to hypercellular marrow with erythroid hyperplasia and normal/nearly normal morphology or hypocellular/aplastic marrow

Differential Diagnosis

  • Aplastic anemia
  • Other hemolytic diseases or processes
  • Myelodysplastic syndromes
  • Congenital dyserythropoietic anemia (CDA type II, ie, hereditary erythroblastic multinuclearity with positive acidified serum lysis test [HEMPAS])
  • Leukemia (AML, ALL)

Monitoring

  • Serial flow cytometry testing – determine when to initiate treatment or anticoagulation therapy
    • Patients with 10% deficient granulocytes have 40% risk of thrombosis
    • Patients with >50% deficient granulocytes have thrombosis within 10 years after diagnosis
  • Flow cytometry testing – monitor response to monoclonal antibody therapy directed against C5
    • Therapy success associated with increased ratios of erythrocyte clones to granulocyte clones

Clinical Background

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal disorder caused by the nonmalignant clonal expansion of one or more stem cell lines. PNH results in a deficiency of cell-surface glycophosphatidylinositol (GPI)-anchored proteins, including complement pathway regulatory proteins. PNH is associated with several phenotypes or phenotype combinations, including intravascular hemolysis, thrombotic complication (especially Budd-Chiari syndrome and mesenteric thrombosis), and aplastic anemia.

Epidemiology

  • Incidence – 1.3/million
  • Age – median onset is 40 years
  • Sex – M:F, equal

Pathophysiology

  • Mutation of PIGA gene results in the deficiency or absence of GPI-anchored cell membrane proteins
  • Pathophysiology of hemolysis involves abnormal RBC sensitivity to complement lysis
  • Pathophysiology of bone marrow failure and thromboses is not known
  • Association between acquired aplastic anemia and PNH
    • Majority of patients with PNH will eventually develop some degree of bone marrow failure
  • Three PNH cell types based on RBC analysis
    • Type I – normal levels of CD59
    • Type II – reduced levels of CD59
    • Type III – absent levels of CD59

Clinical Presentation

  • Chronic hemolysis – patient reports episodic occurrence of dark urine (in minority), jaundice, and hemosiderinuria (in virtually all PNH patients with hemolysis)
  • Symptoms of anemia (fatigue, pallor, weakness)
  • Thrombophilia – occurs in up to 40% of patients
    • Thromboses at unusual sites – abdominal veins most common (Budd-Chiari)
      • Leading cause of death in PNH
  • Aplastic anemia due to bone marrow failure or may be associated with overlap syndromes and aplastic anemia

  • Other signs and symptoms may include abdominal pain, iron deficiency anemia,  jaundice, smooth muscle dysfunction, male impotence, and dysphagia

Treatment

  • Based on level of signs and symptoms
    • Minimal – folic acid and thrombosis prophylaxis
    • Intravascular hemolysis of PNH can be controlled with eculizumab, a humanized monoclonal antibody that blocks formation of cytolytic membrane attack complex of complement
      • Eculizumab is palliative and has no effect on the underlying disease process
    • Mutant clone can be eradicated and normal hematopoiesis restored by allogenic hematopoietic stem cell transplant
  • Subclinical PNH population in aplastic anemia or other bone marrow failure disorder may correlate with a positive immunotherapeutic response

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Paroxysmal Nocturnal Hemoglobinuria Panel, RBC and WBC 2005006
Method: Quantitative Flow Cytometry

Diagnosis of PNH and quantification of PNH clones

Monitoring in confirmed PHH

Analyzes both RBCs and WBCs; cells stained with markers

WBCs

  • CD15 and CD33 (lineage-specific markers for granulocytes and monocytes, respectively)
  • CD14, CD24, and FLAER (to identify PNH cells)

RBCs

  • Glycophorin A (lineage marker)
  • CD59 (to identify PHN cells)
    • Type II and III PNH cells reported when ≥1%

Analytical sensitivity – limit of detection is 0.1% for WBCs and 0.005% for RBCs

Accuracy may be compromised through

  • Significant neutropenia
  • Gross hemolysis
  • Samples lacking CD15, CD33, or glycophorin A expression
  • Recent RBC transfusions
 
Paroxysmal Nocturnal Hemoglobinuria, High Sensitivity, RBC 2004366
Method: Quantitative Flow Cytometry

Individual testing for PNH cell type – not preferred test

Both PNH RBC and PNH WBC are recommended for initial diagnosis of PNH

RBCs stained with markers

  • Glycophorin A (lineage marker)
  • CD59 (to identify PHN cells)
    • Type II and III PNY cells reported when ≥1%

Accuracy may be compromised through

  • Significant neutropenia
  • Gross hemolysis
  • Samples lacking  glycophorin A expression
  • Recent RBC transfusion
 
Paroxysmal Nocturnal Hemoglobinuria, WBC 2005003
Method: Quantitative Flow Cytometry

Individual testing for PNH cell type – not preferred test

Both PNH WBC and PNH RBC are recommended for initial diagnosis of PNH

WBCs stained with markers

  • CD15 and CD33 (lineage-specific markers for granulocytes and monocytes, respectively)
  • CD14, CD24, and FLAER (to identify PNH cells)

Accuracy may be compromised through

  • Significant neutropenia
  • Gross hemolysis
  • Samples lacking CD15, CD33
 
Bone Marrow Services

Determine results of biopsy

   
Additional Tests Available
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential

Evaluate for hemolysis, thrombocytopenia, and leukopenia; aid in the diagnosis of PNH

Reticulocytes, Percent & Number 0040022
Method: Flow Cytometry

Aid in diagnosis of PNH

Lactate Dehydrogenase, Serum or Plasma 0020006
Method: Quantitative Enzymatic

Aid in diagnosis of PNH

Bilirubin, Total, Serum or Plasma 0020032
Method: Spectrophotometry

Aid in diagnosis of PNH

Haptoglobin 0050280
Method: Quantitative Immunoturbidimetry

Aid in diagnosis of PNH

Acid Hemolysin (Ham Test) 0049010
Method: Visual Identification

Obsolete for the diagnosis of PNH; preferred initial diagnostic test for PNH is RBC and WBC PNH panel 

In the rare instance when congenital dyserythropoietic anemia type II is suspected, this is the preferred initial test