Paroxysmal Nocturnal Hemoglobinuria - PNH

  • Diagnosis
  • Algorithms
  • Monitoring
  • Background
  • Lab Tests
  • References
  • Related Content

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
    • Direct Coombs
  • Secondary testing if suspicion exists based on primary tests
    • Flow cytometry analysis of granulocytes and erythrocytes – evaluate for presence of GPI-linked antigens
      • Gold standard for diagnosis of PNH in the presence of appropriate clinical presentation
      • Combined WBC and RBC testing recommended for initial diagnosis
        • WBC testing – most sensitive to monitor the PNH clone size
          • Fluorescein-labeled proaerolysin  (FLAER) included in WBC testing
        • RBC testing – more sensitive for detecting minor PNH clones
          • PNH type II and type III quantification
    • PIGA gene mutation – confirms flow cytometry results but seldom necessary
    • 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

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)
  • 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

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. It is associated with several phenotypes or phenotype combinations, including intravascular hemolysis, thrombotic complication (especially Budd-Chiari syndrome and mesenteric thrombosis), and aplastic anemia (bone marrow failure).

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
    • Deficiency of CD55 and CD59 leads to hemolysis
    • Hemolysis involves abnormal RBC sensitivity to complement lysis
    • Bone marrow failure and thromboses is not known
  • Association between acquired aplastic anemia and PNH
    • ~60% of individuals with acquired aplastic anemia have detectable PNH cells
  • Three PNH types based on presentation
    • Classical PNH – hemolysis and thrombosis
    • PNH in the context of primary bone morrow disorders
    • Subclinical PNH – PNH clones but no evidence of hemolysis
  • 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
  • Genetics
    • X-linked inheritance for PIGA
    • Single mutation is sufficient to produce PNH phenotype

Clinical Presentation

  • Chronic hemolysis and hemoglobinuria
    • Jaundice
    • Dark urine
    • Anemia – fatigue, pallor, weakness
  • Thrombophilia – occurs in ~40% of individuals
    • Venous thromboses at unusual sites
      • Hepatic veins (Budd-Chiari)
      • Cerebral veins
    • Leading cause of mortality in PNH
  • Aplastic anemia is common due to
    • Bone marrow failure
    • May be associated with overlap syndromes and aplastic anemia
  • Other signs and symptoms – may include abdominal pain, iron deficiency anemia, smooth muscle dysfunction, male impotence, dysphagia, chronic kidney disease

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

Paroxysmal Nocturnal Hemoglobinuria (PNH), High Sensitivity, RBC and WBC 2005006
Method: Quantitative Flow Cytometry

Limitations

Compromised accuracy

  • Significant neutropenia
  • Gross hemolysis
  • Samples lacking CD15, CD64, or glycophorin A expression
  • Recent RBC transfusions

Paroxysmal Nocturnal Hemoglobinuria, High Sensitivity, RBC 2004366
Method: Quantitative Flow Cytometry

Limitations

Compromised accuracy

  • Significant neutropenia
  • Gross hemolysis
  • Samples lacking CD15, CD64, or glycophorin A expression
  • Recent RBC transfusions

Paroxysmal Nocturnal Hemoglobinuria, High Sensitivity, WBC 2005003
Method: Quantitative Flow Cytometry

Limitations

Compromised accuracy

  • Significant neutropenia
  • Gross hemolysis
  • Samples lacking CD15, CD64, or glycophorin A expression

Bone Marrow Services

Additional Tests Available

CBC with Platelet Count and Automated Differential 0040003
Method: Automated Cell Count/Differential

Comments

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

Reticulocytes, Percent & Number 0040022
Method: Flow Cytometry

Comments

Aids in diagnosis of PNH

Lactate Dehydrogenase, Serum or Plasma 0020006
Method: Quantitative Enzymatic

Comments

Aids in diagnosis of PNH

Bilirubin, Total, Serum or Plasma 0020032
Method: Spectrophotometry

Comments

Aids in diagnosis of PNH

Haptoglobin 0050280
Method: Quantitative Immunoturbidimetry

Comments

Aids in diagnosis of PNH

General References

Brodsky R. Paroxysmal nocturnal hemoglobinuria. Blood. 2014; 124(18): 2804-11. PubMed

Craig F, Foon K. Flow cytometric immunophenotyping for hematologic neoplasms. Blood. 2008; 111(8): 3941-67. PubMed

Luzzatto L, Gianfaldoni G, Notaro R. Management of paroxysmal nocturnal haemoglobinuria: a personal view. Br J Haematol. 2011; 153(6): 709-20. PubMed

Madkaikar M, Gupta M, Jijina F, Ghosh K. Paroxysmal nocturnal haemoglobinuria: diagnostic tests, advantages, & limitations. Eur J Haematol. 2009; 83(6): 503-11. PubMed

Parker C. Paroxysmal nocturnal hemoglobinuria. Curr Opin Hematol. 2012; 19(3): 141-8. PubMed

Perkins S. Paroxysmal Nocturnal Hemoglobinuria. In Kjeldsberg C. Practical Diagnosis of Hematologic Disorders, 5th ed. Chicago: ASCP Press, 2006.

Preis M, Lowrey C. Laboratory tests for paroxysmal nocturnal hemoglobinuria. Am J Hematol. 2014; 89(3): 339-41. PubMed

Savage W, Brodsky R. New insights into paroxysmal nocturnal hemoglobinuria. Hematology. 2007; 12(5): 371-6. PubMed

References from the ARUP Institute for Clinical and Experimental Pathology®

Borowitz M, Craig F, Digiuseppe J, Illingworth A, Rosse W, Sutherland R, Wittwer C, Richards S, Clinical Cytometry Society. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010; 78(4): 211-30. PubMed

Inoue N, Izui-Sarumaru T, Murakami Y, Endo Y, Nishimura J, Kurokawa K, Kuwayama M, Shime H, Machii T, Kanakura Y, Meyers G, Wittwer C, Chen Z, Babcock W, Frei-Lahr D, Parker C, Kinoshita T. Molecular basis of clonal expansion of hematopoiesis in 2 patients with paroxysmal nocturnal hemoglobinuria (PNH). Blood. 2006; 108(13): 4232-6. PubMed

Liew M, Farley M, Andreasen J, Parker C, Wittwer C. Rare event counting of CD59- red cells in human blood: A 47-month experience using PNH consensus guidelines for WBC and RBC testing in a reference lab. Cytometry B Clin Cytom. 2015; 88(4): 261-9. PubMed

Yaish H, Christensen R, Agarwal A. A neonate with Coombs-negative hemolytic jaundice with spherocytes but normal erythrocyte indices: a rare case of autosomal-recessive hereditary spherocytosis due to alpha-spectrin deficiency. J Perinatol. 2013; 33(5): 404-6. PubMed

Medical Reviewers

Last Update: December 2015