Paroxysmal Nocturnal Hemoglobinuria - PNH

  • Diagnosis
  • Algorithms
  • Monitoring
  • Background
  • Lab Tests
  • References
  • Related Topics
  • Videos

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


  • 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).


  • Incidence – <1/million (Devalet, 2015)
  • Age – median onset is 30-40 years
  • Sex – M:F, equal


  • 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
      • Usually only express small PNH clone
    • Subclinical PNH – PNH clones but no evidence of hemolysis
      • ​Clone <1% and no 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 (due to hemoglobinuria)
    • Anemia – fatigue, pallor, weakness
  • Thrombophilia – occurs in ~40% of individuals (Devalet, 2015)
    • Venous thromboses at unusual sites
      • Hepatic veins (Budd-Chiari)
      • Cerebral veins
    • Arterial thromboses can occur
    • Leading cause of mortality in PNH
    • Increased risk of thromboses during pregnancy and postpartum
  • Aplastic anemia is common due to
    • Bone marrow failure
    • May be associated with overlap syndromes and aplastic anemia
  • Worsening hemolysis with infection
  • Malignancy risk for acute myeloid leukemia, myelodysplastic syndromes
  • Other signs and symptoms – may include abdominal pain, iron deficiency anemia, smooth muscle dysfunction, male impotence, dysphagia, chronic kidney disease
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


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


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


Compromised accuracy

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

Bone Marrow Services

General References

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

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

Devalet B, Mullier F, Chatelain B, Dogné J, Chatelain C. Pathophysiology, diagnosis, and treatment of paroxysmal nocturnal hemoglobinuria: a review. Eur J Haematol. 2015; 95(3): 190-8. PubMed

Höchsmann B, Schrezenmeier H. Congenital CD59 Deficiency. Hematol Oncol Clin North Am. 2015; 29(3): 495-507. 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 CJ. 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 CH. Laboratory tests for paroxysmal nocturnal hemoglobinuria. Am J Hematol. 2014; 89(3): 339-41. PubMed

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

References from the ARUP Institute for Clinical and Experimental Pathology®

Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland R, Wittwer CT, Richards SJ, 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 CJ, 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 CJ, Wittwer CT. 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 HM, Christensen RD, 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

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Last Update: September 2017