Hematology

Hemolytic anemia

Paroxysmal nocturnal hemoglobinuria (PNH)



Topic Completed: 19 July 2021

Minor changes: 19 July 2021

Copyright: 2021, PathologyOutlines.com, Inc.

PubMed Search: Paroxysmal nocturnal hemoglobinuria[title] pathology "last 5 years"[DP]

Anna Sarah Erem, M.D.
Saja Asakrah, M.D., Ph.D.
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Page views in 2021 to date: 847
Cite this page: Erem AS, Asakrah S. Paroxysmal nocturnal hemoglobinuria (PNH). PathologyOutlines.com website. https://www.pathologyoutlines.com/topic/hematologyPNH.html. Accessed September 16th, 2021.
Definition / general
  • Acquired autosomal clonal mutation of hematopoietic stem cells (HSCs) characterized by altered surface protein expression of hematopoietic elements and increased hemolysis, risk of thrombosis and impaired bone marrow function
Essential features
  • Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired somatic mutation in the X linked phosphatidylinositol glycan class A (PIGA) gene, which leaves hematopoietic cells unable to produce the glycosylphosphatidylinositol (GPI) anchor that links cell surface proteins to the plasma membrane (Hematol Transfus Cell Ther 2020 Jul 6 [Epub ahead of print], Blood 2014;124:2804)
  • PNH leads to episodic hemolytic anemia, recurrent thrombosis and bone marrow failure and cytopenias (EJIFCC 2019;30:355)
  • Despite the name, PNH patients may experience hemoglobinuria at night, during the day or not at all (EJIFCC 2019;30:355)
  • PNH clones frequently appear in patients with aplastic anemia and myelodysplastic syndromes (Int J Lab Hematol 2017;39:329)
Terminology
ICD coding
  • ICD-10: D59.5 - paroxysmal nocturnal hemoglobinuria (Marchiafava-Micheli)
  • ICD-11: 3A21.0 - paroxysmal nocturnal hemoglobinuria
Epidemiology
Sites
Pathophysiology
Etiology
Diagrams / tables

Images hosted on other servers:

Suggested pathophysiology of PNH

Suggested pathophysiology of hemolysis in PNH

Complement action in healthy subjects and PNH patients

Clinical features
Diagnosis
Laboratory
Prognostic factors
Case reports
Treatment
Microscopic (histologic) description
Peripheral smear description
Flow cytometry description
  • At least 2 different hematopoietic cell types should be evaluated using antibodies against GPI anchored cell surface marker or using FLAER, which binds to all GPI anchored molecules (Int J Lab Hematol 2016;38:5)
  • Because the majority of potential PNH cases will be negative, an initial screen with fewer antibodies can be performed before using a diagnostic confirmation assay using a full antibody panel (EJIFCC 2019;30:355)
    • Screens may be accomplished with a simpler 2 color panel consisting of FLAER and CD15 labeling of neutrophils, although this is not a substitute for a full diagnostic assay (Int J Lab Hematol 2020;42:589)
  • PNH clones will appear with these immunophenotypes:
  • Different types of RBC clones (Ann Hematol 2019;98:1083):
    • Type I: presence of GPI-APs (CD55 and CD59; i.e. normal, non-PNH RBCs)
    • Type II: partial absence of GPI-APs
    • Type III: complete absence of GPI-APs
    • 10% of patients present with both type II and type III clones (Ann Hematol 2019;98:1083)
  • Analyze the size of PNH clone; if 1 - 5% of a given cell type is GPI deficient, treatment should be considered (Int J Lab Hematol 2016;38:5)
    • Quantitation of the PNH clone is not as accurate when measuring RBCs if the patient has received blood transfusions containing normal RBCs or has had recent hemolytic crises (EJIFCC 2019;30:355)
    • Quantification of RBCs is more accurate when adding anti-CD71 antibodies to the flow cytometry assay and using immature RBC populations, which are resistant to destruction by the complement system (Cytometry B Clin Cytom 2020;98:179)
    • Because diagnosis relies on absence rather than the presence of specific markers, care should be taken to exclude apoptotic cells using cell viability dye (EJIFCC 2019;30:355)
    • Using an 8 color panel can more accurately detect PNH clones compared with a 4 color panel; the sensitivity of the optimized 8 color panel is 0.01% when measuring granulocytes and 0.05% when measuring monocytes, after acquiring 100,000 events (Biomed Rep 2018;8:224)
Flow cytometry images

Contributed by Saja Asakrah, M.D.

High sensitivity PNH flow cytometry

Sample pathology report
  • Peripheral blood sample
    • Paroxysmal nocturnal hemoglobinuria (PNH) (see comment)
    • Comment:
      • Flow cytometric analysis of the submitted blood sample demonstrates the presence of population(s) of white blood cells (neutrophils or monocytes) or red blood cells (RBCs) with deficiency of tested glycosylphosphatidyl inositol (GPI) linked proteins.
      • Percentages of the GPI deficient cell populations are:
        • Total GPI deficient RBCs (type III plus type II): 13.8%
        • Type III (GPI deficient) RBCs (CD235a+ CD59-): 10.2%
        • Type II (partial GPIdeficient) RBCs (CD235a+ CD59- intermediate): 3.6%
        • GPI deficient neutrophils (CD15+ CD24- FLAER-): 28.7%
        • GPI deficient monocytes (CD64+ CD14- FLAER-): 59.4%
      • The flow cytometric findings are consistent with a diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). However, PNH cells may be seen in PNH, aplastic anemia, myelodysplastic syndromes and other conditions. Correlation with clinical and laboratory findings is suggested.
      • Note: Discordance between the size of GPI deficient RBC and WBC populations may be due to hemolysis or transfusion.
      • The lower limit of quantification (lower limit of enumeration) for the RBC assay is 0.01%, for the neutrophil assay is 0.05% and for the monocyte assay is 0.5%. Below these levels, PNH cells may be reported as detected but below the level of quantification. For leukopenic patients, the neutrophil and monocyte assay sensitivity may be decreased.
      • References: Cytometry B Clin Cytom 2018;94:16, Cytometry B Clin Cytom 2018;94:23, Cytometry B Clin Cytom 2018;94:49, Cytometry B Clin Cytom 2018;94:67, CLSI: H52-A2 - Red Blood Cell Diagnostic Testing Using Flow Cytometry, 2nd Edition, 2014, EJIFCC 2019;30:355, Int J Lab Hematol 2019;41:73
Differential diagnosis
Board review style question #1
Which of the following tests is considered the gold standard test for paroxysmal nocturnal hemoglobinuria (PNH) detection?

  1. Bone marrow biopsy
  2. Coombs test
  3. Flow cytometry
  4. Serum haptoglobin level
Board review style answer #1
Board review style question #2
Which of the following is a glycosylphosphatidylinositol (GPI) anchored protein and is part of the paroxysmal nocturnal hemoglobinuria (PNH) flow cytometry panel?

  1. CD11b
  2. CD11c
  3. CD14
  4. CD64
Board review style answer #2
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