PNH testing methodologies

High-sensitivity flow cytometry

High-sensitivity flow cytometry is the current ‘gold standard’ diagnostic test for paroxysmal nocturnal haemoglobinuria (PNH) as it provides the most quantitative and qualitative information.1,2 Using a combination of specific antibodies, fluorescent aerolysin (FLAER) and flow cytometry gating strategies, the presence and size of a PNH clone can be determined.3-7 Monoclonal antibodies, such as anti-CD59, bind to the glycosylphosphatidylinositol (GPI)-linked protein, whereas FLAER, a fluorescently labelled, inactive bacterial toxin, targets the GPI anchor on the cell surface directly, selectively revealing the presence or absence of GPI-anchored proteins.8

High-sensitivity flow cytometry is the current ‘gold standard’ diagnostic test for PNH

Adapted from Robbins and Cotran Pathologic Basis of Disease, Seventh Edition. 2005, Aster JC, “Red blood cell and bleeding disorders”, page 636. ©2005 Elsevier. Reproduced with permission from Elsevier.
FLAER, fluorescent aerolysin; GPI, glycosylphosphatidylinositol

Flow cytometry provides a direct measure of the expression of GPI anchor and GPI-linked proteins (such as CD59) on all haematopoietic cell populations, allowing identification of three distinct PNH clonal populations:3,9

  • Type I cells: normal expression of GPI-anchored proteins
  • Type II cells: intermediate expression of GPI-anchored proteins
  • Type III cells: no detectable expression of GPI-anchored proteins

As PNH is a rare condition (up to 15.9 cases/million),10 many laboratories have never seen a positive case. Variability in testing can be introduced at every step of the process, including sample preparation, antibody selection, instrument set-up, gating strategy, interpretation of results, selection of suitable quality controls and reporting of results. To avoid inter- and intra-laboratory variations in testing procedures, specialised expertise in flow cytometry is required in combination with standardised testing procedures. Further information and guidance on high-quality PNH testing can be found in PNH testing guidelines.

Genetic testing

Recently, mutations of the phosphatidylinositol glycan class A gene, PIG-A, have been identified as the underlying genetic cause of PNH.11 Genetic sequencing of the PIG-A gene could be used to confirm the presence of PNH clones following flow cytometry, but should not be applied to routine testing as >100 mutations of the PIG-A gene have been identified12 with no clear association to PNH subtype or disease severity.

Genetic testing should not be used for diagnosis of, or routine testing for, PNH

  • References
    1. Kelly R et al. Ther Clin Risk Manag 2009; 5: 911-921.
    2. Sharma VR. Clin Adv Hematol Oncol 2013; 11: 1-11.
    3. Hall SE, Rosse WF. Blood 1996; 87: 5332-5340.
    4. Piedras J, López-Karpovitch X. Cytometry 2000; 42: 234-238.
    5. Richards SJ et al. Cytometry 2000; 42: 223-233.
    6. Hernández-Campo PM et al. Transfusion 2008; 48: 1403-1414.
    7. Richards SJ et al. Cytometry B Clin Cytom 2009; 76B: 47-55.
    8. Aster JC. Robbins and Cotran Pathologic Basis of Disease. Philadelphia, PA: Elsevier Saunders; 2004.
    9. Borowitz MJ et al. Cytometry B Clin Cytom 2010; 78B: 211-230.
    10. Hill A et al. Blood 2006; 108: abs 985.
    11. Parker C et al. Blood 2005; 106: 3699-3709.
    12. Johnson RJ, Hillmen P. Mol Pathol 2002; 55: 145-152.

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