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.8,9
In some parts of the world, however, FLAER can be expensive and/or difficult to obtain. In cases where FLAER may not be available, PNH neutrophils and monocytes can be successfully evaluated using an alternative non-FLAER based assay with anti-CD157, a monoclonal antibody that binds to GPI-linked proteins on both neutrophils and monocytes.10,11
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:3,12
- Type I cells: normal expression of GPI-anchored proteins
- Type II cells: partial GPI-antigen deficiency.
- Type III cells: complete GPI-antigen deficiency.
As PNH is a rare condition (up to 15.9 cases/million),13 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.
Recently, mutations of the phosphatidylinositol glycan class A gene, PIG-A, have been identified as the underlying genetic cause of PNH.14 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 identified15 with no clear association to PNH subtype or disease severity.
Genetic testing should not be used for diagnosis of, or routine testing for, PNH
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- Kumar V et al. Robbins and Cotran Pathologic Basis of Disease. Philadelphia, PA: Elsevier Saunders; 2004.
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