Paroxysmal Nocturnal Hemoglobinuria Testing by Flow Cytometry: Brief Overview for Clinicians
Keywords:paroxysmal nocturnal hemoglobinuria, flow cytometry, fluorescent aerolysin, glycophosphatidylinositol, PIGA
Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare, acquired hematopoietic stem cell disorder, caused by a somatic mutation in the PIG-A gene. This mutation is responsible for the synthesis of glycophosphatidylinositol (GPI) anchor that attaches a number of proteins to the cell surface. The mutant gene leads to partial deficiency or absence of all proteins normally linked to the cell membrane by GPI anchor. The primary clinical manifestations of PNH are complement mediated hemolytic anemia, thrombosis in atypical locations and blood cytopenias. Flow cytometry has become the gold standard method for PNH clone detection due to its high sensitivity and specificity and due to the ability to examine multiple GPI-linked proteins on red and white blood cells surface. It is the method of choice for the detection of very small PNH clones in subclinical PNH that often accompanies aplastic anemia and other bone marrow disorders. PNH clone detection traditionally involves the analysis of CD55 and CD59 on red and white blood cells. Other markers such as CD14, CD16, CD24, CD66b and CD157 are suitable to detect GPI-linked proteins in the surface of granulocytes and monocytes. The most useful reagent to assess white blood cell PNH clones is Fluorescent Aerolysin (FLAER) which is a mutated form of proaerolysin conjugated with a fluorochrome. Its advantage in PNH clones detection is due to the ability to bind directly to the glycan portion of the GPI anchor. Flow cytometry is a sophisticated method and a useful tool for clinical cell analysis. However, PNH is a clinical diagnosis and flow cytometric results should always be interpreted with respect to clinical manifestations and other laboratory findings.
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