Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation i n the gene PIGA which encodes an enzyme essential for the synthesis of glyc osylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absenc e or marked deficiency of more than a dozen proteins on PNH blood cells. Cu rrent flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. Howev er because no single GPI anchor protein is always expressed in all cell lin eages, no one monoclonal antibody can be used with confidence to diagnose P NH. We describe a new diagnostic test for PNH, based on the ability of a fl uorescently labeled inactive variant of the protein aerolysin (FLAER) to bi nd selectively to GPI anchors. We compared CPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59, In all cases, FLAER detecte d similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER co uld detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the G PI anchor deficit in PNH.