title: Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin.
abstract: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could 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 GPI anchor deficit in PNH.
authors.affiliation: Department of Oncology, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Brodsky
authors.forename: G L
authors.initials: GL
authors.lastname: Mukhina
authors.forename: S
authors.initials: S
authors.lastname: Li
authors.forename: K L
authors.initials: KL
authors.lastname: Nelson
authors.forename: P L
authors.initials: PL
authors.lastname: Chiurazzi
authors.forename: J T
authors.initials: JT
authors.lastname: Buckley
authors.forename: M J
authors.initials: MJ
authors.lastname: Borowitz
mesh_terms: D000741:Anemia, Aplastic / Q000097:blood / Q000175:diagnosis; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D018957:CD59 Antigens / Q000378:metabolism; D002460:Cell Line / Q000378:metabolism; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000172:deficiency / Q000378:metabolism*; D006098:Granulocytes / Q000378:metabolism; D006457:Hemoglobinuria, Paroxysmal / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D008565:Membrane Proteins / Q000172:deficiency / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D009000:Monocytes / Q000378:metabolism; D052899:Pore Forming Cytotoxic Proteins; D012680:Sensitivity and Specificity
chemical_list: D001427:Bacterial Toxins; D018957:CD59 Antigens; D017261:Glycosylphosphatidylinositols; D007473:Ion Channels; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; C080086:phosphatidylinositol glycan-class A protein; C032676:aerolysin