title: Rare event counting of CD59- red cells in human blood: A 47-month experience using PNH consensus guidelines for WBC and RBC testing in a reference lab.
abstract: BACKGROUND
  Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder characterized by increased complement-mediated lysis of erythrocytes (RBCs) because of low/absent glycophosphatidylinositol (GPI) anchors of numerous cell surface proteins.
  
  
  METHODS
  Rare event analysis was applied to 120 million RBCs (12 normal individuals) and 102 million RBCs (102 normal individuals) to establish a reference range and verify a methodology for rare event analysis. Patient PNH testing (n = 10,984) was performed over 47 months using the 2010 consensus guidelines with CD59-PE and glycophorin A-FITC for RBCs and FLAER-Alexa 488, CD33-PerCP-Cy5.5, CD15-APC, CD14-APC-Cy7, and CD24-PE for WBCs.
  
  
  RESULTS
  The distribution of CD59- RBCs in the normal population was asymmetric with a mean of 5.9, median between 3 and 4, and mode of 2 per million RBCs. The normal range of CD59- RBCs was 0-17/million RBCs. A natural cutoff of 2.5% of the peak expression of CD59 delineates CD59+ from CD59- populations. The incidence of GPI- samples received by the laboratory was 6-7% without correlation to age (P = 0.35) or sex (P = 0.45). The percentage of GPI- neutrophils and monocytes were strongly correlated (R(2) = 0.96) and usually greater than the percentage of GPI- RBCs.
  
  
  CONCLUSIONS
  PNH RBC testing is a good example of rare event analysis applied to clinical cytometry—only 2.5 min are required to collect 1 million RBCs. With an established normal range of CD59- RBCs, the correlation between total cell count and sensitivity for detecting an abnormal population can be calculated using Poisson statistics.
authors.affiliation: Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah, 84108.
authors.forename: Michael
authors.initials: M
authors.lastname: Liew
authors.affiliation: Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah, 84108.
authors.forename: Marjorie
authors.initials: M
authors.lastname: Farley
authors.affiliation: Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah, 84108.
authors.forename: John
authors.initials: J
authors.lastname: Andreasen
authors.affiliation: Division of Hematology, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah, 84132.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Parker
authors.affiliation: Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah, 84108.|Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, 84132.
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
mesh_terms: D018957:CD59 Antigens / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006457:Hemoglobinuria, Paroxysmal / Q000175:diagnosis* / Q000473:pathology; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism
chemical_list: D018957:CD59 Antigens; D008565:Membrane Proteins; D010716:Phosphatidylinositols
keywords: CD59; FLAER; PNH; glycophorin a