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

Introduction 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 (R2 = 0.96) and usually greater than the percentage of GPI- RBCs. Conclusion 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. © 2015 International Clinical Cytometry Society