A simple flow cytometric assay for routine paroxysmal nocturnal hemoglobinuria testing based on immature reticulocytes and granulocytes

Background: The aim of this study was to test an easy-to-perform flow cytometric (FCM) assay for the routine investigation for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH), through the simultaneous detection of PNH clones on immature reticulocytes (i-RET) and granulocytes. Methods: During the last 5 years, eight patients were diagnosed with PNH in our laboratory, among 90 patients prospectively studied for PNH. The determination of glycosylphosphatidylinositol (GPI) deficient cells on the erythroid lineage was made with a two-color FCM assay of CD71 and CD59, evaluating the PNH clone on i-RET. Three color combinations based on CD66b/CD16/CD45 and CD59/CD24/CD45 were used for the determination of GPI-deficient granulocytes. Results: In all the patients with PNH, the PNH clones determined with CD71(+)CD59(-) red blood cells (RBC) were nearly identical to the respective clones determined with CD16(dim/-)/CD66b(-) and CD59(-)/CD24(-) granulocytes, in contrast to the clones determined with CD59-deficient erythrocytes only, which were significantly lower. Conclusions: Our results indicate that the simultaneous assessment of the PNH clone on CD71(+)/CD59(-)i-RET and CD16(dim/-)/CD66b(-) granulocytes, could offer a reliable method of two series PNH screening, at low cost and with ease of application. (c) 2012 International Clinical Cytometry Society