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. © 2012 International Clinical Cytometry Society