High‐sensitivity 5‐, 6‐, and 7‐color PNH WBC assays for both Canto II and Navios platforms

Background Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco‐Phosphatidyl‐Inositol (GPI)‐linked cell surface structures. Fluorescent proaerolysin (FLAER‐Alexa488) is increasingly used to detect GPI‐deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER‐based and non‐FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER. Methods We have used a single tube approach with a 7‐color assay comprising FLAER‐CD157‐CD15‐CD64‐CD24‐CD14‐CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10‐color Navios or 8‐color FACSCanto II platforms. The 6‐color (minus CD14) and 5‐color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5‐color assay comprising FLAER‐CD157PE‐CD64ECD‐CD15PC5‐CD45PC7. Results/Conclusions CD15‐gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64‐gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER‐based plots derive virtually identical data to the non‐FLAER plot for neutrophils ( R 2 = 1) and monocytes ( R 2 = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7‐, 6‐, and 5‐color versions of the assay. Assessment of non‐PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches.