Evaluation of Fluorescently Labeled Aerolysin as a New Kind of Reagent for Flow Cytometry Tests: Optimization of Use of FLAER, Hints, and Limits.

Objectives: Diagnostic tests for paroxysmal nocturnal hemoglobinuria (PNH) are currently based on flow cytometry techniques. Typically, these tests use antibodies against glycosylphosphatidylinositol (GPI)–anchored proteins, but a new approach has been described recently, using a novel reagent named FLAER (fluorescently labeled aerolysin). In this work, we evaluate the performance and highlight the peculiarities of using this new reagent. Results: We investigated the general conditions of staining and explored optimal labeling settings. We found that the kinetics of the FLAER labeling is slightly different from that of antibodies. Our results led us to select a 30-minute incubation period at room temperature using 50 nmol/L as a final concentration of FLAER. As the nonspecific binding was dependent on the balance between FLAER and its ligand, the number of target cells was also found critical. In addition, sample preparation affected FLAER staining, and the lyse-before-stain preparation was preferred. Interestingly, FLAER affinity seems restricted to certain types of GPI anchors, making it unsuitable for exploration of RBCs. Finally, we aimed to evaluate FLAER as a possible single diagnostic tool; we studied cellular background in non-PNH samples and found a limit of detection close to 0.01% in optimal conditions. Conclusions: The performance of the FLAER labeling on leukocytes proves that this reagent is a valuable tool for PNH diagnosis and particularly appropriate for high-sensitivity tests in laboratories aiming to detect minor PNH clones.