Evaluation of the Eosin‐5′‐Maleimide Flow Cytometry Test for the Diagnosis of Hereditary Spherocytosis

INTRODUCTION: Hereditary spherocytosis (HS) is a congenital hemolytic anemia caused by erythrocyte membrane protein defects. While the eosin-5'-maleimide (EMA) binding test by flow cytometry is the established first-line assay, this study evaluates its optimal cut-offs and performance within a large, real-world cohort. METHODS: We retrospectively analyzed 181 consecutive samples (2017-2024) with suspected HS. EMA binding was performed by flow cytometry using conventional and standardized protocols; the latter (implemented in 2023) utilized fixed erythrocyte counts and FL1 bead calibration. Accuracy was assessed against a composite clinical-laboratory reference standard. ROC analysis determined sensitivity, specificity, likelihood ratios, and AUC. RESULTS: HS was confirmed in 50/181 cases (27.6%). EMA fluorescence reduction was significantly greater in HS (median 23.0%) than in non-HS samples (9.0%). The optimal cut-off (16.5%) yielded 96.0% sensitivity, 96.9% specificity, and AUC = 0.990 (95% CI 0.979-0.998). Both conventional and standardized protocols showed equivalent accuracy (AUC 0.988 vs. 0.992). False positives were mainly associated with severe microcytosis or autoimmune hemolytic anemia. CONCLUSIONS: In a consecutive, real-world cohort of patients with suspected hereditary spherocytosis referred to a single reference laboratory receiving samples from across the country, the EMA test demonstrated high diagnostic accuracy consistent with previous reports. Importantly, implementation of a standardized protocol resolved reproducibility issues in fluorescence intensity measurement without compromising diagnostic performance, supporting its robustness and applicability in routine clinical laboratories.