Diagnostic performance of flow cytometric osmotic fragility testing in hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common congenital red blood cell membrane disorder, characterized by structural protein defects that lead to hemolytic anemia. Although several diagnostic tests exist, including osmotic fragility tests (OFTs), acidified glycerol lysis test (AGLT), and the EMA-binding test (EMA), each presents specific limitations regarding sensitivity, specificity, or technical requirements. Flow cytometric osmotic fragility testing (OFT-FCM) emerges as a promising complementary assay, offering a standardized workflow and rapid turnaround time. We conducted a retrospective study including 106 subjects (20 HS patients and 86 healthy controls) recruited at Hospital Clínic de Barcelona between September 2024 and September 2025. Clinical and laboratory data were collected, and all participants underwent OFT, AGLT, EMA, and OFT-FCM using two acquisition protocols (300 and 214 s). Logistic regression and receiver operating characteristic curve analysis were performed to evaluate diagnostic performance and determine optimal cut-off values. HS patients exhibited significantly altered hematologic parameters compared with controls, including higher reticulocyte counts, red cell distribution width, and mean corpuscular hemoglobin. The EMA-binding test demonstrated high specificity (100%) but lower sensitivity (57.9%). OFT achieved high sensitivity (>97%) but low specificity (<47%). AGLT showed balanced accuracy (sensitivity 68.4%, specificity 96.1%). OFT-FCM yielded areas under the curve of 0.85 for both protocols, with optimal thresholds providing specificities of 95-100% and sensitivities of 57-59%. No significant differences were observed between OFT-FCM and EMA performance. OFT-FCM effectively discriminates HS patients from healthy controls and showed diagnostic performance comparable to EMA and favorable relative to classical OFT and AGLT in this cohort, while offering practical advantages in terms of workflow simplicity and turnaround time, and supporting its use as a complementary flow-cytometric assay within the diagnostic work-up of HS.