biostudies-literatureUnknown16(1)Fırat ISOsmotic fragility (OF) is widely used to evaluate red blood cell (RBC) membrane stability, water transport dynamics and hemoglobinopathies, traditionally via spectrophotometric, visual, or flow cytometric techniques. Here, we present a novel flow chamber-based platform integrated with a proprietary imaging software providing AI-driven RBC detection for automated and reproducible OF analysis (BioExP). To assess agreement, we compared MCF₅₀ values from four healthy donors using classical and flow chamber methods. Protocol optimization included determining the "satiation time" (optimal incubation required to induce maximal hemolysis without overexposure) and hemolysis kinetics. Biological sensitivity was tested using two modulators: HgCl₂ (40 µM) to inhibit aquaporin (AQP) channels and lipopoScientific reports7175https://www.ebi.ac.uk/biostudies/studies/S-EPMC12920795biostudies-literatureA novel AI-coupled flow chamber method quantifying erythrocyte osmotic fragility.PMC12920795Damiati SFırat ISArtmann ATArtmann GMCreutz TAlacayır OfalseA novel AI-coupled flow chamber method quantifying erythrocyte osmotic fragility.Osmotic fragility (OF) is widely used to evaluate red blood cell (RBC) membrane stability, water transport dynamics and hemoglobinopathies, traditionally via spectrophotometric, visual, or flow cytometric techniques. Here, we present a novel flow chamber-based platform integrated with a proprietary imaging software providing AI-driven RBC detection for automated and reproducible OF analysis (BioExP). To assess agreement, we compared MCF₅₀ values from four healthy donors using classical and flow chamber methods. Protocol optimization included determining the "satiation time" (optimal incubation required to induce maximal hemolysis without overexposure) and hemolysis kinetics. Biological sensitivity was tested using two modulators: HgCl₂ (40 µM) to inhibit aquaporin (AQP) channels and lipopo2026-01-01T00:00:00Z2026 Feb2026-07-16T13:02:55.027Z2026-07-09T10:56:45.46ZS-EPMC129207954170297310.1038/s41598-026-38322-z