Comparison of flow cytometric osmotic fragility test between kinetic and endpoint assay principle.

To screen hereditary spherocytosis (HS), we first introduced the novel flow cytometric osmotic fragility test (FC-OFT) based on the two-point kinetic assay principle (FC-OFTKinetic). With the introduction of FC systems with automatic tube loaders, we updated the FC-OFT protocol to follow the endpoint assay principle (FC-OFTEndpoint). This study aims to evaluate the updated FC-OFT protocol (FC-OFTEndpoint) and compare its assay performance with that of FC-OFTKinetic. We investigated factors influencing the FC-OFTEndpoint assay, optimized its protocol, and defined the cutoff index using 152 negative or artificially positive control samples. We then compared the assay performance with that of FC-OFTKinetic in 25 patients with anemia, including 14 with spherocytosis-8, 4, 1, and 1 with HS, autoimmune hemolytic anemia, burn injury, and liver cirrhosis, respectively. To optimize FC-OFTEndpoint, we adopted phosphate-buffered saline as the red cell suspension medium, 50% deionized water for hypotonic osmotic pressure in adults, and a 3-min standby time. This FC-OFTEndpoint was more accurate than FC-OFTKinetic in identifying spherocytosis in the 25 patients with anemia (p = 0.0313). FC-OFTEndpoint is a viable alternative to conventional OFT or FC-OFTKinetic for HS screening in clinical laboratories, as automatic FC enhances assay performance. These findings warrant validation in future multicenter studies with larger sample sizes.