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-OFT_Kinetic). With the introduction of FC systems with automatic tube loaders, we updated the FC-OFT protocol to follow the endpoint assay principle (FC-OFT_Endpoint). This study aims to evaluate the updated FC-OFT protocol (FC-OFT_Endpoint) and compare its assay performance with that of FC-OFT_Kinetic. We investigated factors influencing the FC-OFT_Endpoint 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-OFT_Kinetic 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-OFT_Endpoint, 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-OFT_Endpoint was more accurate than FC-OFT_Kinetic in identifying spherocytosis in the 25 patients with anemia (p = 0.0313). FC-OFT_Endpoint is a viable alternative to conventional OFT or FC-OFT_Kinetic for HS screening in clinical laboratories, as automatic FC enhances assay performance. These findings warrant validation in future multicenter studies with larger sample sizes.