Kinetics of Eosin-5-maleimide Binding to Red Blood Cells

Introduction/Objective The binding of eosin-5-maleimide (EMA) to red blood cells in different pH conditions has not been well-investigated. The current standard protocol uses phosphate buffered saline at pH 7.0 to 7.5 for the binding reaction. The reaction typically uses a sixty-minute incubation. However, the effect of pH on EMA binding remains to be determined, nor have the reaction times at different pH conditions. This study utilizes optimal pH conditions of 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 to characterize EMA binding. The two parameters, time, and pH were investigated to extensively describe the binding kinetics of eosin-5-maleimide. Methods/Case Report The working EMA solutions in phosphate buffered saline at different pHs (specifically 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5) were incubated in the dark with 1 µL of washed red blood cells and 25 µL of working EMA solution. The red blood cells and working EMA solution were vigorously mixed before the specified incubation time. The binding was recorded at specified time points, namely 1, 2, 4, 8, 16, 32, and 64 minutes for each pH condition. The binding reaction was terminated by washing three times with 0.1% bovine serum albumin (BSA) in PBS and centrifuged at 400 g for 5 minutes. The red blood cells were then resuspended with 500 µL of PBS and ran in the BD FACSLyric™ flow cytometer. Results (if a Case Study enter NA) The current EMA binding protocol utilizes a. neutral pH of 7.0 to 7.5 recorded at 64 min. Based on our data, better discrimination of hereditary spherocytosis red blood cells from normal red blood cells may be obtained at a more basic pH of 8.5. Thus, a statistical t-test was performed for the comparison between the current setup and the apparent optimal pH conditions at shorter reaction times of 16 min and 32min at pH 8.5. Conclusion The resulting data suggest that enhanced discrimination of HS red blood cells can be achieved in a more basic pH. This was evidently observed at pH 8.5, where the highest percentage decrease is recorded, and the most significant difference compared to the other pH conditions. In conclusion, the study identifies that better discrimination of hereditary spherocytosis red blood cells from normal red blood cells can be determined using pH 8.5 with a shorter incubation time of 32 min. The pH 8.5 at 16 min condition could also be considered if the clinical batch size allows precise incubation of 15 min.