A method to measure permeability of red blood cell membrane to water and solutes using intrinsic fluorescence

• A technique to measure changes in RBC volume using RBC autofluorescence is described.
• Stopped-flow spectroscopy was used to measure RBC volume changes in anisotonic solutions.
• Self-quenching of hemoglobin autofluorescence at high intracellular concentrations results in fluorescence changes.
• We found that a change in RBC volume results in a proportional change in RBC autofluorescence.
• Measurement of cell osmotic permeability using this method has numerous clinical applications.

BackgroundDesigning effective cryopreservation procedures for cells requires knowledge of permeability of cell membrane to water and solutes. To determine cell membrane permeability, one needs to measure the rate of cell volume changes in anisotonic environment. Red blood cells (RBCs) respond very quickly to changes in extracellular solutes concentration, which complicates the use of traditional methods. Preservation of RBCs from umbilical cord blood for neonatal transfusions is currently broadly discussed in the literature, but data on osmotic permeability of cord RBCs is controversial. Therefore, alternative methods to determine osmotic membrane permeability of these cells are warranted. We describe a technique to measure rapid changes in RBC volume through changes in the intensity of RBC autofluorescence.MethodsTo induce osmotically-driven changes in RBC volume, we rapidly mixed human RBCs with anisotonic solutions in a stopped-flow spectroscopy system and the intensity of intrinsic RBC fluorescence was measured.ResultsWe found that change in RBC volume cause a proportional change in the intensity of RBC autofluorescence. This phenomenon occurs due to the self-quenching of RBC hemoglobin autofluorescence at high intracellular concentrations.ConclusionsThis novel method to determine osmotic permeability of RBCs overcomes the limitations of traditional techniques and has numerous clinical applications.