Fluorescein and acidosis. Implications for flap perfusion studies

The visual fluorescein technique underpredicts survival of a skin flap during the first 24 hours after raising the flap. This problem limits its use as a research and clinical tool. There is no compelling explanation for this observation, but two facts are known: fluorescein is a derivative of phthalein, a pH indicator, and ischemic tissues become acidotic, with the pH falling below 7.0. These observations lead to the hypothesis that acidosis quenches fluorescence in distal skin flaps. No data could be found regarding this effect. Therefore, the effect of acidosis on sodium fluorescein was studied in vitro. A spectrophotometer was used to measure the intensity of the fluorescence of sodium fluorescein in buffered solutions of different hydrogen ion concentrations. Two studies were performed at different concentrations of fluorescein. At a concentration of 10(-5) g/mL, there is a drop of 26% in fluorescence intensity from pH 7.5 to 7.0, and 51% between pH 7.5 and 6.5. At 10(-7) g/mL, there is a 43% decrease in fluorescence between pH 7.5 and 6.5. This study supports our hypothesis that acidosis quenches the fluorescence of fluorescein. This effect must be considered when interpretating basic studies of skin flap microcirculation.