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A. Schwartz, C. Marques, G. Abreu, R. Benardes, L. Jolin, E. Snow, L. Wang, A. Gaigalas, W. Carter, J. Cunha-Vaz; Quantitative Characteristics of Fluorescein for Fluorescence Angiograms. Invest. Ophthalmol. Vis. Sci. 2007;48(13):157. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Quantitation of image intensities from fluorescence angiograms could provide reliable information if they were independent of the instrument and the operator. The first step to obtaining such independent quantitative data is to ensure that the characteristics of fluorescein are consistent from different sources and traceable back to an accepted standard.
The present study involved five different sources of fluorescein; one clinical injectable grade, three different "pure" sources from different companies, and the Standard Reference Material fluorescein (SRM 1932) from the National Institute of Standards and Technology. Moreover, sets of data were independently obtained from three separate laboratories; AIBILI in Coimbra, Portugal, Erie Scientific in Portsmouth, NH, and NIST in Gaithersburg, MD. Solutions of fluorescein (10-7 - 10-11 M) from each source were made in PBS at pH 7.4 and emission data were obtained with spectrofluorometers in each laboratory.
The intra-laboratory data from the five sources of fluorescein all described a single calibration line, as well as the same sigmoidal response as a function of pH from 5.0 to 9.0. Inter-laboratory data all described a single calibration line after being normalized, due to differences in the instruments, to the highest fluorescein concentration (800 ng/ml) that maintained linear response in the spectrofluorometer.
Since all data from the three locations were equivalent, this indicates that fluorescein from different sources, including the injectable fluorescein, is traceable back to the NIST SRM 1932. To date, interpretation of fluorescent image intensities has been limited since it has been difficult to compare angiograms across laboratories and time. This multi-site, multi-source study is significant because it provides confidence that quantitative fluorescent measurements based on fluorescein standards made from different sources can provide comparable quantitative results.
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