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A. Schwartz, R. Bernardes, S. Carvajal-Gonzalez, L. Jolin, D. Moore, U. Chakravarthy, G. Staurenghi, G. Coscas, J. Cunha-Vas; An Instrument Performance Survey of HRA Spectralis Scanning Laser Ophthalmoscopes Using Quantitative Standards for Fluorescein, ICG and Autofluorescence. Invest. Ophthalmol. Vis. Sci. 2009;50(13):317.
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This survey was conducted to compare the quantitative fluorescence instrument performance measuring fluorescein, ICG and autofluorescence of the HRA Spectralis SLO (Heidelberg Engineering, Heidelberg, Germany) in four different locations: Coimbra, Portugal; Belfast, Northern Ireland; Milan, Italy and Paris, France.
A quantitative fluorescence cell was developed which contained serial dilutions of fluorochromes in specialized media that give rise to equivalent spectra and quantum efficiency of fluorescein in serum, fluorescein in vitreous humor, ICG and fluorescein for autofluorescence. The cell was inserted into an adaptor that was placed over the eyepiece of the instruments. Images in angiography mode of the standards in the cell were taken at the automatic gain settings of the instrument. The images were analyzed with software developed to perform linear regression calibration plots and calculate the linearity as Average Residual Percent, Detection Threshold in ng/ml, Dynamic Range in decades/scale. Inter-instrument performance parameters were compared across the four sites.
Across all four instruments and all measured fluorochromes, the Average Residual Percent was 4.8 +/- 2.5 (equivalent to r2 >0.995). The average Dynamic range for all fluorochromes was found to be 2.98 +/- 0.37 decades. The average Detection Threshold across the four instruments for each fluorochrome was different (23 ngm/ml for autofluorescence, 122 ngm/ml for fluorescein in serum, 37 ngm/ml for fluorescein in vitreous humor and 12 ngm/ml for ICG), These variations were expected due to the differences in extinction coefficients and quantum efficiencies. However, the average Coefficient of Variation across Detection Thresholds for each of the fluorochromes measured was 45%.
Comparison of the instrument performance (linearity, detection threshold, and dynamic range) was demonstrated. These results suggest that comparison of these parameters can be used to indicate the general level of performance for different makes and models of instruments. Furthermore, this system of standards and software could serve as a quality control tool for studies that involve quantitative fluorescence intensity measurements across patients, time and laboratories.
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