The retinal oximeter consists of a fundus camera with an attached image splitter, as well as a digital camera (Oxymap T1 device connected to Topcon TRC50-DX fundus camera; Oxymap ehf., Reykjavik, Iceland). The device captures images at two wavelengths, one sensitive to oxyhemoglobin (600 nm) and one isosbestic (570 nm), where the absorption spectra of oxyhemoglobin and hemoglobin cross. Computer software detects retinal vessels and uses relative light intensities inside and outside a vessel to calculate the optical density (light absorbance) of a vessel at both wavelengths. The hemoglobin oxygen saturation (SO
2) of a vessel can therefore be calculated, because the optical density ratio at these wavelengths has been shown to have an approximately inverse linear relationship with SO
2.
1
Prior to image capture, patients' eyes were dilated with one drop each of 1% tropicamide and 2.5% phenylephrine. Using the oximeter, trained retinal photographers obtained images of both retinas, centered on the optic disc. The images were 1200 × 1600 pixels and covered a 50° field of the central retina.
For each patient, one eye was randomly selected for analysis. Images were analyzed in a masked fashion by two independent observers with the Oxymap Analyzer software (version 2.3.2; Oxymap ehf.), which automatically detects vessels greater than eight pixels in diameter, using the ring-method protocol previously described by this research group (
Fig. 1).
8 The analysis area was selected by centering quadrant lines on the optic disc. An initial central circle was used to delineate the optic disc. Two additional measurement circles, an inner and outer circle, two and four times the diameter of the central circle, respectively, were then demarcated. To exclude the peripheral retina beyond the outer circle and the optic nerve, only the area between the circles centered on the optic disc were analyzed. The width of this ring was two disc diameters. Vessels beyond the area of analysis, certain areas where vessel detection would prove inaccurate (branching, overlapping, or intersecting), and segments of vessels less than 19 pixels in length were excluded. Vessels to be measured were selected manually. Measurements were first taken to yield arteriolar SO
2 (SaO
2) by quadrant. This was repeated for venules to yield venular SO
2 (SvO
2) by quadrant. Overall saturation was computed by averaging saturation values of each quadrant. Because Oxymap software measurements are calibrated to nondiabetic young individuals, results are relative to that calibration, occasionally resulting in SO
2 measurements greater than 100%. These values were not truncated to 100%, per established oximetry protocol.
5,8,14
Statistical analyses were implemented with Statistical Package for the Social Sciences (SPSS) software (version 20.0; SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) with Tukey's post hoc test was used to compare means, with DR stage as the independent variable and SO2 as the dependent variable.