Validity of endothelial cell analysis methods and recommendations for calibration in topcon SP-2000P specular microscopy

Purpose: To report on the calibration of the Topcon SP-2000P specular microscope and the Endothelial Cell Analysis Module of the IMAGEnet 2000 software, and to establish the validity of the different endothelial cell density (ECD) assessment methods available in these instruments. Methods: Using an external microgrid, we calibrated the magnification of the SP-2000P and the IMAGEnet software. In both eyes of 36 volunteers, we validated 4 ECD assessment methods by comparing these methods to the gold standard manual ECD, manual counting of cells on a video print. These methods were: the estimated ECD, estimation of ECD with a reference grid on the camera screen; the SP-2000P ECD, pointing out whole contiguous cells on the camera screen; the uncorrected IMAGEnet ECD, using automatically drawn cell borders, and the corrected IMAGEnet ECD, with manual correction of incorrectly drawn cell borders in the automated analysis. Validity of each method was evaluated by calculating both the mean difference with the manual ECD and the limits of agreement as described by Bland and Altman. Results: Preset factory values of magnification were incorrect, resulting in errors in ECD of up to 9%. All assessments except 1 of the estimated ECDs differed significantly from manual ECDs, with most differences being similar (<= 6.5%), except for uncorrected IMAGEnet ECD (30.2%). Corrected IMAGEnet ECD showed the narrowest limits of agreement (-4.9 to +19.3%). Conclusions: We advise checking the calibration of magnification in any specular microscope or endothelial analysis software as it may be erroneous. Corrected IMAGEnet ECD is the most valid of the investigated methods in the Topcon SP-200OP/IMAGEnet 2000 combination.