Specular microscopy ancillary study methods for donor endothelial cell density determination of Cornea Donor Study images

Abstract

Purpose: To describe reliable methods for determining central corneal endothelial cell density (ECD) in a multicenter eye bank study.

Methods: The Specular Microscopy Reading Center utilized a dual-grading procedure and adjudication process to classify image quality and determine ECD for a subset of donor endothelial images obtained in the Specular Microscopy Ancillary Study, which is part of the Cornea Donor Study. Two certified readers classified images as analyzable (excellent, good, fair) or unanalyzable and determined the ECD using a variable frame technique. An adjudicator also evaluated the images if quality classifications by the two readers differed by one grade, if any reader found the image unanalyzable, and/or if the ECD determination between the two readers was >or= 5%.

Results: Image quality categorization by the two readers was identical for 441 (64%) of 688 donor images. The ECD differed by < 5% for 442 (69%) of the 645 analyzable images. The ECD determined by the adjudicator was < 5% different than the ECD determined by at least one reader for 193 (95%) of the 203 remaining images.

Conclusions: The dual-grading and adjudication procedures produce reliable, reproducible assessments of image quality and ECD. The importance of two independent readings is evident in that image quality ratings differed between the two readers by one grade in 36% of all images and ECD counts differed by >or=5% for 31% of analyzable images.

FIGURE 1

The Specular Microscopy Reading Center corneal endothelial image classification. Analyzable—Excellent: All cell borders, boundaries, and centers across a single image of the endothelium are distinct excluding the peripheral edges of the image. The single image has a sufficient number of cells to count at least 50 and as many as 150 cells contiguous to each other. Good: A sufficient number of distinct cell borders, boundaries, and centers from a single image of the endothelium to count at least 50 and as many as150 cells from variable frames encompassing a minimum of 15 cells contiguous to each other for each variable frame. Fair: A sufficient number of cell borders, boundaries, and centers from a single image of the endothelium to count at least 50 cells from variable frames encompassing a minimum of 15 cells contiguous to each other for each variable frame. The borders, boundaries and centers of up to 25% of analyzed cells within the variable frames may be indistinct, but sufficient to estimate their location to conduct the analysis. Unanalyzable—Less than 50 cells with distinct borders, boundaries, and centers from a single image of the endothelium to count from variable frames encompassing a minimum of 15 cells contiguous to each other of each variable frame. The borders, boundaries and centers of greater than 25% of potentially analyzable cells within the variable frames are indistinct, and therefore insufficient to estimate their location to conduct the analysis.

FIGURE 2

Decision-making flow chart for the Specular Microscopy Reading Center corneal endothelial image classification.

FIGURE 3

The Specular Microscopy Ancillary Study dual grading and adjudication of eye bank images by the SMRC. RAD = relative absolute difference calculated as [absolute value of (second – primary)/primary] or as [absolute value of (reader – adjudicator)/adjudicator].