On the regional variability of averaged cell area estimates for the human corneal endothelium in relation to the extent of polymegethism

On the regional variability of averaged cell area estimates for the human corneal endothelium in relation to the extent of polymegethism M. J. Doughty 0 Morphometry Non-contact 0 0 M. J. Doughty (&) Department of Vision Sciences, Glasgow-Caledonian University , Cowcaddens Rd, Glasgow G4 OBA , UK Purpose To assess variability in the coefficient of variation (COV) in cell area estimates when using different numbers of cells for endothelial morphometry. Methods Using non-contact specular microscopy images of the corneal endothelium, 4 sets of 20 cases were selected that included 200 cells and had overall (global) COV values of less than 30 (group 1), 31-40 (group 2), 41-50 (group 3) and over 50% (group 4). Subjects could be normal, or had ophthalmic disease (such as diabetes), a history of rigid or soft contact lens wear or were assessed after cataract surgery. A stepwise analysis was undertaken, 20 cells at a time, of the variability in cell area estimates when using different numbers of cells for the calculations. Results Variability in the average cell area values was higher if only 20-60 cells were used in the calculations and then tended to decrease. The standard deviation values on these average cell area values and the calculated COV showed the same overall trends and were more than twice as large for endothelia with marked polymegethism. Using more than 100 cells/ image in markedly polymegethous endothelia only increased the variability in the calculations. Corneal endothelium; Cell areas; Human; Polymegethism specular microscopy Introduction As viewed in vivo by specular microscopy, the corneal endothelium of young healthy adults appears as a mosaic of cells having uniform size and shape [ 1, 2 ]. The cell size, as reported in the most endothelial assessments, is assessed as the endothelial cell density (ECD), in cells/mm2, and provides a very useful indicator of the status of the endothelial cell layer [3]. However, when even some of this uniformity is reduced, then actual considerations of the variation in cell size (area) has been considered important. A specific term was introduced to describe the nonuniformity (i.e. heterogeneity) to the endothelial mosaic, namely polymegethism. This estimates the increased variation in cell areas, reported as the coefficient of variation or COV [ 3 ]. In early studies [ 4 ], it was noted that substantial differences in cell area variation could exist and that any estimates of ECD could be very much dependent on the overall (global) COV assigned to an endothelial cell layer (assessed by wide-field specular microscopy). For the COV estimates themselves, a later retrospective analysis of published endothelial images indicated that the reliability of any COV calculations would be predictably less if cell area heterogeneity appeared to be present, i.e. whether images were subjectively considered to be normal (homogeneous) or showing some evidence of heterogeneity (polymegethism) [ 5 ]. The analysis was, however, limited by the fact that relatively few images were available for analysis and some included somewhat fewer cells than others, often less than 100/image. As a result it was not possible to systematically assess how much the reliability in COV calculations might be reduced according to the extent (or severity) of the perceived polymegethism. The essential basis of determining the extent of polymegethism is to measure the areas of the cells and then to calculate the average value of the cell area and the standard deviation (SD) of this average area value. It is this SD value that is then used to estimate the area variability as a standardised variance, generally known as the coefficient of variation (abbreviated as CV or COV) based on SD/average cell area calculation. This relative variance can be presented as a fraction (e.g. 0.5 for a moderately polymegethous endothelium) or (more usually) as a percentage (e.g. 50%). In general terms, an increased COV could result from the presence of even a few rather larger cells or small cells, or a combination of both [ 6 ]. Stated another way, from a theoretical perspective it could be that parts of an endothelial image could be largely normal with only small regions (or portions) of the mosaic showing larger or smaller cells. The overall estimates of COV have been reported to be dependent on the number of cells measured and therefore included in the calculations [ 5, 7 ]. Small field endothelial images taken from normal corneas of young adults with modern day instruments can be expected to include over 100 cells [ 8–11 ], while in evaluation of corneas after surgical interventions it has been noted that a good quality image should contain at least 75 cells [6]. Assessments of published images indicated that measuring this number of cells (i.e. 75/image) should give reasonably reliable data in terms of predicted variability in cell areas [ 5 ]. Notwithstanding, a systematic analysis of the region (...truncated)