In Vivo Corneal Cell Density Measurement by Scanning Slit Confocal Microscopy: Method and Validation

Abstract

Abstract: : Background: Corneal confocal microscopy (CCM) produces high resolution in vivo images of thin corneal layers enabling the identification of structures in conscious humans and without depending on conventional histology techniques. The acceptance of CCM as a method for cell size and density measurements implies validation of the results against equivalent values obtained by histological techniques and knowledge about the reliability and repeatability of the confocal measurements. Purpose:To develop and validate a technique for quantifying cell density in vivo in six defined corneal layers with a scanning slit confocal microscope (SSCM) as a basis of further investigations. Methods: Six corneal layers were considered. Layer classification was validated by measuring the axial position of the measurement volume in the cornea. Cells (superficial, epithelial, endothelial) or nuclei (keratocytes) were manually counted on the videotape–registered confocal images using an unbiased frame. Surface cell density was calculated from the on–screen image while volumetric density was obtained using stereologic methods. Density measurements were validated by comparing CCM results with those obtained by histology. Inter–session and intra–session repeatability was determined by the Bland and Altman method. Cell densities were calculated for 20 healthy controls. Results: All of the visually identified layers were within their expected depth range, except for the in vivo measured basal layer which was 3% below the range limit. The difference between CCM and histology measurements was on average –1.3 % and ranged between –24.1% (posterior stroma) and +16.4% (basal layer). The average intra–session repeatability coefficient was 5.8 % (range: 0.4–10.2%) and the average inter–session repeatability coefficient was 8.3% (1.9–16.9%). Density measurements in 20 healthy controls were in accordance with those of other investigators. Conclusions:The SSCM can produce repeatable quantitative measurements of corneal cell density in conscious humans.

Keywords: microscopy: confocal/tunneling • cornea: basic science • imaging/image analysis: clinical