Standard microlithographic mosaics to assess endothelial cell counting methods by light microscopy in eye banks using organ culture

Abstract

Purpose: To develop standard microscopic hexagonal mosaics mimicking the human corneal endothelium for quality control of endothelial cell density (ECD) measurement and verification of cell counting strategy by light microscopy in eye banks using organ culture.

Methods: A standard slide, the Keratotest, was developed with 10 laser-engraved mosaics and different predetermined "cell" densities representing the range of ECDs observed routinely. Horizontal and vertical micrometric scales were etched adjacently to each mosaic, and a standard microscopy resolution test pattern was included. The Keratotest was applied to assess the reliability of a computer-assisted analyzer developed for corneal endothelial evaluation based on light microscopy images.

Results: The Keratotest consisted of 10 microlithographic homogeneous mosaics of 1-mm2 printed area and 1.2-microm cell boundary thickness. The micrometric scale associated with each mosaic aided in simultaneous verification of microscope calibration, and the test pattern aided in checking the microscope resolution. The design was unalterable and reproducible, and the glass slide incorporated in a carbon fiber support ensured easy handling and safe transport. Evaluation of the Keratotest mosaics by the computer-assisted analyzer found a high level of agreement (error margin between +0.12 and -0.46%) with the laser-engraved cell density.

Conclusions: This prototype device enabled assessment of reliability of ECD measurement in eye banks. It also allowed verification of the calibration and resolution of light microscopes. Periodic validation of counting procedure in eye banks with mosaics of known "cell" densities should be useful for standardization of donor corneal tissue quality control.