ABSTRACT
PURPOSE: Until now, organ-cultured corneal endothelial mosaic has been assessed in France by cell counting using a calibrated graticule, or by drawing cells on a computerized image. The former method is unsatisfactory because it is characterized by a lack of objective evaluation of the cell surface and hexagonality and it requires an experienced technician. The latter method is time-consuming and requires careful attention. We aimed to make an efficient, fast and easy to use, automated digital analyzer of video images of the corneal endothelium. METHODS: The hardware included a PC Pentium III ((R)) 800 MHz-Ram 256, a Data Translation 3155 acquisition card, a Sony SC 75 CE CCD camera, and a 22-inch screen. Special functions for automated cell boundary determination consisted of Plug-in programs included in the ImageTool software. Calibration was performed using a calibrated micrometer. Cell densities of 40 organ-cultured corneas measured by both manual and automated counting were compared using parametric tests (Student's t test for paired variables and the Pearson correlation coefficient). RESULTS: All steps were considered more ergonomic i.e., endothelial image capture, image selection, thresholding of multiple areas of interest, automated cell count, automated detection of errors in cell boundary drawing, presentation of the results in an HTML file including the number of counted cells, cell density, coefficient of variation of cell area, cell surface histogram and cell hexagonality. The device was efficient because the global process lasted on average 7 minutes and did not require an experienced technician. The correlation between cell densities obtained with both methods was high (r=+0.84, p<0.001). The results showed an under-estimation using manual counting (2191+/-322 vs. 2273+/-457 cell/mm(2), p=0.046), compared with the automated method. CONCLUSIONS: Our automated endothelial cell analyzer is efficient and gives reliable results quickly and easily. A multicentric validation would allow us to standardize cell counts among cornea banks in our country.
Subject(s)
Endothelium, Corneal/cytology , Aged , Autoanalysis/methods , Calibration , Computers , Humans , Middle Aged , Organ Culture Techniques/methods , Postmortem ChangesABSTRACT
BACKGROUND: Endothelial examination of organ culture stored corneas is usually done manually and on several mosaic zones. Some banks use an image analyser that takes account of only one zone. This method is restricted by image quality, and may be inaccurate if endothelial cell density (ECD) within the mosaic is not homogeneous. The authors have developed an analyser that has tools for automatic error detection and correction, and can measure ECD and perform morphometry on multiple zones of three images of the endothelial mosaic. METHODS: 60 human corneas were divided into two equal groups: group 1 with homogeneous mosaics, group 2 with heterogeneous ones. Three standard microscopy video images of the endothelium, graded by quality, were analysed either in isolation (so called mono-image analysis) or simultaneously (so called tri-image analysis), with 50 or 300 endothelial cells (ECs) counted. The automated analysis was compared with the manual analysis, which concerned 10 non-adjacent zones and about 300 cells. For each analysis method, failures and durations were studied according to image quality. RESULTS: All corneas were able to undergo analysis, in about 2 or 7.5 minutes for 50 and 300 ECs respectively. The tri-image analysis did not increase analysis time and never failed, even with mediocre images. The tri-image analysis of 300 ECs was always most highly correlated with the manual count, particularly in the heterogeneous cornea group (r=0.94, p<0.001) and prevented serious count errors. CONCLUSIONS: This analyser allows reliable and rapid analysis of ECD, even for heterogeneous endothelia mosaics and mediocre images.
