In vivo confocal microscopy of the corneal endothelium: comparison of three morphometry methods after corneal transplantation

Abstract

Purpose: The purpose of this study was to assess the endothelium of corneal grafts by in vivo confocal microscopy (IVCM), and to evaluate an automated endothelial software system in comparison with a manual cell count and planimetry.

Patients and methods: Overall, 40 corneal grafts (20 deep anterior lamellar keratoplasties (DALKs) and 20 penetrating keratoplasties (PKs)) were assessed by scanning-slit IVCM. The endothelial cell density (ECD) was estimated with the automated and the manual cell count method of the instrument's Nidek Advanced Vision Information System (NAVIS) software. The results were compared with planimetry as the reference method, and the agreement was assessed.

Results: The mean (±SD) automated ECD was 2278±524 cells/mm(2) (range 1167-3192 cells/mm(2)), whereas the manual cell count method gave significantly lower ECDs with a mean of 1213±677 cells/mm(2) (range 218-2440 cells/mm(2); P<0.001). The manual cell counts were also significantly lower than those by planimetry, with a mean ECD of 1617±813 cells/mm(2) (range 336-2941, P<0.001). Bland-Altman analyses indicated that the limits of agreement (LoA) between the automated and the planimetry method were -671 and +1992 cells/mm(2), whereas they were -1000 and +202 cells/mm(2) when comparing the manual cell counts with planimetry.

Conclusion: Following keratoplasty, the NAVIS automated method is likely to overestimate endothelial cell counts due to oversegmenting of the cell domains. Automated ECDs are substantially higher than those by the manual counting method or planimetry. The differences are considerably larger post-keratoplasty than for normal corneas, and the methods should not be used interchangeably.

Figure 1

(A) Representative set of IVCM images for a corneal graft with a relatively high cell count. (a) Confocal image showing cells with cell-cell borders marked for planimetry. (b) Image illustrating the NAVIS automated cell count analysis for the same cells. Scale bar=50 μm. (B) Representative set of IVCM images for a corneal graft with a relatively low cell count. (a) Confocal image showing cells with cell-cell borders marked for planimetry. (b) Image illustrating the NAVIS automated cell count analysis for the same cells. Scale bar=50 μm.

Figure 2

Boxplots illustrating the distribution of the ECD for 40 corneal grafts. The length of the boxes represents the interquartile range. The horizontal line in the middle of the box represents the median value. The whiskers represent the minimum and maximum values.

Figure 3

Agreement between the NAVIS manual cell count method and planimetry for 40 corneal grafts. The scatterplot shows the difference vs the mean of the two methods (Bland–Altman analysis). Reference lines are provided for the mean difference (−405 cells/mm²) and at ±1.96 × SD of the difference, which represent the 95% LoA.

Figure 4

Agreement between the NAVIS automated method and planimetry for 40 corneal grafts. The scatterplot shows the differences vs the mean of the two methods (Bland–Altman analysis). The output of the automated analysis was not manually corrected. Reference lines are provided for the mean difference (661 cells/mm²) and at ±1.96 × SD of the difference, which represent the 95% LoA.