The aim of this study was to assess data quality from unsupervised endothelial cell counting in the multicentric setting.
We performed an endothelial cell counting trial with two fictitious trial sites. The trial protocol simply demanded for marking 30 cells for analysis. Analyses were performed with the cell counting tool as built into the Topcon SP-3000P specular microscope. The first centre consequently dotted 30 cells. The other centre continuously dotted more cells until 30 cells were included in the cell counting analysis. Both sites analysed the same 89 eyes of corneal outpatients and heathy volunteers. Both sites used a dedicated Topcon SP-3000P microscope. The image pairs from both sites were eventually printed, scanned and re-evaluated with a programme that evaluated all visible cells ("reading centre"). The agreement between both sites was statistically assessed by means of Pearson's correlation and Bland-Altman analysis. The same statistical assessments were also performed for the image pairs as analysed in the reading centre.
The determined cell densities as reported by both trial sites differed by -65 % to 42 %. Furthermore, we also observed a systematic deviation between both sites. Consequently, the coefficient of determination from Pearson's correlation was only 0.947. However, the agreement was as high as 0.997 when the image pairs were analysed in the reading centre. Here the difference between the cell densities of the image pairs ranged from merely -15 % to 9 % with no systematic deviation.
Unsupervised endothelial cell counting does not result in sufficiently objective endothelial cell denstiy estimations. Furthermore, the built-in analysis tools can introduce systematic errors. Both drawbacks can be overcome by a reading centre that evaluates all visible cells on the images. For this reason, we recommend the involvement of a reading centre in multicentric clinical trials on the corneal endothelium.
"The ethics committee of the Albert-Ludwigs-University of Freiburg expressed a favorable opinion and approved our study. The underlying dataset has been described elsewhere : briefly, we acquired multiple SM images in corneal outpatients and healthy volunteers with two different Topcon SP-3000P microscopes. The repeated recordings had been taken within a 10-minute timeframe. "
[Show abstract][Hide abstract] ABSTRACT: Modern specular microscopes (SM) robustly depict the same central area of the corneal endothelium at different time points through a built-in fixation light. However, repeated image acquisitions slightly shift and rotate because of minute changes in head position in the chin and forehead rest. This prevents the manual retrieval of individual corneal endothelial cells (CECs) in repeated measurements because SM images usually lack obvious landmarks. We devised and validated an image registration algorithm that aligns SM images from the same eye to make corresponding CECs coincide.
We retrospectively selected 27 image pairs for the presence of significant image overlap. Each image pair had been recorded on the same day and of the same eye. We applied our registration method in each image pair. Two observers independently validated, by means of alternation flicker, that the image pairs had been correctly aligned. We also repeatedly applied our registration method on unrelated image pairs by randomly drawing images and making certain that the images did not originate from the same eye. This was done to assess the specifity of our method.
All automated registrations of the same-day and same-eye image pairs were accurate. However, one single image incorrectly failed to trigger the non-match diagnosis twice in 81 registration attempts between unrelated images. As it turned out, this particular image depicted only 73 CECs. The average number of CECs was 253 (range 73-393).
Repeated non-contact SM images can be automatedly aligned so that the corresponding CECs coincide. Any successful alignment can be considered as proof of the retrieval of identical CECs as soon as at least 100 CEC centroids have been identified. We believe our method is the first to robustly confirm endothelial stability in individual eyes.
PLoS ONE 03/2013; 8(3):e59261. DOI:10.1371/journal.pone.0059261 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
ReLEx®flex is a corneal refractive procedure performed by removing corneal lenticules with a femtosecond (fs) laser system. Using electron microscopy, tissue parameters of extracted lenticules were analysed for potential correlations to the refractive results. Furthermore, the effect of previous contact lens (CL) wear on refractive stability (regression) was tested.
Patients and methods:
19 lenticules from 11 patients (age 24-56 years, 8 f, 3 m) were prepared for EM. The central areas of the samples were photographed and the distance between the collagen fibres and their diameters were digitally measured. ANOVA analysis was used to correlate postoperative refractional stability with time of preoperative CL use, fibre diameter and the coefficient of variation (CV) of fibre distance.
14 of 19 lenticules were from patients who had worn CL preoperatively. The cumulative duration of CL wear averaged around 31.2 ± 35.5 thousand hours. Preoperative CL use significantly influenced the postoperative regression: the longer time patients had worn CL, the greater was the regression towards myopia (p = 0.01). Additionally, the morphological parameters collagen fibre diameter (p = 0.09) and CV of fibre distance (p = 0.07) had an impact on regression.
Prolonged CL use and alterations in ultrastructural patterns affected the refractive stability after ReLExflex. Although the pathophysiological relationships between CL use, corneal morphological parameters, and refractive stability are still poorly understood, these findings could potentially be used as prognostic markers for postoperative refraction after ReLExflex.
[Show abstract][Hide abstract] ABSTRACT: Measurement of corneal endothelial cell density is important both for clinical diagnosis as well as clinical studies. Since endothelial cell loss is considered irreversible in humans, even small changes in endothelial cell density are relevant. Therefore it is important to know whether different instruments for endothelial cell density measurements give the same results and can thus be used interchangeably. In this study we compare corneal endothelial cell density and morphometry measurements from two widely used non-contact specular microscopes, the Topcon SP3000P and Konan Noncon Robo SP8000.
Endothelial cell measurements were performed with both the Topcon SP3000P and Konan Noncon Robo SP8000 on 34 eyes of 18 consecutive patients of our cornea clinics with poor image quality being the only exclusion criterion. Images were obtained using the auto-focussing method and manual cell selection. Endothelial cell density (ECD), hexagonal cell ratio (HEX) and coefficient of value (CV) of the endothelial cell layer were calculated by the instruments' built-in software.
ECD values calculated by the Konan were systematically higher than Topcon values: in 94 % of eyes Konan gave a higher value than Topcon, leading to a mean difference in ECD between the instruments of 187 cells/mm(2) (P < 0.001 in paired Wilcoxon test). HEX showed a broad range of values and differed greatly with only weak correlation between the two instruments. CV values for Konan mostly exceeded Topcon values, and only showed a weak correlation between the two instruments as well.
Values for ECD between the Konan and the Topcon do correlate well, but the ECDs calculated by the Konan are systematically higher than Topcon values. Both HEX and CV vary greatly and do not correlate sufficiently. Thus we recommend not to use the Konan and the Topcon instrument interchangeably.
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