Figure 1 shows the flow diagram of the study design. Two hundred and sixty eyes of 191 patients were classified into 14 groups (Groups 0-13) based on ECD-Ce at 250 cells/mm2 intervals. We excluded 15 subjects from four groups due to the small sample sizes of the subgroups. Consequently, we included 245 eyes from 183 patients in the final analysis.
Intra-individual reproducibility of data was studied in six right eyes of six control subjects by repeating examinations five times, and the intraclass correlation coefficient (ICC) and 95% confidence interval (CI), and the coefficient of variation (%) were calculated and is listed in Table 1. The ICC (1,5) of the ECD, CV, and HEX by the automated method (0.980, 0.777, and 0.706, respectively) and ECD-Ce (0.973) were greater than 0.7, showing good reproducibility, while those for HEX (ICC (1,5) =0.514) and CV (ICC (1,5) =0.412) by the center method showed fair reproducibility (Table 1).
Table 2 presents the demographic characteristics of the glaucoma and control groups. The type of glaucoma based on guidelines [8] was 78 primary open-angle glaucoma, 16 primary angle-closure glaucoma, 49 neovascular glaucoma, 42 exfoliation glaucoma, and 8 childhood glaucoma. Two eyes with traumatic glaucoma underwent blunt trauma 12 months and 7 years before the measurement date, respectively.
Figure 2 shows the number of participants in each group. Corneal endothelial damage was graded according to the Japanese corneal society guidelines [9] as follows: normal, ≥ 2000 cells/mm2; grade 1, 1000-2000 cells/mm2; and grade 2, 500-1000 cells/mm2. When the ECD was low, ECD-Au was greater than ECD-Ce. None of the 245 eyes was grade 2 using the automated method (ECD-Au less than 1000 cells/mm2), while 44 eyes were classified as Grade 2 (ECD-Ce 500-1000 cells/mm2) using the center method. When difference in ECD-Ce and ECD-Au was plotted against ECD-Ce, a regression formula obtained was; ECD-Au - ECD-Ce=1028.5-0.397*ECD-Ce, r=-0.815, and R2=0.664, P<0.001 (Figure 3), The difference was 0 when ECD-Ce=2593 /mm2.
Table 3 and Figure 4 present the ECD data for each group. Compared with ECD-Ce, ECD-Au was significantly higher in Groups 1-8 (ECD-Ce: 500-2499 cells/mm2). The P value was < 0.001 for Groups 1-7 and P = 0.006 for Group 8. The respective between-method differences in the median ECD values were 860, 731, 558, 476, 275, 198, 176, and 127 cells/mm2, with the differences being larger in low cell-density groups. When the ECD-Ce exceeded 2500 cells/mm2, there were no between-method differences in ECD. This suggests that the critical ECD value where the automated method becomes reliable is 2500 cells/mm2.
Figure 5 presents the Bland-Altman plots of the between-method comparisons of the ECD value, which showed that the ECD-Au overestimated the ECD by a mean difference of +328 cells/mm2 (P < 0.001). Most of the values were not included within the limits of agreement. The differences greatly exceeded the upper limit of agreement for the low-ECD groups. An inclination in the scatter of the dots is indicative of a proportional bias.
Figure 6a presents a scatter graph showing the correlations between ECD-Au and ECD-Ce in glaucomatous eyes. The correlation coefficient was r = 0.91, R2 = 0.8205, P < 0.001, and the linear regression equation was ECD-Au = 0.6033 × ECD-Ce + 1028.5 cells/mm2. Although there were significant between-method differences in the ECD values, there was a close correlation in the ECD values of both methods. When the correlation between the ECD-Ce and ECD-Au was studied in the 36 control eyes, the correlation coefficient was high again (r= 0.861, R2=0.742, P<0.001). The linear regression equation was ECD-Au = 0.5871 × ECD-Ce + 1103.1 cells/mm2. This equation was nearly equal to that in the glaucomatous eyes (Fig 6b). Thus, close correlation and overestimation of ECD in eyes with low ECD is a common finding in both glaucoma and control subjects.
Table 4 shows the age, IOP, CCT, CV, and HEX values in each group. The Mann-Whitney U test showed significant between-method differences in the CV and HEX values. In all groups, the automated method yielded higher CV values and lower HEX values than those obtained using the center method (P < 0.001).
Figure 7 shows an increasing tendency of the between-method CV deviation with decreasing ECD-Ce values. Figure 8 demonstrates the increased between-method differences in HEX with decreased ECD-Ce. Between-method differences in CV and HEX were negatively correlated with ECD-Ce (r= -0.49, P < 0.001 and r = -0.25, P < 0.001, respectively). This indicates that the differences in CV and HEX were larger at lower ECD values.
Figure 9 shows representative corneal endothelial images where significant between-method differences in the ECD values were observed. Images obtained from
a 70-year-old-man in Group 2 revealed ECD values of 867 cells/mm2 and 1669 cells/mm2 and a CV of 28% and 51% using the center and automated methods, respectively. The automated method misidentified the cell border and erroneously divided one large cell into many small cells (Image A). Image B was obtained from a 65-year-old woman in Group 7 with ECD values of 2128 cells/mm2 and 2315 cells/mm2 by the center and automated methods, respectively. Even though the between-method deviation in the ECD decreased with increasing ECD-Ce, the between-method difference was still significant in Group 7 (P < 0.001).