CLINICAL SCIENCE
Central and Peripheral Corneal Endothelial Cell Analysis
With Slit-Scanning Wide-Field Contact Specular
Microscopy: Agreement With Noncontact Specular
Microscopy
Jinhee Lee, MD, Yosai Mori, MD, PhD, Miyuki Ogata, Keiichiro Minami, PhD, and
Kazunori Miyata, MD, PhD
Purpose: The prospective case series aimed to examine the
agreement between the use of a slit-scanning contact specular
microscope and a noncontact specular microscope in corneal
endothelial cell (CEC) analysis and to evaluate the differences
between the central and peripheral regions in normal corneas.
Methods: After confirming normal corneal endothelium with slit-
lamp microscopy, CEC images of 56 eyes of 56 cataractous patients
were analyzed in the central and 4 peripheral regions using a slit-
scanning contact specular microscope. A noncontact specular
microscope was used for the analysis in the central region. The
endothelial cell density (ECD), the percentage of hexagonal shape
cells (HEX), and the coefficient of variation (CV) in the central
region were compared. Differences between central and peripheral
CECs were also evaluated.
Results: The mean ECD was 2778 cell/mm2 and was not
different from the results using the noncontact specular micro-
scope (2736 cell/mm2, P = 0.051). There was a significant
correlation (P , 0.001, R2 = 0.72). The analysis of HEX resulted
in larger values with the slit-scanning contact microscope
(53.13% vs. 48.89%, P , 0.001), whereas there was no
difference in the CV (38.48 vs. 38.04, P = 0.56). On comparing
the central and peripheral regions, there was no significant
difference in the ECD, whereas significant differences were
found in the superior region in the HEX and CV (P , 0.001) and
in the nasal region in CV (P = 0.023).
Conclusions: The analysis of ECD with the use of the slit-scanning
contact specular microscope did not differ from the noncontact
specular microscope, and the results demonstrated no difference
between the central and peripheral ECD.
Received for publication January 28, 2019; revision received March 14, 2019;
accepted March 16, 2019. Published online ahead of print May 3, 2019.
From the Miyata Eye Hospital, Miyazaki, Japan.
The authors have no funding or conflicts of interest to disclose.
Correspondence: Kazunori Miyata, MD, PhD, Miyata Eye Hospital, 6-3
Kurahara-cho, Miyakonojo, Miyazaki, 885-0051, Japan (e-mail: kmiyata@
miyata-med.ne.jp).
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Cornea  Volume 38, Number 9, September 2019
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Key Words: corneal endothelial cell, peripheral, slit-scanning,
cataract
(Cornea 2019;38:1137–1141)
T he specular microscope has been used for evaluating the
morphology and function of corneal endothelial cells
(CECs) noninvasively, and the endothelial cell density
(ECD), the percentage of hexagonal shape cells (HEX), and
coefficient of variation (CV) are determined for analysis.1
Conventionally, a noncontact specular microscope is com-
prehensively used for the analysis of the central CECs,1,2
which are essential for corneal clarity. However, continuous
effects on the peripheral CECs, which are observed after
implantations of an anterior supported intraocular lens3,4 or
shunt devices5,6 in the anterior chamber, result in an
accelerated decrease of the central CECs. Although the
evaluation of peripheral CECs is important, it is difficult
and impractical to examine peripheral CECs using conven-
tional noncontact specular microscopes. A contact specular
microscope2,7–9 and confocal microscope10,11 have been used
for peripheral CEC analysis; however, these require experi-
enced skill and are not suitable for use in clinical practice.
Recently, a slit-scanning wide-field contact specular
microscope has been developed.12 By using contact and
confocal microscopy image capturing, wide-field and high-
quality CEC images can be analyzed. Peripheral regions are
also observable by placing the cone at the place of interest.
Nevertheless, the agreements with conventional noncontact
specular microscopes have not been examined. The objectives
of this prospective and observational case series study were to
examine the agreement between the slit-scanning wide-field
contact specular microscope and a noncontact specular
microscope in the central CEC analysis and to evaluate the
analysis in the peripheral regions in normal corneas.
MATERIALS AND METHODS
This study was approved by the institutional review
board of Miyata Eye Hospital and was performed according
to the tenets of the Declaration of Helsinki. Written informed
consent was obtained from the patients before examinations.
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Lee et al
Cataractous patients who visited Miyata Eye Hospital from
September 2017 to February 2018 were enrolled. Normal
corneal endothelium was confirmed in a slit-lamp microscopy
examination. Patients with the history of ocular surgery,
trauma, contact lens wear, corneal endothelial dysfunction, or
diseases affecting the CEC were excluded. For bilateral
cataract patients, the eye first examined was selected for
analysis. The sample size required for examining the linear
correlation between 2 measurements in the central region was
31 eyes for a significance level of 0.05 and detection power of
0.9 when the R2 was 0.25. The study was planned to exceed
the calculated sample size.
CEC Analysis
CEC images were obtained using a slit-scanning
contact specular microscope, CellChek C (version 1.0.0.5;
Konan Medical, Nishinomiya, Japan), and noncontact spec-
ular microscope, CellChek SL (version 30.00; Konan Med-
ical), on the same day. The former specular microscope used
alternative confocal microscopy optics, which was similar to
the conventional confocal microscope except for the use of
a slit-scanning technology, for enabling wider field observa-
tion of the cornea. After administering topical anesthesia
using 0.4% oxybuprocaine, the probe with an applanation
diameter of 4 mm was placed at the apex of the cornea for
capturing sequential images of the central CEC (width of
0.65 mm and a height of 0.48 mm). In the same manner,
images at 4 peripheral regions (superior, nasal, inferior, and
temporal) were captured. The distance from the center to the
peripheral regions was approximately 5 mm. The noncontact
specular microscope was also used for obtaining the central
CEC image (width of 0.24 mm and a height of 0.40 mm) by
targeting the corneal apex for making the CEC images within
the relevant images with the slit-scanning contact specular
microscope. The image capturing process was repeated until
acceptable quality images were confirmed. Figure 1 shows the
typical CEC images obtained using the 2 specular micro-
scopes: the image sizes were 3.25 times different.
From the captured CEC images, the ECD, HEX, and
CV of individual cell areas were analyzed. The analysis was
performed by a single experienced examiner (M.O.) for mini-
mizing the variance of the analyzed results.1 From the
sequential images, an image in which all CECs could be
FIGURE 1. Typical images obtained using a slit-scanning
contact (left) and noncontact (right) specular microscope in
the central cornea. The image sizes were 0.65 · 0.48 and 0.24
· 0.40 mm, respectively.
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Cornea  Volume 38, Number 9, September 2019
sharply identified was selected. After tracing individual CEC
borders automatically, the examiner corrected improper
tracing manually in an area of 0.60 · 0.40 mm. Then, the
ECD, HEX, and CV were obtained using CellChek software.
For the noncontact measurement, the same procedure and
analysis were conducted for an image with sharper CEC
borders. The numbers of CEC used for analysis were also
recorded for the 2 measurements.
Statistical Analysis
The agreement between the 2 specular microscopes was
examined with the central ECD, HEX, and CV. After
confirming their normality using the Shapiro–Wilk test, the
differences and correlations were examined using a paired t
test and linear regression analysis, respectively.
The intraobserver test was examined for ECD, HEX,
and CV using the intraclass correlation coefficient (ICC).
Bland–Altman analysis13 was also used for evaluating the
95% limits of agreement. In the slit-scanning contact specular
microscope measurements, ECD, HEX, and CV were com-
pared between the center and the peripheral regions using
a paired t test with the Holm correction.
Statistical analyses were performed using R version
3.4.1. P values less than 0.05 were considered statistically
significant.
RESULTS
Fifty-six eyes of 56 patients (mean age: 61.2 years)
were included in the study. Table 1 shows the demographic
data of the subjects. For the sample size, the power in
detecting the correlation between the 2 measurements was
anticipated to be over 0.99. The number of CECs used for
analysis with the slit-scanning contact specular microscope
was 4.1 times more than that with the noncontact specular
microscope, although the CEC image size was 2.5 times
larger. In 5 of 56 images (8.9%), the ECDs were calculated
with CECs being less than 100 in the noncontact
measurement.
Table 2 shows the analysis results of the central CEC
images. The mean ECD measured with the slit-scanning
contact specular microscope was not significantly different
from that measured with the noncontact specular microscope
(P = 0.051, paired t test), and the mean difference (1.61%)
was neglectable. Significant and strong correlation, high ICC,
and the 95% limits of agreement indicated the agreement in
the central ECD analyses. The mean HEX was significantly
larger with the slit-scanning contact specular microscope (P
, 0.001). There was no significant difference in the mean CV
between the 2 specular microscopes (P = 0.56). The R2 and
ICC in the HEX and CV were relatively low.
Figure 2 shows the ECD, HEX, and CV measured with
the slit-scanning contact specular microscope at the central
and peripheral regions, respectively. In the ECD, there was no
significant difference between them (P = 1.00, paired t test
with Holm correction). The HEXs in the superior region were
significantly lower than those in the central region (P ,
0.001). The CVs in the superior and nasal regions were
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Cornea  Volume 38, Number 9, September 2019 Central and Peripheral Corneal Endothelial Cell

endothelium, it was hard to identify the sampling differences

TABLE 1. Demographic Data of Subjects
Specular Microscope Slit-Scanning Contact Noncontact
N 56 eyes of 56 patients
Age, yr 61.2 6 9.3 [26–74]
Gender Male: 23, female: 33
Eye Right: 22, left: 34
Keratometry, diopter 44.00 6 1.79 [40.50–48.75]
Image size, mm 0.65 · 0.48 0.24 · 0.40
No. CECs used for analysis 550 6 84 [343–726] 133 6 20 [77–164]
Mean 6 SD [range].
significantly higher than in the central region (P , 0.001,
0.023, respectively).
DISCUSSION
In the current study, the central ECD, HEX, and CV
were measured with slit-scanning contact and noncontact
specular microscopes in cataract eyes. The central ECD
values were in good agreement with each other, which
coincided with the previous comparison between noncon-
tact specular and conventional contact microscopes.2,9 The
comparison between noncontact and confocal measure-
ments by Kitzmann et al14 showed reproducibility in the
ECD analysis. Together with the previous findings, it was
demonstrated that measurement using the slit-scanning
contact specular microscope was not different from con-
ventional ECD examination. Furthermore, the number of
CECs used for analysis increased with the use of the slit-
scan contact measurement. The mean ECD indicated that
about 84.5% of CECs in the wide-field images were used
for analysis. On the other hand, only 50% of CECs were
used in the noncontact measurement. The confocal optics
and wide-field measurement could analyze more CECs with
higher resolution. Further assessments are necessary to
confirm this.
In the analysis of the HEX and CV, agreement was not
obtained. One of the factors might be the differences in the
quality and size of CEC images, which were superior in the
slit-scanning contact microscope compared with the non-
contact specular microscope (Fig. 1). Although CEC images
of the slit-scanning contact microscope could include the
smaller images of the noncontact microscope, there was a risk
of sampling differences. As the current study analyzed normal
from the CEC images.
The ECDs in the peripheral regions were not signifi-
cantly different from those in the central regions. These
results coincided with the previous results.7,12,15 However,
the distribution of ECD has not been confirmed yet. In some
studies, higher ECD was observed in the peripheral
region.8,16 It is anticipated that higher peripheral ECDs in
normal corneas would be advantageous for Descemet mem-
brane endothelial keratoplasty and Descemet stripping auto-
mated endothelial keratoplasty.17 Wiffen et al18 reported that
ECD was lower in the peripheral region. The methodology
used in the previous studies has not been established: various
instruments, such as the use of contact specular micros-
copy,7,8,12,16,18 confocal microscopy,15 and the analysis area
sizes8,16 and ECD analysis methods, were used previously.
Further assessments of peripheral ECD using advanced and
wider field specular microscope are important.
The current results showed lower HEX and higher CV
of the superior region. Peripheral morphology in more than 4
regions had rarely been evaluated.19 Previous reports showed
that HEX of the inferior region15 was lower and CV of the
temporal region was higher than the center region,18 whereas
Amann et al8 showed that CV was not different between the
central and peripheral regions. In the current study, both
HEX and CV of the central region did not agree well
between the 2 measurements. The previous studies ad-
dressed that the morphology of the CEC could alter with
the devices used.19,20 Hence, it was hard to compare these
with the previous results.
There were some limitations to this study. First, the
specular microscope examinations were taken once for each
measurement. The SD of ECD was 1.6% to 1.8% in
measurements using the noncontact specular microscope.21
With the high correlation, ICC suggested no difference in
the central ECD analysis; however, interobserver testing is
required for confirming the repeatability. Next, in the
current comparison for normal endothelium, it was hard to
confirm that both CEC images were overlapped. The image
positions in the central cornea examination would be close
by aiming the probe position at the corneal apex, and the
images of the slit-scanning contact specular microscope
were sufficiently larger. Further evaluation of endothelium
with abnormal morphology would be required. In addition,
interobserver testing is necessary for confirming the repeat-
ability of the newly developed specular microscope. Last,
the exclusion criteria consisted of diseases that may affect
CECs. Subjects who are primary angle-closure suspects
with iridotrabecular contact,22,23 suspended debris in the

TABLE 2. Analysis Results of Central CECs

Slit-Scanning Contact, Noncontact, 95% Limits
Mean 6 SD [Range] Mean 6 SD [Range] P Correlation ICC of Agreement
ECD 2778 6 300 cells/mm2 [2104 to 3426] 2736 6 242 cells/mm2 [2203 to 3425] 0.051 R2 = 0.72 (P , 0.001) 0.82 2271 to 357 cells/mm2
HEX 53.13% 6 5.44% [41 to 65] 48.89% 6 7.40% [35 to 72] ,0.001 R2 = 0.093 (P = 0.022) 0.24 211.07% to 19.54%
CV 38.48 6 5.50 [35 to 73] 38.04 6 5.17 [35 to 74] 0.56 R2 = 0.18 (P = 0.0012) 0.42 211.71 to 10.82

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Lee et al
FIGURE 2. Comparison of ECD, HEX, and CV in the central
and the peripheral regions by using a paired t test with the
Holm correction. There was no significant difference in ECD
between these (P = 1.00). The HEX in the superior region were
significantly lower than those in the central regions (P ,
0.001). The CVs in the superior and nasal regions were sig-
nificantly higher than those in the central region (P , 0.001,
0.023, respectively).
aqueous humor and breakdown of the blood–aqueous bar-
rier after laser peripheral iridotomy,24,25 and exfoliation
syndrome,26 which might differentiate the CEC and its
distribution from normal eyes, might be included. Addi-
tional examinations would be necessary to diagnose corneal
disorders before recruiting subjects.
In conclusion, the slit-scanning contact specular micro-
scope was not different from the noncontact specular
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Cornea  Volume 38, Number 9, September 2019
microscope for ECD analysis. There was no difference
between the central and peripheral ECD in patients with
cataract. The current results demonstrated that the slit-
scanning contact specular microscope examination would be
effective and advanced for central and peripheral CEC
examinations.
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