BASIC INVESTIGATION
Possible Sampling Error in Corneal Specular Microscopy:
Can Central Endothelial Data Generated by Specular
Microscope Represent the Real Clinical Condition of the
Whole Cornea?
Arthur Buffara van den Berg, MD,*†§ Ricardo Holzchuh, MD, PhD,†‡ and Richard Yudi Hida, MD†‡§
Purpose: To estimate the minimum number of cells required to
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obtain reliable data in a specular microscope, which could possibly
represent the real clinical condition of the corneal endothelium.
Methods: A cross-sectional study of 122 eyes of 61 individuals
submitted to noncontact specular microscope was conducted. Data
generated by the manufacturer’s software were uploaded to specific
statistical software for sampling relative error calculation. When
relative error was above 5%, new images were acquired and more
cells counted until the desired relative error was reached. Data
analyzed in this study for the desired relative error were number of
cells marked by the examiner for each eye (marked cells), number of
cells used for data analysis (analyzed cells), endothelial area used for
analysis, sampling error, and absolute number of images used for
each eye.
Results: The average number of marked cells required to obtain a
relative sampling error of less than 5% was 425.2 6 102.2 cells. The
average number of analyzed cells used by the specular microscope to
generate the data was 247.4 6 51.6 cells. The average endothelial area
of the analyzed cells was 0.43 6 0.08 mm2. The mean sampling error
was 3.7% 6 0.6%, and an average of 2.95 6 0.74 images was needed
to obtain a relative sampling error of less than 5%.
Conclusions: We conclude that, theoretically, a minimum of 425.2
cells from 2.95 images must be marked to obtain reliable results,
which could possibly represent the real endothelial clinical condition
of the whole cornea.
Key Words: specular microscopy, cornea, endothelium
(Cornea 2020;39:779–781)
Received for publication August 12, 2019; revision received December 12,
2019; accepted December 25, 2019. Published online ahead of print
February 22, 2020.
From the *HOLondrina- Hospital de Olhos, São Paulo, Brazil; †Department of
Ophthalmology, Santa Casa de Sao Paulo, São Paulo, Brazil; ‡Department
of Ophthalmology, Universidade de São Paulo (USP), São Paulo, Brazil;
and §Department of Ophthalmology, Universidade Federal de São Paulo
(UNIFESP), São Paulo, Brazil.
The authors have no funding or conflicts of interest to disclose.
A. B. van den Berg and R. Holzchuh contributed equally to this work.
Correspondence: Richard Y. Hida, MD, Department of Ophthalmology,
Universidade Federal de São Paulo (UNIFESP), Rua Afonso de Freitas,
488 apt 61, São Paulo, Brazil 04006-052 (e-mail: ryhida@gmail.com).
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Cornea  Volume 39, Number 6, June 2020
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I ntraocular surgical trauma can lead to loss of corneal
endothelial cells. Studies evaluating intraocular surgical
techniques and drugs generally use specular microscopy to
determine the corneal endothelial cell density (ECD).1–4
Several types of specular microscopes are available in
the market, each with its own calibration, methods of image
capture, magnifications, and software for the processing of the
collected semiological data.
The main shortcoming of the current technology is that
no commercially available device can specify the number of
cells required to obtain a representative sample of corneal
endothelial cells. Thus, clinical management is commonly
based on inaccurate data which is not truly representative of
the endothelial mosaic because very little area and cells are
being used to represent the corneal endothelium profile.
Most specular microscope devices and software perform
counting of the visible cells (automated or not), ignore all
peripheral cells of the marked area, and analyze, mathemati-
cally, the distance and number of dots around each dot. There
is a great possibility for some sampling and counting bias
involved in all data generated from the software of the device.
Previous reports have shown an increase in ECD during
follow-up.5–13 ECD is unlikely to increase after surgical
trauma, suggesting a possible sampling error during examina-
tion, as described in earlier studies.3,14,15
The purpose of this study was to estimate the minimum
number of cells required to obtain reliable data (relative error
less than 5%) using a specular microscope, which could possibly
represent the real clinical condition of the corneal endothelium.
MATERIAL AND METHODS
This cross-sectional study was approved by the Institu-
tional Review Board and Research Ethics Committee of our
institution (University of São Paulo). Informed consent was
obtained from all participants after the nature and possible
consequences of the study had been explained in detail.
One hundred twenty-two eyes of 61 individuals (30 men,
63.9 6 7.9 years old) indicated for cataract surgery were
randomly selected and submitted to corneal specular micros-
copy at the cataract outpatient clinic of our institution.
The exclusion criteria were any corneal disease (eg corneal
guttata, dystrophy, and iridocorneal endothelial syndrome),
history of ocular trauma, use of contact lenses, glaucoma, ocular
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van den Berg et al
surface changes, structural changes of the eyelids (blepharoptosis,
entropion, and ectropion), uveitis, history of any ocular surgery,
diabetes mellitus, and any inappropriate or abnormal endothelial
cell image during examination.
Endothelial images were captured with a noncontact
semiautomatic high-magnification specular microscope (Konan
Noncon Robo SP-8000, with KSS-300 software). The device
captures endothelial images automatically from the 2-mm
central area of the cornea randomly as the images are being
recognized by the software. All data acquisition, processing,
and endothelial tissue sampling were performed by a single
examiner. Routine endothelial data were collected as control
for each specular microscopy image: ECD, coefficient of
variation, mean cell area (mm2), and percentage of hexagonal
cells (HEX).
Each endothelial cell was marked in the center with a dot
by using a mouse, followed by marking adjacent cells
according to the center-to-center method.15,16 Each dot was
placed at the center of each cell. All possible cells were marked
for each specular image. For this study, the absolute total
number of cells marked by the examiner were considered
marked cells. Manufacturer’s software automatically excludes
all peripheral cells of the image, so only cells completely
surrounded by other cells were included for data analysis and
considered in this study as analyzed cells.17
To calculate the sampling error, data generated by
manufacturer’s software were analyzed using a statistical
software (Cells Analyzer; Technical, Brazil) designed specif-
ically to analyze the relative error for endothelial cell count-
ing.18,19 One or more images were captured depending on data
generated by the software. For this study, the confidence
interval was set at 95%, and the sampling relative error was set
at less than 5% (P , 0.05).
When relative error was above 5%, indicating insuffi-
cient sample size, new images were acquired and more cells
counted until the desired relative error was reached. For this
study, a relative error less than 5% indicated a sufficient
sample size.
To estimate the minimum number of cells required to
obtain relative error less than 5%, the following outcome
measurements were analyzed: 1) absolute number of cells
marked by the examiner for each eye (marked cells), 2) absolute
number of cells used for data analysis (after the manufacturer’s
software has discarded the peripheral cells) (analyzed cells), 3)
endothelial area used for analysis (mm2), 4) sampling error (%),
and 5) absolute number of images used for each eye (for relative
error less than 5%).
RESULTS
The average number of marked cells required to obtain a
relative sampling error of less than 5% was 425.2 6 102.2 cells.
The average number of analyzed cells used by the specular
microscope to generate the data (counted cells) was 247.4 6 51.6
cells. This can be an indication that the manufacturer’s device can
discard most of the marked cells for a reliable calculation.
The average endothelial area of the analyzed cells was
0.43 6 0.08 mm2 with the mean sampling error of 3.7%
6 0.6%. The average of 2.95 6 0.74 images was needed to
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Cornea  Volume 39, Number 6, June 2020
obtain a relative sampling error of less than 5%. The mean
control ECD was 2395.3 6 294.3 cells/mm2, the mean total cell
area was 423.6 6 51.0 mm2, the coefficient of variation was
0.40 6 0.04, and HEX was 54.7% 6 4.1%.
DISCUSSION
Corneal specular microscopy has a number of uses in
clinical and surgical practice.3,20,21 For example, it is useful in
the identification of transient and chronic changes in endothelial
cell morphology in contact lens users, surgical procedures, and
donated corneas.3,22–25 However, results obtained by a specular
microscopy have limited reproducibility, reliability, and val-
idity. Differences between devices (image quality, specular area
of analysis, calibration, and number of marked and analyzed
cells) constitute a potential source of inconsistency and bias,
already shown by other authors.15,26,27 Furthermore, no device
provides population reference ranges per age group or indicates
how many cells are required to confer statistical validity.
According to Laing et al., 30 counted endothelial cells are
required to obtain accurate results,2 whereas others have
proposed at least 75 27 or 100 cells28 per evaluation. Binder
et al29 proposed to increase the sample by counting the
maximum number of cells per image and using 3 images from
different regions (central and paracentral) of the cornea in
each evaluation.
In our study, we included the greatest possible number of
counted cells per image, capturing as many images as
necessary to reach the desired level of sampling error
(#5%), as specified by the software (Cells Analyzer), with a
95% confidence interval. When more than one image was
required, we included all the cells identified in each image.
Currently available specular microscopes use only some of the
marked cells in the analysis, especially when automated cell
marking is programed. Many cells are excluded by the
manufacturer’s software because the center-to-center method
requires the counted cells to be fully surrounded by other
cells,17 even in normal corneas. Thus, when one cell is marked
along with the 6 adjacent cells, the result of the analysis is
expressed as the area of a 6-sided cell. In other words, 7 cells
are used to determine the area of one. The smaller the number
of marked cells, the smaller the percentage of counted cells and
the greater the percentage of excluded cells.
Most specular microscopes uses 70 cells for every 100
marked cells for data analysis in both manual and automated
systems. This eventual corneal endothelium analysis possibly
is not a true representative sample of endothelial cell profile. In
some studies, fewer than 50 marked cells were used6,8,9,30 or
no information on marked cells was provided.5,7,10–12
Additional studies with different devices are necessary to
determine the percentage of cells excluded by each type of
equipment and calculate how many marked cells are needed for
reliable results in normal eyes. Further studies must be
performed to understand the impact of marked cells on abnormal
corneas after we understand the normal endothelial patterns
or profile.
One of the most relevant limitations of this study is the
analysis of only normal eyes with normal ECD. However,
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Cornea  Volume 39, Number 6, June 2020
other studies are being performed to analyze the best ways to
quantify the number of counted cells in abnormal corneas.
Our results revealed a small sampling error (3.7% 6
0.6%) when approximately 425 cells were marked manually or
automatically in 2 or 3 images; however, this quantity may not
be suitable in our clinical practice on a daily routine basis.
Further devices or software must be designed to provide a
reliable endothelial cell profile.
We conclude that a minimum of approximately 425.2
6 102.2 cells from 2.95 6 0.74 images must be marked to
obtain reliable results in a specular microscope, which could
possibly represent the real endothelial clinical condition of
the whole cornea.
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