Descemet Complex in Fuchs’ Endothelial Cell Corneal Dystrophy

Journal Pre-proof Diagnostic Performance of Three-Dimensional Thickness of Endothelium/Descemet Complex in Fuchs’ Endothelial Cell Corneal Dystrophy Taher Eleiwa, MD, MSc, Amr Elsawy, MSc, Mohamed Tolba, MD, William Feuer, Sonia Yoo, MD, Mohamed Abou Shousha, MD, PhD PII:

S0161-6420(20)30046-4

DOI:

https://doi.org/10.1016/j.ophtha.2020.01.021

Reference:

OPHTHA 11078

To appear in:

Ophthalmology

Received Date: 25 July 2019 Revised Date:

6 January 2020

Accepted Date: 10 January 2020

Please cite this article as: Eleiwa T, Elsawy A, Tolba M, Feuer W, Yoo S, Shousha MA, Diagnostic Performance of Three-Dimensional Thickness of Endothelium/Descemet Complex in Fuchs’ Endothelial Cell Corneal Dystrophy, Ophthalmology (2020), doi: https://doi.org/10.1016/j.ophtha.2020.01.021. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc. on behalf of the American Academy of Ophthalmology

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Title: Diagnostic Performance of Three-Dimensional Thickness of Endothelium/Descemet Complex in Fuchs’ Endothelial Cell Corneal Dystrophy Authors: Taher Eleiwa, MD, MSc1,2, Amr Elsawy, MSc1,3, Mohamed Tolba, MD1,5, William Feuer,1 Sonia Yoo, MD1, Mohamed Abou Shousha MD, PhD1,3,4 1 Bascom Palmer Eye Institute, Miller School of Medicine, University of Miami, Miami, FL 2 Department of Ophthalmology, Faculty of Medicine, Benha University, Egypt 3 Electrical and Computer Engineering, University of Miami, Miami, FL. 4 Biomedical Engineering, University of Miami, Miami, FL. 5 International medical center, Egyptian Armed Forces, Cairo, Egypt Corresponding Author: Mohamed Abou Shousha, MD, FRSC, PhD Bascom Palmer Eye Institute University of Miami Miller School of Medicine 900 NW 17 Street Miami, Florida 33136 [email protected] Declarations: • Ethics approval and consent to participate: This study was approved by the University of Miami Institutional Review Board. • Availability of data and material: The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. • Keywords: Optical coherence tomography, Descemet’s membrane, corneal endothelium, Fuchs endothelial dystrophy. List of Abbreviations: • FECD: Fuchs’ endothelial Corneal Dystrophy; En/DM: Endothelium-Descemet’s complex; En/DMT: Endothelium-Descemet’s complex thickness; TCT: total corneal thickness; HD-OCT: High-definition Optical Coherence Tomography; AS-OCT: Anterior segment Optical Coherence Tomography; ROC: Receiver Operating Characteristics; AUC: Area under curve; PK: Penetrating keratoplasty; EK: Endothelial Keratoplasty; DSAEK: Descemet Stripping Automated Endothelial Keratoplasty; SD: standard deviation; GEE: Generalized Estimating Equations; 2D: two-dimensional; 3D: three-dimensional. Conflicts of Interest and Source of Funding: Taher Eleiwa, Amr Elsawy, William Feuer and Mohamed Tolba- None to declare Sonia Yoo and Mohamed Abou Shousha: United States Non-Provisional Patents (Application No. 8992023 and 61809518), and PCT/US2018/013409. Patents and PCT are owned by University of Miami and licensed to Resolve Ophthalmics, LLC. Mohamed Abou Shousha and Sonia Yoo are equity holders and sit on the Board of Directors for Resolve Ophthalmics, LLC, and are consultants of Avedro. Sonia H. Yoo is a consultant of CARL Zeiss Meditec and Dompe. Financial Support: This study was supported by a NEI K23 award (K23EY026118), NEI core center grant to the University of Miami (P30 EY014801), and Research to Prevent Blindness (RPB). The funding organization had no role in the design or conduct of this research. Authors' contributions: All authors attest that they meet the current ICMJE criteria for Authorship. Running head: Regional Analysis of Endothelium/Descemet’s membrane Thickness in Fuchs’ Dystrophy

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Abstract

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Purpose: To describe the diagnostic accuracy of the regional three-dimensional (3D)

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endothelium/Descemet’s membrane complex thickness (En/DMT) in Fuchs’ endothelial corneal

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dystrophy (FECD), and to determine its potential role as an objective index of disease severity.

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Design: Observational case-control study.

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Participants: 104 eyes of 79 subjects (64 eyes of 41 FECD patients, and 40 eyes of 38 healthy

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age- and gender-matched controls).

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Methods: All participants were imaged using HD-OCT device (Envisu R2210, Bioptigen,

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Buffalo Grove, IL, USA). FECD was clinically classified into early (without edema) and late-

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stage (with edema). Automatic and manual segmentation of the corneal layers was performed

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using a custom-built segmental tomography algorithm to generate 3D-thickness maps of total

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cornea thickness (TCT) and En/DMT of the central 6 mm cornea. Regional En/DMT, regional

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TCT, and central to peripheral total corneal thickness ratio (CPTR) were evaluated and

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correlated to the clinical severity of the disease. Intraclass Correlation Coefficients (ICC), and

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Bland-Altman plots were used to assess the reliability of the repeated measurements in all eyes.

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Main Outcome Measures: CPTR, average En/DMT and TCT of central, paracentral and

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peripheral regions.

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Results: In FECD, there was a significant increase in En/DMT, CPTR, and TCT as compared to

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controls (P < 0.001). For identifying FECD, average En/DMT of paracentral and peripheral

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regions achieved 94% sensitivity and 100% specificity (Cutoffs, 19 µm and 20 µm, respectively),

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while CPTR had 94% sensitivity with a specificity of 73% (Cutoff, 0.97). For discriminating

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early-stage FECD from controls, average En/DMT of central zones achieved 92% sensitivity and

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97% specificity (Cutoff, 18 µm), while CPTR had 90% sensitivity and 88% specificity (Cutoff,

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0.97). The average En/DMT of central, paracentral and peripheral regions was highly correlated

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with FECD clinical stage (Spearman’s rho = 0.813, 0.793, and 0.721; all P < 0.001,

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respectively), compared to (0.672 and 0.481; P < 0.001) for CPTR and mean TCT of paracentral

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zones, respectively. ICC values ranged from 0.98 (En/DMT) to 0.99 (TCT) with a good

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agreement between the automatic and manual measurements.

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Conclusion: Regional 3D-En/DMT is a novel diagnostic tool of FECD that can be used to

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quantify the disease severity with excellent reliability.

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Introduction

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Fuchs endothelial corneal dystrophy (FECD) is one of the most common indications of

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corneal transplantation.1-5 FECD is defined as a progressive bilateral asymmetric disease of the

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corneal endothelium characterized by endothelial cell loss, endothelial barrier disruption,

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thickening of the Descemet’s membrane (DM), and formation of excrescences known as guttae

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that may result in corneal edema and decreased vision.6 When penetrating keratoplasty (PK) was

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considered the only treatment for FECD, traditional grading of the disease, based on the presence

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of clinically detectable corneal edema and extent of guttae, was appropriate. However, the recent

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progress in the surgical and non-surgical interventions for FECD, makes the accurate diagnosis

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of FECD before the development of irreversible ultrastructural changes in the host tissue

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paramount to the best visual outcomes.7-9 Therefore, future trials for FECD should include

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measures of endothelial morphology, endothelial function, and visual impact regardless of the

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intervention.5

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The current diagnostic tools, including slit lamp examination (SLE), corneal pachymetry,

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specular microscopy and confocal microscopy, fall short of detecting the natural course of the

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disease or predicting its progression, especially after cataract surgery. Although subjective

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clinical evaluation using SLE might be the gold standard, SLE does not account for the presence

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of subclinical edema.10 Regarding pachymetry, isolated measurement of central corneal thickness

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and corneal volume is not always representative of corneal edema or disease severity.10 Repp et

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al. described the relative central to peripheral corneal thickening in two meridians, but it is not

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always effective to assess the extent of the disease.10 Furthermore, potential sampling errors with

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narrow-field specular and confocal microscopy, and regional variations between guttate areas

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and visible cells render the corneal measurements inaccurate.11, 12 Recently, Sun et al. reported a

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new classification of FECD severity by evaluating pachymetry map and posterior corneal

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curvature patterns measured with Scheimpflug tomography.4 They reported loss of parallel

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isopachs in ≤42% of FECD eyes without clinical edema compared to ≥81% in FECD eyes

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suspicious for corneal edema.4 However, they also reported the presence of the tomographic

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features of interest in 7% of the control eyes. Thus, it is important to consider coexisting subtle

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corneal pathologies prior to interpreting the tomographic maps.4, 5

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Previously, Abou Shousha et al. used high-definition optical coherence tomography (HD-

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OCT) to characterize endothelium/Descemet’s membrane complex (En/DM) and to measure its

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central thickness in FECD.13 It is also worth mentioning that these measurements were two-

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dimensional (2D), hence, were not representative of the whole cornea and can easily miss an

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ongoing pathology. In this study, we used an automated custom-built segmental tomography

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algorithm to segment the corneal boundaries and generate three-dimensional (3D) thickness

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maps of endothelium/Descemet’s membrane complex (En/DMT), and total corneal thickness

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(TCT) from the captured HD-OCT images of the central 6-mm cornea. Compared to manual

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segmentation, this algorithm has been proven to be able to automatically segment all corneal

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layers in healthy and pathological corneas with as good as accuracy, though with significantly

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less time and significantly better repeatability as well.14, 15 Using 3D-thickness maps, we

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compared the diagnostic performance of regional En/DMT, regional TCT, and the quotient of

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mean central TCT and mean peripheral TCT at 4-6 mm from the center (CPTR) between FECD

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corneas with variable severity to healthy age-matched controls. Moreover, we determined the

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relationship between these parameters and disease severity.

121 122

Materials and Methods:

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Study design and participants

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This study was approved by the University of Miami Institutional Review Board. All

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participants provided written informed consent before enrollment. The study design complied

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with the Health Insurance Portability and Accountability Act (HIPAA), and followed the tenets

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of the Declaration of Helsinki for biomedical research.

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One hundred and four eyes of 79 individuals (64 clinically diagnosed FECD eyes, and 40

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healthy controls) were prospectively and consecutively recruited from June 2018 to September

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2019 at Bascom Palmer Eye Institute, University of Miami. FECD was diagnosed clinically by

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the presence of central or paracentral guttae, with or without clinically detectable edema. Eyes

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were either phakic or pseudophakic with a posterior chamber intraocular lens implant in the

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capsular bag in the studied groups, without any history of ocular inflammation. Subjects were

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excluded from the study if they had glaucoma, ocular hypertension, inflammatory eye diseases,

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ocular surface diseases, and systemic diseases with ocular involvement. In addition, we excluded

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patients with history of ocular surgery (except uneventful cataract surgery with endocapsular

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intraocular lens insertion at least 6 months prior to enrollment), contact lens wear, and the use of

138

topical medications (except artificial tears) or systemic medications that could affect the cornea.

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Because of the asymmetrical nature of the disease between the two eyes of the same patient, all

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FECD eyes were included unless an exclusion criterion was detected. Slit lamp examination was

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performed on each eye by a masked cornea specialist in order to assign and the examined cornea

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into either a healthy cornea or FECD. Moreover, FECD eyes were clinically graded at the slit-

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lamp according to the following guidelines: grade 1: non-confluent guttae; grade 2: presence of

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any area of confluent guttae, but without clinical edema; grade 3: confluent guttae with clinical

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edema; grade 4: edema associated with whitening or haze.16 We categorized early-stage FECD as

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grade 1 and 2 and late-stage FECD as grade 3 and 4.12 Clinical progression was defined as a

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subsequent decision to proceed to endothelial keratoplasty (EK) based on occurrence of

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clinically evident edema associated with visual impairment reported by the patient.4 Later HD-

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OCT imaging and thickness measurements were reviewed if available.

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Test methods

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Image Acquisition and Analysis

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Anterior segment high-definition optical coherence tomography (HD-OCT; Envisu

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R2210, Bioptigen, Buffalo Grove, IL, USA) was performed to each participant, using a 6 mm

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radial scan pattern centered on the corneal vertex. This device has an axial optical resolution of 3

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µm, a transversal width of 6 mm, a corrected depth of 1.58 mm using the approximate refractive

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index for the whole cornea of 1.376,17 and a scanning speed of 32,000 A-scans per second. Each

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participant was asked to look at a central fixation target and the presence of a visible specular

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reflection in all images of the scan confirmed an optimal centration.18, 19 Then, a custom-built

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segmental tomography algorithm was utilized to automatically segment the corneal epithelium,

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Descemet’s membrane and the endothelium. Then, an experienced and masked observer (TE)

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manually corrected the automated segmentation of images from corneas with late-stage FECD

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because the pathological changes in the disease reduce the accuracy of segmenting the En/DM

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boundary by the automated method. In addition, manual segmentation was performed twice by a

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masked trained manual operator [MT] to test the intra-operator repeatability and compare the

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automatic and manual measurements to determine the accuracy of the algorithm. Elsawy et al.

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had described and validated this algorithm against 5 trained manual operators by measuring the

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reproducibility between the manual operators, the reproducibility between the manual and

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automatic methods, and the repeatability of the automatic and manual methods. Additionally, a

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subjective test was performed by 2 corneal specialists to assess both the manual and automatic

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measurements. They disclosed that the automatic measurements were comparable to the manual

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ones with significantly higher repeatability and less running time per image.14 Random Sample

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Consensus (RANSAC)20 method with a polynomial model was used to estimate the corneal layer

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boundaries from candidate boundary points obtained from thresholding the OCT image followed

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by flattening the corneal layers and subsequent vertical projection to detect relative locations of

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Descemet's layer from the endothelium (Figure 1).14 Then, bi-cubic interpolation of the

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segmented different frames of the scan is used to reconstruct the surfaces of the corneal layers.21

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Finally, the inter-surface distances were corrected using 3D ray-tracing algorithm by applying

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the vector form of Snell’s law at the interface between each 2 consecutive layers to generate the

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average 3D thickness values in each region of the bulls-eye map (Figure 2-a).22, 23 The 6 mm

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bulls-eye map was divided into 14 regions; 2 central, 6 paracentral and 6 peripheral (figure 2-b).

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The mean thickness of the central, paracentral and peripheral regions was calculated and used for

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further analysis.

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Diagnostic indices

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Central, paracentral, and peripheral En/DMT were compared to the corresponding

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regional TCT using the 3D-thickness maps. Using custom-built segmental tomography

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algorithm, thickness values are the measured inter-surface distances, in the OCT reflectivity

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profile, between the endothelial and Descemet’s membrane peaks for En/DMT, and between the

188

epithelial and the endothelial peaks for TCT (Figure 1). Besides, CPTR was calculated as the

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quotient of the mean central TCT and the mean peripheral TCT at 4-6 mm zone from the center

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(Table 1).

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Statistical Analysis

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Statistical analyses were performed using SPSS software version 22.0 (SPSS, Chicago,

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IL, USA). Continuous data were summarized with means and standard deviations while

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dichotomous data were summarized with proportions. Comparisons between groups were

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performed using Generalized Estimating Equations (GEE) methods to account for the correlation

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between two eyes of the same patient.24 Residuals of the fitted models were examined to assess

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model performance and Box-Cox methods were used to identify appropriate transformations to

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effect normality for significance testing when necessary.25

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The sensitivity and specificity of regional En/DMT, CPTR and TCT in differentiating

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between studied groups were determined by generating receiver operating characteristic curves

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(ROC). In addition to area under ROC curves (AUCs), we have provided for each parameter

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sensitivities and specificities, and the parameter cutoffs used to create them. To facilitate

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comparisons between parameters, we chose the cutoffs that provided maximum diagnostic

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accuracy for each parameter. For the objective assessment of FECD severity, Spearman rho

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correlation coefficients were used to quantify correlations between thickness parameters and the

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subjective clinical severity of the disease. Pearson correlations between regional En/DMT and

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CPTR were also calculated. Two-sided p-values less than 0.05 were considered to be statistically

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significant.

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We summarized the reliability of manually segmented measurements with the intraclass

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correlation coefficient (ICC).26 An ICC <0.4 constitutes poor agreement, an ICC >0.75

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constitutes excellent reliability, while an ICC between 0.4 and 0.7 represents fair to good

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reliability.26 Agreement between automated and manually segmented measurements was

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assessed with the Bland-Altman method.27 The two manual segmentations were averaged for the

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Bland-Altman analyses.

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Results:

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Participants

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Our study included 104 eyes of 79 participants; the breakdown included 64 eyes of 41

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FECD patients, and 40 eyes of 38 healthy subjects of similar age and gender. Three dimensional-

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En/DM thickness maps were successfully generated from all included eyes.

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Test Results

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Manually segmented measurements demonstrated excellent reliability for both En/DMT

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and TCT with ICCEn/DMT=0.98 and ICCTCT=0.99. Figure 3 displays the Bland-Altman plot of

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automated versus manual measurements for En/DMT and TCT. Regarding En/DMT, the Bland-

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Altman Limits of Agreement (LOA) ranged from -1.2 to +1.3, about 5% of the range of

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measurement. There was no systematic difference between the two techniques (mean = 0.02,

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paired t-test P=0.74) and no evidence of a differential effect related to measurement magnitude.

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For TCT, the Bland-Altman results were similar with LOA ranging from -3.6 to 3.4, about 4% of

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the range of measurement. Similarly, there was no systematic difference between techniques

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(mean = -0.1, paired t-test P=0.57) and no evidence of a differential effect with measurement

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magnitude.

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Table 2 summarizes the different characteristics of both groups. With the exception of

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peripheral TCT, there were highly significant differences between healthy and FECD eyes in

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TCT and En/DMT measurements. Accounting for pseudophakia in these analyses did not change

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the results (Figure 4). Table 3 compares early with late-stage FECD. Age was not statistically

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significant in any of the models (P >0.4). Late-stage FECD eyes had significantly higher central

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TCT and En/DMT in all locations than did early-stage eyes (P ranging from 0.048 to <0.001).

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CPTR did not differ significantly between stages.

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The qualitative and quantitative differences between the healthy and FECD corneas are

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demonstrated in figure 5. In healthy eyes, the En/DM layer was visualized in the HD-OCT as a

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band formed by 2 smooth regular hyper-reflective lines with a hypo-reflective space in between.

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On the other hand, the posterior line had a wavy irregular appearance with areas of focal

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thickenings in FECD corneas. Using 3D-thickness maps, En/DMT, central TCT and CPTR were

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significantly higher in FECD group compared to controls (P<0.001, Table 2).

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Table 4 summarizes the diagnostic performance of regional En/DMT, CPTR, and TCT.

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For identifying FECD, En/DMT parameters had specificity of 100% and sensitivity ≥92%, while

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TCT parameters had specificity of 73% with sensitivities ranging from 77% to 94%. For

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discriminating between healthy and early-stage disease, En/DMT parameters had specificity

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>92% with sensitivity of 92%, while TCT parameters had specificities ranging from 59% to 88%

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with sensitivities ranging from 80% to 90% (Figure 6, Table 4).

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Objective assessment of FECD severity determined by central En/DMT, paracentral

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En/DMT and peripheral En/DMT showed stronger correlations with subjective clinical grading,

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(Spearman’s rho = 0.813, 0.793, and 0.721; all P < 0.001respectively), compared to Spearman’s

253

rho = 0.672 and 0.481 for CPTR and paracentral TCT, respectively (all P <0.001). Peripheral

254

En/DMT was highly correlated with CPTR (R=0.721, P<0.001, Figure 7).

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During the consultation at which HD-OCT images were acquired, 25 late-stage FECD

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eyes were recommended to have EK. All of them had thicker En/DMT than both healthy and

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early-stage FECD eyes. Of the 39 early-stage FECD eyes, ≥6 months follow-up data were

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available for 14 eyes. No eyes were recommended for EK during the initial consultation at which

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HD-OCT images were acquired. Within the next 14 months, 1 eye was recommended to have

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DSAEK due to the development of clinically evident edema and subjective visual impairment

261

(Figure 8).

262

Discussion:

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FECD is a disease of the corneal endothelium with secondary changes in Descemet’s

264

membrane, stroma, sub-basal nerve plexus and the epithelium.28 Ex-vivo studies after corneal

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transplantation, in FECD eyes, showed that Descemet’s membrane thickening, guttae, and

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abnormal endothelium were correlating to clinical corneal edema.6, 13, 29 A better understanding

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of these changes might help determine when and how to intervene, especially as newer

268

treatments enable earlier intervention.9, 30, 31 Currently, with the advent of HD-OCT, it has

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become possible to carry out non-invasive in-vivo imaging to analyze the corneal microstructure

270

at a quasi-histologic level.15, 32 However, there is still a gap bringing this technology to the

271

clinical practice due to the lack of automated analysis software for rapid and accurate

272

quantification of corneal layers with high repeatability.33 To address this gap, our group has

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developed and validated an automated custom-built algorithm to segment OCT B-scans,

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reconstruct the 3D corneal surfaces using bicubic interpolation, and subsequently generate the

275

3D thickness maps.14, 15 Our findings support that 3D-En/DMT has the highest sensitivity and

276

specificity in diagnosing and grading FECD. Excellent reliability was found for repeated

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measures of TCT and En/DMT, with a good agreement between both manual and automatic

278

methods.

279

Clinical diagnosis of FECD is rarely in doubt, but clinical grading of the disease severity

280

is highly confusing,10 suggesting that a more objective metric of severity is mandated. The wide

281

variation in the normal TCT complicates its use, especially, to detect subclinical edema at one

282

point in time.10, 34 Besides, corneal thickness is not constant and can be expected to vary when it

283

is measured at different times of the day.35 Using pachymetry, Repp et al. described the CPTR as

284

an objective index to assess the disease severity.10 However, that meridional CPTR can miss a

285

focal paracentral edema, thus, not always effective in evaluating the severity of the disease.

286

Previously, Abou Shousha et al. had described the cross-sectional central En/DMT in FECD

287

using manual measurement, but they were not able to highlight peripheral localized changes that

288

could be earlier signs of the disease secondary to the 2D measurement. In our study, we used the

289

generated 3D-thickness maps, from HD-OCT scans, to compare the diagnostic performance of

290

regional En/DMT, regional TCT, and calculated CPTR in FECD. This more objective 3D-

291

thickness analysis is more robust than the 2D measurements, since the data are interpreted from a

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larger region of the cornea rather than only 2 meridians; thus, it is less susceptible to missing

293

minor changes in the optical scan.17 Moreover, we used the peripheral regional thickness as an

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internal reference when measuring central thickness in the same cornea to highlight the relative

295

central corneal swelling in early disease.10, 36 In FECD, all regional En/DMT, central TCT, and

296

paracentral TCT were significantly higher than normal, while the TCT in the peripheral 4-6 mm

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zone of the map was not. Based on the area under the ROC curves, 3D-En/DMT parameters

298

provided excellent detection of FECD, followed by 3D-CPTR. In this cohort, age had no

299

significant impact on En/DMT. However, it should be noted that controls and FECD patients

300

recruited for our study were all elderly. Thus, the impact of age on En/DMT in our study might

301

be limited to this age group and not essentially representative of younger subjects.

302

Although there was a very strong positive correlation between increasing subjective

303

grade and regional En/DMT, this relationship between the regional En/DMT and clinical grade

304

should be interpreted with caution, because this subjective grading may not be the ideal method

305

to quantify the severity, and it is credible that corneas with mild edema could be on the threshold

306

of requiring a corneal transplant. Hence, we investigated the objective relationship between the

307

3D-En/DMT and 3D-CPTR; we found the strongest positive correlation for the peripheral

308

En/DMT, followed by paracentral En/DMT and central En/DMT. This correlation could be an

309

indirect indicator of disease progression, suggesting that regional En/DMT changes possibly

310

before the onset of a clinically evident increase in corneal thickness. This study was not able to

311

identify the exact chronological changes in the En/DM, but we hypothesize that these changes

312

might occur before, and contribute to the secondary subclinical edema (Figure 7).

313

Our study is not without limitations. First, although encouraging, the present results stem

314

from a limited number of patients; however, our study demonstrates a substantial, statistically

315

significant ability to discriminate between healthy eyes and FECD eyes as well as healthy and

316

early FECD (Table 4). In this context, we have included the cutoff values and 95% confidence

317

intervals around the AUCs for further studies to replicate our results and bring this tool into

318

clinical practice. Second, imaging was limited to the central 6 mm of the cornea as the tele-

319

centric probe has a reduced axial resolution and signal intensity in peripheral regions.37 Third,

320

the segmental tomography used in the present study significantly contributed to a better

321

characterization of the En/DM complex in FECD eyes; however, possible concomitant changes

322

in Bowman’s layer and the corneal epithelium were not evaluated. Future studies are required to

323

explore the tomographic features of these layers in FECD. The cross-sectional nature of our

324

study is another limitation, thus the long-term changes in En/DM with advancing FECD weren’t

325

examined. Further, the possibility that cases of mild edema were misclassified as the labeling of

326

FECD into early and late stages was reliant on clinically evident edema using slit-lamp

327

examination. Future studies using more objective labelling parameters such as those reported by

328

Sun et al. using Scheimpflug tomography are required not to misclassify mild disease.4

329

Moreover, time of the HD-OCT imaging was not standardized in this cohort and this may

330

contribute to the slightly lower performance TCT based measurements. It has been reported that

331

afternoon central TCT measures were lower compared to values acquired from early morning;38,

332

39

333

observed within their subject groups over the working day40, 41. Interestingly, the slightly higher

334

performance of CPTR in this study could be attributed to the relative central swelling in FECD

335

corneas,36 contrasted with the relative peripheral thickening in healthy ones42. Further studies

336

are required to explore the effect of diurnal variations on the diagnostic performance of both

337

regional TCT and En/DMT in FECD. Finally, we presented quantitative regional analysis of

338

TCT and En/DMT, but we could not address the functional status of the endothelium. Hence,

339

future prospective, longitudinal larger studies using wider field segmental tomography of all

340

corneal layers are warranted to do so.

however, others noted that no time-dependent difference in central TCT measures were

341

In summary, our study disclosed the three-dimensional En/DMT maps as a potential

342

objective diagnostic tool that can be used to grade the severity of FECD in addition to CPTR

343

obtained from the three-dimensional TCT maps and more subjective modalities such as

344

morphological grading. The concurrent increase in En/DMT in early-stage FECD may

345

potentially interact with endothelial function and subsequently play a role in the development of

346

corneal edema. A strong correlation between En/DMT values and FECD severity points to the

347

potential utility of En/DMT in guidance of treatment decisions and prediction for surgical

348

intervention. Finally, further studies by other teams are needed to replicate these results in their

349

own patient populations and allow to objectively distinguish healthy corneas, corneas with guttae

350

but no edema from FECD cases, and from the subclinical edema as well.

351

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References

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31. Okumura N, Koizumi N, Kay EP, et al. The ROCK inhibitor eye drop accelerates corneal endothelium wound healing. Invest Ophthalmol Vis Sci. 2013;54(4):2493-502. 32. Ang M, Baskaran M, Werkmeister RM, et al. Anterior segment optical coherence tomography. Prog Retin Eye Res. 2018;66:132-56. 33. Ang M, Chong W, Huang H, et al. Comparison of anterior segment optical tomography parameters measured using a semi-automatic software to standard clinical instruments. PLoS One. 2013;8(6):e65559. 34. Kopplin LJ, Przepyszny K, Schmotzer B, et al. Relationship of Fuchs endothelial corneal dystrophy severity to central corneal thickness. Arch Ophthalmol. 2012;130(4):433-9. 35. Doughty MJ, Zaman ML. Human Corneal Thickness and Its Impact on Intraocular Pressure Measures: A Review and Meta-analysis Approach. Surv Ophthalmol. 2000;44(5):367-408. 36. Brunette I, Sherknies D, Terry MA, et al. 3-D characterization of the corneal shape in Fuchs dystrophy and pseudophakic keratopathy. Invest Ophthalmol Vis Sci. 2011;52(1):206-14. 37. Podoleanu A, Charalambous I, Plesea L, et al. Correction of distortions in optical coherence tomography imaging of the eye. Phys Med Biol. 2004;49(7):1277. 38. Hirji N, Larke J. Thickness of human cornea measured by topographic pachometry. Am J Optom Physiol Opt. 1978;55(2):97-100. 39. Fujita S. Circadian rhythm of human corneal thickness (author's transl). Nippon Ganka Gakkai Zasshi. 1980;84(9):1232. 40. Bron A, Chapard J, Creuzot-Garcher C, et al. Is corneal thickness measurement reliable and useful? J Fr Ophtalmol. 1999;22(2):160-8. 41. Wolfs RC, Klaver CC, Vingerling JR, et al. Distribution of central corneal thickness and its association with intraocular pressure: The Rotterdam Study. Am J Ophthalmol. 1997;123(6):767-72. 42. Read SA, Collins MJ. Diurnal Variation of Corneal Shape and Thickness. Optom Vis Sci. 2009;86(3):170-80.

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Legends:

448

Figure 1-a: High-definition optical coherence tomography image of a cornea with Fuchs

449

Endothelial Corneal Dystrophy (FECD). The presence of specular reflection (SR) confirms

450

adequate centration. The preset shows a magnified image of the posterior part of the

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corresponding cornea with the red arrows pointing at Descemet’s membrane (DM), and the blue

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arrows at the corneal endothelium (En). Bars are 100 µm.

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Figure 1-b: Segmental tomography of the OCT image in Figure 1-a after flattening of the

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corneal epithelium (Epi) and corneal endothelium (En). Right upper preset shows a magnified

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image of the anterior part of the flattened cornea with the blue line representing the segmented

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anterior boundary of the corneal epithelium (Epi). The left lower preset shows a magnified image

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of the posterior part of the corresponding cornea with the red dashed line representing the

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segmented Descemet’s membrane (DM), and the blue line for the segmented corneal

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endothelium (En).

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Figure 1-c: Vertical projection of the OCT scan lines after flattening of the corneal layers to

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create a reflectivity profile of the OCT image in figure 1-a. The first and last peaks correspond to

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the epithelial (Epi) and endothelial boundaries, respectively. Descemet's membrane (DM) was

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localized as the most prominent peak just before the endothelial (En) peak.

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Figure 2-a: A diagram illustrating the concept of using three-dimensional ray tracing to correct

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the inter-surface distances and generate the thickness maps. The axial distances between the

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initial surface and the uncorrected surfaces (black arrows) represent the optical path length and it

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is converted to geometric distance (red dashed arrows) using the layer refractive index. Three-

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dimensional ray tracing is used at each surface to correct for the refraction in the incident OCT

469

beam by utilizing a generalized vector implementation of Snell’s law at the refractive interface

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between each 2 consecutive layers. Then, the thickness is measured as the shortest distance

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between the initial surface and the corrected consecutive surfaces.

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Figure 2-b: Bulls-eye map of the central 6 mm cornea showing the arrangement of regions for

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quantitative analysis of the layer thickness: Central region (C1, C2) lies within a 2 mm diameter,

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surrounded by 2 concentric paracentral (M1, M2, M3, M4, M5, M6) and outer (O1, O2, O3, O4,

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O5, O6) rings, each with a 2 mm width. N: Nasal; S: Superior; T: Temporal; I: Inferior.

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Figure 3: Bland-Altman plots of automatic versus manually segmented measurements for

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endothelium/Descemet’s membrane complex thickness (En/DMT, left plot), and total corneal

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thickness (TCT, right plot). The difference between the two measurements is represented against

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the mean of them.

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Figure 4: Box-plot distributions showing that there was no statistically significant effect of the

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intraocular lens status (phakic versus pseudophakic) on the changes in the mean regional corneal

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thickness, and median endothelium/Descemet’s membrane complex thickness values between

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the studied groups. It also highlights that there is almost no overlap between FECD eyes and

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healthy controls.

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Figure 5: showing the qualitative and quantitative discrimination between late-stage Fuchs’

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endothelial corneal dystrophy (A), early-stage Fuchs’ endothelial corneal dystrophy (B), and

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healthy cornea (C) with the corresponding color-coded and bulls-eye maps of the three-

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dimensional total corneal thickness (3D-TCT, left column) and endothelium/Descemet’s

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membrane complex thickness (3D-En/DMT, right column). The presets show magnified images

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of the posterior part of the corresponding cornea with the blue arrows pointing at Descemet’s

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membrane (DM), and the yellow arrows at the corneal endothelium (En). In healthy cornea, the

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En/DM layer was visualized in the HD-OCT as a band formed by 2 smooth regular hyper-

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reflective lines with a hyporeflective space in between. In FECD, the posterior line had a wavy

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irregular appearance with areas of focal thickenings. The bulls-eye and color-coded maps of the

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regional TCT and En/DMT demonstrate the quantitative differences between the displayed eyes.

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Note the excellent discrimination between the studied corneas using the 3D-En/DMT maps,

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compared to the 3D-TCT maps.

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Figure 6: Combined receiver operating characteristics (ROC) graph showing substantial increase

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in the area under ROC curve using regional endothelial/Descemet's membrane complex

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thickness (En/DMT) compared to regional total corneal thickness; and central to peripheral total

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corneal thickness ratio (CPTR) in differentiating Fuchs Endothelial Corneal Dystrophy (FECD)

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from healthy cornea (A) and early-stage FECD from healthy corneas (B).

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Figure 7: The top raw shows scatter plots displaying that central, paracentral, and peripheral

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endothelial/Descemet's membrane complex thickness (En/DMT) values have strong positive

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correlation with the subjective clinical grading reported by Adamis et al.16 The bottom raw

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shows scatter plots displaying that central, paracentral, and peripheral endothelial/Descemet's

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membrane complex thickness (En/DMT) values have strong positive linear correlation with

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central to peripheral total corneal thickness ratio (CPTR).

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Figure 8: Three-dimensional total corneal thickness (3D-TCT) and endothelium/Descemet’s

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membrane complex thickness (3D-En/DMT) maps of one eye with early-stage Fuchs Endothelial

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Corneal Dystrophy (FECD) at the first visit (A) and after 14 months (B). At the first visit, the

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cornea did not have clinically evident edema on slit-lamp examination (SLE) as shown in the

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TCT map and OCT image, yet revealed significant thickening of En/DM complex (A). After 14

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months, obvious edema was detected on SLE as shown in the OCT image with subjective visual

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deterioration. The bulls-eye and color-coded maps demonstrate the progressive increase in both

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regional TCT and En/DMT.

Table 1: Diagnostic indices

Diagnostic index Endothelial/Descemet’s Membrane Thickness (En/DMT) Total Corneal Thickness (TCT) Central to peripheral total corneal thickness ratio (CPTR)

Definition The measured inter-surface distance between the endothelial peak and Descemet’s membrane peak in the OCT reflectivity profile using custom-built segmental tomography algorithm. The measured inter-surface distance between the epithelial and the endothelial peaks in the OCT reflectivity profile using custom-built segmental tomography algorithm. The quotient of central TCT and peripheral TCT at 4-6 mm from the center.

FECD clinical grade9 Grade: N (%) of eyes

Phakic eyes Pseudophakic eyes (PC.IOL) Female Male

Number of eyes Gender

Healthy Group N=40

FECD Group N=64

--------------

24 (60%)

1: 18 (28%), 2: 21 (33%), 3: 11 (17%), 4: 14 (22%) 33 (52%)

16 (40%)

31 (48%)

19 (47%) 21 (53%)

40 (63%) 24 (37%)

65 (50-82) years Central TCT (µm) 521±31 Paracentral TCT (µm) 531±32 Peripheral TCT (µm) 548±35 0.95 (0.87CPTR 1.02) Central En/DMT (µm) 16 (15-18) Paracentral En/DMT (µm) 17 (15-19) Peripheral En/DMT (µm) 18 (16-20) Table 2: Characteristics of study groups Age (range) years

•

• •

69 (50-95) years 569±35 577±31 562±39 1.01 (0.941.08) 23 (17-38) 26 (17-37) 29 (20-40)

Difference between group means (95% CI)

Not applicable

p value

p = 0.449* p = 0.078*

4 (-1, +9)

p = 0.118**

48 (32, 63) 45 (31, 60) 13 (-4,+31) 0.06 (0.05, 0.07) 8 (7, 10) 9 (8, 10) 12 (10, 13)

p <0.001** p <0.001** p = 0.124** p <0.001** p <0.001*** p <0.001*** p <0.001***

FECD: Fuchs’ Endothelial Cell Corneal Dystrophy; PC.IOL: Posterior chamber intraocular lens; TCT: Total Corneal thickness; CPTR: the quotient of central TCT and peripheral TCT at 4-6 mm from the center; En/DMT: Endothelial/Descemet’s membrane complex (En/DM) thickness. 95% CI: 95 percent confidence interval on the difference between groups. Values are presented as means ± standard deviation for TCT, and as median (range) for En/DMT, CPTR and age.

* P value is calculated using Generalized Estimating Equations (GEE) with logistic link function and exchangeable correlation matrix. ** P value is calculated using GEE with identity link function and exchangeable correlation matrix.

*** Box-Cox analysis identified inverse power transformations as appropriate for attenuating normality and variance heterogeneity problems. P value calculated on the transformed variable using GEE with identity link function and exchangeable correlation matrix.

FECD group

FECD clinical grade9 Grade: N (%) of eyes Phakic: N (%) of eyes Intraocular lens status Pseudophakic: N (%) of eyes Central TCT (µm) Paracentral TCT (µm)

Early-stage (39 eyes)

Late-stage (25 eyes)

1: 18 (46%) 2: 21 (54%)

3: 11 (44%) 4: 14 (56%)

20 (51%)

13 (52%)

19 (49%)

12 (48%)

559 ± 29 570 ± 27

585 ± 38 589 ± 35

p value* Difference (95% CI)*

Not applicable

0.9

25 (6, 43) <0.001 20 (3, 36) <0.019 18 (0.2, Peripheral TCT (µm) 555 ± 39 572 ± 38 0.048 36) 0.01 CPTR 1.01±0.03 1.02±0.02 (-0.004, <0.138 0.03) Central En/DMT (µm) 21±3 29±5 8 (6, 10) <0.001 Paracentral En/DMT (µm) 23±3 30±4 7 (5, 8) <0.001 Peripheral En/DMT (µm) 27±4 33±4 6 (4, 8) <0.001 Table 3: Comparison of the thickness parameters between the early and late-stage FECD eyes. •

FECD: Fuchs’ Endothelial Cell Corneal Dystrophy

•

TCT: Total Corneal thickness.

•

En/DMT: Endothelial/Descemet’s membrane complex (En/DM) thickness.

•

CPTR: the quotient of central TCT and peripheral TCT at 4-6 mm from the center.

•

95% CI: 95 percent confidence interval on the difference between groups.

Values are presented as means ± standard deviation for TCT, and as median (range) for En/DMT. * Means compared using Generalized Estimating Equations (GEE) with identity link function and exchangeable correlation matrix. Differences, 95% confidence intervals, and p-values were obtained from the GEE model. Age was included as a covariate though it was not statistically significant in any of the models. Box-Cox transformations not necessary for comparing these two groups of eyes with disease.

Table 4: Receiver Operating Characteristic (ROC) curve data which represent the diagnostic performance of regional endothelial/Descemet thickness (En/DMT), and regional total corneal thickness (TCT) in diagnosing Fuchs’ endothelial corneal dystrophy (FECD). All AUC P-values <0.001 for all parameters. In addition to AUCs, we have provided for each parameter sensitivities and specificities, and the parameter cutoffs used to create them.

Detection of FECD (n=104 eyes) Central TCT

Paracentral TCT

CPTR

Central En/DMT

Paracentral En/DMT

Peripheral En/DMT

AUC±SE

0.834±0.039

0.856±0.036

0.937±0.027

0.978±0.012

0.987±0.008

0.996±0.003

( 95% CI)

(0.76, 0.91)

(0.79, 0.93)

(0.88, 0.99)

(0.95, 1.00)

(0.97, 1.00)

(0.99, 1.00)

Sensitivity

77%

81%

94%

92%

94%

94%

Specificity

73%

73%

73%

100%

100%

100%

Cutoff value

538 µm

548 µm

0.97

18 µm

19 µm

20 µm

Discrimination between healthy and early-stage FECD (n=79 eyes) Central TCT

Paracentral TCT

CPTR

Central En/DMT

Paracentral En/DMT

Peripheral En/DMT

AUC±SE

0.801±0.048

0.834±0.044

0.904±0.04

0.972±0.016

0.979±0.013

0.993±0.005

( 95% CI)

(0.71, 0.90)

(0.75, 0.92)

(0.83, 0.98)

(0.94, 1.00)

(0.95, 1.00)

(0.98, 1.00)

Sensitivity

80%

80%

90%

92%

92%

92%

Specificity

70%

59%

88%

97%

97%

93%

Cutoff value

551 µm

560 µm

0.97

18 µm

19 µm

20 µm

•

FECD = Fuchs’ Endothelial Cell Corneal Dystrophy; AUC = area under the curve; SE = standard error; En/DMT= endothelial/Descemet thickness; TCT= total corneal thickness; CPTR= the quotient of central TCT and peripheral TCT at 4-6 mm from the center; 95% CI: 95 percent confidence interval on the difference between groups. Specificity, sensitivity, and cutoff values are chosen to maximize total diagnostic accuracy (minimize total number of errors).

Precis Regional three-dimensional Endothelium/Descemet’s membrane complex thickness is an objective diagnostic tool that can be potentially used to grade the severity of Fuchs’ Endothelial Cell Corneal Dystrophy.