Article
Reliability of manual measurements of corneal thickness obtained from healthy canine eyes using spectral-domain optical coherence tomography (SD-OCT) Anthony F. Alario, Christopher G. Pirie
Abstract The purpose of this study was to manually measure corneal thickness in canine eyes using a spectral-domain optical coherence tomography (SD-OCT) device and to assess intra- and inter-observer reliability of this technique. Twenty healthy dogs with a mean age of 4.7 y were examined. A 6-mm corneal pachymetry protocol was carried out by 1 operator using 1 SD-OCT device in both eyes of each animal. Measurements were obtained manually and in duplicate by 2 independent investigators (. 24 h apart), using the built-in caliper function. Measurements included epithelial thickness (ET), non-epithelial thickness (NET), and central corneal thickness (CCT). The overall mean ET, NET, and CCT for all eyes examined were 72.3 6 4.6 mm, 538.9 6 42.5 mm, and 611.2 6 40.3 mm, respectively. There was no significant difference in ET, NET, or CCT based on the eye examined [oculus dexter (OD) versus oculus sinister (OS)], age, or gender of the animal. There was no significant difference in replicate measurements of ET, NET, or CCT done by the same operator, although a small but significant difference was noted between operators for ET measurements only. The mean difference in ET between operators was 0.6 mm (P = 0.03). The coefficient of variation ranged from 0.5% to 9.27% and intraclass correlation coefficient ranged from 0.35 to 0.97. Based on these results, manual measurements of corneal thickness in canine eyes using a portable SD-OCT device provided ET, NET, and CCT measurements with clinically acceptable intra- and inter-observer reliability.
Résumé L’objectif de la présente étude était de mesurer manuellement l’épaisseur de la cornée des yeux canins en utilisant un appareil à tomographie par cohérence optique du domaine spectral (SD-OCT) et d’évaluer la fiabilité intra- et inter-observateur de cette technique. Vingt chiens en santé d’un âge moyen de 4,7 ans furent examinés. Un protocole de pachymétrie a été mené par un opérateur utilisant un appareil SD-OCT dans les deux yeux de chaque animal. Les mesures ont été obtenues manuellement et en duplicata par deux chercheurs indépendants (. 24 h de délai), en utilisant la fonction de pied à coulisse incluse. Les mesures incluaient l’épaisseur épithéliale (ET), l’épaisseur non-épithéliale (NET), et l’épaisseur au centre de la cornée (CCT). Les moyennes globales d’ET, de NET et de CCT pour tous les yeux examinés étaient respectivement de 72,3 6 4,6 mm, 538,9 6 42,5 mm, et 611,2 6 40,3 mm. Il n’y avait aucune différence significative des valeurs de ET, NET, ou CCT selon l’œil examiné (œil droit versus œil gauche), âge, ou sexe de l’animal. Il n’y avait aucune différence significative dans les mesures répétées de ET, NET, ou CCT faites par le même opérateur, et une petite mais significative différence fut notée entre les opérateurs pour les mesures de ET seulement. La différence moyenne dans les mesures de ET entre les opérateurs était de 0,6 mm (P = 0,03). Le coefficient de variation variait de 0,5 % à 9,27 % et le coefficient de corrélation intra-classe variait de 0,35 à 0,97. En fonction de ces résultats, la mesure manuelle de l’épaisseur de la cornée des yeux de chien à l’aide d’un appareil SD-OCT portatif fournit des données de ET, NET, et CCT avec une fiabilité intra- et inter observateur qui est cliniquement acceptable. (Traduit par Docteur Serge Messier)
Introduction Until recently, clinical evaluation of the cornea in vivo was limited to visual inspection using slit lamp biomicroscopy. High-resolution ultrasound biomicroscopy (UBM) and confocal microscopy have now made more detailed examinations of the cornea possible (1–4).
Unfortunately, these imaging modalities require corneal contact and therefore risk inadvertent damage. Such contact with the corneal surface may also result in altered tissue morphology and imprecise measurements of epithelial thickness (5). Subsequent technological advancements in imaging techniques, however, have led to the development of optical coherence tomography (OCT).
Department of Clinical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA. Address all correspondence to Dr. Chris Pirie; telephone: (508) 839-5395; fax: (508) 887-4363; e-mail: [email protected] This article was presented as a poster at the 43rd Annual Meeting of the American College of Veterinary Ophthalmologists in Portland, Oregon, USA in October 2012. Received April 18, 2013. Accepted August 19, 2013. 2014;78:221–225
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Figure 1. Optical coherence tomography (OCT) scan of a normal canine cornea. Note the demarcation between the epithelium, stroma, and Descemet’s membrane/endothelium. Caliper measurements are shown where a = epithelial thickness, b = non-epithelial thickness, and c = central corneal thickness.
This non-contact imaging modality captures high-resolution, crosssectional images of the cornea by measuring optical reflections (6). Several studies have validated this modality as a highly reliable and repeatable method for measuring corneal and epithelial thickness in both normal and diseased corneas of humans (1,5,7–10). While OCT is gaining popularity in the field of veterinary ophthalmology, normative data for epithelial and corneal thickness measurements using this imaging modality are still lacking. The purpose of this study was to report epithelial, non-epithelial, and central corneal thickness measurements of healthy canine corneas using a portable spectral-domain optical coherence tomography (SD-OCT) device. The intra- and inter-observer reliability of this device was also evaluated.
Materials and methods Animals Twenty client-owned dogs from the Foster Hospital for Small Animals at the Tufts Cummings School of Veterinary Medicine were used for this study. This study was approved by the Clinical Science Review Committee of the Tufts Cummings School of Veterinary Medicine and conformed to the Association for Research in Vision and Ophthalmology’s statement for the use of animals in vision research. Informed consent was obtained from each animal’s owner before enrollment in the study. All animals received a complete physical and ophthalmologic examination by a board-certified veterinary ophthalmologist (CG Pirie) and were deemed to be free from ocular and systemic disease.
Procedures Animals were placed and maintained in sternal recumbency using gentle manual restraint. A 6-mm corneal pachymetry protocol scan was carried out using the Optovue iVue SD-OCT (Optovue, Freemont, California, USA) and a supplemental Cornea/Anterior Module (CAM) attachment on both eyes of each animal by 1 operator. This protocol consists of 8 radial scans (1024 A scans each), 6 mm in length. All scans were taken by aligning the aiming circle at the center of the pupil. The Optovue iVue SD-OCT software has an internal quality control score for each corneal scan. Only scans with a quality score of . 27, centered on the corneal vertex, and free of motion artifact were accepted for data collection. All measurements were obtained from 1 high-resolution B scan of each eye imaged.
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Table I. Mean corneal thickness measurements, including the 95% confidence interval (CI) and coefficient of variation (CV) for each operator Mean thickness User (mm) 1/2 SD ET Operator 1 72.95 1/2 3.73 Operator 2 71.63 1/2 4.10
CCT 95% CI (mm)
CV %
72.12 to 73.78 70.71 to 72.54
3.32 2.83
NET Operator 1 Operator 2
528.94 to 547.73 529.77 to 549.26
0.75 0.50
538.34 1/2 42.02 539.51 1/2 43.58
CCT Operator 1 611.46 1/2 40.14 602.49 to 620.44 Operator 2 610.91 1/2 40.90 601.77 to 620.06 SD — standard deviation; CCT — central corneal thickness; ET — epithelial thickness; NET — non-epithelial thickness.
0.53 0.51
Measurements were generated manually by 2 independent investigators using the caliper function integrated into the OCT software (Figure 1). Each investigator obtained measurements in duplicate (. 24 h apart). Measurements included epithelial thickness (ET), non-epithelial thickness (NET), and central corneal thickness (CCT). All recorded measurements were analyzed to determine both intraand inter-observer reliability.
Statistical analysis The data, which included patient signalment, eye being imaged, operator, ET, NET, and CCT, were entered into Excel 2010 (Microsoft, Redmond, Washington, USA). Statistical analysis was done using commercial software (SAS Version 9.2; SAS Institute, Cary, North Carolina, USA). A mixed effects model was used to examine relationships between corneal measurements and age, gender, observer, replicate, and eye examined. A P-value of , 0.05 was considered to be statistically significant. To test repeatability and reproducibility, the coefficient of variation (CV) 3 100 and intraclass correlation coefficient (ICC) were calculated. Agreement between operators was further investigated using Bland-Altman plots and by calculating the 95% limits of agreement (LoA) of the ET, NET, and CCT measurements between users (11,12).
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Table II. Mean difference in corneal thickness measurements between operators, including intraclass correlation coefficient (ICC), coefficient of variation (CV), and limits of agreement (LoA)
Average NET Operator 1 and 2 (mm)
Difference in ET Operator One–Two (mm)
Average ET Operator 1 and 2 (mm)
Results Twenty dogs (40 eyes) with a mean age of 4.7 1/2 2.5 y (from 9 mo to 10 y) were used in this study. The study population consisted of 11 castrated males, 3 intact males, 5 spayed females, and 1 intact female. Although various breeds were used, many were mixed breed dogs (n = 8). The overall mean ET, NET, and CCT measurements for all eyes examined were 72.3 1/2 4.6 mm, 538.9 1/2 42.5 mm, and 611.2 1/2 40.3 mm, respectively. There was no significant difference in ET, NET, or CCT based on the eye examined [oculus dexter (OD) versus oculus sinister (OS)], age, or gender of the animal. There was no significant difference in ET, NET, or CCT between replicate measurements done by the same operator. A small but significant difference was noted between operators for ET only. The mean difference in ET between operators was 0.6 mm (P = 0.03). The mean ET, NET, and CCT for each operator, including the 95% confidence interval (CI) and CV, are listed in Table I. The mean differences between operators in corneal thickness measurements, including the ICC and CV, are presented in Table II. The LoA and Bland-Altman plot for each corneal measurement were constructed using the collected data (Table II and Figures 2 to 4). The constructed Bland-Altman plots (Figures 2 to 4) demonstrate a random distribution of observations, with the majority falling between the LoA, which indicates that the LoA analysis is reliable (Table II).
Figure 3. Bland-Altman plot demonstrating differences in canine nonepithelial thickness (NET) measurements between 2 operators. Mean difference (solid line), in addition to the upper and lower limits of agreement (dashed lines), are represented.
Difference in CCT Operator One–Two (mm)
Figure 2. Bland-Altman plot demonstrating differences in canine epithelial thickness (ET) measurements between 2 operators. Mean difference (solid line), in addition to the upper and lower limits of agreement (dashed lines), are represented.
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Limits of agreement Width (lower to upper) (mm) 13.40 (27.30 to 6.11) 15.20 (29.93 to 5.25) 18.51 (210.57 to 7.93) NET — non-epithelial
Difference in NET Operator One–Two (mm)
Mean difference (mm) 1 to 2 1/2 SD ICC CV (%) ET 20.59 1/2 3.42 0.35 9.27 NET 22.34 1/2 3.87 0.97 1.40 CCT 21.32 1/2 4.72 0.97 1.51 SD — standard deviation; ET — epithelial thickness; thickness; CCT — central corneal thickness.
Average CCT Operator 1 and 2 (mm)
Figure 4. Bland-Altman plot demonstrating differences in canine central corneal thickness (CCT) measurements between 2 operators. Mean difference (solid line), in addition to the upper and lower limits of agreement (dashed lines), are represented.
Discussion The results of this study provide normative data regarding the ET, NET, and CCT of eyes of non-sedated dogs using a portable SD-OCT device. Furthermore, the results demonstrate that this SD-OCT system and the manual measuring technique used in this study provide excellent intra- and inter-observer reliability for NET
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and CCT measurements. Although more variability was noted in the ET measurements, specifically between users, clinically acceptable intra- and inter-observer reliability was also obtained. The overall mean canine ET measurement obtained in this study (72.3 mm 1/2 4.6 mm) was thicker than what has been demonstrated in humans. The mean thickness of the human corneal epithelium is reported to range from 48.3 to 58.6 mm depending on the individual study and imaging modality used (1,4,5,8). Similarly, it was noted that NET and CCT measurements obtained in the current study were thicker than values obtained in humans using SD-OCT. The mean NET and CCT of canine eyes in this study was 538.9 mm 1/2 42.5 mm and 611.2 mm 1/2 40.3 mm respectively, whereas, the reported mean NET and CCT in human eyes ranges from 456.7 to 465.2 mm and 519 to 530 mm, respectively (1,13). The discrepancy between ET, NET, and CCT measurements in the current study and previous human studies represents species differences and should be considered by researchers using dogs as an animal model for corneal diseases. Similarly, the overall mean ET and NET measurements obtained in this study were thicker than those obtained in a recent study evaluating anesthetized dogs (n = 8). Using an SD-OCT device, Famose reported mean ET and NET to be 55 mm and 480 mm, respectively (14). Values for CCT of the canine cornea have been well-documented, ranging from 535 to 598 mm using various imaging modalities (3,14–17). These values are comparable to the results of the current study. Variability in published measurements of central corneal thickness may reflect differences in the populations of animals examined or in the method by which measurements were acquired. As recently reported by our group, while CCT measurements using an ultrasonic pachymeter were highly correlated to those obtained using a SD-OCT device, it was noted that the velocity of sound employed by the ultrasonic pachymeter affected the magnitude of the differences obtained using these 2 modalities (15). The SD-OCT system used in this study demonstrated excellent intra- and inter-observer reliability for manually measuring the NET and CCT. There was no statistically significant difference between replicates for either operator and the CV for each user was below 1%. Comparing inter-observer reliability, there was no significant difference in measurements for NET or CCT between operators and the measurements between users agree almost perfectly (ICC . 0.95). Regarding ET measurements, intra-observer reliability was excellent. There was no statistically significant difference between replicates for either operator and the CV for each user was below 5%. Comparing inter-observer reliability, there was a small but statistically significant difference between users and the ICC was substantially lower than the NET and CCT measurements. Despite the lower ICC values, the width of the LoA for ET was narrow, with 95% of observations from 1 observer likely to fall within 7 mm of the other. This variation demonstrates a clinically acceptable inter-observer reliability for this device and is comparable to that found in human studies (1). Several factors may have contributed to the variability in the manual measurements of corneal thickness obtained in this study, including variation in the location of the measurement from the true corneal vertex and differences in the subjective delineation between corneal layers. Another factor to consider is the increments by which the caliper function measures. In the current format, a
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change of 1 unit of measure using the caliper function equates to a 4-mm difference. For thicker structures such as the NET or CCT, a difference of a single unit of measure between examinations and/or examiners, is clinically insignificant (0.65% of the mean CCT). This difference becomes proportionally larger (5% of the mean ET) for thinner structures, such as the corneal epithelium, however, which may explain the relatively lower reliability of manual measurements found for ET in this study. Further advancements in the measurement algorithm may help reduce this variability. For example, the addition of automated vertex identification, computer-assisted segmentation of corneal layers, and a more detailed caliper with increments of 0.5 mm or less would likely improve consistency between measurements. The primary limitation of the current study was the small number of animals examined. Despite this, however, the results provide evidence that manual corneal thickness measurements with SD-OCT are highly reliable both within and between observers. Although the use of non-sedated animals could be considered a limitation of this study, the results demonstrating clinically acceptable intra- and inter-user reliability would suggest that this was not a significant factor. Additionally, stringent quality control criteria were adhered to, for example, any scan showing evidence of motion artifact, poor corneal vertex, or reduced image quality was rejected and only high quality scans were used for corneal measurements. In summary, this study provides normative data regarding ET, NET, and CCT measurements in healthy canine eyes using a portable SD-OCT device. Furthermore, this device demonstrated excellent intra- and inter-operator repeatability, with clinically acceptable differences in measurements between operators.
Acknowledgments The authors thank Dr. Bruce Barton for his help with statistical analysis. Thanks also to Optovue in Freemont, California, USA for providing the Optovue iVue SD-OCT device on loan.
References 1. Prakash G, Agarwal A, Mazhari AI, et al. Reliability and reproducibility of assessment of corneal epithelial thickness by Fourier domain optical coherence tomography. Invest Ophthalmol Vis Sci 2012;53:2580–2585. 2. Nolan W. Anterior segment imaging: Ultrasound biomicroscopy and anterior segment optical coherence tomography. Curr Opin Ophthalmol 2008;19:115–121. 3. Kafarnik C, Fritsche J, Reese S. In vivo confocal microscopy in the normal corneas of cats, dogs and birds. Vet Ophthalmol 2007;10:222–230. 4. Reinstein DZ, Archer TJ, Gobbe M, Silverman RH, Coleman DJ. Epithelial thickness in the normal cornea: Three-dimensional display with Artemis very high-frequency digital ultrasound. J Refract Surg 2008;24:571–581. 5. Francoz M, Karamoko I, Baudouin C, Labbe A. Ocular surface epithelial thickness evaluation with spectral-domain optical coherence tomography. Invest Ophthalmol Vis Sci 2011;52: 9116–9123.
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6. Huang D, Swanson EA, Lin CP, et al. Optical coherence tomography. Science 1991;254:1178–1181. 7. Li Y, Tan O, Brass R, Weiss JL, Huang D. Corneal epithelial thickness mapping by Fourier-domain optical coherence tomography in normal and keratoconic eyes. Ophthalmology 2012;119: 2425–2433. 8. Sin S, Simpson TL. The repeatability of corneal and corneal epithelial thickness measurements using optical coherence tomography. Optom Vis Sci 2006;83:360–365. 9. Correa-Perez ME, Lopez-Miguel A, Miranda-Anta S, IglesiasCortinas D, Alio JL, Maldonado MJ. Precision of high definition spectral-domain optical coherence tomography for measuring central corneal thickness. Invest Ophthalmol Vis Sci 2012;53: 1752–1757. 10. Haque S, Jones L, Simpson T. Thickness mapping of the cornea and epithelium using optical coherence tomography. Optom Vis Sci 2008;85:E963–976. 11. Bunce C. Correlation, agreement, and Bland-Altman analysis: Statistical analysis of method comparison studies. Am J Ophthalmol 2009;148:4–6. 12. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307–310.
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13. Ishibazawa A, Igarashi S, Hanada K, et al. Central corneal thickness measurements with Fourier-domain optical coherence tomography versus ultrasonic pachymetry and rotating Scheimpflug camera. Cornea 2011;30:615–619. 14. Famose F. Assessment of the use of spectral domain optical coherence tomography (SD-OCT) for evaluation of the healthy and pathological cornea in dogs and cats. Vet Ophthalmol 2013; 17:12–22. 15. Alario AF, Pirie CG. Central corneal thickness measurements in normal dogs: A comparison between ultrasound pachymetry and optical coherence tomography. Vet Ophthalmol 2013; E-pub ahead of print: DOI:10.1111/vop.12074. 16. Gilger BC, Whitley RD, McLaughlin SA, Wright JC, Drane JW. Canine corneal thickness measured by ultrasonic pachymetry. Am J Vet Res 1991;52:1570–1572. 17. Park YW, Jeong MB, Kim TH, et al. Effect of central corneal thickness on intraocular pressure with the rebound tonometer and the applanation tonometer in normal dogs. Vet Ophthalmol 2011;14:169–173.
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Reliability of manual measurements of corneal thickness obtained from healthy canine eyes using spectral-domain optical coherence tomography (SD-OCT) Anthony F. Alario, Christopher G. Pirie
Abstract The purpose of this study was to manually measure corneal thickness in canine eyes using a spectral-domain optical coherence tomography (SD-OCT) device and to assess intra- and inter-observer reliability of this technique. Twenty healthy dogs with a mean age of 4.7 y were examined. A 6-mm corneal pachymetry protocol was carried out by 1 operator using 1 SD-OCT device in both eyes of each animal. Measurements were obtained manually and in duplicate by 2 independent investigators (. 24 h apart), using the built-in caliper function. Measurements included epithelial thickness (ET), non-epithelial thickness (NET), and central corneal thickness (CCT). The overall mean ET, NET, and CCT for all eyes examined were 72.3 6 4.6 mm, 538.9 6 42.5 mm, and 611.2 6 40.3 mm, respectively. There was no significant difference in ET, NET, or CCT based on the eye examined [oculus dexter (OD) versus oculus sinister (OS)], age, or gender of the animal. There was no significant difference in replicate measurements of ET, NET, or CCT done by the same operator, although a small but significant difference was noted between operators for ET measurements only. The mean difference in ET between operators was 0.6 mm (P = 0.03). The coefficient of variation ranged from 0.5% to 9.27% and intraclass correlation coefficient ranged from 0.35 to 0.97. Based on these results, manual measurements of corneal thickness in canine eyes using a portable SD-OCT device provided ET, NET, and CCT measurements with clinically acceptable intra- and inter-observer reliability.
Résumé L’objectif de la présente étude était de mesurer manuellement l’épaisseur de la cornée des yeux canins en utilisant un appareil à tomographie par cohérence optique du domaine spectral (SD-OCT) et d’évaluer la fiabilité intra- et inter-observateur de cette technique. Vingt chiens en santé d’un âge moyen de 4,7 ans furent examinés. Un protocole de pachymétrie a été mené par un opérateur utilisant un appareil SD-OCT dans les deux yeux de chaque animal. Les mesures ont été obtenues manuellement et en duplicata par deux chercheurs indépendants (. 24 h de délai), en utilisant la fonction de pied à coulisse incluse. Les mesures incluaient l’épaisseur épithéliale (ET), l’épaisseur non-épithéliale (NET), et l’épaisseur au centre de la cornée (CCT). Les moyennes globales d’ET, de NET et de CCT pour tous les yeux examinés étaient respectivement de 72,3 6 4,6 mm, 538,9 6 42,5 mm, et 611,2 6 40,3 mm. Il n’y avait aucune différence significative des valeurs de ET, NET, ou CCT selon l’œil examiné (œil droit versus œil gauche), âge, ou sexe de l’animal. Il n’y avait aucune différence significative dans les mesures répétées de ET, NET, ou CCT faites par le même opérateur, et une petite mais significative différence fut notée entre les opérateurs pour les mesures de ET seulement. La différence moyenne dans les mesures de ET entre les opérateurs était de 0,6 mm (P = 0,03). Le coefficient de variation variait de 0,5 % à 9,27 % et le coefficient de corrélation intra-classe variait de 0,35 à 0,97. En fonction de ces résultats, la mesure manuelle de l’épaisseur de la cornée des yeux de chien à l’aide d’un appareil SD-OCT portatif fournit des données de ET, NET, et CCT avec une fiabilité intra- et inter observateur qui est cliniquement acceptable. (Traduit par Docteur Serge Messier)
Introduction Until recently, clinical evaluation of the cornea in vivo was limited to visual inspection using slit lamp biomicroscopy. High-resolution ultrasound biomicroscopy (UBM) and confocal microscopy have now made more detailed examinations of the cornea possible (1–4).
Unfortunately, these imaging modalities require corneal contact and therefore risk inadvertent damage. Such contact with the corneal surface may also result in altered tissue morphology and imprecise measurements of epithelial thickness (5). Subsequent technological advancements in imaging techniques, however, have led to the development of optical coherence tomography (OCT).
Department of Clinical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA. Address all correspondence to Dr. Chris Pirie; telephone: (508) 839-5395; fax: (508) 887-4363; e-mail: [email protected] This article was presented as a poster at the 43rd Annual Meeting of the American College of Veterinary Ophthalmologists in Portland, Oregon, USA in October 2012. Received April 18, 2013. Accepted August 19, 2013. 2014;78:221–225
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Figure 1. Optical coherence tomography (OCT) scan of a normal canine cornea. Note the demarcation between the epithelium, stroma, and Descemet’s membrane/endothelium. Caliper measurements are shown where a = epithelial thickness, b = non-epithelial thickness, and c = central corneal thickness.
This non-contact imaging modality captures high-resolution, crosssectional images of the cornea by measuring optical reflections (6). Several studies have validated this modality as a highly reliable and repeatable method for measuring corneal and epithelial thickness in both normal and diseased corneas of humans (1,5,7–10). While OCT is gaining popularity in the field of veterinary ophthalmology, normative data for epithelial and corneal thickness measurements using this imaging modality are still lacking. The purpose of this study was to report epithelial, non-epithelial, and central corneal thickness measurements of healthy canine corneas using a portable spectral-domain optical coherence tomography (SD-OCT) device. The intra- and inter-observer reliability of this device was also evaluated.
Materials and methods Animals Twenty client-owned dogs from the Foster Hospital for Small Animals at the Tufts Cummings School of Veterinary Medicine were used for this study. This study was approved by the Clinical Science Review Committee of the Tufts Cummings School of Veterinary Medicine and conformed to the Association for Research in Vision and Ophthalmology’s statement for the use of animals in vision research. Informed consent was obtained from each animal’s owner before enrollment in the study. All animals received a complete physical and ophthalmologic examination by a board-certified veterinary ophthalmologist (CG Pirie) and were deemed to be free from ocular and systemic disease.
Procedures Animals were placed and maintained in sternal recumbency using gentle manual restraint. A 6-mm corneal pachymetry protocol scan was carried out using the Optovue iVue SD-OCT (Optovue, Freemont, California, USA) and a supplemental Cornea/Anterior Module (CAM) attachment on both eyes of each animal by 1 operator. This protocol consists of 8 radial scans (1024 A scans each), 6 mm in length. All scans were taken by aligning the aiming circle at the center of the pupil. The Optovue iVue SD-OCT software has an internal quality control score for each corneal scan. Only scans with a quality score of . 27, centered on the corneal vertex, and free of motion artifact were accepted for data collection. All measurements were obtained from 1 high-resolution B scan of each eye imaged.
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Table I. Mean corneal thickness measurements, including the 95% confidence interval (CI) and coefficient of variation (CV) for each operator Mean thickness User (mm) 1/2 SD ET Operator 1 72.95 1/2 3.73 Operator 2 71.63 1/2 4.10
CCT 95% CI (mm)
CV %
72.12 to 73.78 70.71 to 72.54
3.32 2.83
NET Operator 1 Operator 2
528.94 to 547.73 529.77 to 549.26
0.75 0.50
538.34 1/2 42.02 539.51 1/2 43.58
CCT Operator 1 611.46 1/2 40.14 602.49 to 620.44 Operator 2 610.91 1/2 40.90 601.77 to 620.06 SD — standard deviation; CCT — central corneal thickness; ET — epithelial thickness; NET — non-epithelial thickness.
0.53 0.51
Measurements were generated manually by 2 independent investigators using the caliper function integrated into the OCT software (Figure 1). Each investigator obtained measurements in duplicate (. 24 h apart). Measurements included epithelial thickness (ET), non-epithelial thickness (NET), and central corneal thickness (CCT). All recorded measurements were analyzed to determine both intraand inter-observer reliability.
Statistical analysis The data, which included patient signalment, eye being imaged, operator, ET, NET, and CCT, were entered into Excel 2010 (Microsoft, Redmond, Washington, USA). Statistical analysis was done using commercial software (SAS Version 9.2; SAS Institute, Cary, North Carolina, USA). A mixed effects model was used to examine relationships between corneal measurements and age, gender, observer, replicate, and eye examined. A P-value of , 0.05 was considered to be statistically significant. To test repeatability and reproducibility, the coefficient of variation (CV) 3 100 and intraclass correlation coefficient (ICC) were calculated. Agreement between operators was further investigated using Bland-Altman plots and by calculating the 95% limits of agreement (LoA) of the ET, NET, and CCT measurements between users (11,12).
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Table II. Mean difference in corneal thickness measurements between operators, including intraclass correlation coefficient (ICC), coefficient of variation (CV), and limits of agreement (LoA)
Average NET Operator 1 and 2 (mm)
Difference in ET Operator One–Two (mm)
Average ET Operator 1 and 2 (mm)
Results Twenty dogs (40 eyes) with a mean age of 4.7 1/2 2.5 y (from 9 mo to 10 y) were used in this study. The study population consisted of 11 castrated males, 3 intact males, 5 spayed females, and 1 intact female. Although various breeds were used, many were mixed breed dogs (n = 8). The overall mean ET, NET, and CCT measurements for all eyes examined were 72.3 1/2 4.6 mm, 538.9 1/2 42.5 mm, and 611.2 1/2 40.3 mm, respectively. There was no significant difference in ET, NET, or CCT based on the eye examined [oculus dexter (OD) versus oculus sinister (OS)], age, or gender of the animal. There was no significant difference in ET, NET, or CCT between replicate measurements done by the same operator. A small but significant difference was noted between operators for ET only. The mean difference in ET between operators was 0.6 mm (P = 0.03). The mean ET, NET, and CCT for each operator, including the 95% confidence interval (CI) and CV, are listed in Table I. The mean differences between operators in corneal thickness measurements, including the ICC and CV, are presented in Table II. The LoA and Bland-Altman plot for each corneal measurement were constructed using the collected data (Table II and Figures 2 to 4). The constructed Bland-Altman plots (Figures 2 to 4) demonstrate a random distribution of observations, with the majority falling between the LoA, which indicates that the LoA analysis is reliable (Table II).
Figure 3. Bland-Altman plot demonstrating differences in canine nonepithelial thickness (NET) measurements between 2 operators. Mean difference (solid line), in addition to the upper and lower limits of agreement (dashed lines), are represented.
Difference in CCT Operator One–Two (mm)
Figure 2. Bland-Altman plot demonstrating differences in canine epithelial thickness (ET) measurements between 2 operators. Mean difference (solid line), in addition to the upper and lower limits of agreement (dashed lines), are represented.
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Limits of agreement Width (lower to upper) (mm) 13.40 (27.30 to 6.11) 15.20 (29.93 to 5.25) 18.51 (210.57 to 7.93) NET — non-epithelial
Difference in NET Operator One–Two (mm)
Mean difference (mm) 1 to 2 1/2 SD ICC CV (%) ET 20.59 1/2 3.42 0.35 9.27 NET 22.34 1/2 3.87 0.97 1.40 CCT 21.32 1/2 4.72 0.97 1.51 SD — standard deviation; ET — epithelial thickness; thickness; CCT — central corneal thickness.
Average CCT Operator 1 and 2 (mm)
Figure 4. Bland-Altman plot demonstrating differences in canine central corneal thickness (CCT) measurements between 2 operators. Mean difference (solid line), in addition to the upper and lower limits of agreement (dashed lines), are represented.
Discussion The results of this study provide normative data regarding the ET, NET, and CCT of eyes of non-sedated dogs using a portable SD-OCT device. Furthermore, the results demonstrate that this SD-OCT system and the manual measuring technique used in this study provide excellent intra- and inter-observer reliability for NET
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and CCT measurements. Although more variability was noted in the ET measurements, specifically between users, clinically acceptable intra- and inter-observer reliability was also obtained. The overall mean canine ET measurement obtained in this study (72.3 mm 1/2 4.6 mm) was thicker than what has been demonstrated in humans. The mean thickness of the human corneal epithelium is reported to range from 48.3 to 58.6 mm depending on the individual study and imaging modality used (1,4,5,8). Similarly, it was noted that NET and CCT measurements obtained in the current study were thicker than values obtained in humans using SD-OCT. The mean NET and CCT of canine eyes in this study was 538.9 mm 1/2 42.5 mm and 611.2 mm 1/2 40.3 mm respectively, whereas, the reported mean NET and CCT in human eyes ranges from 456.7 to 465.2 mm and 519 to 530 mm, respectively (1,13). The discrepancy between ET, NET, and CCT measurements in the current study and previous human studies represents species differences and should be considered by researchers using dogs as an animal model for corneal diseases. Similarly, the overall mean ET and NET measurements obtained in this study were thicker than those obtained in a recent study evaluating anesthetized dogs (n = 8). Using an SD-OCT device, Famose reported mean ET and NET to be 55 mm and 480 mm, respectively (14). Values for CCT of the canine cornea have been well-documented, ranging from 535 to 598 mm using various imaging modalities (3,14–17). These values are comparable to the results of the current study. Variability in published measurements of central corneal thickness may reflect differences in the populations of animals examined or in the method by which measurements were acquired. As recently reported by our group, while CCT measurements using an ultrasonic pachymeter were highly correlated to those obtained using a SD-OCT device, it was noted that the velocity of sound employed by the ultrasonic pachymeter affected the magnitude of the differences obtained using these 2 modalities (15). The SD-OCT system used in this study demonstrated excellent intra- and inter-observer reliability for manually measuring the NET and CCT. There was no statistically significant difference between replicates for either operator and the CV for each user was below 1%. Comparing inter-observer reliability, there was no significant difference in measurements for NET or CCT between operators and the measurements between users agree almost perfectly (ICC . 0.95). Regarding ET measurements, intra-observer reliability was excellent. There was no statistically significant difference between replicates for either operator and the CV for each user was below 5%. Comparing inter-observer reliability, there was a small but statistically significant difference between users and the ICC was substantially lower than the NET and CCT measurements. Despite the lower ICC values, the width of the LoA for ET was narrow, with 95% of observations from 1 observer likely to fall within 7 mm of the other. This variation demonstrates a clinically acceptable inter-observer reliability for this device and is comparable to that found in human studies (1). Several factors may have contributed to the variability in the manual measurements of corneal thickness obtained in this study, including variation in the location of the measurement from the true corneal vertex and differences in the subjective delineation between corneal layers. Another factor to consider is the increments by which the caliper function measures. In the current format, a
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change of 1 unit of measure using the caliper function equates to a 4-mm difference. For thicker structures such as the NET or CCT, a difference of a single unit of measure between examinations and/or examiners, is clinically insignificant (0.65% of the mean CCT). This difference becomes proportionally larger (5% of the mean ET) for thinner structures, such as the corneal epithelium, however, which may explain the relatively lower reliability of manual measurements found for ET in this study. Further advancements in the measurement algorithm may help reduce this variability. For example, the addition of automated vertex identification, computer-assisted segmentation of corneal layers, and a more detailed caliper with increments of 0.5 mm or less would likely improve consistency between measurements. The primary limitation of the current study was the small number of animals examined. Despite this, however, the results provide evidence that manual corneal thickness measurements with SD-OCT are highly reliable both within and between observers. Although the use of non-sedated animals could be considered a limitation of this study, the results demonstrating clinically acceptable intra- and inter-user reliability would suggest that this was not a significant factor. Additionally, stringent quality control criteria were adhered to, for example, any scan showing evidence of motion artifact, poor corneal vertex, or reduced image quality was rejected and only high quality scans were used for corneal measurements. In summary, this study provides normative data regarding ET, NET, and CCT measurements in healthy canine eyes using a portable SD-OCT device. Furthermore, this device demonstrated excellent intra- and inter-operator repeatability, with clinically acceptable differences in measurements between operators.
Acknowledgments The authors thank Dr. Bruce Barton for his help with statistical analysis. Thanks also to Optovue in Freemont, California, USA for providing the Optovue iVue SD-OCT device on loan.
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