Cornea
Normative Values for Corneal Densitometry Analysis by Scheimpflug Optical Assessment Sorcha N´ı Dhubhghaill,1 Jos J. Rozema,1,2 Sien Jongenelen,1 Irene Ruiz Hidalgo,1 Nadia Zakaria,1,2 and Marie-Jos´e Tassignon2 1 2
Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium Department of Medicine and Health Sciences, Antwerp University, Wilrijk, Belgium
Correspondence: Sorcha N´ı Dhubhghaill, Department of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium; [email protected]. Submitted: September 10, 2013 Accepted: December 2, 2013 Citation: N´ı Dhubhghaill S, Rozema JJ, Jongenelen S, Ruiz Hidalgo I, Zakaria N, Tassignon M-J. Normative values for corneal densitometry analysis by Scheimpflug optical assessment. Invest Ophthalmol Vis Sci. 2014;55:162–168. DOI:10.1167/iovs. 13-13236
PURPOSE. To describe the normative data for corneal Scheimpflug densitometry based on a cohort of normal participants. METHODS. A total of 445 healthy participants were recruited for assessment (794 eyes). Left and right eyes were considered separately. All participants were assessed using the corneal densitometry analysis add-on to the standard software of the Oculus Pentacam. Densitometry measurements were obtained and expressed in standardized grayscale units (GSU). RESULTS. All participants were Caucasian; 42% were male and 58% were female. The mean age was 48.0 6 15.3 years (range, 20.2–84.2 years). Mean corneal densitometry over the 12-mmdiameter area was 19.74 6 3.89 GSU. When divided by radial zone, densitometry values were lowest in the central zone (16.76 6 1.87 GSU) and highest in the periphery (27.36 6 7.47 GSU). There was no difference between central zone and the surrounding 2- to 6-mm annulus (P > 0.05), though the 6- to 10-mm and the 10- to 12-mm zones displayed higher densitometry values (P < 0.001). When divided by depth, the anterior layer displayed the highest densitometry reading of 25.81 6 5.14 GSU, which was significantly higher than that of both the central (P < 0.001) and the posterior layers (P < 0.001). Changes in corneal densitometry were correlated with age, though not within the central 6-mm-diameter ring. No sex difference was seen within the cohort. CONCLUSIONS. This add-on to the standard imaging software allows rapid and objective assessment of the corneal densitometry. We provide normative data that may be used as a reference facilitating research and complementing clinical examination. Keywords: cornea, Pentacam, Scheimpflug, densitometry
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oss of clarity is the cornea’s natural response to a wide range of pathological insults, such as infections, corneal dystrophies, and degeneration. Assessment and monitoring of the resulting corneal haze is therefore an essential element of ophthalmological examination. Clinically, this is frequently performed as a standard slit-lamp examination with documentation of findings, which may be augmented with a descriptive scale of severity.1 These methods, even when supplemented with photography, are subject to variations between observers. The development of an objective and reproducible method of measuring corneal haze would therefore provide a more reliable means of monitoring pathology and interventions. Corneal haze affects the transmission of light through the cornea, resulting in backward scattering of light (i.e., toward the observer), and has been assessed using a number of techniques.2–6 Using such techniques to augment standard clinical assessment, higher levels of backscatter have been detected even in corneas that were considered clinically clear,7 indicating the potential for objective measurements to surpass slit-lamp examinations. The use of Scheimpflug imaging was first reported by Smith et al.,8 although at the time the applications were limited.9,10 Modern devices employ charge-coupled device (CCD) chips that facilitate rapid data acquisition and analysis.11 Noninvasive Scheimpflug analysis of the anterior segment simultaneously Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783
detects backscattered light, from which a deeper optical analysis can be performed to generate maps of corneal topography, pachymetry, and anterior chamber depth. It is also possible to compose a map of the amount of backscattered light in the different regions of the cornea, called a corneal densitometry map. In practice, corneal densitometry has been described in infectious keratitis,12,13 corneal dystrophies,14,15 keratoconus,16–18 and post-LASIK19,20 and corneal graft surgeries.21–24 Assessment protocols vary, however, so a direct comparison is not feasible. The aim of this study was to describe the standardized Scheimpflug densitometry analysis add-on to the latest software of the Oculus Pentacam. Furthermore, normative values for corneal densitometry are provided, based on measurements over a large group of Caucasian participants.
PATIENTS
AND
METHODS
Participants Four hundred forty-five patients were recruited into the study in the Antwerp University Hospital, and both eyes were assessed where possible. Participants did not report a history of corneal pathology and were not using topical drops that would impair corneal function. The exclusion criteria included 162
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FIGURE 1. Screen data output of the Scheimpflug optical densitometry assessment.
any corneal scarring pathology such as infections, dystrophies, trauma, and ectatic conditions like keratoconus. Any previous history of cornea or intraocular surgery was also considered grounds for exclusion. Peripheral limbal corneal degenerations that are associated with aging were not considered exclusion criteria. Systemic conditions that were considered exclusion criteria were diabetes mellitus, multiple sclerosis, and uncontrolled hypertension. Patients were not excluded on the basis of systemic medications unless they were known to induce corneal changes. Only participants over the age of 20 were included in the data analysis. The study protocol was reviewed and approved by the institutional ethics committee (ref 10/36/ 241). Informed consent was obtained, and the research was
conducted according to the tenets of the Declaration of Helsinki.
Measurement Technique The corneal densitometry analysis is provided as an add-on to the standard software of the Pentacam Scheimpflug device (Oculus Optikger¨ate GmbH, Wetzlar, Germany). The measurement protocol takes a series of 25 images (1003 3 520 pixels) over different meridians with a uniform blue light source. The acquisition protocol takes approximately 2 seconds to complete. The study was performed in a windowless clinical assessment room with a uniform ambient light level of 4 lux as
TABLE 1. Repeatability and Reproducibility Based on Three Repeated Measurements of Eight Right Eyes by Two Operators 0–2 mm
2–6 mm
6–10 mm
10–12 mm
Total
0.65 (2.8) 0.35 (1.5)
0.42 (2.0) 0.14 (0.7)
0.87 (3.3) 0.10 (0.4)
3.33 (9.3) 0.72 (2.0)
0.93 (3.6) 0.31 (1.2)
0.31 (2.1) 0.15 (1.0)
0.24 (1.8) 0.10 (0.7)
0.51 (2.6) 0.17 (0.8)
1.29 (5.2) 0.71 (2.9)
0.43 (2.5) 0.19 (1.1)
0.39 (3.0) 0.06 (0.4)
0.26 (2.2) 0.16 (1.3)
0.43 (2.4) 0.16 (0.9)
0.95 (4.4) 0.67 (3.1)
0.40 (2.6) 0.17 (1.1)
0.36 (2.1) 0.22 (1.3)
0.29 (1.8) 0.08 (0.5)
0.60 (2.8) 0.12 (0.5)
1.76 (6.4) 0.75 (2.7)
0.57 (2.9) 0.22 (1.1)
Anterior Repeat Reprod Center Repeat Reprod Posterior Repeat Reprod Total Repeat Reprod
Values within parentheses represent the corresponding percentages of the values.
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TABLE 2. Mean and Standard Deviation Values of Corneal Densitometry in 395 Right Eyes (OD) and 399 Left (OS) Eyes and Pearson Correlation (Corr) Between the Two Eyes 0–2 mm
2–6 mm
6–10 mm
10–12 mm
Total
22.87 6 2.91 22.85 6 2.90 0.963
21.32 6 2.80 21.21 6 2.70 0.965
26.89 6 8.68 26.37 6 8.67 0.967
35.69 6 11.22 37.18 6 11.96 0.829
25.81 6 5.14 25.78 6 5.03 0.949
14.63 6 1.62 14.59 6 1.57 0.954
13.83 6 1.80 13.69 6 1.69 0.968
19.68 6 6.78 19.14 6 6.75 0.972
24.64 6 6.68 24.90 6 6.50 0.835
17.70 6 3.74 17.47 6 3.60 0.958
12.77 6 1.46 12.66 6 1.46 0.881
12.27 6 1.65 12.03 6 1.55 0.916
17.62 6 5.13 16.92 6 5.10 0.968
21.75 6 5.74 22.08 6 5.57 0.867
15.70 6 3.10 15.39 6 2.99 0.947
16.76 6 1.87 16.70 6 1.84 0.952
15.81 6 1.97 15.64 6 1.86 0.960
21.39 6 6.76 20.81 6 6.74 0.971
27.36 6 7.47 28.05 6 7.51 0.844
19.74 6 3.89 19.55 6 3.76 0.955
Anterior OD OS Corr Center OD OS Corr Posterior OD OS Corr Total OD OS Corr
All values represented in grayscale units.
measured by a luxmeter (ISO-Tech; RS Components, Corby, UK). In the analysis, the program automatically locates the corneal apex and analyzes an area around it with a diameter of 12 mm. The output is expressed in grayscale units (GSU). The GSU scale is calibrated by proprietary software, which defines a minimum light scatter of 0 (maximum transparency) and maximum light scatter of 100 (minimum transparency). For the purposes of local densitometry analysis, the 12-mmdiameter area is subdivided into four concentric radial zones. The first, central zone is 2 mm in diameter and centered on the apex. The second zone is an annulus extending from 2 mm to a 6-mm-diameter circle. The third zone annulus extends from 6 to 10 mm with the final zone extending from 10 mm to a 12mm-diameter circle. These topographical zones are available
preset in the software. The output can also be subdivided based on corneal depth into anterior, central, and posterior layers rather than based on specific anatomical layers of the cornea; the anterior layer corresponds to the anterior 120 lm, and the posterior layer to the most posterior 60 lm of the cornea. The central corneal layer has no fixed thickness but is defined by subtraction of the anterior and posterior layers from the total thickness. Furthermore, the layer densitometry may be calculated for each of the concentric radial zones, which allows more detailed analysis based on both area and depth. The reproducibility and repeatability assessments were performed on eight participants who underwent the measurement protocol three times by two different examiners.
FIGURE 2. Corneal densitometry measurements subdivided by (A) surface area and (B) corneal layer; *** refers to a statistical significance of P < 0.001.
Corneal Densitometry in the Normal Population
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FIGURE 3. Corneal densitometry based on topographic area plotted against age in (A) the central 2 mm, (B) 2 mm to 6 mm, (C) 6 mm to 10 mm, and (D) 10 mm to 12 mm.
Statistical Processing Data were analyzed using Excel 2008 for Macintosh (version 12.1.0; Microsoft, Redmond, WA) and GraphPad Prism 4 Statistics for Macintosh (GraphPad, Inc., La Jolla, CA) using standard repeatability, reproducibility, t-test, one-way ANOVA, and Pearson’s correlation coefficient (version 4.0b; GraphPad, Inc.). Sample size and power calculations were performed using PASS (version 11; NCSS, Inc., Kaysville, UT), and linear regression modeling required SPSS (version 19; IBM Corp., New York, NY). Results were subjected to a Bonferroni correction to account for a inflation. As densitometry values between right and left eyes of a subject are highly correlated, eyes of the same subject were treated separately and not combined for statistical analysis. In the Results section we report the findings for right eyes only except where otherwise stated.
RESULTS Patient Demographics This study included 445 healthy participants, resulting in data for 794 eyes (395 right eyes, 399 left eyes). All participants were Caucasian; 42% were male and 58% were female. The mean age was 48.0 6 16.7 years (range, 20.2–84.2 years). The
mean age of men was 50.5 6 15.65 years and of women was 44.98 6 15.06 years. Based on predicted standard deviation of 4, the sample size of 395 eyes results in a mean with a 95% confidence interval width of 0.4, indicating an adequate sample size.
Corneal Densitometry An example of the data output for a single densitometry assessment is shown in Figure 1. The results for the repeatability (i.e., within subject) and reproducibility (i.e., between examiners) are summarized in Table 1. The average repeatability was 3.3 6 1.8%, while the average reproducibility was 1.3% 6 0.8%. The lowest degree of repeatability was detected in the 10- to 12-mm limbal region (6.4%), particularly in the anterior layer (9.3%). The lowest reproducibility was also found in the 10- to 12-mm limbal region, but at the posterior layer (3.1%). Mean densitometry values for the right eyes (n ¼ 395) and left eyes (n ¼ 399) are presented in Table 2, along with the Pearson correlation between eyes of the same subject. Right and left eye densitometry values were highly correlated (all Pearson correlation coefficients were higher than 0.851), so right and left eyes were analyzed and processed separately to avoid artificially reduced standard deviations.
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FIGURE 4. Corneal densitometry based on layer plotted against age.
The mean corneal densitometry over the entire 12-mmdiameter area was 19.74 6 3.83 GSU (right eyes) and 19.55 6 3.76 GSU (left eyes) (Table 1). When considered by radial zone, densitometry values were lowest in the central zone (16.76 6 1.87 OD) and highest in the periphery (27.36 6 7.47 OD) (Fig. 2A). There was no significant difference between the densitometry values of the central 2-mm zone and the surrounding 2- to 6-mm annulus (one-way ANOVA, P > 0.05), though values for the 6- to 10- and the 10- to 12-mm zones were significantly higher (P < 0.001). When densitometry values are
divided by depth, the anterior layer displayed the highest degree of backscatter, 25.81 6 5.14 OD GSU, which was significantly higher than for both the central (P < 0.001) and the posterior layers (P < 0.001; Fig. 2B).
Corneal Densitometry in Relation to Age Total corneal densitometry (right eye) was significantly correlated with age (Pearson r ¼ 0.560, P < 0.0001). The relationship between corneal densitometries and age is
TABLE 3. Corneal Densitometry Values in 389 Right Eyes Divided by 10-Year Age Group Increments
Number Mean age, y SD, y 0–2 mm 2–6 mm 6–10 mm 10–12 mm Anterior layer Center layer Posterior layer Total anterior to posterior, 0–12 mm
20–30 y
30–40 y
40–50 y
50–60 y
60–70 y
70–80 y
65 25.6 2.23 16.6 6 1.78 14.9 6 1.61 14.6 6 2.08 20.9 6 4.29 21.6 6 2.61 14.2 6 1.66 12.6 6 1.51 16.1 6 1.82
51 34.1 2.83 16.9 6 1.87 15.4 6 1.78 17.5 6 1.79 24.6 6 6.06 23.8 6 4.06 15.8 6 2.50 13.9 6 2.09 17.9 6 2.77
93 45.5 2.80 17.2 6 1.95 16.0 6 1.89 20.3 6 3.78 27.0 6 5.3 25.6 6 3.69 17.38 6 2.42 15.5 6 2.11 19.5 6 2.60
78 54.1 2.86 16.4 6 1.77 15.8 6 1.86 23.3 6 6.35 29.8 6 8.42 26.7 6 5.31 18.7 6 3.85 16.7 6 3.07 20.7 6 3.99
77 64.2 2.76 16.5 6 1.83 16.1 6 2.11 25.9 6 6.59 30.9 6 7.37 28.3 6 5.39 19.8 6 3.62 17.5 6 2.98 21.9 6 3.87
25 73.5 2.83 16.9 6 1.87 17.0 6 2.55 29.7 6 7.83 31.9 6 7.67 30.5 6 6.46 21.7 6 4.36 18.7 6 3.15 23.6 6 4.47
All densitometry values represented in grayscale units.
Corneal Densitometry in the Normal Population displayed in Figures 3 and 4. There was no significant correlation with age within the central 2-mm area (Pearson r ¼ 0.031, P ¼ 0.540), though correlation with age increased progressively as the zones moved toward the limbus. A statistically significant but minor increase in densitometry in the 2- to 6-mm region was observed (r ¼ 0.224, P < 0.0001). The 6- to 10-mm annulus and the 10- to 12-mm annulus were significantly correlated with age, with higher increases observed (r ¼ 0.655, P < 0.001 and r ¼ 0.467, P < 0.001, respectively). When subdivided by layer, all three layers were significantly correlated with age, by r ¼ 0.484 (P < 0.001), r ¼ 0.584 (P < 0.001), and r ¼ 0.600 (P < 0.001) for the anterior, central, and posterior layers, respectively. Corneal densitometry values based on age range are shown in Table 3.
Corneal Densitometry in Relation to Sex There was a significantly higher proportion of women than of men in the cohort, particularly in the category of subjects older than 60 years. To adjust for this and for the effect of age on densitometry previously noted, a linear regression model was constructed to determine the effect of sex. The regression model was performed on all right eye densitometry values. After correction for age, there was no significant influence of sex on densitometry (P ¼ 0.975).
DISCUSSION In this study we present a normative database for corneal Scheimpflug densitometry values in the largest cohort of normal participants yet reported. The challenge posed by a new assessment modality is to ensure a standardized protocol that facilitates comparison between published data and those generated in the clinical setting. Previous reports have described the backscatter profiles of small specific points,6 individual layers,3 and surface areas varying from 4 to 10 mm in diameter.8,12,16 This is the first paper to report normal backscatter values for all of these corneal parameters out to a diameter of 12 mm. Corneal densitometry was found to be lowest in the central 6 mm, increasing significantly as it reached the limbus, consistent with clinical observations. When divided by layer, the anterior 120 lm showed the highest degree of backscatter, with the lowest backscatter occurring at the posterior 60 lm. This is contrary to confocal densitometry analyses, where the highest backscatter occurs in the posterior layers.6 This discrepancy is in part due to the noncontact nature of the Scheimpflug analysis, resulting in greater reflection at the interfaces between layers with different refractive index, for example, the corneal endothelium, Bowman’s membrane, and the air–cornea interface. In confocal microscopy, on the other hand, the reflection at the air–cornea interface is eliminated by the use of contact gels. A second reason for the difference in signal intensity at the endothelial layer between Scheimpflug and confocal microscopy is differences in illumination and image acquisition. Scheimpflug systems illuminate the cornea perpendicularly and analyze the corneal cross section from an angle of 6458. Confocal microscopy, on the other hand, both illuminates and images the cornea perpendicularly, which results in higher amounts of specularly reflected light than with Scheimpflug. The layered structure of the cornea itself may also account for the differences in reflection, as the organization of the corneal lamellae displays a weaving pattern in the anterior stroma while the posterior stroma shows a higher degree of organization with lamellae lying regularly in the plane of the cornea.25 The density of keratocytes in the anterior cornea is also higher than in the central and posterior cornea.26
IOVS j January 2014 j Vol. 55 j No. 1 j 167 The earliest report of corneal backscatter assessments concluded that there was a significant increase in corneal density with age.2 These findings have been both contradicted3,8,12 and supported6 by subsequent reports. Both observations may in fact be correct, as we noted that while there was no age-related change in the central 6 mm of the cornea, a clear increase is seen in the zone adjacent to the limbus. This increase in densitometry is most significant in the 6- to 10-mm zone, and likely represents the development of age-related corneal limbal degenerations such as farinata, arcus senilus/ lipoides, crocodile shagreen, Vogt’s white limbal girdle, and Hassal-Henle bodies, all of which have been characterized clinically but have no significant effect on the patient’s visual experience.27,28 These limbal-based degenerative conditions are so benign and common in the elderly population that they were not considered exclusion criteria. It is important to reiterate that the values presented for the 10- to 12-mm zone must be interpreted with caution, as repeatability and reproducibility in this region were the weakest (repeatability 6.4%, reproducibility 2.7%). Normal variations in the white-to-white corneal diameter29 mean that some participants have corneas smaller than the 12-mm analysis limit. Portions of the limbus and sclera can therefore be included in the assessment of the outermost zone, resulting in higher backscatter values. Even small changes in the position of the limbus can have a significant effect on the 10to 12-mm densitometry results, though this does not occur in the more central annuli. Due to this effect, the significant increase in scatter with age in this 10- to 12-mm region cannot be definitively interpreted nor directly correlated with aging. Hillenaar et al.6 also reported a sex difference of 3.5% in corneal backscatter by in vivo confocal microscopy. That observation was not reproduced in this cohort nor in a cohort assessed by scatterometry.3 Clinically, the added value of Scheimpflug densitometry is in the generation of a rapid quantitative value for corneal backscatter that may be compared with this normative cohort. There are a number of limitations, however. The relationship between corneal backscatter and forward scatter (i.e., toward the retina) is not clear,30 and an increased corneal densitometry does not necessarily correlate with a reduction in visual quality. Scheimpflug optical densitometry is also not intended as a substitute for in vivo confocal microscopy, which provides a degree of magnification and resolution not paralleled by the Scheimpflug device, albeit for only 0.14% of the total cornea.6 In addition, these facilities and the hardware are not widely available. Scheimpflug optical densitometry devices, on the other hand, are widespread across corneal clinics, and the addition of the densitometry analysis protocol does not lengthen duration of examination. In conclusion, we present an automated systematic means of rapidly assessing corneal backscatter using a Scheimpflug device and describe corneal densitometry values for the largest cohort of normal corneas yet reported. There was a significant increase in corneal densitometry with age, though this was confined to the peripheral cornea. These values for corneal densitometry measurements, including subdivisions based on surface area and layer, may provide a standardized platform for further studies and facilitate greater use of this analysis in clinical practice.
Acknowledgments The authors thank Kristein Wouters for her assistance in the statistical analysis for this study. Supported in part by an unrestricted grant from the Flemish Agency for Innovation by Science and Technology (IWT-TBM 110684). The Oculus Corporation provided a small hardware grant
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Corneal Densitometry in the Normal Population to acquire the data. The authors alone are responsible for the content and writing of the paper. Disclosure: S. N´ı Dhubhghaill, Oculus Optikger¨ate GmbH (F); J.J. Rozema, Oculus Optikger¨ate GmbH (F); S. Jongenelen, Oculus Optikger¨ate GmbH (F); I. Ruiz Hidalgo, Oculus Optikger¨ate GmbH (F); N. Zakaria, Oculus Optikger¨ate GmbH (F); M.-J. Tassignon, Oculus Optikger¨ate GmbH (F)
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Normative Values for Corneal Densitometry Analysis by Scheimpflug Optical Assessment Sorcha N´ı Dhubhghaill,1 Jos J. Rozema,1,2 Sien Jongenelen,1 Irene Ruiz Hidalgo,1 Nadia Zakaria,1,2 and Marie-Jos´e Tassignon2 1 2
Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium Department of Medicine and Health Sciences, Antwerp University, Wilrijk, Belgium
Correspondence: Sorcha N´ı Dhubhghaill, Department of Ophthalmology, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium; [email protected]. Submitted: September 10, 2013 Accepted: December 2, 2013 Citation: N´ı Dhubhghaill S, Rozema JJ, Jongenelen S, Ruiz Hidalgo I, Zakaria N, Tassignon M-J. Normative values for corneal densitometry analysis by Scheimpflug optical assessment. Invest Ophthalmol Vis Sci. 2014;55:162–168. DOI:10.1167/iovs. 13-13236
PURPOSE. To describe the normative data for corneal Scheimpflug densitometry based on a cohort of normal participants. METHODS. A total of 445 healthy participants were recruited for assessment (794 eyes). Left and right eyes were considered separately. All participants were assessed using the corneal densitometry analysis add-on to the standard software of the Oculus Pentacam. Densitometry measurements were obtained and expressed in standardized grayscale units (GSU). RESULTS. All participants were Caucasian; 42% were male and 58% were female. The mean age was 48.0 6 15.3 years (range, 20.2–84.2 years). Mean corneal densitometry over the 12-mmdiameter area was 19.74 6 3.89 GSU. When divided by radial zone, densitometry values were lowest in the central zone (16.76 6 1.87 GSU) and highest in the periphery (27.36 6 7.47 GSU). There was no difference between central zone and the surrounding 2- to 6-mm annulus (P > 0.05), though the 6- to 10-mm and the 10- to 12-mm zones displayed higher densitometry values (P < 0.001). When divided by depth, the anterior layer displayed the highest densitometry reading of 25.81 6 5.14 GSU, which was significantly higher than that of both the central (P < 0.001) and the posterior layers (P < 0.001). Changes in corneal densitometry were correlated with age, though not within the central 6-mm-diameter ring. No sex difference was seen within the cohort. CONCLUSIONS. This add-on to the standard imaging software allows rapid and objective assessment of the corneal densitometry. We provide normative data that may be used as a reference facilitating research and complementing clinical examination. Keywords: cornea, Pentacam, Scheimpflug, densitometry
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oss of clarity is the cornea’s natural response to a wide range of pathological insults, such as infections, corneal dystrophies, and degeneration. Assessment and monitoring of the resulting corneal haze is therefore an essential element of ophthalmological examination. Clinically, this is frequently performed as a standard slit-lamp examination with documentation of findings, which may be augmented with a descriptive scale of severity.1 These methods, even when supplemented with photography, are subject to variations between observers. The development of an objective and reproducible method of measuring corneal haze would therefore provide a more reliable means of monitoring pathology and interventions. Corneal haze affects the transmission of light through the cornea, resulting in backward scattering of light (i.e., toward the observer), and has been assessed using a number of techniques.2–6 Using such techniques to augment standard clinical assessment, higher levels of backscatter have been detected even in corneas that were considered clinically clear,7 indicating the potential for objective measurements to surpass slit-lamp examinations. The use of Scheimpflug imaging was first reported by Smith et al.,8 although at the time the applications were limited.9,10 Modern devices employ charge-coupled device (CCD) chips that facilitate rapid data acquisition and analysis.11 Noninvasive Scheimpflug analysis of the anterior segment simultaneously Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783
detects backscattered light, from which a deeper optical analysis can be performed to generate maps of corneal topography, pachymetry, and anterior chamber depth. It is also possible to compose a map of the amount of backscattered light in the different regions of the cornea, called a corneal densitometry map. In practice, corneal densitometry has been described in infectious keratitis,12,13 corneal dystrophies,14,15 keratoconus,16–18 and post-LASIK19,20 and corneal graft surgeries.21–24 Assessment protocols vary, however, so a direct comparison is not feasible. The aim of this study was to describe the standardized Scheimpflug densitometry analysis add-on to the latest software of the Oculus Pentacam. Furthermore, normative values for corneal densitometry are provided, based on measurements over a large group of Caucasian participants.
PATIENTS
AND
METHODS
Participants Four hundred forty-five patients were recruited into the study in the Antwerp University Hospital, and both eyes were assessed where possible. Participants did not report a history of corneal pathology and were not using topical drops that would impair corneal function. The exclusion criteria included 162
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FIGURE 1. Screen data output of the Scheimpflug optical densitometry assessment.
any corneal scarring pathology such as infections, dystrophies, trauma, and ectatic conditions like keratoconus. Any previous history of cornea or intraocular surgery was also considered grounds for exclusion. Peripheral limbal corneal degenerations that are associated with aging were not considered exclusion criteria. Systemic conditions that were considered exclusion criteria were diabetes mellitus, multiple sclerosis, and uncontrolled hypertension. Patients were not excluded on the basis of systemic medications unless they were known to induce corneal changes. Only participants over the age of 20 were included in the data analysis. The study protocol was reviewed and approved by the institutional ethics committee (ref 10/36/ 241). Informed consent was obtained, and the research was
conducted according to the tenets of the Declaration of Helsinki.
Measurement Technique The corneal densitometry analysis is provided as an add-on to the standard software of the Pentacam Scheimpflug device (Oculus Optikger¨ate GmbH, Wetzlar, Germany). The measurement protocol takes a series of 25 images (1003 3 520 pixels) over different meridians with a uniform blue light source. The acquisition protocol takes approximately 2 seconds to complete. The study was performed in a windowless clinical assessment room with a uniform ambient light level of 4 lux as
TABLE 1. Repeatability and Reproducibility Based on Three Repeated Measurements of Eight Right Eyes by Two Operators 0–2 mm
2–6 mm
6–10 mm
10–12 mm
Total
0.65 (2.8) 0.35 (1.5)
0.42 (2.0) 0.14 (0.7)
0.87 (3.3) 0.10 (0.4)
3.33 (9.3) 0.72 (2.0)
0.93 (3.6) 0.31 (1.2)
0.31 (2.1) 0.15 (1.0)
0.24 (1.8) 0.10 (0.7)
0.51 (2.6) 0.17 (0.8)
1.29 (5.2) 0.71 (2.9)
0.43 (2.5) 0.19 (1.1)
0.39 (3.0) 0.06 (0.4)
0.26 (2.2) 0.16 (1.3)
0.43 (2.4) 0.16 (0.9)
0.95 (4.4) 0.67 (3.1)
0.40 (2.6) 0.17 (1.1)
0.36 (2.1) 0.22 (1.3)
0.29 (1.8) 0.08 (0.5)
0.60 (2.8) 0.12 (0.5)
1.76 (6.4) 0.75 (2.7)
0.57 (2.9) 0.22 (1.1)
Anterior Repeat Reprod Center Repeat Reprod Posterior Repeat Reprod Total Repeat Reprod
Values within parentheses represent the corresponding percentages of the values.
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TABLE 2. Mean and Standard Deviation Values of Corneal Densitometry in 395 Right Eyes (OD) and 399 Left (OS) Eyes and Pearson Correlation (Corr) Between the Two Eyes 0–2 mm
2–6 mm
6–10 mm
10–12 mm
Total
22.87 6 2.91 22.85 6 2.90 0.963
21.32 6 2.80 21.21 6 2.70 0.965
26.89 6 8.68 26.37 6 8.67 0.967
35.69 6 11.22 37.18 6 11.96 0.829
25.81 6 5.14 25.78 6 5.03 0.949
14.63 6 1.62 14.59 6 1.57 0.954
13.83 6 1.80 13.69 6 1.69 0.968
19.68 6 6.78 19.14 6 6.75 0.972
24.64 6 6.68 24.90 6 6.50 0.835
17.70 6 3.74 17.47 6 3.60 0.958
12.77 6 1.46 12.66 6 1.46 0.881
12.27 6 1.65 12.03 6 1.55 0.916
17.62 6 5.13 16.92 6 5.10 0.968
21.75 6 5.74 22.08 6 5.57 0.867
15.70 6 3.10 15.39 6 2.99 0.947
16.76 6 1.87 16.70 6 1.84 0.952
15.81 6 1.97 15.64 6 1.86 0.960
21.39 6 6.76 20.81 6 6.74 0.971
27.36 6 7.47 28.05 6 7.51 0.844
19.74 6 3.89 19.55 6 3.76 0.955
Anterior OD OS Corr Center OD OS Corr Posterior OD OS Corr Total OD OS Corr
All values represented in grayscale units.
measured by a luxmeter (ISO-Tech; RS Components, Corby, UK). In the analysis, the program automatically locates the corneal apex and analyzes an area around it with a diameter of 12 mm. The output is expressed in grayscale units (GSU). The GSU scale is calibrated by proprietary software, which defines a minimum light scatter of 0 (maximum transparency) and maximum light scatter of 100 (minimum transparency). For the purposes of local densitometry analysis, the 12-mmdiameter area is subdivided into four concentric radial zones. The first, central zone is 2 mm in diameter and centered on the apex. The second zone is an annulus extending from 2 mm to a 6-mm-diameter circle. The third zone annulus extends from 6 to 10 mm with the final zone extending from 10 mm to a 12mm-diameter circle. These topographical zones are available
preset in the software. The output can also be subdivided based on corneal depth into anterior, central, and posterior layers rather than based on specific anatomical layers of the cornea; the anterior layer corresponds to the anterior 120 lm, and the posterior layer to the most posterior 60 lm of the cornea. The central corneal layer has no fixed thickness but is defined by subtraction of the anterior and posterior layers from the total thickness. Furthermore, the layer densitometry may be calculated for each of the concentric radial zones, which allows more detailed analysis based on both area and depth. The reproducibility and repeatability assessments were performed on eight participants who underwent the measurement protocol three times by two different examiners.
FIGURE 2. Corneal densitometry measurements subdivided by (A) surface area and (B) corneal layer; *** refers to a statistical significance of P < 0.001.
Corneal Densitometry in the Normal Population
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FIGURE 3. Corneal densitometry based on topographic area plotted against age in (A) the central 2 mm, (B) 2 mm to 6 mm, (C) 6 mm to 10 mm, and (D) 10 mm to 12 mm.
Statistical Processing Data were analyzed using Excel 2008 for Macintosh (version 12.1.0; Microsoft, Redmond, WA) and GraphPad Prism 4 Statistics for Macintosh (GraphPad, Inc., La Jolla, CA) using standard repeatability, reproducibility, t-test, one-way ANOVA, and Pearson’s correlation coefficient (version 4.0b; GraphPad, Inc.). Sample size and power calculations were performed using PASS (version 11; NCSS, Inc., Kaysville, UT), and linear regression modeling required SPSS (version 19; IBM Corp., New York, NY). Results were subjected to a Bonferroni correction to account for a inflation. As densitometry values between right and left eyes of a subject are highly correlated, eyes of the same subject were treated separately and not combined for statistical analysis. In the Results section we report the findings for right eyes only except where otherwise stated.
RESULTS Patient Demographics This study included 445 healthy participants, resulting in data for 794 eyes (395 right eyes, 399 left eyes). All participants were Caucasian; 42% were male and 58% were female. The mean age was 48.0 6 16.7 years (range, 20.2–84.2 years). The
mean age of men was 50.5 6 15.65 years and of women was 44.98 6 15.06 years. Based on predicted standard deviation of 4, the sample size of 395 eyes results in a mean with a 95% confidence interval width of 0.4, indicating an adequate sample size.
Corneal Densitometry An example of the data output for a single densitometry assessment is shown in Figure 1. The results for the repeatability (i.e., within subject) and reproducibility (i.e., between examiners) are summarized in Table 1. The average repeatability was 3.3 6 1.8%, while the average reproducibility was 1.3% 6 0.8%. The lowest degree of repeatability was detected in the 10- to 12-mm limbal region (6.4%), particularly in the anterior layer (9.3%). The lowest reproducibility was also found in the 10- to 12-mm limbal region, but at the posterior layer (3.1%). Mean densitometry values for the right eyes (n ¼ 395) and left eyes (n ¼ 399) are presented in Table 2, along with the Pearson correlation between eyes of the same subject. Right and left eye densitometry values were highly correlated (all Pearson correlation coefficients were higher than 0.851), so right and left eyes were analyzed and processed separately to avoid artificially reduced standard deviations.
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FIGURE 4. Corneal densitometry based on layer plotted against age.
The mean corneal densitometry over the entire 12-mmdiameter area was 19.74 6 3.83 GSU (right eyes) and 19.55 6 3.76 GSU (left eyes) (Table 1). When considered by radial zone, densitometry values were lowest in the central zone (16.76 6 1.87 OD) and highest in the periphery (27.36 6 7.47 OD) (Fig. 2A). There was no significant difference between the densitometry values of the central 2-mm zone and the surrounding 2- to 6-mm annulus (one-way ANOVA, P > 0.05), though values for the 6- to 10- and the 10- to 12-mm zones were significantly higher (P < 0.001). When densitometry values are
divided by depth, the anterior layer displayed the highest degree of backscatter, 25.81 6 5.14 OD GSU, which was significantly higher than for both the central (P < 0.001) and the posterior layers (P < 0.001; Fig. 2B).
Corneal Densitometry in Relation to Age Total corneal densitometry (right eye) was significantly correlated with age (Pearson r ¼ 0.560, P < 0.0001). The relationship between corneal densitometries and age is
TABLE 3. Corneal Densitometry Values in 389 Right Eyes Divided by 10-Year Age Group Increments
Number Mean age, y SD, y 0–2 mm 2–6 mm 6–10 mm 10–12 mm Anterior layer Center layer Posterior layer Total anterior to posterior, 0–12 mm
20–30 y
30–40 y
40–50 y
50–60 y
60–70 y
70–80 y
65 25.6 2.23 16.6 6 1.78 14.9 6 1.61 14.6 6 2.08 20.9 6 4.29 21.6 6 2.61 14.2 6 1.66 12.6 6 1.51 16.1 6 1.82
51 34.1 2.83 16.9 6 1.87 15.4 6 1.78 17.5 6 1.79 24.6 6 6.06 23.8 6 4.06 15.8 6 2.50 13.9 6 2.09 17.9 6 2.77
93 45.5 2.80 17.2 6 1.95 16.0 6 1.89 20.3 6 3.78 27.0 6 5.3 25.6 6 3.69 17.38 6 2.42 15.5 6 2.11 19.5 6 2.60
78 54.1 2.86 16.4 6 1.77 15.8 6 1.86 23.3 6 6.35 29.8 6 8.42 26.7 6 5.31 18.7 6 3.85 16.7 6 3.07 20.7 6 3.99
77 64.2 2.76 16.5 6 1.83 16.1 6 2.11 25.9 6 6.59 30.9 6 7.37 28.3 6 5.39 19.8 6 3.62 17.5 6 2.98 21.9 6 3.87
25 73.5 2.83 16.9 6 1.87 17.0 6 2.55 29.7 6 7.83 31.9 6 7.67 30.5 6 6.46 21.7 6 4.36 18.7 6 3.15 23.6 6 4.47
All densitometry values represented in grayscale units.
Corneal Densitometry in the Normal Population displayed in Figures 3 and 4. There was no significant correlation with age within the central 2-mm area (Pearson r ¼ 0.031, P ¼ 0.540), though correlation with age increased progressively as the zones moved toward the limbus. A statistically significant but minor increase in densitometry in the 2- to 6-mm region was observed (r ¼ 0.224, P < 0.0001). The 6- to 10-mm annulus and the 10- to 12-mm annulus were significantly correlated with age, with higher increases observed (r ¼ 0.655, P < 0.001 and r ¼ 0.467, P < 0.001, respectively). When subdivided by layer, all three layers were significantly correlated with age, by r ¼ 0.484 (P < 0.001), r ¼ 0.584 (P < 0.001), and r ¼ 0.600 (P < 0.001) for the anterior, central, and posterior layers, respectively. Corneal densitometry values based on age range are shown in Table 3.
Corneal Densitometry in Relation to Sex There was a significantly higher proportion of women than of men in the cohort, particularly in the category of subjects older than 60 years. To adjust for this and for the effect of age on densitometry previously noted, a linear regression model was constructed to determine the effect of sex. The regression model was performed on all right eye densitometry values. After correction for age, there was no significant influence of sex on densitometry (P ¼ 0.975).
DISCUSSION In this study we present a normative database for corneal Scheimpflug densitometry values in the largest cohort of normal participants yet reported. The challenge posed by a new assessment modality is to ensure a standardized protocol that facilitates comparison between published data and those generated in the clinical setting. Previous reports have described the backscatter profiles of small specific points,6 individual layers,3 and surface areas varying from 4 to 10 mm in diameter.8,12,16 This is the first paper to report normal backscatter values for all of these corneal parameters out to a diameter of 12 mm. Corneal densitometry was found to be lowest in the central 6 mm, increasing significantly as it reached the limbus, consistent with clinical observations. When divided by layer, the anterior 120 lm showed the highest degree of backscatter, with the lowest backscatter occurring at the posterior 60 lm. This is contrary to confocal densitometry analyses, where the highest backscatter occurs in the posterior layers.6 This discrepancy is in part due to the noncontact nature of the Scheimpflug analysis, resulting in greater reflection at the interfaces between layers with different refractive index, for example, the corneal endothelium, Bowman’s membrane, and the air–cornea interface. In confocal microscopy, on the other hand, the reflection at the air–cornea interface is eliminated by the use of contact gels. A second reason for the difference in signal intensity at the endothelial layer between Scheimpflug and confocal microscopy is differences in illumination and image acquisition. Scheimpflug systems illuminate the cornea perpendicularly and analyze the corneal cross section from an angle of 6458. Confocal microscopy, on the other hand, both illuminates and images the cornea perpendicularly, which results in higher amounts of specularly reflected light than with Scheimpflug. The layered structure of the cornea itself may also account for the differences in reflection, as the organization of the corneal lamellae displays a weaving pattern in the anterior stroma while the posterior stroma shows a higher degree of organization with lamellae lying regularly in the plane of the cornea.25 The density of keratocytes in the anterior cornea is also higher than in the central and posterior cornea.26
IOVS j January 2014 j Vol. 55 j No. 1 j 167 The earliest report of corneal backscatter assessments concluded that there was a significant increase in corneal density with age.2 These findings have been both contradicted3,8,12 and supported6 by subsequent reports. Both observations may in fact be correct, as we noted that while there was no age-related change in the central 6 mm of the cornea, a clear increase is seen in the zone adjacent to the limbus. This increase in densitometry is most significant in the 6- to 10-mm zone, and likely represents the development of age-related corneal limbal degenerations such as farinata, arcus senilus/ lipoides, crocodile shagreen, Vogt’s white limbal girdle, and Hassal-Henle bodies, all of which have been characterized clinically but have no significant effect on the patient’s visual experience.27,28 These limbal-based degenerative conditions are so benign and common in the elderly population that they were not considered exclusion criteria. It is important to reiterate that the values presented for the 10- to 12-mm zone must be interpreted with caution, as repeatability and reproducibility in this region were the weakest (repeatability 6.4%, reproducibility 2.7%). Normal variations in the white-to-white corneal diameter29 mean that some participants have corneas smaller than the 12-mm analysis limit. Portions of the limbus and sclera can therefore be included in the assessment of the outermost zone, resulting in higher backscatter values. Even small changes in the position of the limbus can have a significant effect on the 10to 12-mm densitometry results, though this does not occur in the more central annuli. Due to this effect, the significant increase in scatter with age in this 10- to 12-mm region cannot be definitively interpreted nor directly correlated with aging. Hillenaar et al.6 also reported a sex difference of 3.5% in corneal backscatter by in vivo confocal microscopy. That observation was not reproduced in this cohort nor in a cohort assessed by scatterometry.3 Clinically, the added value of Scheimpflug densitometry is in the generation of a rapid quantitative value for corneal backscatter that may be compared with this normative cohort. There are a number of limitations, however. The relationship between corneal backscatter and forward scatter (i.e., toward the retina) is not clear,30 and an increased corneal densitometry does not necessarily correlate with a reduction in visual quality. Scheimpflug optical densitometry is also not intended as a substitute for in vivo confocal microscopy, which provides a degree of magnification and resolution not paralleled by the Scheimpflug device, albeit for only 0.14% of the total cornea.6 In addition, these facilities and the hardware are not widely available. Scheimpflug optical densitometry devices, on the other hand, are widespread across corneal clinics, and the addition of the densitometry analysis protocol does not lengthen duration of examination. In conclusion, we present an automated systematic means of rapidly assessing corneal backscatter using a Scheimpflug device and describe corneal densitometry values for the largest cohort of normal corneas yet reported. There was a significant increase in corneal densitometry with age, though this was confined to the peripheral cornea. These values for corneal densitometry measurements, including subdivisions based on surface area and layer, may provide a standardized platform for further studies and facilitate greater use of this analysis in clinical practice.
Acknowledgments The authors thank Kristein Wouters for her assistance in the statistical analysis for this study. Supported in part by an unrestricted grant from the Flemish Agency for Innovation by Science and Technology (IWT-TBM 110684). The Oculus Corporation provided a small hardware grant
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Corneal Densitometry in the Normal Population to acquire the data. The authors alone are responsible for the content and writing of the paper. Disclosure: S. N´ı Dhubhghaill, Oculus Optikger¨ate GmbH (F); J.J. Rozema, Oculus Optikger¨ate GmbH (F); S. Jongenelen, Oculus Optikger¨ate GmbH (F); I. Ruiz Hidalgo, Oculus Optikger¨ate GmbH (F); N. Zakaria, Oculus Optikger¨ate GmbH (F); M.-J. Tassignon, Oculus Optikger¨ate GmbH (F)
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